Résumé&en&Français&:&

Rôle& des& lymphocytes& T& gamma& delta& dans& l'induction& de& la& réponse&immunitaire&adaptative&dans&un&contexte&d'infection& par&Plasmodium*falciparum

14!

List&of&Illustrations& Figures:&

for 78% of all deaths. Insecticide resistance, especially to pyrethroids, is widespread in malaria vectors. Of the 65 countries reporting any monitoring data for 2010-2013, 53 reported resistance to at least one insecticide in one malaria vector from one collection site (Figure 3.10), and 41 countries reported resistance to two or more insecticide classes. Pyrethroid resistance was the most commonly reported (Figure 3.9); in 2013, two thirds of the countries monitoring this class reported resistance.

Recommendations of the GPIRM are slowly being implemented at the country level. In 2013, 14 of 63 countries reported the use of multiple insecticides of different classes for IRS. Seven countries that used pyrethroids for IRS in 2011 or 2012 used an alternative class with a different mode of action in 2013. In six cases this change was associated with a decline in IRS coverage, possibly due to the higher cost of the alternative. The GPIRM recommends that country programmes develop long-term plans for insecticide resistance monitoring and management that include full costing of activities. Development of these plans has only just begun: seven countries in the WHO African Region have such plans in place while there has been limited progress in development of plans in other WHO regions.

International initiatives in support of GPIRM have commenced. In 2013, WHO established a Vector Control Advisory Group to facilitate the development of new tools, approaches and technologies, and to shorten the process of bringing these to market. To improve affordability of existing and new tools, initiatives such as pooled procurements, improved global forecasting, long-term contracts and tax incentives are being explored. WHO is also supporting the development of comprehensive regional and national databases on insecticide resistance. Information will be used to inform locally appropriate vector control, guide policy for managing insecticide resistance and facilitate resource mobilization for implementation. Plasmodium-infected Anopheles mosquito inoculates human hosts with sporozoites in the dermis of skin. The sporozoites move into the bloodstream, and parasitize hepatocytes. Invaded sporozoites develop into merozoites with replication (liver-stage). Then, merozoites are released from hepatocytes.

Merozoites parasitize red blood cells (RBCs), replicate, and then rupture the RBCs, leading to new infection of normal RBCs (nRBCs; blood-stage).

Merozoites have several developmental phases: the ring form (early trophozoite), trophozoite, and schizont. Furthermore, some merozoites mature into female and male gametocytes.

related to adaptive immunity. gd T cells play critical roles in protective immune responses against protozoan parasites, bacteria, and viruses that are associated with various infectious diseases (26)(27)(28)(29)(30)(31)(32). This review focuses on the protective abilities of gd T cells and IFN-g in the response against malaria infection.

IFN-g MEDIATES PROTECTIVE IMMUNITY AGAINST BLOOD-STAGE PLASMODIUM PARASITES

Mice (on a C57BL/6 or CBA background) that are genetically IFN-g-deficient or IFN-g receptor (IFN-gR)-deficient or that are treated with anti-IFN-g antibody and infected with blood-stage P. berghei are unable to control the infecting parasite (33)(34)(35). In the cases of infection with blood-stage P. chabaudi and P. yoelii parasites, genetically IFN-g-deficient or IFN-g receptor (IFN-gR)deficient mice or anti-IFN-g antibody-treated mice on a C57BL/6 or CBA background show delayed elimination of the parasites (36)(37)[START_REF] Desakorn | Stage-dependent production and release of histidine-rich protein 2 by Plasmodium falciparum[END_REF][START_REF] Raj | Antibodies to PfSEA-1 block parasite egress from RBCs and protect against malaria infection[END_REF]. These experimental malaria models demonstrate that IFN-g is a key pro-inflammatory cytokine for controlling bloodstage Plasmodium parasites (Table 1). IFN-g is produced by many cell types and involved in many steps of immune responses. ab T cells, NK cells, NKT cells, and gd T cells have been shown to produce IFN-g after infection with Plasmodium parasites. The contributions of producers of IFN-g to protective immunity against Plasmodium parasites are complicated (Figure 2). Thus, more detailed experiments using IFN-g signaling-deficient models should be performed to determine the mechanism(s) underlying the involvement of IFN-g in immune protection against Plasmodium parasites.

IFN-g PRODUCERS AND THEIR ACTIVATION IN PLASMODIUM INFECTION CD4 + ab T CELLS AND CD8 + ab T CELLS

Given that MHC class II-deficient mice on a C57BL/6 background are unable to control P. yoeliiand P. chabaudi-infected RBCs (iRBCs), CD4 + ab T cells respond to Plasmodium-iRBCs after priming with malarial antigens in an MHC class II context on DCs [START_REF] Jauhiainen | Analysis of endogenous ATP analogs and mevalonate pathway metabolites in cancer cell cultures using liquid chromatography-electrospray ionization mass spectrometry[END_REF]. CD4 + ab T cells strongly increase their ability to produce IFN-g after infection with P. berghei (32). High proportions of CD4 + ab T cells from P. falciparum-infected human subjects respond to iRBCs and produce IFN-g, compared to CD4 + ab T cells from naïve human subjects (4). Moreover, some studies have shown that CD4 + ab T cell-depleted C57BL/6 or CBA mice treated with anti-CD4 antibody are unable to control blood-stage P. chabaudi or P. berghei (32,33,[START_REF] Lang | Early activation of human V gamma 9V delta 2 T cell broad cytotoxicity and TNF production by nonpeptidic mycobacterial ligands[END_REF][START_REF] Bozdech | The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum[END_REF]. These lines of evidence Figure&5& :&Proposed&time/exposure]dependent&changes&in&antibody&longevity&and&plasma& cell&composition& [START_REF] Hviid | ! Trying! to! remember:! immunological! B! cell! memory! to! malaria[END_REF].&& Exposure! (arrows)! to! Plasmodium! falciparum parasites! appears! to! result! in! formation! of! adequate!B!cell!memory!and!antibodySsecreting!shortSlived!plasma!cells!(SLPC)!early!on.!As! protective! immunity! is! acquired,! the! establishment! of! increasing! numbers! of! longSlived! plasma!cells!(LLPC)!is!facilitated!(perhaps!as!a!result!of!decreased!intensity!and!frequency!of! malariaSassociated! inflammation),! thereby! reducing! the! reliance! on! SLPC,! and! hence! increasing!the!overall!longevity!of!the!antibody!responses!(wavy!line).

tional antibody-secreting plasma cells and memory B cells. Germinal center-derived plasma cells secrete high-affinity, appropriately class-switched antibodies, whereas the memory B cells encode such antibodies but do not secrete them. The large majority of the germinal center-derived plasma cells following a primary response are also [29]. Furthermore, some studies have measured significant antibody levels in the absence of detectable B cell memory [30], while others have reported durable B cell memory in the absence of circulating antibody [31,32]. Much of this apparent inconsistency is likely explainable by differences in the time-point the responses were measured relative to

AnƟbody level Time

Naive B cell

Key:

Memory B cell SLPC LLPC

TRENDS in Parasitology

Figure 1. Proposed time/exposure-dependent changes in antibody longevity and plasma cell composition. Exposure (arrows) to Plasmodium falciparum parasites appears to result in formation of adequate B cell memory and antibody-secreting short-lived plasma cells (SLPC) early on. As protective immunity is acquired, the establishment of increasing numbers of long-lived plasma cells (LLPC) is facilitated (perhaps as a result of decreased intensity and frequency of malaria-associated inflammation), thereby reducing the reliance on SLPC, and hence increasing the overall longevity of the antibody responses (wavy line).

Human&Vγ9Vδ2&T&cells&

γδ!T!cells!are!T!lymphocytes!with!a!T!cell!receptor!(TCR)!made!up!of!a!gamma!and!a!delta! chain! (as! opposed! to! the! alpha! and! beta! chains! for! αβ! T! cell! TCRs).! They! constitute! a! lymphocyte!minority!in!the!circulating!blood,!but!are!the!majority!in!epithelial!tissues!(lung,! gut! and! genital! tract)! (Bonneville! et! al.! 2010).! They! can! display! both! innate! and! adaptive! characteristics! and! different! subsets! (bearing! different! gamma! and! delta! chain! combinations)!recognise!different!species!of!ligand!(See! Table!1).!The!Vγ9Vδ2!T!cell!subset!is! the!focus!of!this!project,!and!therefore!the!topic!of!this!section.!Vγ9Vδ2!T!cells!make!up!0.5S 10%!or!peripheral!blood!lymphocytes.!They!are!found!only!in!primates!and!recognise!small,! phosphorylated! molecules! called! phosphoantigens! (see! section! 1.

R E L A T I O N S H I P B E T W E E N S T R U C T U R E, A N T I G E N I C I T Y A N D I M M U N O G E N I C I T Y
In order to understand the interaction of antibodies with MSP1 it is important to understand some of the structural features of the molecule that are relevant to the biology of the parasite and how antibodies that bind to MSP1 may interfere with the processes involved.

MSP1 is synthesized from the onset of schizogony (Holder and Freeman, 1982) as a precursor that rapidly associates with MSP7 (Pachebat et al. 2001, 2007) and the complex is transported to the surface of the intracellular parasite where it is retained as a result of its glycosyl phosphatidyl inositol (GPI) anchor. At the end of schizogony merozoite release (or egress) from the infected red blood cell is accompanied by proteolytic processing of the complex by the protease subtilisin 1 (Sub 1) (Koussis et al. 2009). This so-called primary processing produces a complex of polypeptides held together by noncovalent interactions on the surface of the merozoite (Holder et al. 1987 ;Lyon et al. 1987 ;McBride and Heidrich, 1987). MSP1 gives rise to 4 fragments that have been named based on their apparent size in SDS-polyacrylamide gel electrophoresis, an N-terminal 83 kDa fragment (MSP1 83 ), internal 30 and 38 kDa fragments (MSP1 30 and MSP1 38 ) and a C-terminal 42 kDa fragment (MSP1 42 ). The product of a third gene (MSP6) is also part of this complex on the merozoite surface (Trucco et al. 2001) and both MSP7 and MSP6 are also processed. MSP1 is also dimeric (Sanders et al. 2007), an association in part mediated by sequences within MSP1 42 (Babon et al. 2007). The significance of the primary processing of the MSP1 complex is still obscure, although it is possible that it causes conformational changes leading to acquisition or change of function for the protein that is now on the surface of the free merozoite. An even more profound change to the structure of the MSP1 complex occurs at the time of merozoite invasion of red blood cells. The parasite protease subtilisin 2 (Sub2) (Barale et al. 1999 ;Hackett et al. 1999 ;Harris et al. 2005) . As a consequence of this secondary processing MSP1 33 is shed from the surface with the rest of the MSP1 complex, and MSP1 19 is retained by its GPI anchor on the surface of the invading parasite (Blackman et al. 1990). This entire process is summarized in Fig. 1. Very quickly MSP1 19 is then internalized and is the first known marker of the developing food vacuole; interestingly, it remains in the food vacuole for the remainder of the parasite's intracellular development and may have a function in this location (Dluzewski et al. 2008).

M

S P 7 Merozoite Surface Protein 1 complex

Path less traveled

The precise pathway of γδ T cell development remains less well defined, in part because of a failure to identify cell surface markers unique to γδ T cell progenitors. At first glance, that suggests that only late in differentiation do cells committed to the γδ T lineage arise from a common precursor 14 . The cells' predominant CD4 -CD8 -phenotype in the periphery is consistent with their emergence directly from DN cells, contingent on productive rearrangement of genes encoding TCRγ and TCRδ chains. Indeed, in studies of mice in which a 'fate-tracking marker' was used, there was no evidence of maturation of γδ T cells through the DP stage 15 , and γδ T cell differentiation shows no cell-autonomous requirement for either the pre-TCR or any putative 'γδ pre-TCR' 16 . Nonetheless, other studies have shown positive selection for at least some γδ T cells expressing particular TCRγδ heterodimers 17,18 , although unlike conventional αβ T cell selection, it does not involve polymorphic MHC molecules.

Once selected, the first-formed fetal γδ T cells, displaying an almost monoclonal variable (V) γ 5V δ 1 TCR repertoire, emigrate to the skin; the second wave, expressing a closely related V γ 6V δ 1 TCR repertoire, populates the genital tract and tongue; and the third wave (at around birth and thereafter), mostly displaying V γ 7 + or V γ 1 + TCRs, populates the small intestinal epithelium (Fig. 2). Subsequently, many V γ 4 + cells populate the peripheral blood and lymphoid tissues 19 . In humans, γδ T cell development also shows ontogenetic variation in the homing patterns of emigrating thymocytes, albeit to a lesser extent 19 . In mouse skin, homing is partly attributable to the chemokine receptor CCR10, which is upregulated after positive selection in the thymus 17,18 , but the selective tissue-homing patterns are not yet fully understood.

Although selective tissue homing is often cited as a characteristic feature of γδ T cell development, some naive αβ T cells may also localize directly to the epithelia 20,21 (Fig. 2). Thus, the criteria by which γδ and αβ T cells are commonly distinguished are seldom absolute in vivo.

Notably, mouse αβ T cells expressing a CD8αα homodimer rather than a CD8αβ heterodimer do not seem to follow the prototypic pathway of αβ T cell differentiation and may, after agonist-mediated positive selection, home directly to epithelial sites 2,15,22,23 (Fig. 2). In such sites, the cells' gene expression and resulting functional abilities more closely parallel those of colocalized γδ T cells than those of colocalized 'conventional' CD8αβ + αβ T cells 24,25 . Thus, whereas thymocyte differentiation seems at first glance to proceed along one of two paths, toward αβ or γδ T cell maturation, 'enigmatic' variations on that theme are becoming evident. It is therefore essential to understand how the 'choice' of pathway for each cell is regulated.

Notch: providing critical competences

A consideration of 'fate choice' inevitably involves the Notch family of signaling proteins, which have key effects on many developmental pathways 26 . In mammals, four known Notch transmembrane receptors (Notch 1-Notch4) can engage five known ligands (Delta-like 1 (DL1), DL3 and DL4, and Jagged1 and Jagged2). Those receptor-ligand interactions promote proteolytic cleavage of cell-surface Notch receptors and thereby release the intracellular domain to translocate to the nucleus. There, the intracellular domain forms heterodimers with RBP-Jκ, converting it from a transcriptional repressor to a transcriptional activator and resulting in the transcription of Notch target genes such as Hes1. Notably, the activation of Notch targets might directly determine a developmental outcome or might make cells competent to respond to other factors. Interestingly, each of those mechanisms is cited as the basis for different proposals for how Notch influences T cell differentiation.

Notch is essential to the commitment to become a T cell, and the first progenitor cells to enter the thymus express Hes1, indicating that Notch signaling has already occurred 27 . Consistent with that idea, Notch1-deficient bone marrow precursors adopt a B cell fate, even in the thymus 28 , whereas bone marrow precursors overexpressing a constitutively active Notch1 intracellular domain differentiate as immature T cells rather than B cells, even in the bone marrow 29 . But the function of Notch goes further (Fig. 1). Both fresh ex vivo thymic stromal cell monolayer cultures and OP9 bone marrow stromal cells support the differentiation which are functional and four of which are pseuodogenes, and the remaining three families (II, III, and IV) each consist of a single gene. The remaining two genes are VA, which lies 5! to the TCRGVII, and VB, which lies 5! to TCRGVIII. 24 25 The TCRD locus is located within the TCRA locus between the AV and AJ gene segments. It is flanked by TCRD deleting elements ("JA and #REC) such that rearrangements of the deleting elements to each other or rearrangement of AV to AJ gene segments causes deletion of the intervening TCRD gene locus. The germline encoded TCRD locus consists of eight V segments, three D segments, four J segments, and a single C segment. 14 TCRDV2 is located immediately upstream of the D and J segments, whereas TCRDV3 is located downstream of the TCRDJ cluster in an inverted orientation. TCRDV1 is located among the TCRAV genes 360 kb upstream of the TCRDC gene cluster, whereas the remaining five TCRDV genes (described as TCRAV/DV) are also found among the TCRAV genes. 14

V(D)J RECOMBINATION

The T lymphocyte has a unique ability to recognise a vast array of non-self antigens and rearrangement of multiple V, D, and J gene segments accounts for the diversity and specificity necessary for this function. During TCR gene rearrangement the V, D (only in TCRB and TCRD chain rearrangement), and J segments are brought together into a continuous V-D-J coding block (fig 2). 15 These events are mediated by recombination activating genes, RAG1 and RAG2, 26 27 and the enzyme terminal deoxynucleotidyl transferase (TdT). 28 The TCR gene segments, as with the immunoglobulin genes, are flanked by recombination signal sequences (RSS), which act as recognition sites for the VDJ recombinase and mark the points at which somatic recombination occurs. The RSS comprises two conserved sequences, a heptamer (CACA/TGTG) separated by 12 or 23 random nucleotides from a consensus nonamer sequence (ACAAAAACC or GGTTTTTGT). Recombination may only take place between gene segments flanked by different spacers, 29 30 and this phenomenon is known as the "12-23" rule. 31 Therefore, V gene segments flanked 3! by a 23 bp spacer can recombine with D segments or directly with J segments, both of which are flanked 5! by a 12 bp spacer. The rearrangement of TCRB and TCRD is a two step process, in which a D gene first recombines with a J gene and then a V gene recombines with the D-J block (fig 2). Because of the absence of D genes in TCRA and TCRG, only V-J recombination occurs in these two chains.

"Diversity of the T cell repertoire is not only a consequence of different V, D, J recombinations but also results from nucleotide insertion and deletion at junctional sites" Figure 1 Germline organisation of the T cell receptor (TCR) genes. The TCRA, TCRB, and TCRG loci are located on the chromosomes at positions 14q11-12, 7q32-35, and 7p15, respectively. The TCRD loci are also situated on chromosome 14, within the TCRA loci. The gene segments are designated using the World Health Organisation standards for TCR nomenclature. 7 The shaded boxes indicate the type of gene segment: V, variable (black); D, diversity (white); J, joining (dark grey); C, constant (light grey). wave of T cells that appears But, as the development of mbers increase while those dult animals, and especially ls make up only a small perd lymph node T cells. These nts and chickens. γ δ T cells of various organs, such as eproductive tract [reviewed ithin the mouse skin, there on of γ δ T cells where one l. Although some γ δ T cells s, such as producing tissuemouse skin [4], most γ δ T milar to those produced by olytic responses upon being . Significantly, gene expresells that were isolated from e small intestines indicated y transcribe similar effector dies suggest that γ δ and αβ nctions and that ligand recificantly to the unique role ost immune competence. ]. Yet, other than within the mouse skin, there is no known anatomic location of γ δ T cells where one cannot find αβ T cells as well. Although some γ δ T cells may have specialized functions, such as producing tissuespecific growth factors in the mouse skin [4], most γ δ T cells can secrete cytokines similar to those produced by αβ T cells and can mount cytolytic responses upon being triggered through the γ δ TCR. Significantly, gene expression profiles of γ δ and αβ T cells that were isolated from the epithelial layer of murine small intestines indicated that both types of cell actively transcribe similar effector function genes [5]. These studies suggest that γ δ and αβ T cells have similar effector functions and that ligand recognition may contribute significantly to the unique role of γ δ T cells in maintaining host immune competence.

γ δ and αβ TCRs recognize different antigens for these chains to contact both MHC and bound peptide.

In γ δ TCRs, the γ chain CDR3 loops are short, with narrow length distributions similar to Ig light chains, and the γ δ T cells comprise the first wave of T cells that appears during fetal thymic ontogeny. But, as the development of αβ T cells progresses, their numbers increase while those of γ δ T cells remain stable. In adult animals, and especially in mice and humans, γ δ T cells make up only a small percentage (∼1-5%) of spleen and lymph node T cells. These numbers are higher in ruminants and chickens. γ δ T cells also line the epithelial layers of various organs, such as the small intestine, liver and reproductive tract [reviewed in refs. 2,3]. Yet, other than within the mouse skin, there is no known anatomic location of γ δ T cells where one cannot find αβ T cells as well. Although some γ δ T cells may have specialized functions, such as producing tissuespecific growth factors in the mouse skin [4], most γ δ T cells can secrete cytokines similar to those produced by αβ T cells and can mount cytolytic responses upon being triggered through the γ δ TCR. Significantly, gene expression profiles of γ δ and αβ T cells that were isolated from the epithelial layer of murine small intestines indicated that both types of cell actively transcribe similar effector function genes [5]. These studies suggest that γ δ and αβ T cells have similar effector functions and that ligand recognition may contribute significantly to the unique role of γ δ T cells in maintaining host immune competence.

γ δ and αβ TCRs recognize different antigens for these chains to contact both MHC and bound peptide.

In γ δ TCRs, the γ chain CDR3 loops are short, with narrow length distributions similar to Ig light chains, and the [START_REF] Bozdech | The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum[END_REF]. The last few amino acids from the TCR V gene segments also contribute to CDR3 diversity, but their effects are negligible and are not included in the calculation.

%"# ! ! 36!

yδ&T&cell&development&

During!foetal!development,!γδ!T!cells!develop!alongside!αβ!T!cells!in!the!thymus!from!a! common!progenitor! [START_REF] Petrie | ! Commitment! to! the! T! cell! receptorSalpha! beta! or! S gamma! delta! lineages! can! occur! just! prior! to! the! onset! of! CD4! and! CD8! expression! among! immature!thymocytes[END_REF]!Dudley!et!al.!1995).!They!pass!through!several!stages,! and!most!of!the!work!to!define!the!differentiation!steps!has!been!done!in!mice.!Both!γδ!and! αβ!T!cells!start!as!CD4SCD8S!double!negative!(DN)!T!cells,!then!the!now!committed!αβ!T!cells! become! CD4+CD8+! double! positive! (DP)! and! finally! CD4/CD8+! single! positive! (SP)! lymphocytes.! γδT! cells! separate! from! the! αβ! T! cells! at! the! DN! stage! (See!Figure ! 7).! At! this! stage,!TCR!genes!undergo!somatic!rearrangement!to!produce!a!wide!spectrum!of!different! TCRs.! TCR's! are! heterodimers,! and! each! chain! is! made! up! of! a! membrane! bound! constant! region!and!a!variable!region.!There!are!four!possible!types!of!chain!-!α,!β,!γ!and!δ.!Each!gene! consists! of! multiple! variable! (V),! junction! (J)! and,! for! the! β! and! δ! genes,! diversity! (D)! segments! (Figure ! 8).! These! segments! can! be! rearranged! in! an! almost! infinite! number! of! combinations.! The! δ! gene! lies! within! the! α! gene,! therefore! their! expression! is! mutually! exclusive.!There!are!fewer!γ!and!δ!chains!than!there!are!α!and!β.!Though!γ!and!δ!genes!show! less!diversity!in!the!V!region,!there!is!a!large!potential!junctional!variability! [START_REF] Kazen | ! Evolution! of! the! V,! D,! and! J! gene! segments! used! in! the! primate! gammadelta! TScell! receptor! reveals! a! dichotomy! of! conservation! and! diversity.! Proceedings! of! the!National!Academy!of!Sciences!of!the[END_REF].!! During!TCR!rearrangement!clonal!lymphocyte!populations!with!γδ!TCRs!of!same!γδ!chain! pairings! are! produced! in! 'waves'! from! about! the! 8 th ! week! of! pregnancy,! although! lymphocytes! with! diverse! γδ! TCRs! more! akin! to! αβ! TCRs! are! also! produced! [START_REF] Mcvay | ! The! generation! of! human! gammadelta! T! cell! repertoires! during! fetal! development[END_REF].! It! is! still! being! debated! whether! γδ! T! cell! linage! is! fixed! before! TCR! gene! rearrangement,!or!if!successful!γδ!TCR!production!drives!γδ!lineage,!or!if!the!strength!of!preS TCR!and!γδ!TCR!signalling!determines!cell!fate! [START_REF] Lee | ! Towards! a! molecular! understanding! of! the! differential! signals! regulating! alphabeta/gammadelta!T!lineage!choice[END_REF][START_REF] Hayes | !Beyond!alphabeta/gammadelta!lineage!commitment:!TCR! signal! strength! regulates! gammadelta! T! cell! maturation! and! effector! fate[END_REF][START_REF] Coffey | !The!TCR!ligandSinducible!expression!of!CD73!marks!γδ!lineage!commitment!and! a! metastable! intermediate! in! effector! specification.! The![END_REF]. DP! αβ! T! cells! undergo! stringent! selection! in! the! thymus! for! the! ability! of! their! TCR! to! firstly!bind!selfSMHCs!(positive!selection)!and!then!to!not!bind!selfSAg!(negative!selection).! However,! as! γδ! TCRs! are! independent! of! MHC! presentation! for! Ag! recognition,! it! is! as! yet! unknown! how! or! whether! γδ! T! cells! undergo! equivalent! positive! and! negative! selection.! Once!committed!to!the!γδ!T!cell!lineage!they!leave!the!thymus!and!migrate!to!the!tissues,! with!different!TCRs!enriched!in!different!locations.!For!example!Vδ1+T!cells!predominate!in! the!gut!epithelium,!whereas!Vγ9Vδ2!T!cells!enter!the!peripheral!blood!circulation.!!

Vγ9Vδ2&TCR&and&ligand&recognition&

Different! γδ! TCR! pairings! are! linked! to! specific! localisation,! ligand! recognition! and! function.! In! mice,! certain! subsets! appear! to! be! 'preSprimed'! in! the! thymus! by! their! TCR! to! recognise! pathogen! and! danger! associated! molecular! patterns! (PAMPs! and! DAMPs,! respectively)! in! a! manner! reminiscent! of! innate! immune! cells.! The! Vδ2! TCR! chain! mostly! pairs!with!the!Vγ9!TCR!chain,!and!as!mentioned!above,!Vγ9Vδ2!T!cells!are!activated!by!small,! nonSpeptidic,!phosphorylated!molecules!called!phosphoantigens!(PSAgs).!

P]Ag&and&the&DOX]P&isoprenoid&biosynthesis&pathway&

Vγ9Vδ2! T! cells! were! noticed! to! be! activated! and! expanded! in! response! to! certain! infections! including! tuberculosis! [START_REF] Balbi | !TSlymphocytes!with!gamma!delta+!V!delta!2+!antigen!receptors!are!present!in! increased! proportions! in! a! fraction! of! patients! with! tuberculosis! or! with! sarcoidosis.! The! American!review!of!respiratory!disease[END_REF],! leprosy! [START_REF] Modlin | ! Lymphocytes! bearing! antigenSspecific! gamma! delta! TScell! receptors! accumulate!in!human!infectious!disease!lesions[END_REF],! salmonellosis! [START_REF] Hara | !Predominant!activation!and!expansion!of!V!gamma!9Sbearing!gamma!delta!T!cells! in! vivo! as! well! as! in! vitro! in! Salmonella! infection.! The![END_REF],!brucellosis! [START_REF] Bertotto | ! Lymphocytes! bearing! the! gamma! delta! T! cell! receptor! in! acute! Brucella! melitensis!infection[END_REF],!meningitis! (Raziuddin!et!al.! 1994),! malaria! (Ho! et! al.! 1990;[START_REF] Roussilhon | Responses! of! T! cells! from! sensitized! donors! to! recombinant! and! synthetic! peptides! corresponding! to! sequences! of! the! Plasmodium! falciparum! SERP! antigen[END_REF][START_REF] Schwartz | ! Delayed! expansion! of! V! delta! 2+! and! V! delta! 1+! gamma! delta! T! cells! after! acute!Plasmodium!falciparum!and!Plasmodium!vivax!malaria.!The!Journal!of!allergy!and!clinical! immunology[END_REF],! toxoplasmosis! [START_REF] Scalise | !Lymphocytes!bearing!the!gamma!delta!TScell!receptor!in!acute!toxoplasmosis[END_REF]!and!leishmaniasis! [START_REF] Russo | ! AntigenSreactive! gamma! delta! T! cells! in! human! leishmaniasis[END_REF] When we isolated IPP from mycobacteria, it was clear that there were additional antigens present in our preparations. Earlier studies had found four major peaks of antigenic activity on high-performance liquid chromatography analysis, which are antigenic activity for Vg2Vd2 T cells and their use of the pathway (13,14). Support for this theory was reported Jomaa et al. (15), based on the antigenicity of different bact species. Jomaa's laboratory (16) went on to show that dele of two upstream enzymes in the pathway abolished ant nicity, whereas deletion of the terminal enzyme LytB gre Fig. 1. MEP and mevalonate pathways for isoprenoid biosynthesis. The MEP pathway i found in most Eubacteria (with the notable exception of Gram-positive cocci), apicomple protozoa, and chloroplasts, whereas the meva nate pathway is found in Archaebacteria, eukaryo and the cytoplasm of plants. Genes for MEP enzymes are also termed ispC (dxr), ispD (ygbP), (ychB), ispF (ygbB), ispG (gcpE), and ispH (lytB).

Immunological Reviews 215/2007

affect activity as compared with the co pyrophosphate (3,30,31). A number of (Fig. 3) that are capable of stimulating Vg2 synthesized , including mono-ethyl phosp (7), bromohydrin pyrophosphate (BrHP phostim (32)(33)(34), and 2-methyl-3-bute (31). The latter two are being used for cl Unlike protein antigens and many recognition of prenyl pyrophosphate antig antigen entry, antigen processing, or intrac antigen-presenting molecule. Thus, we f hyde fixation of antigen-presenting cells (A peptide and lipid antigens from entering th the ability of the APC to present prenyl pyro human gd T cells (35). Also, activation w calcium flux (<1.5 min) on IPP stimu metabolic acidification (within 10 s) on Br These findings led us to propose that p antigens are directly presented to Vg2Vd2

One previously unanswered question is butyl pyrophosphate (3FBPP) (Fig. 3, [1 proposed as the structure for the 26 mycobacteria (14) prior to the discovery exists in nature. To answer this question, Eric Oldfield's lab, we have now synthesi for bioactivity (36). Synthetic 3FBPP was equilibrium with 4,4 diol-3-methyl-bu A!common,!Vγ9Vδ2!T!cellSspecific!and!TCRSdependent,!ligand!was!variously!found!to!be! protease! resistant,! [START_REF] Pfeffer | ! Primary! responses! of! human! T! cells! to! mycobacteria:! a! frequent! set! of! gamma/delta! T! cells! are! stimulated! by! proteaseSresistant! ligands.! European![END_REF][START_REF] Constant | ! Stimulation! of! human! gamma! delta! T! cells! by! nonpeptidic! mycobacterial! ligands.!Science![END_REF]! of! low! molecular! weight,! [START_REF] Pfeffer | !A!lectinSbinding,!proteaseSresistant!mycobacterial!ligand!specifically!activates!V! gamma!9+!human!gamma!delta!T!cells[END_REF][START_REF] Constant | ! Stimulation! of! human! gamma! delta! T! cells! by! nonpeptidic! mycobacterial! ligands.!Science![END_REF][START_REF] Tanaka | ! Nonpeptide! ligands! for! human! gamma! delta! T! cells[END_REF]!and!phosphorylated! [START_REF] Constant | ! Stimulation! of! human! gamma! delta! T! cells! by! nonpeptidic! mycobacterial! ligands.!Science![END_REF][START_REF] Bürk | !Human!V!gamma!9SV!delta!2!cells!are!stimulated!in!a!crossS reactive!fashion!by!a!variety!of!phosphorylated!metabolites[END_REF][START_REF] Behr | ! Plasmodium! falciparum! stimuli! for! human! gammadelta! T! cells! are! related! to! phosphorylated!antigens!of!mycobacteria.!Infection!and!immunity[END_REF],with!the!phosphate!group!essential!for!activity! [START_REF] Schoel | !Phosphate!is!essential!for!stimulation!of!V!gamma!9V! delta! 2! T! lymphocytes! by! mycobacterial! low! molecular! weight! ligand.! European![END_REF].!A!product!of!isoprenoid!biosynthesis,!Isopentenylpyrophosphate!(IPP),! was! the! first! natural! PSAg! identified! [START_REF] Tanaka | !Natural!and!synthetic!nonSpeptide!antigens!recognized!by!human!gamma!delta! T!cells[END_REF].! Isoprenoids! are! essential! for.cell! metabolism!and!are!building!blocks!for!the!biosynthesis!of!cholesterol,!ubiquinone,!steroid! hormones!and!other!products.!! At! the! time! of! IPP! discovery! only! the! Mevalonate! pathway! had! been! identified! for! isoprenoids!biosynthesis.!After!the!discovery!of!a!second!isoprenoid!biosynthesis!pathway,! the!1SdeoxySDSxylulose!5Sphosphate!(DOXSP)!(or!2SCSmethylSDSerythritol!4Sphosphate,!MEP)! pathway,! [START_REF] Rohmer | ! Isoprenoid! biosynthesis! in! bacteria:! a! novel! pathway! for! the! early! steps! leading!to!isopentenyl!diphosphate.!The!Biochemical!journal[END_REF][START_REF] Rohmer | !The!discovery!of!a!mevalonateSindependent!pathway!for!isoprenoid!biosynthesis! in!bacteria,!algae!and!higher!plants.!Natural!product!reports[END_REF][START_REF] Kuzuyama | !Mevalonate!and!nonmevalonate!pathways!for!the!biosynthesis!of!isoprene!units[END_REF]! Eisenreich! et! al.! 2004) [START_REF] Morita | ! Recognition! of! nonpeptide! prenyl! pyrophosphate! antigens! by! human! gammadelta!T!cells.!Microbes!and!infection!/!Institut!Pasteur[END_REF]!Belmant!et!al.!1999).!It!was!further!noted!that!IPP!concentrations!in!one!Vγ9Vδ2!T!cellS activating!E.coli!strain!were!below!the!threshold!necessary!for!activation,!thereby!suggesting! another! PSAg! was! responsible! [START_REF] Jomaa | Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs[END_REF].! Therefore! as! the! DOXSP! pathway! was! outlined,! other! Vγ9Vδ2! T! cell! activating! PSAgs! were! sought. [START_REF] Hintz | Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human gammadelta T cells in Escherichia coli[END_REF].!HMBPP!was!shown!to!be!10,000!times!more!potent!than!IPP!at!activating!Vγ9Vδ2!T! cells! [START_REF] Morita | ! Structural! Features! of! Nonpeptide! Prenyl! Pyrophosphates! That! Determine! Their!Antigenicity!for!Human!γ!δ!T!Cells[END_REF].! The! structure! and! reactivity! of! 6! natural! prenyl! PSAgs! have! since! been! outlined,! as! well! as! several! synthetic! PSAgs! including! bromohydrin! pyrophosphate! (BrHPP)!(Figure!10)! [START_REF] Morita | !Nonpeptide!antigens,!presentation!mechanisms,!and!immunological!memory! of! human! Vgamma2Vdelta2! T! cells:! discriminating! friend! from! foe! through! the! recognition! of! prenyl!pyrophosphate!antigens[END_REF].!! Both!isoprenoid!synthesis!pathways!produce!IPP,!but!only!the!DOXSP!pathway!produces! HMBPP.! Thus! it! was! proposed! that! the! differing! reactivitys! of! Vγ9Vδ2! T! cells! to! IPP! and! HMBPP!served!as!an!identifier!of!external!threats!(from!bacteria!and!parasites!which!use!the! DOXSP! pathway)! and! of! stressed! self! (metabolically! altered! cells! producing! excess! IPP)! (Gober!et!al.!2003;[START_REF] Morita | !Nonpeptide!antigens,!presentation!mechanisms,!and!immunological!memory! of! human! Vgamma2Vdelta2! T! cells:! discriminating! friend! from! foe! through! the! recognition! of! prenyl!pyrophosphate!antigens[END_REF]

! (see! Figure!9),!it!was!noted!that!Vγ9Vδ2!T!cellSactivating!bacteria!used!this!pathway!
! It! was! shown! that! E.coli! which! had! been! genetically! modified! to! bypass! the! MEP! pathway! did! not! activate! Vγ9Vδ2T! cells! (Altincicek!et!al.!2001)!but!that!E.coli!with!downstream!MEP!pathway!enzymes!knocked!out! were! highly! activatory! to! Vγ9Vδ2! T! cells! (Eberl,! Altincicek,! et! al.! 2002)! (Figure! 9).! A! low! molecular! weight! PSAg! metabolite! of! the! DOXSP! pathway! was! pinpointed,! and! finally! characterised! as! (E)S4SHydroxyS3SmethylSbutS2Senyl! pyrophosphate! (HMBPP)!
.! A!class!of!drugs!called!bisphosphonates!has!also!been!found!to!stimulate!Vγ9Vδ2!T!cells.! Bisphosphonates! are! used! to! treat! osteoporosis,! Paget's! disease! and! tumourSassociated! bone! disorders.! They! function! by! inhibiting! the! Dimethylallyl! pyrophosphate! (DMAPP)! downstream! processing! enzyme! farnesyl! pyrophosphate! synthase! (FPPS).! This! disruption! results! in! osteoclast! and! tumour! cell! apoptosis,! but! also! in! an! increased! concentration! of! cellular!IPP!that!becomes!sufficient!to!activate!Vγ9Vδ2!T!cells.!!

P]Ag&presentation&]&Butyrophilin&

Vγ9Vδ2!T!cell!PSAgSmediated!activation!requires!speciesSspecific!cellScell!contact! [START_REF] Morita | !Direct!presentation!of!nonpeptide!prenyl!pyrophosphate!antigens!to!human! γδ!T!cells[END_REF][START_REF] Kato | !Requirement!of!speciesSspecific!interactions!for!the!activation!of!human!gamma! delta!T!cells!by!pamidronate[END_REF][START_REF] Green | ! Recognition! of! nonpeptide! antigens! by! human! V! gamma! 9V! delta! 2! T! cells! requires! contact! with! cells! of! human! origin.! Clinical! and! experimental! immunology[END_REF]!with!nonSfixed!presenting!cells! [START_REF] Morita | !Direct!presentation!of!nonpeptide!prenyl!pyrophosphate!antigens!to!human! γδ!T!cells[END_REF].!Furthermore,!Vγ9Vδ2!TCRSdependent!activation!by!PSAg!involves!the!complementary! determining!region!(CDR)!loops!of!the!TCR!-!an!area!which!is!much!larger!than!a!PSAg! (Wang! et!al.!2010;[START_REF] Allison | !Structure!of!a!human!gammadelta!TScell!antigen!receptor[END_REF]

.!This!property,!together!with!the!lack!of!success!in!isolating!PS Ag!bound!to!Vγ9Vδ2!TCR!pointed!to!the!existence!of!a!PSAg!presenting!molecule.!Many!cells! are! able! to! present! PSAg! to! Vγ9Vδ2! T! cells,! which! indicate! that! the! presenting! molecule! is! functionally! nonSpolymorphic! and! ubiquitously! expressed.! Very! recently,! a! butyrophilin! (BTN)!molecule!BTN3A1!has!been!identified!as!a!PSAg!presenting!molecule.!! ! ! Figure&11&:&Simplified&schematics&depicting&the&allosteric&model&and&the&antigen]presenting& model&of&BNT3A1&P]Ag&presentation&(Karunakaran!&!Herrmann!2014).& !(A)!Sketches!representing!the!allosteric!model!describing!possible!events!involved!in!PSAgS mediated! activation! of! Vγ9Vδ2! TCR.! X! -! represents! unidentified! molecules! that! might! be! involved! in! the! mechanism.! (B)! Schematic! representation! of! antigenSpresentation! model.! Note!that!both!models!can!

THE ANTIGEN-PRESENTING MODEL

Vavassori et al. used a different approach to identify the speciesspecific factor mandatory for PAg-mediated activation and describe BTN3A1 as an antigen-presenting molecule (95). Their approach takes advantage of the fact that mouse lacks a BTN3 ortholog. At first, murine reporter cells expressing a Vg9Vd2 TCR were used to screen mouse-human hybrid cell lines for their capacity to mediate PAg-dependent stimulation with the aim to map this trait to a part of the human genome. By analysis of several of such mouse-hybrid cell lines the telomeric 3-27 Mb region of the human chromosome 6p was found to be mandatory for PAg-presentation. This region comprises the entire MHC as well as the BTN3A1 and BTN3A2 but not the BTN3A3 gene. Thus, genomic localization of the mandatory gene(s) is fully consistent with previously published data that BTN3A1 is mandatory for PAg-mediated activation. The genetic evidence for BTN3A1 as candidate for the molecule involved was further confirmed by knock down and over-expression experiments.

Interestingly, the reporter cells used in this study were not Vg9Vd2 TCR-transduced murine hybridoma cells as described above but Vg9Vd2 T lymphocytes generated from RAG knock-out mice transgenic for the Vg9Vd2 TCR B2G9, which were matured in vivo by administration of anti-CD3 mAb (95,96). An important difference between data obtained with primary murine reporter cells expressing the Vg9Vd2 TCR B2G9 and Vg9Vd2 TCR-MOP transduced reporter cells is that the agonistic mAb 20.1 was not stimulatory but inhibitory for the transgenic mouse cells. First results of our group obtained with TCR transductants suggest that this difference reflects variation of the TCR clonotypes, which stands against the idea of mAb 20.1 being a general activator of Vg9Vd2 T cells. Nevertheless, to our knowledge, there is no published data on determination of frequencies of mAb 20.1 vs. PAg-reactive cells or direct comparison of sensitivity of different TCR clonotypes for either stimulus supporting this notion. If TCR clonotypes do indeed differ in their sensitivity to both types of stimuli, it would affect models on PAg or mAb 20.1 action. Our interpretation of the presumed clonal differences would rely on substrate competition and inherent qualities of different TCR clonotypes. In the former case, we hypothesize that upon treatment of cells with PAg or mAb 20.1 BTN3A1 adopts a new conformation, which somehow allows binding of Vg9Vd2 TCR to BTN3-ED-PAg or mAb complex or to BTN3-ED-associated cell surface molecules(s). This conformation could differ to some extent after exposure of the cell to PAg or mAb 20.1 whereby mAb 20.1 might inhibit conversion into the PAg induced conformation. As a result, some TCR clonotypes cannot bind to the mAb 20.1-induced conformation. Indeed, one could imagine that mAb 20.1-binding "freezes" BTN3-ED in a conformation (93), which is distinct from the PAg-induced one (93, 95). Considering inherent qualities of TCR clonotypes as the basis for their differential capacity in recognizing BTN3A1-ED-PAg complex or BTN3-mAb complex, we propose or speculate that some Vg9Vd2 TCR, e.g., TCR B2G9 preferentially bind to a complex of PAg bound to the BTN3A1-ED, whereas others would preferentially bind to the conformationally changed BTN3A1 whose ED does not need to be in complex with the PAg. Consistent with this model would be that the area covered by the mAb 20.1 is rather near to the hypothetical PAg-binding site discussed in the next paragraph. Consequently for some TCR mAb 20.1 would compete with binding of the Vg9Vd2 TCR to a BTN3A1-PAg complex while for others mAb 20.1 would still be stimulatory.

De Libero and coworkers (95) provide also a wealth of data in favor of a direct binding of PAg to BTN3A1-ED and of binding of BTN3A1-PAg complexes to the Vg9Vd2 TCR: (i) IPP and HMBPP induce a substantial IFNg secretion by the murine reporter cells cultured in BTN3A1-V domain coated culture plates. (ii) Mass spectrometry data of BTN3A1-V incubated with IPP is consistent with a BTN3A1-IPP complex of 1:1 stoichiometry. (iii) Plasmon resonance analysis of PAg binding to BTN3A1-V domain allowed calculation of K d values. These are considerably lower than that of MHC-peptide complexes: 66.9 ⇥ 10 6 M for binding of BTN3A1-V to IPP and 3.06 ⇥ 10 6 M for binding of BTN3A1-V to HMBPP. (iv) Crystal structures of complexes generated from BTN3A1-V and IPP or HMBPP, respectively, identify a shallow Ag-binding groove. ing, suggesting a role for gd T cells in the graft-versusleukaemia (GVL) effect [14].

Anti-tumour responses for gd T cells have also been obtained after stimulation with different compounds, such as bisphosphonates or synthetic phosphoantigens. These molecules induced great expansion and activation of gd T cells, in healthy donors or patients with lymphoma or multiple myeloma [13]. These activated gd T cells exhibited cytotoxicity against different malignant haematological cell lines and multiple myeloma cells [15].

Modulating&Vγ9Vδ2&T&cell&activation:&Co]receptors&and&their&ligands&

Besides! the! TCR,! Vγ9Vδ2! T! cells! express! many! other! receptors! that! mediate! their! immune!responses!(Figure !12).!!

Adhesion*partner*molecules*

The!ability!to!stably!interact!with!a!target!cell!is!vital!for!cellSmediated!cytotoxic!activity.!CD8! CTLs!express!lymphocyte!functionSassociated!antigen!(LFA)!S1!and!CD2!and!the!interaction!of! these!adhesion!receptors!with!their!ligands!(ICAMS1!and!LFAS3,!respectively)!is!important!for! CD8! CTL! cytotoxic! function! [START_REF] Springer | !The!lymphocyte!functionSassociated!LFAS1,!CD2,!and!LFAS3!molecules:!cell! adhesion!receptors!of!the!immune!system[END_REF][START_REF] Davignon | ! Monoclonal! antibody! to! a! novel! lymphocyte! functionSassociated! antigen! (LFAS1):!mechanism!of!blockade!of!T!lymphocyteSmediated!killing!and!effects!on!other!T!and!B! lymphocyte!functions[END_REF][START_REF] Van!de!wielsvan!kemenade | !Both!LFAS1Spositive!and!Sdeficient!T!cell!clones!require!the! CD2/LFAS3! interaction! for! specific! cytolytic! activation[END_REF].!Vγ9Vδ2!T!cells!express!both!LFAS1!and!CD2,!and!studies!have!shown! a!link!between!this!expression!and!their!targeted!cytotoxic!abilities! [START_REF] Kato | !Requirement!of!speciesSspecific!interactions!for!the!activation!of!human!gamma! delta!T!cells!by!pamidronate[END_REF][START_REF] Wang | ! Different! roles! of! the! CD2! and! LFAS1! TScell! coSreceptors! for! regulating!cytotoxic,!proliferative,!and!cytokine!responses!of!human!V!gamma!9/V!delta!2!T!cells[END_REF].!

Natural*Killer*receptors*(NKR)*

Vγ9Vδ2!T!cells!express!both!the!CStype!lectin!family!receptor!natural!killer!group!(NKG)!S 2A! and! immunoglobulin! receptor! immunoglobulinSlike! transcript! 2! (ILT2).! Both! of! these! deliver! inhibitory! signals! upon! MHCI! binding,! and! thereby! allow! differentiation! between! normal!and!tumour!cells!(which!often!express!lower!levels!of!MHCI)! [START_REF] Nedellec | ! Human! Vgamma9Vdelta2! T! cells:! from! signals! to! functions[END_REF].! They! also! express! the! activatory! receptor! NKG2D,! which! binds! to! MHC! classSI! related! proteins! (MIC)! A! and! B,! and! the! UL16Sbinding! proteins! (ULBP1S4),! both! of! which! are! often! expressed!on!tumour!and!infected!cells! (Das!et!al.!2001;!Corvaisier!et!al.!2005 [START_REF] Wrobel | !Lysis!of!a!broad!range!of!epithelial!tumour!cells!by!human!gamma!delta!T!cells:! involvement! of! NKG2D! ligands! and! TScell! receptorS! versus! NKG2DSdependent! recognition[END_REF].!

;!Wrobel!et!al.! 2007).!NKG2D!is!a!very!important!coSstimulator!of!Vγ9Vδ2!T!cells!and!has!even!been!shown! to!act!independently!of!TCR!engagement!

Toll>like*receptors*(TLR)*

TLRs!recognise!PAMPs!and!are!expressed!on!innate!immune!and!epithelial!cells.!Vγ9Vδ2! T! cells! have! been! shown! to! express! surfaceSlocalised! TLR! 1,! 2! and! 6,! and! endolysosomeS localised!TLR!3!and!7! (Pietschmann!et!al.!2009).!The!TLR2STLR1!and!TLR1STLR6!heterodimers! recognise! bacterial! cell! wall! constituents! (triacyl! and! diacyllipopeptide,! respectively).! TLR3! and! TLR7! recognise! intracellular! viral! products! (double! and! single! stranded! RNA,! respectively)!(reviewed!(Kawai!&!Akira!2010)).!TLR!signalling!increases!Vγ9Vδ2!proliferation,! cytokine!release!and!cytotoxicity! [START_REF] Wesch | !Direct!costimulatory!effect!of!TLR3!ligand!poly(I:C)!on!human!gamma!delta!T! lymphocytes[END_REF]!Shojaei!et!al.!2009).!!

Fc*receptor*

A!Vγ9Vδ2!T!cell!subset!has!been!shown!to!express!the!IgG!Fc!receptor!FcγR11A!(CD16)! after!activation!(Angelini!et!al.!2004;!Lafont!et!al.!2001).!Engagement!of!CD16!leads!to!TNFα! production!(Lafont!et!al.!2001),!and!expression!of!CD16!is!associated!with!a!better!cytotoxic! activity!(Angelini!et!al.!2004).!It!could!also!be!involved!in!the!ADCC!function!of!Vγ9Vδ2!T!cells! against! tumour! cells! treated! with! therapeutic! human! antibodies! (GertnerSDardenne! et! al.! 2009;!Capietto!et!al.!2011).!! 1.3.3 Vγ9Vδ2&T&cell&role&anti]infection& As! previously! mentioned! the! differing! reactivity! of! Vγ9Vδ2! T! cells! to! IPP! and! HMBPP! provides! a! mechanism! for! recognising! threats! from! both! infection! and! dysregulated! self.! Accordingly! Vγ9Vδ2! T! cells! are! implicated! in! the! immune! response! to! numerous! diseases,! both!infections!and!cancers.!Once!activated!Vγ9Vδ2!T!cells!counter!the!infection!or!stressed! self!via!numerous!
!to!different!cytokines!in!vitro!can!push!Vγ9Vδ2!T!cells!into!different!memory! subsets,! for! example! ILS15! exposure! during! BrHPP! stimulation! results! in! a! predominantly! Tcm! population,! while! exposure! to! ILS21! leads! to! a! predominantly! Tem! population! (Eberl,! Engel,!et!al.!2002).!Exposure!to!ILS15!or!ILS21!and!ILS2!increases!cytotoxic!functions!(Chen!et! al.! 2001;! Thedrez! et! al.! 2009).! The! majority! of! circulating! Vγ9Vδ2! T! cells! having! a! Tem! phenotype!means!that!they!can!launch!an!immediate!response!to!infection!or!stress!without! the!need!for!clonal!expansion!or!de!novo!differentiation.!! At!birth!in!humans!there!is!a!greater!percentage!of!Vδ1+!than!Vδ2+!T!cells!in!foetal!cord! blood.!There!follows!a!rapid!expansion!of!the!Vγ9Vδ2!T!cell!subset!in!the!first!years!of!life,! thought!to!be!a!consequence!of!exposure!to!PSAg!producing!microorganisms!(Parker!et!al.! 1990;! Nadia! Caccamo! et! al.! 2006).! Recent! analysis! of! preSterm! foetal! blood! has! shown! an! expansion! of! the! Vγ9Vδ2! T! cell! subset! before! week! 20,! which! diminishes! to! term.! The! authors! suggest! that! this! could! be! due! to! Vγ9Vδ2! T! cell! sequestration! in! foetal! tissues,! as! Vγ9Vδ2! T! cells! are! known! to! be! enriched! in! foetal! intestine! (McVay! et! al.! 1998).! PreSterm! foetal!Vγ9Vδ2!T!cells!express!CSC!chemokine!receptor!(CCR)S5
!and!the!granzymes!A!and!K,! produce! IFNγ! after! PMA! (phorbol! 12Smyristate! 13Sacetate)/ionomycin! stimulation! and! readily!expand!after!HMBPP!or!zoledronate!stimulation! (Dimova!et!al.!2015).!Thus!Vγ9Vδ2!T! cells!are!already!primed!preSbirth!to!respond!to!PSAg!activation!with!a!preSprogramed!Th1! effector!response.!

Effector&functions&

Cytokine*secretion*

Vγ9Vδ2! T! memory! cells! are! predominantly! Th1! effector! subtype,! secreting! IFNγ,! ILS2,! and! TNFα! [START_REF] Goodier | !Cytokine!profiles!for!human!V!gamma!9+!T!cells!stimulated!by!Plasmodium! falciparum[END_REF][START_REF] Lang | !Early!activation!of!human!V!gamma!9V!delta!2!T!cell!broad!cytotoxicity!and!TNF! production! by! nonpeptidic! mycobacterial! ligands[END_REF][START_REF] Subauste | !Preferential!activation!and!expansion!of!human!peripheral!blood!gamma! delta! T! cells! in! response! to! Toxoplasma! gondii! in! vitro! and! their! cytokine! production! and! cytotoxic! activity! against! T.! gondiiSinfected! cells.! The![END_REF][START_REF] García | !SingleScell!cytokine!analysis!of!gamma!delta!T!cell!responses!to!nonpeptide! mycobacterial!antigens.!Journal!of!immunology![END_REF][START_REF] Wang | !Human!V!gamma!2V!delta!2!T!cells!produce!IFNSgamma!and!TNFSalpha!with!an! on/off/on! cycling! pattern! in! response! to! live! bacterial! products[END_REF]!although!they!can!also!be!driven!to!a!Th2!phenotype! [START_REF] Wesch | !Differentiation!of!resting!human!peripheral!blood!gamma! delta!T!cells!toward!Th1S!or!Th2Sphenotype[END_REF].! Th1! cytokine! production! activates! and! potentiates! other! innate! effector! cells! such! as! macrophages,!and!can!polarise!the!response!of!adaptive!cells!such!as!CTLs! [START_REF] Dieli | !Differentiation!of!effector/memory!Vdelta2!T!cells!and!migratory!routes!in!lymph! nodes!or!inflammatory!sites.!The![END_REF].!!

Cytotoxicity*

There!are!two!main!mechanisms!of!cellSmediated!cytotoxicity!-!the!death!receptor!(FasS L)! dependent! pathway! and! the! granule! exocytosis! pathway.! Vγ9Vδ2! T! cells! can! use! them! both.!

FAS>L>mediated*

The! death! receptor! pathway! is! dependent! on! FasSL! binding! to! Fas! on! the! target! cell! (reviewed! [START_REF] Brint | ! Life! in! the! Fas! lane:! differential! outcomes! of! Fas! signaling.!Cellular[END_REF]).!Vγ9Vδ2!T!cells!express!FasSL,!and!have!been!shown!to!use!this! pathway! to! target,! for! example,! Mycobacteria! tuberculosis! [START_REF] De | ! Specific! lytic! activity! against! mycobacterial! antigens! is! inversely! correlated! with! the! severity! of! tuberculosis.! Clinical[END_REF]! and! Brucellauis!infected!cells! (Oliaro!et!al.!2005)

.!! ! ! ! 44!

Perforin/granulysin>mediated*

The! granuleSexocytosis! pathway! involves! the! release! of! granules! containing! soluble! factors,! which! kill! the! target! cell.! Factors! include! perforin,! granzymes! and! granulysin.! Perforin! creates! pores! in! a! cell! membrane,! allowing! entry! of! toxic! granzyme! proteins! and! granulysin!into!the!cell.!Vγ9Vδ2!T!cells!contain!both!perforin!and!granulysin! [START_REF] Troyesblomberg | ! Human! gamma! delta! T! cells! that! inhibit! the! in! vitro! growth! of! the! asexual! blood! stages! of! the! Plasmodium! falciparum! parasite! express! cytolytic! and! proinflammatory!molecules[END_REF]!Farouk!et!al.!2004;!Oliaro!et!al.!2005)!and!this!expression!is!induced!upon!their! differentiation!into!effector!cells! [START_REF] Gertner | !Human!gammadelta!T!lymphocytes!strip!and!kill!tumor!cells!simultaneously[END_REF].!Perforin!and/or!granulysin!have!been! shown!to!be!important!in!PSAgSactivated!Vγ9Vδ2!T!cells!ability!to!inhibit!or!kill!both!tumour! cells! [START_REF] Lang | !Early!activation!of!human!V!gamma!9V!delta!2!T!cell!broad!cytotoxicity!and!TNF! production! by! nonpeptidic! mycobacterial! ligands[END_REF] [START_REF] Groh | ! Human! lymphocytes! bearing! T! cell! receptor! gamma/delta! are! phenotypically! diverse! and! evenly! distributed! throughout! the! lymphoid! system.! The![END_REF][START_REF] Brandes | ! Flexible! migration! program! regulates! gamma! delta! TScell! involvement! in! humoral!immunity[END_REF]

(Agrati!et!al.!2008;!Caccamo!et!al.!2011)!of!neutrophils.!It!has!also!been!shown!that!Vγ9Vδ2! T!cells!induce!neutrophil!differentiation!into!APCs!(Davey!et!al.!2014).!! 1.3.3.2.4.3 B*cells* In!vitro!Vγ9Vδ2!T!cell!exposed!to!ILS21!have!been!shown!to!take!on!a!follicular!helper!T! cell!(Tfh)!phenotype!and!help!B!cell!Ab!production!(Vermijlen!et!al.!2007;!Bansal!et!al.!2012;! N.!Caccamo!et!al.!2006)!accompanied!by!CD4+!Tfh!cells!(Caccamo!et!al.!2012).Vγ9Vδ2!T!cells! have!also!been!shown!to!increase!expression!of!APC!markers!on!B!cells!(Petrasca!&!Doherty! 2014).! In! vivo! Vγ9Vδ2! T! cells! are! found! in! the! T! cell! zone! and! the! germinal! centres! of! secondary!lymphoid!tissues!
.!Together!this!indicates! that! Vγ9Vδ2! T! cells! could! play! a! critical! role! in! germinal! centre! activation! of! AbSmediated! immune!responses.! ! ! ! 45!

αβ*T*cells*

In! vitro! Vγ9Vδ2! T! cells! are! able! to! affect! the! polarisation! of! the! CD4! T! cell! response! generated! by! a! DC.! They! induce! ILS12! but! not! ILS10! production! by! DCs,! thus! resulting! in! a! predominantly!Th1!CD4!T!cell!response! (Ni!et!al.!2012;!Ismaili!et!al.!2002;!Dunne!et!al.!2010;[START_REF] Martino | !Complementary!function!of!gamma!delta!TSlymphocytes!and!dendritic!cells!in! the!response!to!isopentenylSpyrophosphate!and!lipopolysaccharide!antigens[END_REF][START_REF] Martino | ! Enhancement! of! BCGSinduced! Th1! immune! response! through!Vgamma9Vdelta2!T!cell!activation!with!nonSpeptidic!drugs[END_REF].!! Furthermore! Vγ9Vδ2! T! cells! can! themselves! take! on! APC! phenotype! and! function! to! activate!naïve!αβ!T!cells.!This!shall!be!addressed!in!detail!in!the!following!section.! have captured antigens may also act as reservoirs of antigen that can be presented to T cells by other APCs. Therefore, evidence from in vitro studies supports both direct and indirect roles for mast cells in antigen presentation. However, although some mast cells certainly express MHC class II when analysed directly ex vivo, whether they truly present antigen in vivo remains to be determined.

Basophils modulate T cell responses

Basophils are a rare population of granulocytes that comprise ~1% of circulating peripheral leukocytes, and they are structurally and phenotypically related to mast cells. Like mast cells, basophils contain abundant granules with pre-formed inflammatory mediators that can be immediately released upon crosslinking of FcεRI 34-37 . Important new reagents have recently been used to show that basophils and mast cells have functionally unique and non-overlapping roles in IgE-mediated and IgGmediated allergic inflammation in mice 38-43 . In addition, it has been suggested that basophils are central to the differentiation of T helper 2 (T H 2) cells. Indeed, a number of studies have suggested that basophil-derived IL-4 is crucial for the development of T H 2 cell responses to cysteine proteases, allergens and extracellular parasites 4,23,42,44-50 .

Basophils as APCs.

Although basophil-derived IL-4 can skew T cells to a T H 2 cell phenotype, it had been presumed that antigen-specific T cell activation by professional APCs, such as DCs, is still necessary for T H 2 cell induction. To examine the requirement for DCs in antigen presentation during T H 2 cell differentiation, a number of groups have used mice expressing the human diphtheria toxin receptor (DTR) under the Cd11c promoter (Cd11c-DTR mice); in these mice, diphtheria toxin treatment results in the ablation of CD11c-expressing cells. When bone marrow from Cd11c-DTR mice was used to reconstitute irradiated wild-type mice, diphtheria toxin treatment of these chimeric mice did not alter T H 2 cell responses to papain or Trichuris muris 3,4 . Furthermore, papain injection or T. muris infection of mice in which MHC class II expression was restricted to DCs 3,4,51 did not result in a T H 2 cell response. Together, these results suggested that MHC class II expression by DCs alone was neither necessary nor sufficient for T H 2 cell induction. This suggested that another MHC class II-expressing cell type might be required for T H 2 cell induction in these models. Indeed, basophils were found to constitutively express MHC class II, CD80 and CD86, and the expression of these molecules was further upregulated by activation with IL-3 (REF. 23) or papain 3 . Moreover, co-culture of ovalbumin (OVA)-specific naive CD4 + T cells with antigen-pulsed basophils alone was sufficient to induce T cell proliferation and T H 2 cell differentiation in vitro 3,4,23 . Basophils could also present antigens to T cells in vivo, as antigen-specific naive CD4 + T cells displayed a T H 2 cell phenotype in CIITA-deficient mice (which lack MHC class II expression) injected with peptide-pulsed basophils 23 . Basophils were found to effectively incorporate soluble but not particulate antigens by macropinocytosis 3 , which was further enhanced by antigen-specific IgE molecules bound to FcεRI on basophils. IgE-coated basophils efficiently incorporated specific antigens in vivo 52 , and the addition of antigen-specific IgE molecules to CD4 + T cell and basophil co-cultures augmented the induction of T H 2 cells 23 . However, it is unclear whether the augmented T H 2 cell response mediated by IgE was owing to enhanced antigen presentation or increased IL-4 cathepsin S inhibitors) 8,9 . This suggests that antigen processing and loading on MHC class I molecules both occur in endocytic compartments in this pathway. Adding to the confusion, it has been shown recently that the cytosolic (proteasome-dependent) pathway can also occur independently of TAP, possibly through another, as yet unidentified, peptide transporter 10 .

The relative contributions of the cytosolic and vacuolar pathways to cross-presentation are not easy to determine in vitro or in vivo, because MHC class I molecules are retained in the ER and are unstable and/ or scarce in endosomes in both TAP-deficient cells and proteasome inhibitor-treated cells 11-13 . The best The origin of the MHC class I molecules involved in cross-presentation is also a matter of debate. Initially, cross-presenting MHC class I molecules were proposed to originate from the plasma membrane and to recycle to endosomes. A conserved tyrosine residue in their cytosolic tail, which is required for internalization from the cell surface, was shown to be crucial for crosspresentation 15 . However it was shown recently that CD74 promotes the trafficking of newly synthesized MHC class I molecules from the ER to endocytic compartments in DCs and that this routing is required for the crosspresentation of cell-associated antigens 16 . Finally, in relation to MHC class I molecule trafficking, RAB3B and RAB3C -two small GTPases that are involved in regu-Figure 1 | Intracellular pathways for cross-presentation in dendritic cells. After phagocytosis, exogenous antigens can be exported into the cytosol, where they are processed by the proteasome. The processed antigens can then be loaded on MHC class I molecules in the endoplasmic reticulum (ER) (the cytosolic pathway with ER loading) or re-imported into the phagosome to be loaded on MHC class I molecules (the cytosolic pathway with phagosomal loading). The SNARE SEC22B, which localizes in the ER-Golgi intermediate compartment (ERGIC) and interacts with syntaxin 4 on phagosomes, mediates the recruitment of a subset of ER components, including transporter associated with antigen processing (TAP), to phagosomes. Alternatively, exogenous antigens can be degraded into peptides in the phagosome, where they are then loaded on MHC class I molecules (the vacuolar pathway). However, although the collective knowledge of the different steps in MHC class I antigen presentation allows us to determine which peptides are excluded from binding to MHC class I molecules, we cannot predict the actual peptides that are presented. How immunodominant peptides are generated and whether and how they differ from normal peptides is also not known with certainty. This indicates that gaps in our understanding have to be filled in order to move from descriptive to predictive biochemistry of MHC class I antigen presentation.

R E V I E W S

59!

generating!functional!Vγ9Vδ2!TSAPC!lines!from!2!Hodgkin!lymphoma!patients! [START_REF] Landmeier | !Activated!human!gammadelta!T!cells!as!stimulators!of!specific!CD8+!TScell! responses!to!subdominant!Epstein!Barr!virus!epitopes:!potential!for!immunotherapy!of!cancer[END_REF].!! The!Moser!group!have!proposed!a!trial!using!Vγ9Vδ2!TSAPCs! (Khan,!Eberl,!et!al.!2014).& Briefly,! 100mL! patient! blood! is! taken! from! cancer! patients,! and! PBMCs! are! purified! and! stimulated! with! zoledronate,! ILS2! and! ILS15! for! 14! days.! Vγ9Vδ2! TSAPCs! are! then! loaded! 24hrs!with!carefully!selected!tumour!antigens!or!autologous!tumour!cell!extracts,!frozen!and! shipped!to!the!hospital.!AgSloaded!Vγ9Vδ2!TSAPCs!are!injected!intravenously!into!the!patient! (timing! and! number! of! doses! to! be! worked! out),! with! perhaps! a! follow! up! treatment! of! zoledronate! after! 24hrs! to! increase! IFNγ! and! TNFα! production,! and! also! promote! lymph! node!homing!by!the!Vγ9Vδ2!T!cells!by!augmenting!CCR7!expression.! However! amongst! the! promise! there! are! also! causes! for! concern.! Activated! Vγ9Vδ2! T! cells!release!large!quantities!of!IFNγ!and!TNFα!that!could!induce!potentially!harmful!acute! stage! immune! responses.! Therefore! careful! testing! for! residual! cytokine! production! preS injection! is! necessary.! Acute! stage! responses! can! also! be! reduced! by! paracetamol! or! acetaminophen! (Reid!et!al.!2010).!

The!2012!paper!of!the!Rossig!group!suggests!another!potential!pitfall.!They!showed!that! peptideSloaded! Vγ9Vδ2! TSAPCs! were! unable! to! induce! CTLs! capable! of! lysing! target! cells! transduced!with!fullSlength!protein! (Altvater!et!al.!2012).!Although!DCs!were!likewise!unable,! it!is!clear!that!more!work!needs!to!be!done!to!find!the!optimal!Vγ9Vδ2!T!cell!loading!and!CTL! expansion!conditions.!

Vγ9Vδ2+& T& cells& in& the& immune& response& to& P.& falciparum&infection&

Vγ9Vδ2!T!cells!are!heavily!implicated!in!the!immune!response!to!P.!falciparum!malaria! infection.! There! is! also! evidence! of! their! involvement! in! the! immune! response! to! P.! vivax! infection! [START_REF] Perera | ! Transient! increase! in! circulating! gamma/delta! T! cells! during! Plasmodium! vivax!malarial!paroxysms.!The![END_REF]!Chuangchaiya!et!al.!2010)!however!the!focus!of!this!thesis!is!P.! falciparum,!and!therefore!this!section!concerns!the!role!of!Vγ9Vδ2!T!cells!in!response!to!P.! falciparum.!!

Vγ9Vδ2& T& cell& activation& in& response& to& P.* falciparum& exposure& 1.5.1.1 Early& Evidence& that& Vγ9Vδ2& T& cells& play& a& role& in& the& immune& response& to& P.* falciparum& ]& Activation& of& Vγ9Vδ2& T& cells& in& primary& infections&

It!was!observed!that!γδ!T!cells!are!expanded!in!vivo!in!the!peripheral!blood!of!primary! infected! P.! falciparum! malaria! patients! (Ho! et! al.! 1990;[START_REF] Roussilhon | Responses! of! T! cells! from! sensitized! donors! to! recombinant! and! synthetic! peptides! corresponding! to! sequences! of! the! Plasmodium! falciparum! SERP! antigen[END_REF][START_REF] Chang | !Subset!heterogeneity!among!gamma!delta!T!cells!found!in!peripheral!blood! during!Plasmodium!falciparum!malaria[END_REF][START_REF] Roussilhon | !Human!TcR!gamma!delta+!lymphocyte!response!on!primary!exposure!to! Plasmodium!falciparum.!Clinical!and!experimental!immunology[END_REF]! and! remain! elevated! for! up! to! two! months! [START_REF] Schwartz | ! Delayed! expansion! of! V! delta! 2+! and! V! delta! 1+! gamma! delta! T! cells! after! acute!Plasmodium!falciparum!and!Plasmodium!vivax!malaria.!The!Journal!of!allergy!and!clinical! immunology[END_REF].! In! vitro! studies! involving! PBMC! stimulation! by!P.! falciparum! extract! showed!that!Vγ9Vδ2!T!cells!made!up!the!major!fraction!of!the!expanding!γδ!T!cells! [START_REF] Goerlich | !Plasmodium!falciparum!merozoites!primarily!stimulate!the!V!gamma!9!subset! of!human!gamma/delta!T!cells[END_REF]! Behr! &! Dubois! 1992;[START_REF] Goodier | !Quantitative!analysis!of!the!response!of!human! T!cell!receptor!V!gamma!9+!cells!to!Plasmodium!falciparum.!European![END_REF][START_REF] Goodier | !Human!peripheral!blood!gamma!delta!T!cells!respond!to!antigens!of! Plasmodium!falciparum[END_REF][START_REF] Roussilhon | !Human!TcR!gamma!delta+!lymphocyte!response!on!primary!exposure!to! Plasmodium!falciparum.!Clinical!and!experimental!immunology[END_REF]!Jones!et!al.!1996).!This!was!confirmed!in!vivo! [START_REF] Schwartz | ! Delayed! expansion! of! V! delta! 2+! and! V! delta! 1+! gamma! delta! T! cells! after! acute!Plasmodium!falciparum!and!Plasmodium!vivax!malaria.!The!Journal!of!allergy!and!clinical! immunology[END_REF].! Vγ9Vδ2! T! cells! were! also! found! to! be! increased! in! human! spleens! during! infection! [START_REF] Bordessoule | ! Preferential! localisation! of! human! lymphocytes! bearing!gamma!delta!T!cell!receptors!to!the!red!pulp!of!the!spleen[END_REF][START_REF] Nakazawa | ! MalariaSinduced! increase! of! splenic! gamma! delta! T! cells! in! humans,! monkeys,!and!mice[END_REF],!a!phenomenon!that!was!confirmed!in!both! mouse! [START_REF] Langhorne | !Phenotypic!characterization!of!splenic!T!cells!from!mice! infected! with! Plasmodium! chabaudi! chabaudi.! Scandinavian! journal! of! immunology[END_REF][START_REF] Van!der!heyde | !Expansion!of!the!CD4S,!CD8S!gamma!delta!T!cell!subset!in!the!spleens! of! mice! during! nonSlethal! bloodSstage! malaria[END_REF][START_REF] Nakazawa | ! MalariaSinduced! increase! of! splenic! gamma! delta! T! cells! in! humans,! monkeys,!and!mice[END_REF]!and!monkey! models! [START_REF] Nakazawa | ! MalariaSinduced! increase! of! splenic! gamma! delta! T! cells! in! humans,! monkeys,!and!mice[END_REF].!The!rapid!expansion!of!Vγ9Vδ2!T!cells!during!infection!and! ! ! 60! their! homing! to! sites! of! known! importance! in! parasite! clearance! indicated! that! Vγ9Vδ2! T! cells!played!a!role!in!the!response!to!infection.!

Mechanism&of&activation&

Vγ9Vδ2!T!cells!were!historically!found!to!be!activated!in!vitro!by!schizonts!and!merozoite! erythrocyte! stage! parasites! [START_REF] Goerlich | !Plasmodium!falciparum!merozoites!primarily!stimulate!the!V!gamma!9!subset! of!human!gamma/delta!T!cells[END_REF]! Jones! et! al.! 1996;! Costa! et! al.! 2011),! and! one!study!also!showed!that!gametocyte!stage!exoSantigens!can!activate!γδ!T!cells! (Ramsey! et!al.!2002).! At! the! time! when! Vγ9Vδ2! T! cells! were! first! implicated! in! the! immune! response! to! P.! falciparum!infection,!the!Ag!recognised!by!Vγ9Vδ2!T!cells!was!unknown.!The!P.!falciparum! Ag! was! reported! as! superSantigen! like! (Behr! &! Dubois! 1992;! Goodier,! KrauseSJauer,! et! al.! 1992),!not!a!heatSshockSrelated!nor!a!glucoseSregulated!protein! [START_REF] Ho | ! Polyclonal! expansion! of! peripheral! gamma! delta! T! cells! in! human! Plasmodium! falciparum!malaria.!Infection!and!immunity[END_REF],!resistant!to! acid,! alkaline,! high! temperatures! and! protease! treatment! [START_REF] Pichyangkul | !Activation!of!gammadelta!T!cells!in!malaria:!interaction!of!cytokines!and!a! schizontSassociated!Plasmodium!falciparum!antigen.!The!Journal!of!infectious!diseases[END_REF],! phosphorylated! [START_REF] Pichyangkul | !Activation!of!gammadelta!T!cells!in!malaria:!interaction!of!cytokines!and!a! schizontSassociated!Plasmodium!falciparum!antigen.!The!Journal!of!infectious!diseases[END_REF][START_REF] Behr | ! Plasmodium! falciparum! stimuli! for! human! gammadelta! T! cells! are! related! to! phosphorylated!antigens!of!mycobacteria.!Infection!and!immunity[END_REF]! and! soluble! [START_REF] Behr | ! Plasmodium! falciparum! stimuli! for! human! gammadelta! T! cells! are! related! to! phosphorylated!antigens!of!mycobacteria.!Infection!and!immunity[END_REF][START_REF] Lindberg | Vδ2!T!cells!remains!poorly!understood.!It!has!been!recently! shown!that!PSAg!produced!by!tumour!cells!is!presented!to!the!Vγ9Vδ2!TCR!by!BTN3A1![END_REF].! Activation! was! also! shown! to! be! Vγ9Vδ2! TCRSmediated! (Costa! et! al.! 2011;[START_REF] Lindberg | Vδ2!T!cells!remains!poorly!understood.!It!has!been!recently! shown!that!PSAg!produced!by!tumour!cells!is!presented!to!the!Vγ9Vδ2!TCR!by!BTN3A1![END_REF]!but!not!MHCI!or!II!dependent!(van!der!Heyde!et!al.!1993).!After! the!DOXSP!pathway!was!discovered!(see!above!section!1.3.2.1)!Plasmodium!parasites!were! found! to! use! this! pathway! (reviewed! [START_REF] Guggisberg | ! Isoprenoid! Biosynthesis! in! Plasmodium! falciparum[END_REF]),! and! the! putative! PSAg! assumed! to! be! a! bySproduct! of! isoprenoid! biosynthesis.! It! is! anticipated! (though! not! yet! definitively! proven)! that! the! soluble! molecule! released! by! erythrocyte! stage! parasites! is! indeed!a!PSAg!of!the!DOXSP!pathway,!most!likely!HMBPP.! Cytokines,! specifically! ILS2! and! ILS15,! have! been! found! necessary! for! Vγ9Vδ2! T! cell! activation!and!viability!in!vitro!after!P.!falciparum!stimulation!(by!lysate!or!Ag)! [START_REF] Elloso | !Human!gamma!delta!T!cell!subsetSproliferative!response!to!malarial!antigen! in! vitro! depends! on! CD4+! T! cells! or! cytokines! that! signal! through! components! of! the! ILS2R.![END_REF]!Jones!et!al.!1996;[START_REF] Elloso | ! The! effects! of! interleukinS15! on! human! gammadelta! T! cell! responses! to! Plasmodium!falciparum!in!vitro[END_REF].!CD28!signalling!has!also!been!shown!important! for!iRBC!supernatant!(SN)S!activated!human!Vγ9Vδ2!T!cell!viability!and!proliferation!in!vitro! [START_REF] Ribot | !B7SCD28!costimulatory!signals!control!the!survival!and!proliferation!of!murine! and!human!γδ!T!cells!via!ILS2!production[END_REF].! Interestingly! human! Vγ9Vδ2! T! cells,! activated! by! iRBC! SN,! downS regulated! CD28! and! upSregulated! the! CD28! ligands! CD80! and! CD86! surface! expression.! In! mice,!CD28!coSstimulation!of!γδ!T!cells!induces!activation,!proliferation!and!ILS2!production.! The!essential!nature!of!this!signalling!was!demonstrated!in!mice!infected!by!P.!berghei!ANKA,! where!CD28!knock!out!(KO)!resulted!in!blocked!T!cell!expansion,!activation,!and!IFNγ!and!ILS 17!production! [START_REF] Ribot | !B7SCD28!costimulatory!signals!control!the!survival!and!proliferation!of!murine! and!human!γδ!T!cells!via!ILS2!production[END_REF]..!

Difference&between&primary&and&secondary&immune&responses&

The! bulk! of! work! on! Vγ9Vδ2! T! cell! responses! to! P.! falciparum! infection! has! been! on! primary! infected! adult! patients,! usually! Caucasians! living! in! nonSendemic! regions,! rather! than! residents! of! endemic! regions! who! are! exposed! to! numerous! malaria! infections! and! possibly!chronically!infected.!Vγ9Vδ2!T!cell!increase!is!not!observed!in!the!peripheral!blood! from!children!and!adults!living!in!malaria!endemic!areas! [START_REF] Hviid | ! The! gamma/delta! TScell! response! to! Plasmodium! falciparum! malaria! in! a! population!in!which!malaria!is!endemic.!Infection!and!immunity[END_REF] [START_REF] Goodier | !Gamma!delta!T!cells!in!the!peripheral!blood!of!individuals!from!an!area!of! holoendemic!Plasmodium!falciparum!transmission.!Transactions!of!the!Royal!Society!of!Tropical![END_REF][START_REF] Worku | ! Lymphocyte! activation! and! subset! redistribution! in! the! peripheral! blood! in! acute! malaria! illness:! distinct! gammadelta+! T! cell! patterns! in! Plasmodium! falciparum! and! P.! vivax!infections.!Clinical!and!experimental!immunology[END_REF][START_REF] Hviid | !Perturbation!and!proinflammatory!type!activation!of!V!delta!1(+)!gamma!delta!T! cells! in! African! children! with! Plasmodium! falciparum! malaria.! Infection! and! immunity[END_REF].! (Though! care! must! always!be!taken!when!comparing!γδ!T!cells!from!children!and!adults,!as!the!ratio!of!γδ!T!cell! subsets!change!with!age! [START_REF] Parker | ! Evidence! for! extrathymic! changes! in! the! T! cell! receptor! gamma/delta! repertoire.!The![END_REF]!Nadia!Caccamo!et!al.!2006).)! In!a!recent!longitudinal!study!of!Ugandan!children,!the!percentage!of!Vγ9Vδ2!T!cells!in! peripheral!blood!was!inversely!correlated!with!prior!incidences!of!malaria!infections.!Ex!vivo! Vγ9Vδ2! T! cell! proliferation,! cytokine! production! and! immuneSmodulatory! gene! expression! was!also!negatively!associated!with!prior!malaria!episodes!-!indicating!decreased!peripheral! blood!Vγ9Vδ2!T!cell!activity!with!increasing!exposures!to!the!parasite!(P. [START_REF] Jagannathan | Loss and dysfunction of Vdelta2(ϩ) gammadelta T cells are associated with clinical tolerance to malaria[END_REF].!Decreased!peripheral!activity!and!numbers!of!Vγ9Vδ2!T!cells!is!also!found!in!primary! P.! falciparum! infected! adults.! Vγ9Vδ2! T! cells! taken! from! the! peripheral! blood! during! ! ! 61! paroxysms!were!found!to!expand!less!and!produce!less!TNFα!in!response!to!IPP!stimulation! than!Vγ9Vδ2!T!cells!taken!during!recovery,!postStreatment.!It!was!also!found!that!there!are! less!Vγ9Vδ2!T!cells!(particularly!Vγ2Jγ1.2γδ!T!cells!(US!nomenclature),!the!TCR!subset!that!is! particularly! reactive! to! PSAgs)! in! circulation! during! P.! falciparum! paroxysm! than! during! recovery! (Martini!et!al.!2003).! This! could! be! explained! by! homing! of! active! Vγ9Vδ2! T! cells! away! from! the! peripheral! circulation!during!acute!malaria!infection.!Indeed!large!numbers!of!Vγ9Vδ2!T!cells!have!been! observed! in! the! spleen! of! malaria! patient! autopsy! specimens,! indicating! substantial! tissue! sequestration! in! malaria! patients! [START_REF] Bordessoule | ! Preferential! localisation! of! human! lymphocytes! bearing!gamma!delta!T!cell!receptors!to!the!red!pulp!of!the!spleen[END_REF][START_REF] Nakazawa | ! MalariaSinduced! increase! of! splenic! gamma! delta! T! cells! in! humans,! monkeys,!and!mice[END_REF].! Alternatively!Vγ9Vδ2!T!cells!could!be!subject!to!immune!regulation!or!anergy!during!acute! malaria!infection!(Vγ9Vδ2!T!cells!are!very!susceptible!to!activation!induced!cell!death!by!FasS FasSL!interaction!as!demonstrated!for!M.!tuberculosis! [START_REF] Li | ! Involvement! of! the! Fas/Fas! ligand! pathway! in! activationSinduced! cell! death! of! mycobacteriaSreactive!human!gamma!delta!T!cells:!a!mechanism!for!the!loss!of!gamma!delta!T! cells! in! patients! with! pulmonary! tuberculosis[END_REF]).!! It!is!clear!that!much!care!has!to!be!taken!when!extrapolating!results!from!naïve!adults! experiencing! a! primary! infection,! to! adults! living! in! endemic! settings.! However,! interesting! and! relevant! data! on! the! possible! function! of! Vγ9Vδ2! T! cells! can! be! gleaned! from! naïve! settings,!as!primary!infection!can!be!a!relevant!model!to!understand!the!early!response!of! Vγ9Vδ2!T!cells!to!P.!falciparum!infection.! [START_REF] Sanou | !Haematological!parameters,!natural!regulatory!CD4!+!CD25!+!FOXP3+!T!cells! and!γδ!T!cells!among!two!sympatric!ethnic!groups!having!different!susceptibility!to!malaria!in! Burkina!Faso[END_REF].!

;!P.!Jagannathan!et! al.! 2014).! Instead! Vδ1+! T! cells! are! the! major! γδ! T! cell! subset! expanded! in! response! to! infection!

How&does&Vγ9Vδ2&T&cell&activation&affect&disease&outcome?&

It! is! well! established! that! Vγ9Vδ2! T! cells! are! activated! by! P.! falciparum! parasites.! Evidence!of!a!protective!role!of!γδ!T!cells!is!suggested!by!differences!in!γδ!T!cell!populations! between! sympatric! Fulani! and! Mossi! ethnic! groups! in! Burkina! Faso.! The! Fulani! are! more! resistant!to!malaria!than!the!Mossi,!with!lower!incidence!rates!of!clinical!disease!and!lower! parasitemia.!This!resistance!is!accompanied!by!a!small!but!significant!enrichment!of!the!γδ!T! cell!population!in!peripheral!blood!of!Fulani!compared!to!the!Mossi!

Lessons&from&mouse&studies.&

A!study!of!the!relative!importance!of!different!immune!cells!to!an!infection!response!is! very! difficult! to! do! in! humans;! therefore! much! work! has! been! done! in! mice.! A! murine! Vγ9Vδ2! T! cell! equivalent! has! yet! to! be! identified! (if! one! exists! at! all),! however! general! principles!and!ideas!of!avenues!for!investigation!can!be!gleaned.!! !Contrasting! studies! have! shown! both! essential! [START_REF] Tsuji | !Gamma!delta!T!cells!contribute!to!immunity!against!the!liver!stages!of!malaria!in! alpha!beta!TScellSdeficient!mice.!Proceedings!of!the!National!Academy!of!Sciences[END_REF] Beyond! the! debate! on! necessity,! there! is! much! evidence! that! γδ! T! cells! positively! contribute!to!protection.!γδ!T!cells!have!been!found!to!be!able!to!compensate!for!both!B!cell! [START_REF] Van! Der! Heyde | ! Gamma! delta! T! cells! function! in! cellSmediated! immunity! to! acute! bloodSstage! Plasmodium! chabaudi! adami! malaria[END_REF][START_REF] Van!der!heyde | !Expansion!of!the!gammadelta!T!cell!subset!in!vivo!during!bloodstage! malaria!in!B!cellSdeficient!mice[END_REF]!van!der!Heyde!et!al.!2006;[START_REF] Seixas | !gammadelta!T!cells!contribute!to!control!of!chronic!parasitemia!in! Plasmodium!chabaudi!infections!in!mice[END_REF]!and!CD8!and!NK!T!cell! [START_REF] Taniguchi | ! Malaria! protection! in! beta! 2SmicroglobulinSdeficient! mice! lacking! major! histocompatibility! complex! class! I! antigens:! essential! role! of! innate! immunity,! including! gammadelta!T!cells[END_REF]!activity,!help!B!cell!activation! and! class! switching! (F. [START_REF] Kobayashi | ! Plasmodium! berghei! XAT:! contribution! of! gammadelta! T! cells! to! host! defense! against! infection! with! bloodSstage! nonlethal! malaria! parasite[END_REF],! activate/enhance! DC! activities! [START_REF] Inoue | !Enhancement!of!dendritic!cell!activation!via!CD40!ligandSexpressing!γδ!T!cells! is! responsible! for! protective! immunity! to! Plasmodium! parasites.! Proceedings! of! the! National! Academy!of!Sciences!of!the!United!States!of!America[END_REF]!Inoue!et!al.!2014),!contribute!to!cytokine!production! (Weidanz!et!al.!2010;[START_REF] Kopacz | ! γδ! TScells! may! interfere! with! a! productive! immune! response! in! Plasmodium!yoelii!infections[END_REF])! (which! has! also! been! associated! with! cerebral! malaria! (Haque! et! al.! 2001;[START_REF] Yañez | ! Gamma! delta! TScell! function! in! pathogenesis! of! cerebral! malaria! in! mice! infected!with!Plasmodium!berghei!ANKA.!Infection!and!immunity[END_REF]),! and! skew! the! immune! response! to! predominantly! Th1! type! responses! [START_REF] Inoue | !Enhancement!of!dendritic!cell!activation!via!CD40!ligandSexpressing!γδ!T!cells! is! responsible! for! protective! immunity! to! Plasmodium! parasites.! Proceedings! of! the! National! Academy!of!Sciences!of!the!United!States!of!America[END_REF][START_REF] Seixas | !gammadelta!T!cells!contribute!to!control!of!chronic!parasitemia!in! Plasmodium!chabaudi!infections!in!mice[END_REF])!(See!Table&6!for!details).!

;!Kopacz!&!Kumar!1999;!Seixas!&!Langhorne!1999)!γδ!T!cell!function!in!clearing!murine! Plasmodium!infection!(See!Table&5!for!details).!Interestingly!there!is!no!overlap!of!combined! mouse! species,! inoculation! route,! or! parasite! species.! The! most! striking! difference! is! inoculation!route:!those!studies!which!showed!that!γδ!T!cells!are!not!essential!for!parasite! clearance!use!intraSperitoneal!injection!of!blood!
! iRBC! P.!chabaudi! adami! 556KA! B!cell!KO!(J H D)! ! B!cell!KO!(J H D)!+! antiSγδ!mAb! Able!to!clear!infection! yδ!T!cell!expansion.! Unable!to!clear!infection! (van!der! Heyde!et!al.! 1995)! C57/BL6x12 9! IP!1x10 6 ! iRBC! P.!chabaudi! adami! 556KA! B!cell!KO!(J H D!/µS MT)! Increased!γδ!T!cell! expansion!and!activation! compared!to!WT.! (van!der! Heyde!et!al.! 1996)! BALB/c! IP!1x10 6 ! iRBC! P.!chabaudi! adami! 556KA! B!cell!KO!(J H !S/S)! ! B!cell!KO!(J H !S/S)! +antiSCD4!mAb! Able!to!clear!infection.! γδ!T!cell!expansion.! γδ!T!cell!expansion! abrogated.! (van!der! Heyde!et!al.! 2006)! 129SvxC57/ BL6! C57/BL6! IP!1x10 5 ! iRBC! P.!chabaudi! chabaudi! (AS)! B!cell!KO!(µSMT)! ! B!cell!KO!(µSMT)+! TCR!δ(S/S)! Able!to!clear!infection.! γδ!T!cell!expansion.! Unable!to!clear!infection.!! ! (Seixas!&! Langhorne! 1999)! Potential&B& cell&helper.& CBA/JNCrj! IV!1x10 4 ! iRBC! P.!Berghei! XAT! antiSγδ!mAb! Lower!IgG2a,!IgG2b,!

Contribute& to&cytokine& production&

C57/BL6! IV!1x10 5 ! iRBC! P.!chabaudi! adami! 556KA! antiSγδ!mAb! Decreased!ILS10,!IFNγ,! and!(MCP)!S1!expression! in!spleen!cells.! (Weidanz!et! al.!2010)! C57/BL6! IP!1x10 6 ! iRBC! P.!yoelii!17X! NL! TCR!δ(S/S)! Decreased!IFNγ!in!spleen.! (Kopacz!&! Kumar!1999)! Associated& with& Cerebral& Malaria& DBA/2! IP!5x10 5 ! iRBC! P.!yoelii!17X! L!(PYL)! ILS2!induced!CM! ! ! ILS2!+!antiSγδ!mAb! Increased!γδ!T!cell! accumulation!within! cerebral!vessels.! CM!resistance!restored.! (Haque!et!al.! 2001)! C57/BL6x12 9! IP!1x10 6 ! iRBC! P.!berghei! ANKA! antiSγδ!mAb! TCR!δ(S/S)! Prevented!CM! Did!not!prevent!CM! (Yañez!et!al.! 1999)! Skew& immune& response&to& Th1&type.& C57/BL6J! IV!1x10 4 ! iRBC! P.!Berghei! XAT! TCR!δ(S/S)!/!antiSγδ! mAb! Less!IFNγ!in!spleen,!and! lower!proportion!IFNγ+! CD4+!T!cells.! (Inoue!et!al.! 2012)! 129SvxC57/ BL6! C57/BL6! IP!1x10 5 ! iRBC! P.!chabaudi! chabaudi! (AS)! B!cell!KO!(µSMT)! Dominant!expansion!of! Vγ2Vδ4!population! produced!IFNγ!

Vγ9Vδ2&T&cells&release&inflammatory&cytokines&

A! large! feature! of! the! immune! response! to! P.! falciparum! infection! is! the! production! inflammatory! cytokines.! In! in! vitro! studies!of!schizontSactivated! PBMCs! from! naïve! donors,! Vγ9Vδ2! T! cells! have! been! found! to! produce! TNFα! and! be! the! major! source! of! IFNγ,! more! than! NK! cells! or! macrophages! [START_REF] Goodier | !Cytokine!profiles!for!human!V!gamma!9+!T!cells!stimulated!by!Plasmodium! falciparum[END_REF][START_REF] Pichyangkul | !Activation!of!gammadelta!T!cells!in!malaria:!interaction!of!cytokines!and!a! schizontSassociated!Plasmodium!falciparum!antigen.!The!Journal!of!infectious!diseases[END_REF]! Scragg! et! al.! 1999;[START_REF] Scragg! Et! Al.! ; ! D'ombrain | !studies!have!reported!stimulation!of!Vγ9Vδ2!TScells!by!schizont!extracts!/! iRBC!lysates!(Behr!&!Dubois![END_REF].!They!have!also!been!shown!to!express!TNFα,!TGFSβ!and!ILS8,! and!occasionally!ILS10,!ILS2!and!ILS5! [START_REF] Goodier | !Cytokine!profiles!for!human!V!gamma!9+!T!cells!stimulated!by!Plasmodium! falciparum[END_REF].!In!ex!vivo!analysis!of!cord!blood! from! mothers! in! an! endemic! setting! who! had! experienced! malaria! during! pregnancy,! the! Vγ9Vδ2! T! cells! produced! significantly! more! IFNγ! and! TNFα! than! newSborns! from! healthy! mothers,!as!did!Vγ9Vδ2!T!cells!from!the!mother! [START_REF] Engelmann | ! Activation! status! of! cord! blood! gamma! delta! T! cells! reflects! in! utero! exposure! to! Plasmodium! falciparum! antigen.! The![END_REF].!This!inflammatory! cytokine! production! by! Vγ9Vδ2! T! cells! has! been! associated! with! both! protection! and! pathogenesis.!

Cytokine*production*is*associated*with*protection:*

In! humans,! the! group! of! Robert! Sauerwein! has! performed! vaccination! studies! where! healthy,!malaria!naïve,!volunteers!are!exposed!to!three!doses!of!P.!falciparum!(via!the!bite! of! 12S15! infected! mosquitos)! with! the! accompaniment! of! chloroquine! treatment.! This! permits!the!parasite!to!mature!to!blood!stage,!when!it!is!then!swiftly!killed!before!disease! symptoms! can! develop.! The! volunteers! are! later! challenged! by! the! bites! of! 5! infected! mosquitos,!and!their!infection!status!monitored.!The!control!volunteers!who!had!not!been! previously! inoculated! all! contracted! malaria,! while! the! inoculated! volunteers! remained! healthy.!This!experimental!infection!setting!gave!a!unique!opportunity!to!follow!the!in!vivo! immune!response!to!malaria!infection.!Vaccinated!(protected)!volunteers!showed!increased! IFNγ,!TNFα!and!ILS2!production!to!nonSvaccinated!(nonSprotected)!when!PBMCs,!taken!preS challenge! and! 1! day! post! challenge,! were! stimulated! by! iRBC! in! vitro! [START_REF] Roestenberg | ! Protection! against! a! malaria! challenge! by! sporozoite! inoculation.! The! New!England![END_REF]! Teirlinck! et! al.! 2011).! IFNγ! levels! were! also! increased! in! PBMCs! from! vaccinated! volunteers!taken!days!9,!35,!140!and!400!post!challenge!when!stimulated!by!both!iRBC!and! sporozoites! (Teirlinck!et!al.!2011).!γδ!T!cells!were!found!to!be!the!major!IFNγ!contributors,! with! αβ! T! cells! the! next! largest.! The! majority! of! responding! cells! were! effector! memory,! indicating!recall!responses,!and!IFNγ!producing!γδ!T!cells!were!demonstrated!to!be!a!major! contributor!to!parasiteSspecific!recall!responses! (Teirlinck!et!al.!2011).!Thus!IFNγ!production! by! lymphocytes! including! γδ! T! cells,! correlated! with! acquired! immunity! to! P.! falciparum! infection.! In! longitudinal! studies! of! semiSimmune! children! from! Papua! New! Guinea,! the! in! vitro! response! of! PMBCs! to! iRBC! was! measured,! and! subsequent! malaria! incidence! recorded.! Increased! IFNγ! production! by! PBMCs! correlated! with! reduced! risk! of! future! moderate! and! high!density!P.!falciparum!infection.!!Further,!though!there!was!much!donor!heterogeneity,! γδ!T!cells!were!the!predominant!IFNγ!producing!cell!population!(D' Ombrain!et!al.!2008).! ! ! ! 65!

Cytokine*production*is*associated*with*pathogenesis:*

A!different!longitudinal!study!of!children!from!Papua!New!Guinea!suggests!that!γδ!T!cell! cytokine! production! is! involved! in! severe! malaria.! Ex! vivo! stimulation! of! PBMCs! from! children!with!either!severe!or!uncomplicated!malaria!or!healthy!controls!showed!that!γδ!T! cells!and!monocytes!were!responsible!for!inflammatory!cytokines!associated!with!'high!odds'! of!severe!malaria! [START_REF] Stanisic | ␥␦ T cells and CD14ϩ monocytes are predominant cellular sources of cytokines and chemokines associated with severe malaria[END_REF].! A! longitudinal! study! of! Ugandan! children! found! that! decreased! in! vitro! TNFα! and! IFNγ! production! by! γδ! T! cells! after! iRBC! stimulation! was! correlated! with! increased! previous! parasite!exposure.!They!further!found!that!many!of!the!genes

!upregulated!in!low!exposed! compared!to!highly!exposed!children!encoded!cytokines.!In!general,!lower!in!vitro!Vγ9Vδ2!T! cell! responsiveness! to! iRBC! correlated! with! lower! subsequent! incidences! of! symptomatic! infection,!but!to!increased!probability!of!higher!parasitemia.!Thus!suggesting!a!link!between! Vγ9Vδ2! T! cell! cytokine! production! and! disease! severity! -! that! disease! symptoms! stem! in! some! part! from! Vγ9Vδ2! derived! cytokines! -! but! also! to! control! of! parasitemia! (P.! Jagannathan!et!al.!2014).!! High!levels!of!proSinflammatory!cytokines!have!been!associated!with!cerebral!malaria!in! humans! (reviewed! (Angulo! &! Fresno! 2002))! and! IFNγ! producing! γδ! T! cells! have! been! implicated!in!mouse!models!of!cerebral!malaria!(See!Table&6).! Together,! these! studies! indicate! that! while! Vγ9Vδ2! T! cell! inflammatory! cytokine!
responses! can! be! protective,! excess! inflammation! leads! to! pathology.! The! careful! balance! between! inflammatory! and! regulatory! signals! appear! to! be! destabilised! by! the! parasite,! particularly!in!primary!infection.!The!subsequent!ability!to!launch!an!appropriately!regulated! Vγ9Vδ2! T! cell! cytokine! response! likely! depends! on! multiple! factors! including! frequency! of! malaria!infection!and!exposure!to!other!diseases.!

Direct&anti]parasitic&mechanism&in&vitro&

Over!the!last!20!years!in!vitro!studies!have!been!used!to!build!a!picture!of!how!Vγ9Vδ2!T! cells!directly!inhibit!the!erythrocyte!stage!lifeScycle.!The!first!studies!showed!that!Vγ9Vδ2!T! cells!targeted!the!iRBCs!in!a!contact!dependent!manner,!and!that!perhaps!merozoites!were! the! target,! as! inhibition! of! parasite! lifeScycle! was! not! seen! until! after! parasite! reinvasion! [START_REF] Elloso | !Inhibition!of!Plasmodium!falciparum!in!vitro!by!human!gamma!delta!T!cells.! The![END_REF][START_REF] Troyesblomberg | ! Human! gamma! delta! T! cells! that! inhibit! the! in! vitro! growth! of! the! asexual! blood! stages! of! the! Plasmodium! falciparum! parasite! express! cytolytic! and! proinflammatory!molecules[END_REF]!Farouk!et!al.!2004).!Active!granulysin!release! by! the! Vγ9Vδ2! T! cells! was! implied! in! mediating! parasite! growth! inhibition,! as! granulysin! production! correlated! with! lifeScycle! inhibition! (Farouk! et! al.! 2004).! Our! group! has! shown! that!Vγ9Vδ2!T!cell!inhibition!of!parasites!was!indeed!granulysinS,!but!not!perforinSmediated.! In!an!experiment!where!Vγ9Vδ2!T!cells!were!coScultured!with!iRBC!schizonts!and!removed! before! rupture,! we! showed! definitively! that! merozoites! are! the! target,! and! that! schizonts! are!not!affected!by!granulysin!release! (Costa!et!al.!2011).!

Discussion&

Vγ9Vδ2! T! cells! make! up! a! minor! population! in! the! peripheral! blood,! and! expansion! in! response! to! P.! falciparum! infection! is! not! seen! until! after! crisis,! and! during! recovery.! Interestingly,!as!mentioned!above,!antiSγδ!mAb!treatment!of!P.!berghei!XAT!infected!mice!at! different!timepoints!showed!that!while!γδ!T!cells!are!essential!for!clearance!before!D9!post! infection,! after! this! point! they! are! dispensable.! This! indicates! that! γδ! T! cell! amplification,! detected!predominantly!after!D9!in!the!peripheral!blood,!is!not!linked!to!parasite!clearance! and!thus!γδ!T!cell!contribution!to!parasite!clearance!was!not!by!weight!of!numbers! [START_REF] Inoue | !Enhancement!of!dendritic!cell!activation!via!CD40!ligandSexpressing!γδ!T!cells! is! responsible! for! protective! immunity! to! Plasmodium! parasites.! Proceedings! of! the! National! Academy!of!Sciences!of!the!United!States!of!America[END_REF].!How!then!can!such!a!minor!population!play!such!a!large!role?!One!explanation!is! via!their!activation!of!other!immune!cells.!As!described!in!section!1.3.

Project&Rationale&

Malaria! remains! a! major! global! disease! burden,! particularly! in! less! economically! developed!nations.!Effective!treatments!are!available,!but!still!an!estimated!627,000!people! (mostly!children)!die!from!malaria!every!year!although!sustained!global!efforts!have!resulted! in! 42%! drop! in! mortality! rates! since! 2000! (WHO! 2014).! With! the! creeping! spread! of! insecticide! resistant! mosquitoes! and! drug! resistant! parasites,! new! drugs! and! an! effective! vaccine! are! urgently! required.! To! find! new! avenues! for! immune! interventions! and! vaccine! strategies,!research!is!being!focused!on!the!mechanism!of!the!immune!response!to!malaria! infection.! Vγ9Vδ2! T! cells! are! activated! and! expanded! during! erythrocyte! stage! infection! (Roussilhon!et!al.!1990;!Behr!&!Dubois!1992)!and!have!antiSparasitic!properties! (Farouk!et!al.! 2004;! Costa! et! al.! 2011).! They! therefore! represent! a! potentially! useful! immune! effector! to! harness.! Many! aspects! of! their! activation! and! functions! remain! unclear,! particularly! the! mechanisms! of! their! activation! by! erythrocyte! stage! parasites,! and! their! impact! on! the! adaptive!immune!response.!!

This!thesis!project!focused!on!investigating!two!aspects!of!Vγ9Vδ2!T!cellSiRBC!interaction! 1) The!mechanism!of!Vγ9Vδ2!T!cell!activation!by!the!iRBC!!!Project!1.! 2) The!subsequent!potential!antigen!presenting!role!of!Vγ9Vδ2!T!cells!in!inducing! an!adaptive!immune!response!to!P.!falciparum!!!Project!2.! ! 71!
From! this! data! we! propose! a! model! of! Vγ9Vδ2! T! cell! activation! in! which! PSAgs! of! the! DOXSP! pathway! are! released! into! the! surrounding! milieu! upon! parasite! rupture,! and! are! then!able!to!distantly!activate!Vγ9Vδ2!T!cells.! My!specific!contributions!to!this!paper!included!parasite!and!Vγ9Vδ2!T!cell!line!culture! and! purification,! assistance! in! the! planning! and! execution! of! PSAg! release! kinetics! studies,! setting! up! flow! cytometry! based! assessment! of! iRBC:! Vγ9Vδ2! T! cell! conjugate! formation,! statistical!analysis!for!PSAg!dose!response!experiments!and!careful!reading!and!editing!of!the! finished!manuscript.!! For! flow! cytometry! based! assessment! of! iRBC:Vγ9Vδ2! T! cell! conjugate! formation,! a! significant!amount!of!time!was!spent!establishing!the!optimal!iRBC!labelling!and!coSculture! set! up! to! measure! the! formation! of! conjugates! if! present.! Various! strategies! of! labelling,! fixation! and! washing! were! assessed! to! discover! the! optimal! postScoSculture! processing! for! maintenance!of!conjugates.!Different!iRBC!labelling!strategies!were!compared!to!allow!clear! distinction!between!Vγ9Vδ2!T!cell!lines!and!iRBC/uninfected!(ui)RBC;!including!glycophorin!A! (GPA)SAb! surface! staining! GPA! is! expressed! on! RBC! but! not! lymphocytes)! and! violet! cell! tracker!and!CFSE!cytoplasm!labelling.!A!major!difficulty!encountered!was!the!definition!of!a! negative! control! as! uiRBC! physiology! is! not! the! same! as! iRBC.! Therefore,! for! example,! separate! CFSE! labelling! strategies! had! to! be! adopted,! which! led! to! difficulties! in! defining! positive! and! negative! populations.! When! optimal! conditions! had! been! established,! clear! Daudi:Vγ9Vδ2!T!cell!conjugates!(used!as!a!positive!control)!were!visible,!but!no!conjugates! were!found!in!iRBC:Vγ9Vδ2!T!cell!coSculture!above!uiRBC:Vγ9Vδ2!T!cell!conjugate!levels.! I n humans and nonhuman primates, the main peripheral blood ␥␦T-cell subset expresses the V␥9 and V␦2 T-cell receptor (TCR) chains. This V␥9V␦2 T-cell subset accounts for 1 to 10% of total blood T lymphocytes and is expanded in patients upon infection by pathogens such as Plasmodium falciparum (1)(2)(3)(4)(5) or Mycobacterium tuberculosis (6) and in patients with lymphoid malignancies (7). In malaria patients, this expansion may play a dual role, both promoting pathology (3,5) and contributing to the control of parasite density. Indeed, V␥9V␦2 T cells efficiently limit in vitro P. falciparum expansion by granulysin-dependent cytotoxicity (1,(8)(9)(10). In malaria patients, high levels of granulysin-expressing V␥9V␦2 T cells correlate with their ex vivo parasitespecific degranulation capacity, and elevated granulysin concentration in plasma suggests significant discharge during acute P. falciparum malaria (1). As a step toward a better understanding of how V␥9V␦2 T cells target parasites, we recently showed that the antiparasitic activity of V␥9V␦2 T cells targets the extracellular merozoites (1). The intraerythrocytic developmental stages, which appear insensitive to the antiparasitic effect (1), seem to potently trigger V␥9V␦2 T-cell activation and degranulation (1,(11)(12)(13)(14). However, how precisely and which intraerythrocytic developmental stages activate V␥9V␦2 T cells still is unclear.

V␥9V␦2 T cells are activated by so-called phosphoantigens, which are nonpeptidic intermediate metabolites of the isoprenoid production pathway (15; recently reviewed in reference 16). The natural phosphoantigen (E)-4-hydroxy-3-methyl-but-enyl-pyrophosphate (HMBPP) is produced by the DOXP pathway and is 1,000 times more potent for specifically activating V␥9V␦2 T cells than the isopentenyl-pyrophosphate (IPP) molecule, which is produced by both the DOXP pathway and the mevalonate path-way (17,18). Apicomplexa and, notably, Plasmodium spp. do not possess the mevalonate pathway and use the DOXP pathway to produce isoprenoids (19). Although it has been shown that V␥9V␦2 T-cell activation by P. falciparum extracts is abrogated by apyrase treatment (12), the involvement of the parasitic DOXP pathway has never been formally proven, and the potency of the bioactivity of parasitic phosphoantigens on V␥9V␦2 T cells has never been assessed.

In the case of tumor cells, it is well established that cell-to-cell contact is required for V␥9V␦2 T-cell activation, and, like cytotoxic ␣␤ T cells, their activation may be triggered by the formation of a cytotoxic synapse during contact with an activating tumor target cell (20). Recent reports demonstrated a mandatory role for a B7-related butyrophilin (CD277/BTN3A) for the phosphoantigen-dependent activation of V␥9V␦2 T cells by tumor targets or mycobacterium-infected cells (21)(22)(23)(24). One of the proposed models suggests that V␥9V␦2 T cells recognize BTN3A modifications induced by binding the phosphoantigens produced inside the target cells (22). However, phosphoantigens also can be released into the supernatant of microorganisms or infected cell cultures. Furthermore, soluble phosphoantigens can be pulsed onto the surface of noninfected presenting cells (25), which stimulate V␥9V␦2 T cells in a contact-dependent manner. This suggests that V␥9V␦2 T cells can be activated by soluble phosphoantigens at a distance from the producing cell.

In the case of P. falciparum, numerous studies have reported stimulation of V␥9V␦2 T cells by schizont extracts/infected red blood cell (iRBC) lysates (3,11,12,14,26), culture supernatants (26)(27)(28)(29), and/or intact iRBCs cocultivated with V␥9V␦2 T cells (1,5,14,30), suggesting that V␥9V␦2 T-cell contact with iRBC is dispensable. However, at this point, how P. falciparum intracellular stages activate V␥9V␦2 T cells is unknown.

To address these issues and to gain novel insights on V␥9V␦2 T-cell activation by P. falciparum, here we explored the expression of BTN3A by iRBC, the timing of phosphoantigen release by iRBC, and the involvement of the parasite DOXP pathway in phosphoantigen production, and we quantified the P. falciparum bioactivity for V␥9V␦2 T cells.

MATERIALS AND METHODS

P. falciparum culture. FCR3 parasites were cultured in O ϩ red blood cells (RBCs) (Etablissement Français du Sang-EFS-Aquitaine, France) in complete parasite medium (CPM; RPMI 1640 supplemented with 10% human serum, gentamicin, glutamine, and hypoxanthine) and were regularly tested for the absence of Mycoplasma contamination (1). Parasitemia was assessed by hydroethidine staining (31) or examination of Giemsastained smears. Parasite cultures were synchronized by sorbitol (32) and/or heparin treatment (33). When required, midstage schizonts (around 38 to 40 h postinvasion [hpi]) were purified by gel flotation on gelofusin to Ͼ80% parasitemia (31).

Generation of V␥9V␦2 T-cell lines. Short-term lines (␥␦T-cell lines) were generated from healthy donor peripheral blood mononuclear cells (PBMCs; EFS Aquitaine, France) as previously described (1). Briefly, PBMCs were stimulated with 400 nM bromohydrin pyrophosphate (BrHPP) (IPH1101; Innate Pharma) in complete medium (RPMI 1640 supplemented with 10% decomplemented fetal calf serum, glutamine, and antibiotics) in the presence of 300 UI/ml interleukin-2 (IL-2). After 20 days, V␥9V␦2 T-cell purity was evaluated by flow cytometry (FACS Fortessa; BD Biosciences). ␥␦T-cell lines were used when the percentage of V␦2 ϩ CD3 ϩ cells was Ͼ80%, unless indicated otherwise. For each experiment, two ␥␦T-cell lines, freshly generated in parallel from two distinct donors, were used.

BTN3A staining. PBMCs, ␥␦T-cell lines, purified P. falciparum cultures and uninfected red blood cells (uiRBCs) were stained with 15 g/ml anti-butyrophillin 3 (BTN3A) antibody (clone 20.1; kind gift from E. Scotet, Nantes, France) for 45 min at room temperature. Cells were washed once and stained with fluorescein isothiocyanate (FITC)-coupled goat anti-mouse antibody (Beckman-Coulter) for 1 h at room tempera-ture. After washing, PBMCs and ␥␦T-cell lines also were stained with anti-CD3-phycoerythrin (PE) antibody (Becton-Dickinson), and iRBCs were stained with hydroethidine (5 g/ml) to discriminate iRBCs from uiRBCs. Surface marker expression was assessed by flow cytometry.

CD107a degranulation assay. The CD107a degranulation assay was performed as described previously (1). Briefly, 10 5 cells of ␥␦T-cell lines starved overnight of interleukin-2 (IL-2) or freshly collected PBMCs were incubated in the presence of stimulant and anti-CD107a-PE antibody (BD Bioscience) for 4 h. Cells were collected, washed, labeled with anti-V␦2-FITC antibody (Beckman Coulter), and analyzed by flow cytometry. For transwell experiments, cell culture inserts (0.4-m polycarbonate membrane; the Transwell system; Nunc, Roskilde, Denmark) were used by following the manufacturer's recommendations, with ␥␦T cells seeded in the bottom of the wells in the presence of anti-CD107a-PE antibody and midstage schizonts (38 to 40 hpi) in the upper chamber. For antibody blocking experiments, ␥␦T cells were preincubated for 1 h with anti-V␦2 (clone immu389; Beckman Coulter), anti-BTN3A (clone 103.2; kind gift from D. Olive [21]), or anti-NKG2D (clone 149810; R&D Systems) antibody before stimulation as described previously (1).

Preparation of iRBC supernatants. Synchronized midstage schizont cultures (38 to 40 hpi) were washed and adjusted to 5% hematocrit in CPM. Culture supernatants (G, J, K, L, M, and S) were prepared from independent parasite cultures and collected 4 h later from a 4% parasitemia culture. Each culture supernatant was tested on at least two different ␥␦T-cell lines, and at least two different supernatants were used in each assay. Rupture supernatants were collected from cultures at 1.5% parasitemia, when rupture was complete and reinvasion had occurred. Parasite stages were estimated from microscopic examination of Giemsastained smears. All of the collected supernatants were spun down at 870 ϫ g for 5 min, filtered through a 0.22-m Millipore filter, and frozen until use. As a control, supernatant from uiRBCs cultivated in parallel was collected using the same protocol. When indicated, supernatants were ultracentrifuged in a Beckman Optima L-100XP centrifuge in a 90Ti rotor at 197,000 ϫ g for 3 h at 4°C. For pyrophosphatase treatment, we used 0.2 U/ml potato apyrase (Apy) (Sigma-Aldrich) for 1 h as described previously (34), followed by filtration using a 3-kDa-cutoff Centricon filter (Amicon) according to the manufacturer's instructions. Supernatant activity was tested by CD107a assay.

Fosmidomycin treatment. Young trophozoite iRBC cultures (20 hpi) were adjusted to 4% parasitemia, 5% hematocrit in CPM and incubated for 21 h with serial dilutions of fosmidomycin with or without the addition of 1 M farnesyl pyrophosphate (both from Sigma-Aldrich). Duplicate supernatants of treated parasites were frozen until use. ␥␦T-cell lines were treated similarly. In order to assess the effect of fosmidomycin on iRBC viability after 21 h of incubation, iRBC cultures were diluted to 1% parasitemia and 1% hematocrit and allowed to reinvade in fresh CPM. Cycle progression was assessed by Giemsa staining, and parasitemia was determined by hydroethidine staining as described above.

Kinetics of phosphoantigen and PfHRP2 release. Three independent cultures (A, B, and C) of synchronized iRBCs (14 hpi) were washed and adjusted to 1% parasitemia and 5% hematocrit in CPM and dispensed in 24-well plates. Cultures were unfed during the time of study. Supernatants from duplicate wells were collected at different time points, filtered through a 0.22-m filter, and frozen until use. Supernatants from uiRBCs served as controls. Thawed supernatants were tested in duplicate for phosphoantigen bioactivity on two independent ␥␦T-cell lines. iRBC rupture was assessed by monitoring parasite developmental stages using Giemsastained blood smears. P. falciparum histidine-rich protein 2 (PfHRP2) was measured in supernatants using a commercial enzyme-linked immunosorbent assay (ELISA) by following the manufacturer's instructions (Malaria Ag Celisa; Cellabs, Sydney, Australia).

Treatment with the apicomplexan cGMP-dependent protein kinase inhibitor compound C2. Synchronized schizont cultures (around 40 to 44 hpi) were washed once with RPMI, adjusted to 1% parasitemia and 5% hematocrit, and resuspended in CPM containing 2 M compound 2, which was

RESULTS

Plasmodium falciparum-infected red blood cells do not express butyrophilin 3 and activate V␥9V␦2 T cells in the absence of contact.

We first asked whether P. falciparum iRBCs could activate V␥9V␦2 T cells by a mechanism similar to that of cancer cells, through BTN3A binding of intracellular phosphoantigens (21)(22)(23)(24). Using flow cytometry we found that, contrary to T cells, neither uninfected RBC (uiRBC) nor iRBC expressed BTN3A (Fig. 1A). This suggests that iRBCs activate V␥9V␦2 T cells through an alternative mechanism, involving either another phosphoantigen presentation molecule on the iRBC surface or through recognition of phosphoantigens released by iRBCs into the microenvironment.

To examine whether contact of iRBCs with V␥9V␦2 T cells was mandatory for activation, V␥9V␦2 short-term lines (␥␦T-cell lines) were coincubated with iRBCs with or without physical sep-aration by a transwell insert, and their activation was monitored using a CD107a degranulation assay (1). The gating strategy is illustrated in Fig. 1B, showing, as expected, that 87%, 25%, and 28% of V␥9V␦2 T cells activated with HMBPP, Daudi cells, and iRBC, respectively, expressed the CD107a marker of degranulation. Separation by a 0.4-m transwell membrane abrogated V␥9V␦2 T-cell activation by the Daudi cell line, which is known to require cell-cell contact, and did not alter the response to soluble HMBPP. Interestingly, physical separation by the transwell did not abrogate or alter V␥9V␦2 T-cell reactivity to iRBCs (Fig. 1C; also see Fig. S1A in the supplemental material). This indicates that soluble mediators, released by mature iRBCs and diffusing freely across the 0.4-m transwell membrane, activate V␥9V␦2 T cells in the absence of contact with iRBCs. This was confirmed by timelapse confocal microscopy (see Fig. S1B), where almost all V␥9V␦2 T cells formed long-lived conjugates with Daudi cells, while iRBC-V␥9V␦2 conjugates were scarcely observed. Consistent with this, we did not detect conjugates between V␥9V␦2 T cells and iRBCs by flow cytometry. Altogether, these data suggest that triggering of V␥9V␦2 T-cell activation by iRBCs relies on soluble mediators released in the microenvironment.

Molecules released in iRBC supernatant activate V␥9V␦2 T cells in a TCR-and BTN3A-dependent manner and have characteristics of phosphoantigens. P. falciparum is known to produce HMBPP through the DOXP pathway, and iRBC extract has been shown to contain HMBPP; however, little is known about the release of phosphoantigens in iRBC culture supernatants. To gain insight on this aspect, we generated culture supernatants from iRBCs and examined their ability to activate V␥9V␦2 T cells. Consistent with transwell experiments, the iRBC supernatant induced V␥9V␦2 T-cell activation from fresh PBMCs in the same range as intact iRBCs (Fig. 2A,left). This shows that parasite supernatant activation of V␥9V␦2 T cells did not require their prior in vitro priming, expansion, or selection. Supernatants also induced degranulation of ␥␦T-cell lines in a dose-dependent manner (Fig. 2A, right). Moreover, ultracentrifugation of iRBC supernatant did not alter its capacity to activate V␥9V␦2 T cells (Fig. 2B), indicating that the activating mediators are not carried by exosome-like vesicles released by iRBCs (recently described by Regev-Rudzki et al. [36]) that can cross the 0.4-m transwell membrane.

Thus, we investigated whether the activating molecules released in the iRBC supernatant had the reported chemical characteristics of phosphoantigens (12). Supernatants ultrafiltered using 3-kDa-cutoff filters efficiently activated V␥9V␦2 T cells but lost this activity after a prior treatment with apyrase, similar to the prototypic phosphoantigen HMBPP (Fig. 2C). Apyrase treatment did not affect V␥9V␦2 T cells, as activation induced by an anti-CD3 antibody was insensitive to the addition of apyrase-treated supernatants. Therefore, the activating molecules released in the iRBC supernatant have a molecular mass lower than 3 kDa and are terminally pyrophosphorylated. Furthermore, activation of V␥9V␦2 T cells by iRBC supernatant was prevented using anti-V␦2 blocking antibody in a dose-dependent manner, as was the activation by HMBPP (Fig. 2D). This indicates that iRBC supernatant, like HMBPP, stimulates V␥9V␦2 T cells in a TCR-dependent manner. NKG2D, an activating receptor expressed by V␥9V␦2 T cells, also could be involved in these mechanisms. However, including an anti-NKG2D blocking antibody in the activation test had no effect on V␥9V␦2 T-cell activation. ␥␦T-cell lines expressed BTN3A after expansion (Fig. 2E,left). Activation of V␥9V␦2 T cells by phosphoantigens has been shown to be dependent on BTN3A expression by V␥9V␦2 T cells themselves (21). In line with this, V␥9V␦2 T-cell activation by iRBC supernatant was abrogated using neutralizing anti-BTN3A antibody (clone 103.2) (Fig. 2E, right). Thus, the activating molecule(s) released in the culture supernatant have the properties of phosphoantigens.

The activating molecules released by iRBC are intermediates of the DOXP pathway. In order to assess the contribution of the parasites' DOXP pathway to the production of iRBC-released bioactive molecules, we incubated iRBCs in the presence of fosmidomycin, which inhibits DOXP reductase, the first enzyme of this pathway (37) (Fig. 3A). V␥9V␦2 T-cell activation by iRBC culture supernatants was efficiently reduced by fosmidomycin in a dose-dependent manner (Fig. 3B, black line). However, as high doses of fosmidomycin specifically inhibit parasite cycle progression (Fig. 3C), the decrease of V␥9V␦2 T-cell activation might merely reflect a quantitative decrease of parasite maturation and a correlative diminution of phosphoantigen production. In order to circumvent this putative bias, fosmidomycin-treated parasite cultures were complemented with exogenous farnesyl pyrophosphate (FPP), which is produced downstream from the prototypical phosphoantigens HMBPP and IPP (Fig. 3A). In order to determine the optimum dose of FPP able to rescue parasitemia, we monitored the iRBC-treated cultures up to 42 h posttreatment and selected a dose of 1 M FPP (see Fig. S2A in the supplemental material). While 1 M FPP effectively restored the parasite cell cycle progression in the presence of up to 5 M fosmidomycin for 21 h (Fig. 3C, gray line), it did not restore the V␥9V␦2 T-cell activation capacity of the fosmidomycin-treated culture supernatants (Fig. 3B, gray line). Of note, neither fosmidomycin nor FPP at the concentrations used affected V␥9V␦2 T-cell activa- or treated (gray bars) with 0.2 U/ml apyrase at 37°C for 1 h, followed by 3-kDa-cutoff ultrafiltration (Centricon filter). Undiluted iRBC-SNs J and L were tested on ␥␦T-cell lines. Anti-CD3 antibody was used as a phosphatase-insensitive activation control. Data shown are means from duplicates Ϯ SD obtained from a representative experiment using one (␥␦T-cell line 244) out of two independently generated lines. (D) V␥9V␦2 T cells were preincubated with blocking antibodies against V␦2 or NKG2D at concentrations indicated in the caption before activation with either HMBPP (5 nM) or iRBC-SNs K and L. As a negative control, supernatants from uninfected RBC (uiRBC-SNs) were collected and tested in parallel on two independent ␥␦T-cell lines (shown is representative ␥␦T-cell line 227). (E) Expression of BTN3A was assessed on ␥␦T-cell lines after 20 days of expansion (left). V␥9V␦2 T cells then were preincubated with either 1 g/ml anti-V␦2 antibody or 1 g/ml anti-NKG2D antibody or anti-BTN3A blocking antibody at the concentrations indicated before stimulation with either HMBPP (5 nM) or iRBC-SNs L and S (right). Shown are results obtained using representative ␥␦T-cell line 387 (out of 3). S2B). These results clearly demonstrate the obligatory link between phosphoantigen production by the parasite and V␥9V␦2 activation and indicate that the V␥9V␦2 activators are produced upstream from FPP and downstream from DOXP in the DOXP pathway.

V␥9V␦2-stimulating molecules are released in the culture supernatant upon schizont rupture. The results described above were obtained with iRBC culture supernatants collected from synchronized cultures containing mostly mature stages (38 to 40 hpi) of the parasite. To investigate the kinetics of activator molecule release during blood-stage development, supernatants were collected at different time points after invasion and their bioactivity was tested on two ␥␦T-cell lines using the CD107a assay (Fig. 4A). The timing of iRBC schizont rupture was evaluated by monitoring in parallel the release of HRP2 into the iRBC supernatants, which occurs at the time of parasite egress [START_REF] Desakorn | Stage-dependent production and release of histidine-rich protein 2 by Plasmodium falciparum[END_REF]. Bioactivity in the supernatants raised abruptly around 40 hpi and reached a plateau by 50 hpi. Comparable activation kinetic profiles were observed for the two ␥␦T-cell lines, although maximal CD107a reactivity was 20% for ␥␦T-cell line 232 and more than 30% for ␥␦T-cell line 233. The kinetics of HRP2 release were superimposable on those of bioactivity release and were consistent with schizont rupture monitored using Giemsa-stained smears, which showed reinvasion starting at approximatively 40 hpi and being essentially complete by 50 hpi. Altogether, this indicated that the V␥9V␦2 activators were released concomitantly with HRP2, at the very end of the intraerythrocytic cycle, most likely upon parasite egress.

To confirm this conclusion, we blocked parasite egress using compound 2 (C2), a specific inhibitor of the parasite cGMPdependent protein kinase G that regulates parasite egress (35). After checking that the C2 used did not affect V␥9V␦2 T-cell activation by HMBPP, we tested the ability of supernatants from C2-treated cultures to activate V␥9V␦2 T cells (Fig. 4B). Late intraerythrocytic developmental stages were treated with C2 for up to 10 h, and the culture supernatants collected at different time points were tested for HRP2 and V␥9V␦2 T-cell activation capability. We performed experiments with para-sites at approximately 44 hpi (Fig. 4B, top) or parasites at approximately 42 hpi to avoid early schizont rupture events (Fig. 4B, bottom). Under both conditions, blocking schizont rupture by C2 prevented the release of HRP2 into the culture supernatant, as expected, and strongly decreased their bioactivity (Fig. 4B), leading to a C2-induced plateau, contrasting with the sustained increase in the mock-treated culture. C2 treatment was reversible, as parasites resumed egress and simultaneously discharged HRP2 and phosphoantigens into the supernatants after C2 withdrawal (Fig. 4C). There was an almost quantitative recovery of the HRP2 and phosphoantigen bioactivity in the C2-treated culture supernatant after washing out C2 and further culturing the parasites in the absence of C2. Altogether, these data led to the conclusion that the bulk of the activator molecule content is released upon schizont rupture, although we cannot exclude leaking of some bioactivity due to increased permeability of iRBCs at the latest stages of schizogony [START_REF] Raj | Antibodies to PfSEA-1 block parasite egress from RBCs and protect against malaria infection[END_REF].

Quantification of phosphoantigen bioactivity. Due to insufficient sensitivity, we were not able to directly measure phosphoantigen concentrations in the supernatants by standard techniques [START_REF] Jauhiainen | Analysis of endogenous ATP analogs and mevalonate pathway metabolites in cancer cell cultures using liquid chromatography-electrospray ionization mass spectrometry[END_REF]. Therefore, we quantified bioactivity in iRBC supernatants using the CD107a assay with two different ␥␦T-cell lines. We expressed bioactivity as HMBPP equivalents using a calibration curve of V␥9V␦2 T-cell activation obtained with HMBPP. Interestingly, the two ␥␦T-cell lines prepared freshly for this experiment had different dose-response profiles from that for HMBPP (Fig. 5A). ␥␦T-cell line 354 was exquisitely responsive, with a maximal degranulation plateauing at concentrations above 0.39 nM HMBPP and with a calculated 50% maximal response concentration (EC 50 ) of 0.005 nM. ␥␦T-cell line 355 was less responsive, with a calculated EC 50 of 0.571 nM HMBPP and a response threshold of 0.012 nM. The bioactivity of supernatants J, K, and L, collected from late schizont cultures (previously used in experiments shown in Fig. 2), was fitted to these dose-response curves. J, K, and L bioactivities were estimated at 0.029, 0.044, and 0.072 nM HMBPP equivalents, respectively, using ␥␦T-cell line 354, and at 0.042, 0.063, and 0.084 nM HMBPP equivalents, re- spectively, using ␥␦T-cell line 355. This outlined a similar ability to detect phosphoantigens for both ␥␦T-cell lines, despite differing sensitivity and threshold responses.

To evaluate the overall parasite bioactivity, we collected the rupture supernatant from a culture in which schizont rupture, parasite egress, and parasite invasion had proceeded to completion. Bioactivity of this rupture supernatant was titrated with the same two 354 and 355 ␥␦T-cell lines and found to be 0.100 nM and 0.192 nM, respectively (Fig. 5B). This indicated that 80 ϫ 10 4 ruptured schizont iRBCs produce roughly 2.6 to 5.2 pg HMBPP equivalents, i.e., 0.003 to 0.006 fg per iRBC. Altogether, these results show that V␥9V␦2 T cells are extremely sensitive to trace amounts of phosphoantigens.

DISCUSSION

In this paper, we clarify how V␥9V␦2 T cells are activated by blood-stage malaria parasites. We show that iRBC-V␥9V␦2 T-cell contact is dispensable, although we cannot formally exclude a possible interaction with merozoites. This is consistent with the absence of BTN3A on the iRBC surface, and that molecules with the characteristics of phosphoantigens, the V␥9V␦2 T-cell-activating moieties, are produced as soluble molecules from the DOXP pathway and released mostly at the time of P. falciparum erythrocytic egress. V␥9V␦2 T cells are shown to be exquisitely sensitive to phosphoantigen stimulation, although with a substantial variability among donors. The estimated parasite bioactivity and the low threshold of V␥9V␦2 T-cell activation are compatible with a distant activation of V␥9V␦2 T cells by phosphoantigens released in the blood during malarial infection.

As BTN3A is not expressed onto iRBCs, phosphoantigens cannot be presented to V␥9V␦2 T cells by this molecule by the iRBCs, unlike cancer cells (21)(22)(23)(24). The potent activation noted by soluble molecules released in the culture supernatant by iRBCs indicates that presentation by the iRBC itself is fully dispensable. Ultracentrifugation ruled out the possibility of presentation by microvesicles (36), and efficacy of BTN3A blocking on V␥9V␦2 Tcell activation suggested an autopresentation of the captured parasite-derived activator molecules by the V␥9V␦2 T cells that express BTN3A (25,[START_REF] Lang | Early activation of human V gamma 9V delta 2 T cell broad cytotoxicity and TNF production by nonpeptidic mycobacterial ligands[END_REF]. Thus, our overall results showed that the The timing of expression of the DOXP pathway in the apicoplast [START_REF] Bozdech | The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum[END_REF][START_REF] Ralph | Tropical infectious diseases: metabolic maps and functions of the Plasmodium falciparum apicoplast[END_REF] is consistent with the observed stage-dependent bioactivity, as young intracellular stages (ring stages and young trophozoites), in which the apicoplast is poorly active, do not stimulate V␥9V␦2 T cells [START_REF] Pichyangkul | Activation of gammadelta T cells in malaria: interaction of cytokines and a schizont-associated Plasmodium falciparum antigen[END_REF]. The exact chemical composition of parasite stimulants still is uncertain; this is why the quantification of bioactivity was estimated against an HMBPP reference, which is the most active phosphoantigen reported to date. Accordingly, our quantification of soluble bioactivity, expressed as HMBPP equivalents, may underestimate the actual amount of activators if some, such as IPP (15), have a lower specific activity. Nevertheless, our estimates are in accordance with the range of bioactivity (0.1 to 10 nM) measured in supernatants of patient's neutrophils that phagocytosed HMBPP-producing bacteria [START_REF] Davey | Human neutrophil clearance of bacterial pathogens triggers anti-microbial gammadelta T cell responses in early infection[END_REF], with these amounts being sufficient to stimulate V␥9V␦2 T cells.

Essentially similar temporal patterns of HRP2 and V␥9V␦2 T-cell stimulant release into the microenvironment were observed (Fig. 4A). The bulk of these stimulants seem to be released at the end of schizogony, when the parasite egresses from the erythrocyte. Inhibition of their release by C2 was somewhat less efficient when parasites were treated at the very late developmental stages (Fig. 4B,upper). This suggests some leaking of phosphoantigens before egress, likely due to the increased permeability of iRBCs at the later stages of schizogony, as suggested by recent observations showing permeability to immunoglobulins [START_REF] Raj | Antibodies to PfSEA-1 block parasite egress from RBCs and protect against malaria infection[END_REF]. In this case, leakiness would preferentially affect low-molecular-mass metabolites, such as phosphoantigens, while proteins of the size of HRP2 (approximately 35 kDa) would remain intracellular. Several studies, including ours, reported stimulation of V␥9V␦2 T cells by intact mature iRBCs cocultivated with V␥9V␦2 T cells (1,5,9,14,30). We calculated that the amount of stimulants released from as few as 2% of the iRBCs for the first 20 h of parasite culture already could be above the threshold of V␥9V␦2 T-cell activation. This suggests that at least part of stimulation by so-called intact iRBCs occurs through phosphoantigens released from the iRBCs during cocultivation, either upon artifactual, spontaneous lysis of fragile iRBCs or rupture of a few older schizonts in the parasite culture. Nevertheless, the conclusion that the bulk of phosphoantigens are released upon schizont rupture is substantiated by the fact that supernatants collected after complete rupture (Fig. 5B) yielded a 10-fold larger amount of bioactivity than supernatants collected from late developmental stages. As the supernatants were filtered, the potential contribution of direct activation of V␥9V␦2 T cells by egressed merozoites is excluded.

There was some variability of ␥␦T-cell line sensitivity to parasite supernatants, which were used undiluted in most experiments. This differed from the homogeneously maximal activation conveyed by 100 nM HMBPP, used as a positive control. Variability of the response to HMBPP itself was readily unmasked when using concentrations several logs lower than 100 nM (Fig. 5A), i.e., in the range of bioactive phosphoantigens produced by iRBCs. ␥␦T-cell line variability, highlighted at low stimulating concentrations, might be explained by donor-dependent variability of circulating V␥9V␦2 T cells among PBMC (TCR density, differentiation status, immunologic history, and current infections) or could reflect variability generated during the in vitro expansion of V␥9V␦2 T cells for 20 to 22 days. Donor-dependent variability of IFN-␥ production by V␥9V␦2 T cells in response to iRBCs has been reported by d'Ombrain et al. (30).

The experimental conditions used here to investigate the dose response of V␥9V␦2 T cells (10 5 cells and up to 80 ϫ 10 4 iRBCs) allow some extrapolation to clinical situations. In malarial patients, parasite counts in the range of 80 ϫ 10 4 iRBCs/ml (0.02% parasitemia) are frequently observed. Such numbers should release enough phosphoantigens to distantly stimulate patrolling V␥9V␦2 T cells in vivo. As mature P. falciparum intraerythrocytic stages are sequestered in the microvasculature [START_REF] Schofield | Intravascular infiltrates and organ-specific inflammation in malaria pathogenesis[END_REF], schizont burst occurs in anatomically specific niches. We speculate that the elevated sensitivity of V␥9V␦2 T cells allows in vivo activation despite dilution of phosphoantigens in the extracellular milieu and/or in the bloodstream. Triggering of V␥9V␦2 activation could occur in the red pulp of the spleen, where V␥9V␦2 T cells accumulate and young intraeythrocytic stages are retained in the slow open circulation [START_REF] Safeukui | Retention of Plasmodium falciparum ring-infected erythrocytes in the slow, open microcirculation of the human spleen[END_REF]. It also could occur in microvessels, where mature iRBC sequestration reduces the blood flow and provokes infiltrates and inflammation [START_REF] Schofield | Intravascular infiltrates and organ-specific inflammation in malaria pathogenesis[END_REF].

Exploring these hypotheses is complicated by the inappropriate sensitivity of phosphoantigen detection in patient's plasma using mass spectrometry, as the reported limit of quantification for DMAPP and IPP is in the range of 30 nM [START_REF] Jauhiainen | Analysis of endogenous ATP analogs and mevalonate pathway metabolites in cancer cell cultures using liquid chromatography-electrospray ionization mass spectrometry[END_REF], i.e., 2 to 3 orders of magnitude less than that of the V␥9V␦2 T cells in bioassay used here. Our preliminary investigations did not find phosphoantigen bioactivity in malaria patients' plasma. This may reflect the short half-life of phosphoantigens in the peripheral circulation [START_REF] Sicard | ! areas.! One! specific! way! in! which! they! do! this! is! to! inhibit! the! maturation! of! professional! APCs! DCs,! thereby! preventing/diminishing! effective! CD4! and! CD8! T! cell! activation,!and!subsequent!B!cell!help!(Urban!et!al.![END_REF] and does not exclude high concentrations in some tissues.

The results presented here provide a novel framework to understand the activation of V␥9V␦2 T cells during malaria infection and, more generally, infection by microorganisms lacking butyrophilin and releasing or secreting activator molecules such as phosphoantigens into the microenvironment. ! 100! .! We! therefore! concluded! that! the! 10S20%! proliferation! was! due! to! nonSspecific! activation! occurring! in! allogenic! coSculture.! Weak! Vγ9Vδ2! TSAPC! activation! of! naïve! CD4! and! CD8! T! cells! could! be! being!overshadowed!by!this!nonSspecific!activation.!To!minimise!background!activation!we! plan! to! use! negatively! sorted! naïve! CD4! and! CD8! T! cells! as! responder! cells.! The! higher! responder!cell!proliferation!induced!in!three!of!the!MLR!could!be!due!to!chance!optimal!HLA! mismatch.!However,!these!highly!reactive!MLR!matches!were!performed!using!only!Vγ9Vδ2! T!cell!lines,!without!nonSstimulated!Vγ9Vδ2!T!cell!for!comparison.!Therefore!we!cannot!be! certain!that!the!high!proliferation!seen!was!not!due!to!nonSspecific!effects.!We!are!now!HLA! typing!blood!donors!to!ensure!optimal!mismatch!and!testing!corresponding!stimulated!and! nonSstimulated!Vγ9Vδ2!T!cells!to!assess!the!uniqueness!of!activated!Vγ9Vδ2!T!cell!activity!in! highly!reactive!MLRs.!

  Figure!1!:!Countries!with!ongoing!transmission!of!malaria!2013!(WHO!2014).! ......................!22! Figure! 2! :! Countries! reporting! insecticide! resistance! since! 2010,! by! number! of! insecticide! classes!(WHO!2014).!.......................................................................................................!22! Figure! 3! :! Areas,! or! tiers,! of! differing! risks! of! artemisinin! resistance,! SouthSEast! Asia,! December!2014!(WHO!2014).! .........................................................................................!22! Figure!4!:!Life!cycle!of!Plasmodium!falciparum!parasites!(Inoue!et!al.!2013).! ........................!24! Figure!5!:!Proposed!time/exposureSdependent!changes!in!antibody!longevity!and!plasma!cell! composition!(Hviid!et!al.!2015).!......................................................................................!28! Figure!6!:!The!assembly!and!processing!of!the!MSP1!complex!(Holder!2009).!......................!34! Figure!7!:!Stages!of!T!cell!differentiation!(Hayday!&!Pennington!2007).!................................!35! Figure! 8! :! Genetic! organization! and! putative! variability! of! the! γδ! TCR! adapted! from! (Konigshofer!&!Chien!2006;!Bonilla!&!Oettgen!2010).! ....................................................!35! Figure!9!:!MEP!(DOXSP)!and!mevalonate!pathways!for!isoprenoid!biosynthesis.!(Morita!et!al.! 2007)!...............................................................................................................................!37! Figure!10!:!Structure!and!activity!of!prenyl!pyrophosphate!antigens!and!analogs.!(Morita!et! al.!2007)!..........................................................................................................................!37! Figure!11!:!Simplified!schematics!depicting!the!allosteric!model!and!the!antigenSpresenting! model!of!BNT3A1!PSAg!presentation!(Karunakaran!&!Herrmann!2014).! ........................!40! Figure!12!:!γδ!TScell!receptors!(Rey!et!al.!2009).! .....................................................................!40! Figure! 13! :! APC! and! αβ! T! cell! interaction! at! the! synaptic! cleft! (Huppa! &! Davis! 2003;! Kambayashi!&!Laufer!2014).!...........................................................................................!46! Figure! 14! :! Schematic! representation! of! the! class! I! presentation! pathway! (Neefjes! et! al.! 2011).!..............................................................................................................................!48! Figure! 15! :! Schematic! representation! of! the! class! II! presentation! pathway! (Neefjes! et! al.! 2011)!...............................................................................................................................!49! Figure!16!:!Schematic!representation!of!the!crossSpresentation!pathway!(Joffre!et!al.!2012). !........................................................................................................................................!49! Figure!17:!Possible!CD4/CD8!T!cell!activation!by!iRBCSstimulated!Vγ9Vδ2!T!cell!lines!in!MLR. !......................................................................................................................................!103! Figure!18!:!Experiment!outline!to!test!MSP1!crossSpresentation!by!Vγ9Vδ2!T!cells.!...........!105! Figure!19!:!iRBCSstimulated!Vγ9Vδ2!T!cells!express!CD54!and!transiently!express!CD36.!...!107! !

Figure 1 . 1

 11 Figure 1.1 Countries with ongoing transmission of malaria, 2013

Figure 3 . 10 Figure 6 . 9 FIGURE 1 |

 310691 Figure 3.10 Countries reporting resistance since 2010, by number of insecticide classes

  Figure&6&:&The&assembly&and&processing&of&the&MSP1&complex&(Holder!2009).& !MSP1!is!synthesized!and!associates!with!other!proteins,!particularly!MSP7!and!MSP6!on!the! surface! of! the! developing! schizont! and! anchored! to! the! plasma! membrane! by! the! glycosyl! phosphatidyl! inositol! (GPI)! anchor.! At! or! just! before! parasite! egress! and! merozoite! release! the!complex!is!cleaved!into!a!series!of!fragments!by!the!subtilisinSlike!protease!SUB1.!Some! products! of! this! primary! processing! remain! associated! on! the! merozoite! surface.! When! the! merozoite! invades! a! red! cell,! secondary! processing,! mediated! by! another! subtilisin,! SUB2! cleaves! the! membraneSanchored! 42kDa! MSP1! fragment! into! further! 33S! and! 19! kDa! fragments.!The!complex,!including!MSP1 33 ,!is!shed!from!the!merozoite!surface!whilst!the!CS terminal!MSP1 19 ! is!carried!into!the!newly!invaded!red!blood!cell.! ! ! ! Table&1&:&Activating&ligands&for&γδ&T&cells&(Vantourout!&!Hayday!2013).& Subset& Antigen& Reference& Vδ1!(intraepithelial! lymphocyte!(IELs))!

Fig. 1 .

 1 Fig.1. The assembly and processing of the MSP1 complex. MSP1 is synthesized and associates with other proteins, particularly MSP7 and MSP6 on the surface of the developing schizont and linked to the plasma membrane by the glycosyl phosphatidyl inositol (GPI) anchor. At or just before parasite egress and merozoite release the complex is cleaved into a series of fragments by the subtilisin-like protease SUB1 (Koussis et al. 2009); for clarity only the processing of MSP1 is shown but both MSP6 and MSP7 are also processed. Some products of this primary processing remain associated on the merozoite surface. When the merozoite invades a red cell, secondary processing, mediated by another subtilisin, SUB2 cleaves the membrane-anchored 42 kDa MSP1 fragment into further33-and 19 kDa fragments (Harris et al. 2005). The complex, including MSP1 33 , is shed from the merozoite surface whilst the C-terminal MSP1 19 is carried into the newly invaded red blood cell(Blackman et al. 1990). Following invasion MSP1 19 is rapidly internalized into the forming nascent food vacuole, where it persists to the end of the parasite's intracellular development before being discarded in the residual body(Dluzewski et al. 2008)

Figure 1 Figure 24 :

 124 Figure 1 Stages of T cell differentiation. Left, subdivisions of the DN1, DN2 and DN3 stages. Solid black arrows, established transitions; dashed black arrows, uncertain transitions. ISP, immature single-positive; SP, single positive.

Figure 1 .

 1 Figure 1. Potential amino acid sequence diversities of murine immune receptors. The calculated potential amino acid sequence diversities in immunoglobulin (Ig) (without allowance for somatic hypermutation) and αβ and γ δ TCR genes are shown. The first two hypervariable regions of Ig and TCRs (CDR1 and CDR2) are encoded within the V gene segments. The pairing of the random V gene segments (V H and V L , Vα and Vβ, Vγ and Vδ) generates the combinatorial diversity listed as V region combinations. The third hypervariable regions (CDR3s) are encoded within the D and the J gene segments and are listed as junctional combinations. The mechanisms for diversity generation used for this calculation include different D and J gene segment usage, N nucleotide addition of up to six nucleotides at each junction, variability in the 3′ joining position in V and J gene segments and translation of D gene segments in different reading frames. Numbers are corrected for out-of-frame joining, codon redundancy and N region mimicry of germline sequences as detailed in Elliot et al. [42]. The last few amino acids from the TCR V gene segments also contribute to CDR3 diversity, but their effects are negligible and are not included in the calculation.

Figure 1 .

 1 Figure 1. Potential amino acid sequence diversities of murine immune receptors. The calculated potential amino acid sequence diversities in immunoglobulin (Ig) (without allowance for somatic hypermutation) and αβ and γ δ TCR genes are shown. The first two hypervariable regions of Ig and TCRs (CDR1 and CDR2) are encoded within the V gene segments. The pairing of the random V gene segments (V H and V L , Vα and Vβ, Vγ and Vδ) generates the combinatorial diversity listed as V region combinations. The third hypervariable regions (CDR3s) are encoded within the D and the J gene segments and are listed as junctional combinations. The mechanisms for diversity generation used for this calculation include different D and J gene segment usage, N nucleotide addition of up to six nucleotides at each junction, variability in the 3′ joining position in V and J gene segments and translation of D gene segments in different reading frames. Numbers are corrected for out-of-frame joining, codon redundancy and N region mimicry of germline sequences as detailed in Elliot et al.[START_REF] Bozdech | The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum[END_REF]. The last few amino acids from the TCR V gene segments also contribute to CDR3 diversity, but their effects are negligible and are not included in the calculation.

Figure 1 .

 1 Figure 1. Potential amino acid sequence diversities of murine immune receptors. The calculated potential amino acid sequence diversities in immunoglobulin (Ig) (without allowance for somatic hypermutation) and αβ and γ δ TCR genes are shown. The first two hypervariable regions of Ig and TCRs (CDR1 and CDR2) are encoded within the V gene segments. The pairing of the random V gene segments (V H and V L , Vα and Vβ, Vγ and Vδ) generates the combinatorial diversity listed as V region combinations. The third hypervariable regions (CDR3s) are encoded within the D and the J gene segments and are listed as junctional combinations. The mechanisms for diversity generation used for this calculation include different D and J gene segment usage, N nucleotide addition of up to six nucleotides at each junction, variability in the 3′ joining position in V and J gene segments and translation of D gene segments in different reading frames. Numbers are corrected for out-of-frame joining, codon redundancy and N region mimicry of germline sequences as detailed in Elliot et al.[START_REF] Bozdech | The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum[END_REF]. The last few amino acids from the TCR V gene segments also contribute to CDR3 diversity, but their effects are negligible and are not included in the calculation.

  ! pathway! is! found! in! most! Eubacteria! (with! the! notable! exception! of! GramSpositive! cocci),!apicomplexan!protozoa,!and!chloroplasts,!whereas!the!mevalonate!pathway!is!found!in! archaebacteria,! eukaryotes,! and! the! cytoplasm! of! plants.! In! E.coli,! knock! out! or! Dxr! or! GcpE! (highlighted! in! red)! blocked,! while! knock! out! of! lytB! (highlighted! in! green)! greatly! increased! Vγ9Vδ2!T!cell!activation!by!the!mutant!E.coli!strains!(Altincicek!et!al.!2001;!Eberl,!Altincicek,!et! al.!2002).!! ! & Figure&10&:&Structure&and&activity&of&prenyl&pyrophosphate&antigens&and&analogs.&(Morita!et! al.!2007)& The!structure!and!biological!activity!of!various!prenyl!pyrophosphates!is!shown.!Values!are!the! concentration! required! for! halfSmaximum! stimulation! of! proliferation! for! the! Vγ9Vδ2! TScell! clone!12G12.! methyl-D-erythritol 4-phosphate pathways

Fig. 3 .

 3 Fig. 3. Structure and activity of prenyl pyrophosphate antigens and analogs. Structure and biological activity of various prenyl pyrophosphates

FIGURE 2 |

 2 Figure&11&:&Simplified&schematics&depicting&the&allosteric&model&and&the&antigen]presenting& model&of&BNT3A1&P]Ag&presentation&(Karunakaran!&!Herrmann!2014).& !(A)!Sketches!representing!the!allosteric!model!describing!possible!events!involved!in!PSAgS mediated! activation! of! Vγ9Vδ2! TCR.! X! -! represents! unidentified! molecules! that! might! be! involved! in! the! mechanism.! (B)! Schematic! representation! of! antigenSpresentation! model.! Note!that!both!models!can!be!partially!combined.!!

  The amino acids proposed to interact with HMBPP or IPP respectively are marked in Figure 1A. (v) Plasmon resonance analysis revealed a low affinity binding of recombinant Vg9Vd2 TCR multimers (dextramers) (TCR G2B9) to immobilized eukaryotic recombinant BTN3A1-ED (vi) Surface enhanced Raman scattering demonstrates binding of monomeric TCR to BTN3A1 with a K d of 34 ⇥ 10 5 M in the presence and 93 ⇥ 10 5 M in the absence of IPP. All these data can be interpreted www.frontiersin.org December 2014 | Volume 5 | Article 648 | 5

Figure 1 .

 1 Figure 1. NK and gd T-cell receptors. Receptors have been classified as activating receptors (green), which mediate cytolysis, adhesion or co-stimulatory receptors (brown), which contribute to positive signals, and inhibitory receptors (red), which inhibit cytolysis. NK and gd T cells express certain common receptors, such as NKG2D and KIR. Integration of these signals contributes to regulation of NK or gd T-cell activities, which include activation, proliferation, cytokine or chemokine secretion and cytolysis in part by the release of cytotoxic granules containing perforin or granzymes. Bold type indicates the principle receptors.

  ;![START_REF] Viey | !PhosphostimSactivated!gamma!delta!T!cells!kill!autologous!metastatic!renal!cell! carcinoma[END_REF]!and!infectious!targets!such!as!H1N1!influenza!virus! infected!macrophages!(Qin!et!al.!2009)!intracellular!M.!tuberculosis!(Dieli!et!al.!2000;[START_REF] Dieli | ! GranulysinSdependent! killing! of! intracellular! and! extracellular! Mycobacterium! tuberculosis! by! Vgamma9/Vdelta2! T! lymphocytes.! The![END_REF]!and!blood!stage!P.!falciparum!(Farouk!et!al.!2004;!Costa!et!al.!2011).!1.3.3.2.3 T*Regulatory*functions*It! has! been! shown! that! PSAg,! ILS15! and! transforming! growth! factor! (TGF)β! stimulated! Vγ9Vδ2!T!cells!stably!upSregulate!FOX3P!and!inhibit!CD3+/CD28+!induced!peripheral!blood! mononuclear! cell! (PBMC)! proliferation! in! a! contactSdependent! manner.! This! suggests! that! Vγ9Vδ2!T!cells!can!function!similarly!to!CD4+/25+!naturally!occurring!regulatory!T!cell!(Tregs)![START_REF] Casetti | ! Cutting! edge:! TGFSbeta1! and! ILS15! Induce! FOXP3+! gammadelta! regulatory! T! cells! in! the! presence! of! antigen! stimulation[END_REF][START_REF] Wesch | !Human!gamma!delta!T!regulatory!cells!in!cancer:!fact!or! fiction?![END_REF].!1.3.3.2.4 Interactions*with*other*immune*cells*As! well! as! direct! antiSinfection! and! antiScancer! functionality,! Vγ9Vδ2! T! cells! can! also! provide!stimulation!to!cells!of!both!the!innate!and!adaptive!immune!responses.!!1.3.3.2.4.1 Monocytes*and*monocyte>derived*DCs*In!vitro,!Vγ9Vδ2!T!cells!trigger!monocyte!activation!and!maturation!to!DCs!via!IFNγ,!TNFα,!GranulocyteSmacrophage!colonySstimulating!factor (GMSCSF)!and!ILS4!secretion![START_REF] Brandes | CrossIpresenting!human!gammadelta!T!cells!induce!robust!CD8+!alphabeta!T! cell!responses[END_REF].!Also!in!vitro,!they!interact!with!monocyte!derived!DCs!in!a!positive!feedback!loop!via! type!I!IFN!release!by!the!DC![START_REF] Kunzmann | !PolyinosinicSpolycytidylic!acidSmediated!stimulation!of!human!gammadelta! T!cells!via!CD11c!dendritic!cellSderived!type!I!interferons[END_REF][START_REF] Devilder | ! Early! triggering! of! exclusive! IFNSgamma! responses! of! human! Vgamma9Vdelta2!T!cells!by!TLRSactivated!myeloid!and!plasmacytoid!dendritic!cells[END_REF]!and!TNFα!and!IFNγ! release! by! the! Vγ9Vδ2! T! cells!(Ismaili! et! al.! 2002;[START_REF] Conti | ! Reciprocal! activating! interaction! between! dendritic! cells! and! pamidronateS stimulated! gammadelta! T! cells:! role! of! CD86! and! inflammatory! cytokines[END_REF].! This! results! in! APC! maturation,!increased!crossSpresentation!and!decreased!phagocytic!activity!by!the!DC![START_REF] Conti | ! Reciprocal! activating! interaction! between! dendritic! cells! and! pamidronateS stimulated! gammadelta! T! cells:! role! of! CD86! and! inflammatory! cytokines[END_REF][START_REF] Martino | !Complementary!function!of!gamma!delta!TSlymphocytes!and!dendritic!cells!in! the!response!to!isopentenylSpyrophosphate!and!lipopolysaccharide!antigens[END_REF][START_REF] Shrestha | ! Regulation! of! acquired! immunity! by! gamma! delta! TScell/dendriticScell! interactions[END_REF]!Dunne!et!al.!2010).!1.3.3.2.4.2 Neutrophils*Both!Vγ9Vδ2!T!cells!and!neutrophils!are!early!arrivals!at!infection!sites.!Vγ9Vδ2!T!cells! can!maintain!the!viability![START_REF] Davey | !Human!neutrophil!clearance!of!bacterial!pathogens!triggers!antiSmicrobial!γδ! T!cell!responses!in!early!infection[END_REF]!and!induce!both!chemotaxis!and!phagocytosis!

  Figure& 13& :& APC& and& αβ& T& cell& interaction& at& the& synaptic& cleft& (Huppa! &! Davis! 2003;! Kambayashi!&!Laufer!2014).& A.!Profile!view!of!DC!and!CD4+!T!cell!interactions,!with!examples!of!each!of!signals!1,!2!and!3! necessary!for!T!cell!activation.!B.!Face!view!of!the!synaptic!cleft!illustrating!the!central!supraS molecular! activation! complex! (cSMAC,! green),! the! peripheral! SMAC! (pSMAC,! purple),! the! distal!SMAC!(dSMAC,!blue)!and!the!location!of!molecules/ligand!pairs!enriched!in!each!area.!

Box 1 |Figure 2 |

 12 Figure& 14& :& Schematic& representation& of& the& class& I& presentation& pathway&[START_REF] Neefjes | ! Towards! a! systems! understanding! of! MHC! class! I! and! MHC! class! II! antigen! presentation[END_REF].&& End! of! life! proteins! and! defective! ribosomal! products! (DRiPs)! in! the! cytoplasm!are!recycled!into! peptides! and! amino! acids! by! the! proteasome! and,! if! present,! the! immunoproteasome.! Peptides! can! then! be! further! modified! by! aminopeptidases! in! the! cytoplasm! (for! example! by! thimet!oligopeptidase!(TOP)! which! generates! peptides! of! 8S15! amino! acids! long,! and! tripeptidyl! peptidase! II! (TPPII)! which! generates! peptides! >15! amino! acids! long).! It! is! also! possible! for! peptides! from! neighboring! cells!to!enter!via!tight!junctions.!Cytosolic!peptides!are!transported!into!the!endoplasmic!reticulum!(ER)! by! the! transporter! associated! with! antigen! presentation! (TAP).! TAP! associates! with! MHCI! in! the! ER! membrane!together!with!the!peptideSloading!complex!(PLC).!The!PLC!consist!of!the!chaperone!protein! tapasin,! Calreticulin! and! ERp57,! which! stabilise! the! MHCI! molecule! and! facilitate! peptide! loading.! Peptides!can!be!further!processed!in!the!ER!by!ERAP!(ERSresident!peptidase).!Once!associated!with!a! peptide,! the! now! stable! MHCI! is! transported! via! the! Golgi! apparatus! to! the! external! membrane.! For! recycling,! MHCI! are! marked! for! ubiquitinylation! by! the! protein! membraneSassociated! ring! finger! (C3HC4)!1!(MARCH1),!resulting!in!their!internalisation!and!either!recycling!or!lysosomal!degradation.! ! ! ! ! ! ! ! !

Figure 3 |

 3 Figure3| The basic MHC class II antigen presentation pathway. MHC class II αand β-chains assemble in the endoplasmic reticulum (ER) and form a complex with the invariant chain (Ii). The Ii-MHC class II heterotrimer is transported through the Golgi to the MHC class II compartment (MIIC), either directly and/or via the plasma membrane. Endocytosed proteins and Ii are degraded by resident proteases in the MIIC. The class IIassociated Ii peptide (CLIP) fragment of Ii remains in the peptide-binding groove of the MHC class II dimer and is exchanged for an antigenic peptide with the help of the dedicated chaperone HLA-DM (known as H2-M in mice). MHC class II molecules are then transported to the plasma membrane to present antigenic peptides to CD4 + T cells. APC, antigen-presenting cell; TCR, T cell receptor.

  stage!parasites!for!inoculation,!while!those! which! show! γδ! T! cells! are! essential! mostly! use! intraSvenous! inoculation! routes.! Different! parasite! species! have! different! multiplication! rates! and! different! susceptibility! to! cellular! immune! effectors,! and! different! mouse! strains! may! have! differently! polarized! immune! responses.! These! factors! combined! with! differing! inoculation! routes! between! studies,! can! create!very!different!infection!kinetics!and!disease!severity.!As!demonstrated!by!Inoue!et!al,! the! kinetics! of! the! immune! response! relative! to! disease! progression! is! important! for! γδ! T! cellSmediated!protection![START_REF] Inoue | !Enhancement!of!dendritic!cell!activation!via!CD40!ligandSexpressing!γδ!T!cells! is! responsible! for! protective! immunity! to! Plasmodium! parasites.! Proceedings! of! the! National! Academy!of!Sciences!of!the!United!States!of!America[END_REF].!Thus!from!mouse!studies!we!can!learn!that,!in! certain!infection!conditions!γδ!T!cells!are!essential!for!parasite!clearance,!but!not!in!others.!

  !clear!infection.! γδ!T!cell!(and!NK!cell)! expansion.!! Unable!to!clear!infection.!clear!infection.! Lower!cDC!and!pDC! maturation!and! expansion!in!the!spleen.!! Less!ILS12!production.! Able!to!clear!infection.! DC!activation!and! expansion.

  Abbreviations:! KO:! knock! out;! IV:! intravenous! injection;! IP:! intraSperitoneal! injection;! MCP:! macrophage! chemoattractant!protein.!

  3.2.4,!Vγ9Vδ2!T!cells! ! ! 66! are! known! to! interact! with! NK! cells,! monocytes,! DCs,! B! cells! and! αβ! T! cells,! and! DC! interaction! has! already! been! shown! important! in! parasite! clearance! in! mice.! Due! to! their! innate!reactivity!to!the!parasites,!Vγ9Vδ2!T!cells!could!very!quickly!activate!and!recruit!other! components!of!the!immune!system!via!direct!interaction!and!cytokine!release.!Subsequently,! after! having! launched! the! initial! phases! of! a! response,! they! possibly! go! to! lymph! nodes! to! launch!or!boost!the!adaptive!αβ!T!cell!responses!by!acting!as!APCs.!Humanised!mice!would! be!very!useful!to!unpick!the!detailed!mechanisms!of!Vγ9Vδ2!T!cell!function.

  activation and expansion of the V␥9V␦2 subpopulation of ␥␦T cells, which inhibit the Plasmodium falciparum blood cycle through soluble cytotoxic mediators, abrogating merozoite invasion capacity. Intraerythrocytic stages efficiently trigger V␥9V␦2 T-cell activation and degranulation through poorly understood mechanisms. P. falciparum blood-stage extracts are known to contain phosphoantigens able to stimulate V␥9V␦2 T cells, but how these are presented by intact infected red blood cells (iRBCs) remains elusive. Here we show that, unlike activation by phosphoantigen-expressing cells, V␥9V␦2 T-cell activation by intact iRBCs is independent of butyrophilin expression by the iRBC, and contact with an intact iRBC is not required. Moreover, blood-stage culture supernatants proved to be as potent activators of V␥9V␦2 T cells as iRBCs. Bioactivity in the microenvironment is attributable to phosphoantigens, as it is dependent on the parasite DOXP pathway, on V␥9V␦2 TCR signaling, and on butyrophilin expression by V␥9V␦2 T cells. Kinetic studies showed that the phosphoantigens were released at the end of the intraerythrocytic cycle at the time of parasite egress. We document exquisite sensitivity of V␥9V␦2 T cells, which respond to a few thousand parasites. These data unravel a novel framework, whereby release of phosphoantigens into the extracellular milieu by sequestered parasites likely promotes activation of distant V␥9V␦2 T cells that in turn exert remote antiparasitic functions.

  ␥␦T-Cell Activation by Malaria Phosphoantigens October 2015 Volume 83 Number 10 iai.asm.org 3817 Infection and Immunity on September 11, 2015 by UNIVERSITE VICTOR SEGALEN http://iai.asm.org/ Downloaded from 4-[7-[(dimethylamino)methyl]-2-(4-fluorphenyl)imidazo[1,2-a] pyridin-3-yl]pyrimidin-2-amine, or its vehicle, dimethyl sulfoxide (DMSO) (35). iRBC supernatants were collected at various time points. Statistical analysis. Statistical tests used are indicated in the figure legends when applied. When shown, error bars correspond to means Ϯ standard deviations (SD).

FIG 1

 1 FIG 1 Plasmodium falciparum-infected RBC do not express butyrophilin 3 and activate V␥9V␦2 T cells without contact. (A) Plasmodium falciparuminfected (iRBCs) or uninfected red blood cells (uiRBCs) were incubated with anti-butyrophilin3 (BTN3A) antibody (black line) or with an isotypic control (light gray) and analyzed by flow cytometry. BTN3A expression in CD3 ϩ PBMC also was analyzed. Shown are data of BTN3 labeling from one representative experiment out of three. (B) Gating strategy for CD107a degranulation test. V␥9V␦2 short-term lines (␥␦T-cell lines) were incubated with stimulants (medium, HMBPP, Daudi cells, or iRBC at a 10:1 target-to-effector ratio) and PE-labeled anti-CD107a antibody for 4 h, washed, and subsequently incubated with FITC-labeled anti-V␦2 antibody. Degranulated cells are identified by flow cytometry as CD107a-positive cells within the V␦2 ϩ population. (C) Stimulants (100 nM HMBPP, 200 nM BrHPP) or target cells (iRBCs, uiRBCs, or Daudi cells at the indicated target/effector ratios) were either incubated for 4 h with V␥9V␦2 T cells or cultured in the upper chamber of the 0.4-m polycarbonate transwell device. V␥9V␦2 T-cell degranulation was further assessed by CD107a assay after 4 h of incubation in contact with (gray bars) or physically separated from (black bars) stimulants as indicated. Shown are the results from one representative ␥␦T-cell line (␥␦T-cell line 168) out of 4 (complete data are in Fig. S1 in the supplemental material). Midstage schizonts (38 to 40 hpi) were used as iRBCs.

FIG 2

 2 FIG 2V␥9V␦2T-cell activation is mediated by soluble phosphoantigen(s) in a TCR-dependent manner. The reactivity of V␥9V␦2 T cells toward iRBC culture supernatants (iRBC-SNs) collected from different parasite cultures (M, J, K, L, and S) at 4% parasitemia was assayed using the CD107a degranulation test. (A) Reactivity of V␥9V␦2 T cells from fresh PBMCs from 5 malaria-naive donors to intact iRBCs or uiRBCs at the indicated ratios was compared to the reactivity to the corresponding culture supernatants (left) (statistical significance is calculated using one-way analysis of variance [ANOVA]; **, P Ͻ 0.01; ***, P Ͻ 0.0001). Serial 2-fold dilutions of iRBC-SN J were assayed on V␥9V␦2 short-term lines (␥␦T-cell lines). (Right) Means Ϯ SD from duplicates obtained from a representative experiment using one (␥␦T-cell line 226) out of two independently generated ␥␦T-cell lines. (B) Reactivity of ␥␦T-cell lines to iRBC-SN G was tested before (ϪUCF) and after (ϩUCF) ultracentrifugation at 197,000 ϫ g on two ␥␦T-cell lines (depicted are representative data obtained with ␥␦T-cell line 151). (C) V␥9V␦2 T-cell degranulation was measured after incubation with medium alone, HMBPP (100 nM), and iRBC-SNs J and L and were left untreated (black bars) or treated (gray bars) with 0.2 U/ml apyrase at 37°C for 1 h, followed by 3-kDa-cutoff ultrafiltration (Centricon filter). Undiluted iRBC-SNs J and L were tested on ␥␦T-cell lines. Anti-CD3 antibody was used as a phosphatase-insensitive activation control. Data shown are means from duplicates Ϯ SD obtained from a representative experiment using one (␥␦T-cell line 244) out of two independently generated lines. (D) V␥9V␦2 T cells were preincubated with blocking antibodies against V␦2 or NKG2D at concentrations indicated in the caption before activation with either HMBPP (5 nM) or iRBC-SNs K and L. As a negative control, supernatants from uninfected RBC (uiRBC-SNs) were collected and tested in parallel on two independent ␥␦T-cell lines (shown is representative ␥␦T-cell line 227). (E) Expression of BTN3A was assessed on ␥␦T-cell lines after 20 days of expansion (left). V␥9V␦2 T cells then were preincubated with either 1 g/ml anti-V␦2 antibody or 1 g/ml anti-NKG2D antibody or anti-BTN3A blocking antibody at the concentrations indicated before stimulation with either HMBPP (5 nM) or iRBC-SNs L and S (right). Shown are results obtained using representative ␥␦T-cell line 387 (out of 3).

  ␥␦T-Cell Activation by Malaria Phosphoantigens October 2015 Volume 83 Number 10 iai.asm.org 3819 Infection and Immunity on September 11, 2015 by UNIVERSITE VICTOR SEGALEN http://iai.asm.org/ Downloaded from tion by HMBPP (see Fig.

FIG 3

 3 FIG 3 DOXP pathway inhibition abrogates activation of V␥9V␦2 T cells by iRBC-SN. (A) Simplified representation of the steps in the DOXP pathway relevant to our experimental protocol. G3P, glutaraldehyde 3 phosphate; DOXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; HMBPP, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; FPP, farnesyl pyrophosphate; exo. FPP, exogenous FPP. Note that between MEP and HMBPP, several steps have been skipped. Endogenous products are in boldface characters. (B) V␥9V␦2 T-cell reactivity was tested toward iRBC-SNs collected from iRBC cultures treated for 21 h with fosmidomycin in the presence or absence of 1 M FPP. Data shown are means Ϯ SD of duplicates obtained from a representative experiment using ␥␦T-cell line 266. (C) Effect of fosmidomycin treatment and exogenous FPP (1 M) addition on parasite culture progression. IRBC cultures were treated during 21 h with fosmidomycin, with or without 1 M FPP. Parasitemia after reinvasion was evaluated in triplicate wells. Controls in CPM alone without fosmidomycin are shown on the left.

FIG 4

 4 FIG 4 Phosphoantigens are released during iRBC rupture. (A) Synchronized parasites from 3 independent cultures (A, B, and C) at 1% parasitemia were cultured in CPM for 62 h, and iRBC supernatants were collected at the indicated time points (hours postinvasion [hpi]) across the parasite developmental cycle (time zero corresponds to parasitic invasion). The phosphoantigen bioactivity in the various iRBC-SNs was assessed on two independent ␥␦T-cell lines (232 and 233) using CD107a surface expression. HRP2 concentration in the iRBC-SNs was determined by ELISA. Data show the means Ϯ SD from CD107a expression induced by the three independent culture SNs (A, B, and C) and their means Ϯ SD for HRP2 content at each time point. (B) Synchronized iRBC cultures (1% parasitemia) were treated with compound 2 (C2) or a control (DMSO) at the time indicated by the arrow, and supernatants were collected at different time points of treatment. Two independent C2 treatment experiments are shown on 44-hpi schizonts (top) and 42-hpi schizonts (bottom). HRP2 content was measured by ELISA (left), and phosphoantigen concentrations were assessed by CD107a test (right) on 3 ␥␦T-cell lines. Results for representative ␥␦T-cell line 384 are shown. (C) Parasite cultures used for panel B were washed after 6 h of treatment with compound (C2), fresh medium was added, and supernatants were collected at the indicated times postwash and tested for both their HRP2 content (left) and their ability to induce V␥9V␦2 T-cell degranulation (right). Shown are results from one ␥␦T-cell line (the same as that shown in panel B) out of three.

  ␥␦T-Cell Activation by Malaria Phosphoantigens October 2015 Volume 83 Number 10 iai.asm.org 3821 Infection and Immunity on September 11, 2015 by UNIVERSITE VICTOR SEGALEN http://iai.asm.org/ Downloaded from activating molecules have all the characteristics of phosphoantigens.

FIG 5

 5 FIG 5 Measurement of V␥9V␦2 T-cell stimulation bioactivity in iRBC supernatants. Dose-response stimulation of two different ␥␦T-cell lines with either HMBPP expressed as a concentration and corresponding quantities in the assays (A) or supernatant of fully ruptured iRBCs (see Materials and Methods) and the theoretical corresponding number of contributing iRBCs in the assay, extrapolated from the dilution factor (B). After stimulation, CD107a surface expression was assayed. V␥9V␦2 T-cell activation obtained with culture supernatants J, K, and L and uiRBC supernatant are shown. Data represent means Ϯ SD from duplicates. Titration of the rupture supernatant with one ␥␦T-cell line was calculated at half of the maximum of the percent CD107a ϩ V␦2 ϩ , which was obtained after stimulation by both supernatant dilution determined in panel B and the HMBPP concentration determined in panel A. The HMBPP equivalent content of the supernatant corresponds to the HMBPP concentration determined in panel A multiplied by the supernatant dilution factor determined in panel B.

Figure S1 .Figure S2 .Figure 1 ,Figure 1 .

 S1S211 Figure S1. Vγ9Vδ2 T cell activation by iRBCs does not require contact. (A) Vγ9Vδ2 T cell degranulation was assessed by the CD107a assay after 4h-incubation of γδT-cell lines with stimulants present in the same well (grey bars) or separated by a Transwell device (black bars). BrHPP was used as a soluble, freely diffusing positive control. Daudi cells were used as a non diffusing stimulant control. Different iRBC/ γδT-cell ratios were used as indicated. uiRBC refers to uninfected red blood cells. Nd: not done. Experimental results from 3 different γδT-cell lines (59, 61 and 62) are shown. (B) Highly purified Vγ9Vδ2 T cells (>90% of purity) which had been screened for capacity to degranulate in the presence of iRBCs with an average reactivity >50%, were labeled with Lysotracker red and incubated with purified iRBCs (38 hpi) at different effector:target ratios or with Daudi cells (E:T ratio = 1:2) as control. During the 2 hours of observation, almost all Vγ9Vδ2 T cells formed long-lived conjugates with Daudi cells, while iRBC-Vγ9Vδ2 conjugates could scarcely be observed. Freeze screen time-lapse microscopy pictures were captured at 5:00 min (Daudi cells) or 4:04 min (iRBCs) using a Leica DMI6000 TCS SP5, confocal microscope inverted stand and lasers emitting at 488 and 543 nm. Images were processed using ImageJ software.

Figure 2 .

 2 Figure 2. Vγ9Vδ2 T lymphocytes acquire an APC-like phenotype after in vitro stimulation by blood stage P. falciparum. Kinetics of Vδ2+ T cell expansion and HLA-DR, HLA-ABC, CD40, CD80, CD83 and CD86 expression on Vδ2+ T cells stimulated 12 days with 5 x10 6 /ml iRBC (red), 5mil/ml uiRBC (blue), 10nM HDMAPP (green) or Complete Medium (black). (A) Representative donor and (B) Mean of 5 donors. Expressed as Median Fluorescent intensity (MFI) on Vδ2+ T cells and % positive Vδ2+ T cells. Mean is indicated by the horizontal bar. Dotted line indicates MFI positive gating threshold. Statistical significance was calculated using 2-way ANOVA and is indicated for time-course comparison and individual time-points. (***P<0.001, **P<0.01, *P<0.05).

Figure 3 ,Figure 3 . 1 .

 331 Figure 3, Howard et al

2 . 2 . 3 . 1 . 1 . 2

 223112 Abbreviations:!ND=!Not!Done,!NA=Not!Applicable.!FC!:!febrile!controls;!VFR!:!malaria!patients!migrants!to! France!born!in!malaria!endemic!countries!and!visiting!friends!and!relatives!in!malaria!endemic!areas;!TEO!:! malaria!patients,!travellers!of!European!(Caucasian)!origin;!FTEO!:!malaria!patient,!travellers!of!European! (Caucasian)!origin!with!a!history!of!frequent!visits!to!endemic!areas;!*Insufficient!(<200)!Vγ9Vδ2!T!cell! events!recorded!therefore!patient!not!included!in!Figure!1!analysis.! !

Figure&18&

  Figure&18&:&Experiment&outline&to&test&MSP1&cross]presentation&by&Vγ9Vδ2&T&cells.!

  cleaves the C-terminal MSP1 42 into 2 fragments : a N-terminal 33 kDa fragment (MSP1 33 ) and a C-terminal 19 kDa fragment (MSP1

  19

stage surface Red blood cell invasion Merozoite release/egress

	S P 7 M MSP1 MSP1 Schizont surface M S P 6	primary processing (SUB1)	M S P 6 Merozoite surface 83 83 42 42 38 30 30 38	M S P 7 38 38 33 33 30 30 19 83 83 "Ring" secondary M S P 6 processing (SUB2)

  effector!mechanisms!as!outlined!below.! As!previously!mentioned!Vγ9Vδ2!T!cells!rapidly!expand!in!response!to!stimulus.!It!has!been! shown! that! this! expansion! is! greater! and! more! rapid! on! secondary! exposure![START_REF] Shen | ! Adaptive! immune! response! of! Vgamma2Vdelta2+! T! cells! during! mycobacterial! infections.!Science![END_REF][START_REF] Chen | ! Adaptive! immune! response! of! Vgamma2Vdelta2! T! cells:! a! new! paradigm[END_REF]!indicating!a!memory!population!(reviewed!(De!Rosa!et!al.!2004)).! Indeed! it! has! been! proposed! that! Vγ9Vδ2! T! cells! follow! the! same! memory! differentiation!

	Table&2&:&T&cell&memory&subsets&and&their&characteristics&(Battistini!et!al.!2005).& 1.3.3.1 Expansion&and&differentiation&
	Subset&	Identifying!markers!	Characteristics!
	T&naïve&&	CD45RA+!CD27+!	Express! CCR7! and! CD62L,! which! permit! lymphS
				node!homing.!
	Tcm&		CD45RAS!CD27+!	Express! CCR7! and! CD62L,! but! also! the! CD45RO!
	memory! marker.! They! therefore! represent! an! pattern! as! αβ! T! cells,! including! naïve! (Tnaïve),! central! memory! (Tcm),! effector! memory.!
	AgSprimed! population! able! to! traffic! through! (Tem)! and! terminally! differentiated! effector! memory! (Temra)! T! cells! (Dieli! et! al.! 2003)! as!
	the! lymph! nodes! and! ready! to! respond! quickly! outlined!in!and!reviewed!in!(Sallusto!et!al.!2004;!Battistini!et!al.!2005)!
	Tem&&	Exposure	CD45RAS!CD27S!	to!a!stimulus.! DownSregulated! CCR7! and! CD62L! expression.!
				Have! increased! inflammatory! cytokine!
				expression! such! as! CCR2,! CCR5,! CCR6! and!
				CXCR3.! Represent! a! pool! of! AgSprimed! cells!
				patrolling!the!peripheral!tissues.!
	Temra&	CD45RA+!CD27S!	Fail!to!proliferate!in!response!to!stimulation!and!
				produce! low! levels! of! IFNγ.! However! contain!
				large! amounts! of! granulysin! and! perforin.!
				Represent!a!differentiated!cytotoxic!population.!
	!		!
		!		43!
		!

Table 1 ,

 1 Howard et al

	Table&1:&Patient&cohort&information.!
	Patient& Febrile&Infection&	Parasit7&	Details&	Duration&of&stay&in&	Group& Disease&attributes&	Anti7infection&specific&
			aemia&		malaria&endemic&zone.&&&			treatment&
	P1!	Febrile,!non0malarial!	NA!	!	!ND!	FC!	!	!
	P2!	Dengue!fever!	NA!	Non0migrant!	!ND!	FC!	Non0complicated!	Unknown!
	P3!	E!Coli!urinary!infection!	NA!	Non0migrant!	!ND!	FC!	Non0complicated!/!RAS!	Ceftriaxone!
		(compatible!with!
		pyelonephritis)!
	P4!	Prostatitis!0!Klebsiella!	NA!	Migrant,!from!the!Congo! !ND!	FC!	Acute!kidney!dysfunction!	Ceftriaxone,!then!ertapenem!
		pneumonia!BLSE!+!					with!urinary!retention.!	then!sulfamethoxazole!and!
		Lambliasis!(fortuitous!						trimethoprim!
		discovery)!!						(bactrim)+albendazole!
	P5!	EBV!infection!(compatible!	!NA!	Migrant,!regularly!travel!to!	!ND!	FC!	Non0complicated!/!RAS!	Unknown!
		with!infectious!		Tchad.!
		mononucleosis)!
	P6!	P.!falciparum!	0.5!	Non!migrant!	2!weeks!humanitarian!	FTEO! Non0complicated!	DHA0PQP!+!metronidazole!
					mission.!			(blastocytis!hominis!in!feces)!
	P7!	P.!falciparum!	0.6!	Non0migrant,!previous!	2!months!in!Ivory!Coast! FTEO! Non0complicated!/!high!	DHA0PQP!
				malaria!episodes.!			platelets!
	P8!	P.!falciparum!	3!	Military.!	5!months!military!mission!FTEO! Non0complicated!	DHA0PQP!
	P9!	P.!falciparum!	4.4!	Non0migrant,!previous!	7!months!humanitarian!	FTEO! Non0complicated!	DHA0PQP!
				malaria!cases.!	mission!
	P10!	P.!falciparum!	19!	Non0migrant,!frequent!trips!	2!weeks!	FTEO! Severe:!hyperparasitemia,!	Artesunate!then!DHA0PQP!+!
				to!the!Ivory!Coast.!			prostration,!instability,!	ceftriaxone!
							hemodynamic.!
	P11!	P.!falciparum!	<0,01!	Not!specified!	!ND!	TEO! !	!
	P12!	P.!falciparum!	0.05!	Not!specified!	14!days!	TEO! Non0complicated!	Atovaquone/proguanil!
	P13!	P.!falciparum!	0.5!	Non0migrant!	!	TEO! Non0complicated!	DHA0PQP!
	P14!	P.!falciparum!	2.23!	Non0migrant!	1!month!	TEO! Non0complicated!	DHA0PQP!
	P15!	P.!spp!	0!(PCR+)! Migrant,!born!in!the!Ivory!	Internship!	VFR! Non0complicated!	Atovaquone/proguanil!
				Coast.!

malaria! patients,! travellers! of! European! (Caucasian)! origin;! FTEO! :! malaria! patient,! travellers! of! European! (Caucasian)! origin! with! a! history!of!frequent!visits!to!endemic!areas;!Migrant:!migrant!to!France;!Non0migrant:!born!and!resident!in!France.!*Insufficient!(<200)! Vγ9Vδ2!T!cell!events!recorded!therefore!patient!not!included!in!Figure!1!analysis.! ! 97! Figure 2, Howard et al

Supplemental*Table*1:!Percentage*and*total*lymphocyte*counts*of*patient*cohort*on*hospital* admission.*

  SupplementalTable 1, Howard et al

	!	*		*		*		*		*		*
	*	Circulating*	CD3+*total*	CD3+CD4+CD8A*	CD3+CD4ACD8+*	γδ*total*	Vδ2+*
		lymphocytes*
	*	!	Giga/l.* Giga/l.*	%*	Giga/l.*	%*	Giga/l.*	%*	Giga/l.*	%*	Giga/l.*	%*
	Healthy*Average:*	1.6&4!	1,1&1.7!	67&76!	0.7&1.1!	38&46!	0.5&0.9!	31&40!	NA!	NA!	NA!	NA!
	P2*	FC!	0.906!	0.493!	54.43!	0.303!	33.42!	0.152!	16.81!	0.025!	2.71!	0.022!	2.47!
	P3*	FC!	0.775!	0.651!	83.97!	0.501!	64.67!	0.134!	17.24!	0.01!	1.32!	0.003!	0.43!
	P4*	FC!	1.388!	0.529!	38.13!	0.363!	26.16!	0.152!	10.95!	0.018!	1.26!	0.01!	0.74!
	P5*	FC!	3.229!	2.34!	72.48!	1.292!	40.01!	0.888!	27.5!	0.093!	2.89!	0.081!	2.51!
	P9*	FTEO!	0.701!	0.538!	76.8!	0.368!	52.42!	0.132!	18.88!	0.025!	3.61!	0.021!	3.04!
	P10*	FTEO!	0.985!	0.563!	57.14!	0.392!	39.76!	0.116!	11.73!	ND!	ND!	ND!	ND!
	P11*	TEO!	2.516!	1.487!	59.08!	0.746!	29.64!	0.573!	22.79!	0.056!	2.22!	0.041!	1.61!
	P12*	TEO!	2.149!	1.739!	80.92!	1.076!	50.08!	0.608!	28.31!	0.062!	2.89!	0.014!	0.67!
	P13*	TEO!	0.414!	0.361!	87.26!	0.174!	41.92!	0.166!	39.99!	0.01!	2.37!	0.008!	1.82!
	P14*	TEO!	0.249!	0.227!	90.98!	0.151!	60.57!	0.066!	26.56!	0.008!	3!	0.003!	1.1!
	P16*	VFR!	0.803!	0.599!	74.53!	0.384!	47.79!	0.143!	17.84!	0.062!	7.76!	0.035!	4.32!
	P18*	VFR!	1.177!	0.861!	73.12!	0.439!	37.28!	0.305!	25.93!	0.11!	9.31!	0.063!	5.37!
	P19*	VFR!	0.692!	0.57!	82.38!	0.395!	57.05!	0.138!	19.96!	ND!	ND!	ND!	ND!
	P20*	VFR!	1.621!	0.21!	87.06!	1.411!	49.03!	0.795!	33.44!	0.53!	0.053!	0.008!	0.48!
	P22**	VFR!	0.51!	0.407!	79.86!	0.271!	53.12!	0.125!	24.48!	0.007!	1.29!	0.003!	0.51!
	P23*	VFR!	0.857!	0.473!	80.6!	0.361!	61.42!	0.096!	16.33!	0.014!	2.3!	0.011!	1.82!
	P24*	VFR!	2.127!	1.48!	69.57!	1.063!	49.97!	0.315!	14.81!	0.049!	2.32!	0.034!	1.58!
	P25*	VFR!	0.353!	0.277!	78.45!	0.218!	61.63!	0.047!	13.44!	0.005!	1.43!	0.003!	0.75!
	P26*	VFR!	0.938!	0.601!	64.03!	0.366!	39.06!	0.189!	20.16!	0.031!	3.29!	0.011!	1.13!
	P28*	VFR!	0.38!	0.276!	72.7!	0.171!	45!	0.091!	23.87!	0.008!	2.07!	0.005!	1.27!
	P29*	VFR!	0.949!	0.736!	77.57!	0.592!	62.41!	0.122!	12.82!	0.015!	1.59!	0.01!	1.04!
	P30*	VFR!	0.537!	0.351!	65.36!	0.248!	46.25!	0.09!	16.66!	0.015!	2.75!	0.004!	0.66!
	P31*	VFR!	0.334!	0.254!	75.99!	0.155!	46.49!	0.054!	16.07!	0.035!	10.57!	0.01!	2.9!
	P33*	VFR!	0.754!	0.624!	82.76!	0.545!	72.26!	0.091!	12.01!	0.012!	1.53!	0.003!	0.39!
	P34**	VFR!	0.325!	0.247!	76.08!	0.177!	54.45!	0.054!	16.72!	0.008!	2.46!	0.006!	1.79!

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iai.asm.org October 2015 Volume 83 Number 10 Infection and Immunity on September 11, 2015 by UNIVERSITE VICTOR SEGALEN http://iai.asm.org/ Downloaded from

iai.asm.org October 2015 Volume 83 Number 10 Infection and Immunity on September 11, 2015 by UNIVERSITE VICTOR SEGALEN http://iai.asm.org/ Downloaded from

iai.asm.org October 2015 Volume 83 Number 10 Infection and Immunity on September 11, 2015 by UNIVERSITE VICTOR SEGALEN http://iai.asm.org/ Downloaded from

ACKNOWLEDGMENTS

We thank the cytometry core facility of Bordeaux University and Q. Osseman, R. Carmeille, D. Duluc, J. Dalennes, and M. Mayoux for technical assistance and/or for valuable discussions. We thank M. Capone for critical reading of the manuscript. We thank D. Olive and E. Scotet for the kind gift of the anti-BTN3A antibodies.

This work received funding from the Centre National de la Recherche Scientifique (UMR CNRS 5164), the French Research Agency (ANR-12-Blanc-GDSTRESS), the Ligue Contre le Cancer (Comité de la Gironde), and the EviMalar European Community 7th Framework Program (contract 242095 EviMalar). D.B. is supported by a grant from the Wellcome Trust (grant REF 094752). J.H. is a fellow of the EviMalar PhD Program.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We have no financial conflicts of interest.

39!

BTNs! are! immunoglobulin! superfamily! members! made! of! two! immunoglobulinSlike! extracellular!domains!(IgV!and!IgC),!a!transmembrane!domain!and,!for!some,!a!B30.2!family! intracellular!domain!(found!on!proteins!involved!in!innate!immunity)! [START_REF] Compte | ! Frontline:! Characterization! of! BT3! molecules! belonging! to! the! B7! family! expressed!on!immune!cells[END_REF].! The!structure!of!the!IgG!domains!are!consistent!with!a!cell!surface!receptor,!while!the!B30.2! domain! structure! is! consistent! with! proteinSprotein! interactions! (Rhodes! et! al.! 2001).! The! BTN!genes!are!located!on!chromosome!6! (Ruddy!et!al.!1997)!and!have!sequence!similarity! with!B7!family!of!coSstimulators!CD80!and!CD86! (Linsley!et!al.!1994).!The!murine!Skint!family! belongs!to!the!BTNSlike!family,!and!has!sequence!similarity!with!the!BTN!family!genes.!This!is! particularly!interesting!as!Skint!1!is!involved!in!murine!Vγ5Vδ1!T!cell!thymic!differentiation! and!localisation!in!the!skin! (AbelerSDörner!et!al.!2012).! There!are!three!BTN!families!-!BTN1,!BTN2!and!BTN3,!and!there!are!three!forms!of!BTN3! -!A1,!A2!and!A3,!the!extracellular!domains!of!which!cannot!be!discriminated!by!available!Ab.! Only! BTN3A1! and! BTN3A3! contain! the! B30.2! family! domain,! and! BTN3A3! has! a! truncated! version! [START_REF] Compte | ! Frontline:! Characterization! of! BT3! molecules! belonging! to! the! B7! family! expressed!on!immune!cells[END_REF].!Analysis!of!BTN3!expression!showed!that!it!is!present!on!the! surface! of! hematopoietic! and! nonShematopoietic! tumour! cell! lines,! TS,! BS! and! NK! cells,! monocytes,!both!immature!and!mature!DCs!and!IFNγ!and!TNFα!stimulated!endothelial!cells! [START_REF] Compte | ! Frontline:! Characterization! of! BT3! molecules! belonging! to! the! B7! family! expressed!on!immune!cells[END_REF]

was! demonstrated!by!Harly!et!al!(Harly!et!al.!2012).!There!have!since!been!2!models!proposed! for!the!mechanism!of!activation!(See!Figure!11):!the!allosteric!model!proposed!by!the!groups! of!Adams!and!Scotet/Bonneville!(Harly!et!al.!2012;!Harly!et!al.!2014;!Sandstrom!et!al.!2014;! Palakodeti! et! al.! 2012;! Rhodes! et! al.! 2015;! Decaup! et! al.! 2014)! and! the! Ag! presentation! model!put!forward!by!the!group!of!De!Libero!(Vavassori!et!al.!2013).! In! the! allosteric! model,! PSAg! binds! to! the! B30.2! intracellular! domain.! This! leads! to! conformational! change! and! reduced! mobility! of! the! BTN3A1,! which! somehow! leads! to! Vγ9Vδ2!TCR!activation,!(Harly!et!al.!2012;!Palakodeti!et!al.!2012;!Rhodes!et!al.!2015)

.!B30.2! has!a!binding!pocket!for!PSAg,!the!mutation!of!which!blocked!PSAg!binding!and!also!Vγ9Vδ2! T!cell!activation.!Binding!of!HMBPP!was!found!to!be!stronger!than!for!IPP,!lending!a!possible! reason!for!the!increased!potency!of!HMBPP! [START_REF] Sandstrom | The intracellular B30.2 domain of butyrophilin 3A1 binds phosphoantigens to mediate activation of human Vgamma9Vdelta2 T cells[END_REF]. However!despite!this,! most! of! the! allosteric! model! contributors! believe! that! the! Vγ9Vδ2! TCR! probably! does! not! bind!directly!to!BTN3A1!plus!PSAg,!and!that!other!protein!partners!are!involved.!The!team!of! Thomas! Hermann! showed! that! additional! proteins! coded! by! chromosome! 6! are! required! (Riaño!et!al.!2014).!The!cytoskeletal!adaptor!periplakin!has!recently!been!identified!by!the! team!of!John!Trowsdale!as!a!protein!partner!that!interacts!with!the!intracellular!domain!of! BTN3A1! and! is! necessary! for! PSAg! activation! of! Vγ9Vδ2! T! cells! (Rhodes! et! al.! 2015),! thus! linking!the!cytoskeleton!to!Vγ9Vδ2!T!cell!activation.! In!the!Ag!presenting!model,!PSAg!binds!directly!to!the!extracellular!domain!of!BTN3A1,! which!then!binds!to!the!Vγ9Vδ2!TCR! [START_REF] Vavassori | Butyrophilin 3A1 binds phosphorylated antigens and stimulates human gammadelta T cells[END_REF].!A!shallow!binding!site!for!PSAg! was! found! on! the! extracellular! domain,! again! with! greater! affinity! for! HMBPP! than! IPP.! Surface! immobilized! variable! regions! of! BTN3A1! (which! is! included! in! the! extracellular! domain)! were! able! to! activate! transgenic! murine! cells! expressing! Vγ9Vδ2! TCR! when! PSAg! were!included,!and!Vγ9Vδ2!TCR!multimers!could!bind!directly!to!the!immobilised!BTN3A1SV,! albeit!with!low!affinity.! The!debate!over!which!model!is!most!correct!is!ongoing! [START_REF] Harly | ! Molecules! and! Mechanisms! Implicated! in! the! Peculiar! Antigenic!Activation!Process!of!Human!Vγ9Vδ2!T!Cells[END_REF][START_REF] Karunakaran | !The!Vγ9Vδ2!T!Cell!Antigen!Receptor!and!ButyrophilinS3!A1:! Models! of! Interaction,! the! Possibility! of! CoSEvolution,! and! the! Case! of! Dendritic! Epidermal! T! Cells[END_REF][START_REF] Kabelitz | ! Critical! role! of! butyrophilin! 3A1! in! presenting! prenyl! pyrophosphate! antigens! to! human!γδ!T!cells[END_REF][START_REF] Willcox | !Resolving!the!mystery!of!pyrophosphate!antigen! presentation[END_REF][START_REF] De!libero | !Phosphoantigen!Presentation!to!TCR!γδ!Cells,!a!Conundrum! Getting!Less!Gray!Zones[END_REF].!However,!the! majority!of!work!thus!far!completed!supports!the!allosteric!model,!especially!since!Vγ9Vδ2! TCR! binding! to! the! immobilized! BTN3! extracellular! domain! has! not! been! demonstrated! [START_REF] Wang | Butyrophilin 3A1 plays an essential role in prenyl pyrophosphate stimulation of human Vgamma2Vdelta2 T cells[END_REF][START_REF] Sandstrom | The intracellular B30.2 domain of butyrophilin 3A1 binds phosphoantigens to mediate activation of human Vgamma9Vdelta2 T cells[END_REF].!