Pauline Faucher

Frédéric Beuvon

Daniela Fignani

Guido Sebastiani

Georgia Afonso

Zhicheng Zhou

Bertrand Dousset

Christian Boitard

Francesco Dotta

Roberto Mallone

Etienne Larger
email: etienne.larger@aphp.fr

Immunoregulated insulitis and slow-progressing type 1 diabetes after duodenopancreatectomy

Keywords: type 1 diabetes, duodenopancreatectomy, autoimmunity, regulatory T cells, insulin secretion

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INTRODUCTION

Type 1 diabetes is the late consequence of an autoimmune process targeting insulin-producing pancreatic beta cells [START_REF] Atkinson | Type 1 diabetes[END_REF][START_REF] Katsarou | Type 1 diabetes mellitus[END_REF]. The disease occurs on a susceptible genetic background following elusive environmental triggers [START_REF] Craig | Early-life factors contributing to type 1 diabetes[END_REF] and is T-cell mediated, involving both CD4 + and CD8 + T cells that destroy beta cells. The presence of one or more auto-antibodies (aAbs) against GAD, islet antigen (IA)-2 and zinc transporter (ZnT)8 in adults is the hallmark of an ongoing autoimmune process. An improper balance between regulatory T cells (Tregs), which express the lineage marker forkhead box (FOX)P3, and effector T cells (Teffs) is central to the progression of this autoimmune process towards beta-cell destruction [START_REF] Von Herrath | Type 1 diabetes as a relapsing-remitting disease?[END_REF]. Up to the critical threshold that becomes insufficient to maintain euglycaemia, it is unknown whether the progression of beta-cell destruction is linear or follows a relapsing-remitting pattern, possibly reflecting cyclic disruption and restoration of the Treg/Teff balance [START_REF] Von Herrath | Type 1 diabetes as a relapsing-remitting disease?[END_REF]. We report the case of a patient positive for anti-GAD aAbs (GADA), whose diabetes remained well controlled with low-dose long-acting insulin for 8 years after partial pancreatectomy. Surgical pancreas specimens and blood samples were studied to search for histopathological and autoimmune T-cell correlates of this indolent clinical course.

CASE REPORT

A 47-year-old woman was diagnosed with a serous cystadenoma of the uncinate process in 2002. The timeline of the clinical course is summarized in Fig. 1 and detailed in electronic supplementary materials [ESM] Table 1. Diabetes was diagnosed 3 years later (2005), glycaemia was said to be "moderately elevated", at the weight of 96 kg and BMI of 37 kg/m². The patient had no family history of diabetes and no personal history of gestational diabetes at any of her 3 pregnancies. Metformin and gliclazide were started. She also had Hashimoto's thyroiditis that required 112.5 µg/day L-thyroxin. An uneventful duodenopancreatectomy (Whipple procedure with removal of the head of the pancreas) was performed 9 years later (February 2011), because of increasing tumour size (70 mm diameter) accompanied by abdominal pain, signs of vena cava compression with limb oedema and deteriorating diabetes (HbA1c 83 mmol/mol, 9.7%), with add-on insulin glargine started before surgery. Post-operative treatment included metformin, gliclazide, insulin glargine and pancreatic enzyme replacement therapy (lipase, Creon®, 150,000 U/d). Faecal elastase-1 concentration was 18 µg/g (normal values >200), confirming severe pancreatic exocrine failure and the need for replacement therapy. She was referred to our Diabetology unit in July 2012, 17 months after surgery. She had lost 16 kg since surgery, and gliclazide was stopped due to hypoglycaemia. HLA typing was DQB1*03:01/ DRB1*04:01 (neutral for type 1 diabetes susceptibility) and DQB1*05:03/DRB1*14:01, a rare haplotype with unknown association with disease susceptibility or protection. Antithyreoperoxydase aAbs were positive (118 IU/mL; positive threshold >34). GADA were also positive at high titre, IA-2 and ZnT8 aAbs were negative.

Insulin glargine was stopped 3 months later (October 2012), and resumed after 2 months in light of positive GADA and intolerance to metformin doses higher than 1,500 mg/day. With this treatment, blood glucose levels remained at target, with HbA1c values between 43 and 55 mmol/mol (6.1-7.2%) for more than 6 years. In 2015, i.e. 3 years after surgery, fasting C-peptide concentration was 0.27 nmol/L (normal values 0.27-1.27 nmol/L) and raised to 0.35 nmol/L 6 min after a 1-mg intravenous glucagon challenge. Peripheral blood mononuclear cells (PBMCs) were collected for T-cell assays. Another aAb measurement performed 6 years after the initial one (2019) documented increasing GADA titres and the appearance of high-titre anti-ZnT8, both persisting one year later. Antismooth muscle cell aAbs were positive on two occasions 1 year apart (1:2,000, then 1:50; positive threshold >1:20). One year later, fasting C-peptide values were lower than before (0.13 nmol/L), but still increased to 0.21 nmol/L 2 h after stimulation by a mixed meal test, documenting some residual insulin secretion. At the last follow-up in November 2020, fasting C-peptide was 0.10 nmol/L, and 0.15 nmol/L after a mixed meal test. A second PBMC sample was collected. The patient gave written informed consent and the study was approved by the Cochin Hospital Ethics Committee.

METHODS

Immunohistochemistry

Beta-cell area, alpha-cell area and CD8 + , CD4 + and FOXP3 + insulitis were assessed on 5 consecutive 4-μm sections taken from 4 different distal regions of peri-tumoural pancreatic tissue (excluding the uncinate process). Immunohistochemical staining employed an avidin-biotinperoxidase system. Briefly, sections were placed in a Bond III Automated Immunohistochemistry Vision Biosystem (Leica). Tissues were deparaffinised and pre-treated with EDTA buffer, pH 8.8 at 98°C for 20 min. After washing, peroxidase activity was blocked for 10 min using the Bond Polymer Refine Detection Kit DC9800 (Leica). Tissues were washed again and incubated with primary antibodies for 20 min: anti-insulin (RRID:AB_10013624, 1/500), anti-glucagon (RRID:AB_10013726, 1/1000), anti-CD8 (clone C8/144B, RRID:AB_2075537; 1/100), anti-CD4 (clone 4B12, RRID:AB_10554438; 1/200), anti-FOXP3 (clone 236A/E7, RRID:AB_445284; 1/80), as previously described [START_REF] Goux | mRNA Expression levels of genes involved in antitumor immunity: Identification of a 3-gene signature associated with prognosis of muscleinvasive bladder cancer[END_REF]. Tissues were subsequently coated with polymer for 10 min and developed with 3,3'-Diaminobenzidine for 8 min. Slices were washed with distilled water and counterstained with acid hemalum (Mayer). Quantifications were performed with a Hamamatsu Nanozoomer and the NDP.view software. Pancreatic and islet areas on insulin-or glucagon-stained slides were manually defined based on morphology, irrespective of islet size (possibly excluding clusters of few (<10) cells lacking an identifiable islet architecture). Insulin and glucagon area is expressed as the percentage of pancreatic area. Insulitis was scored as positive according to a threshold level of ≥15 CD8 + cells/islet, a more restrictive definition than the consensus criterion of ≥15 CD45 + cells/islet [START_REF] Campbell-Thompson | The diagnosis of insulitis in human type 1 diabetes[END_REF]. CD4 + insulitis was assessed with the same criteria.

Immunofluorescence

To validate FOXP3 staining, CD25 hi CD127 lo Tregs and CD25 -CD127 + Teffs were sorted and expanded as described [START_REF] Putnam | Expansion of human regulatory T-cells from patients with type 1 diabetes[END_REF], fixed and permeabilised for flow cytometry staining with anti-FOXP3 antibody (clone 259D/C7, RRID:AB_11153143), or paraffin-embedded for immunofluorescence staining. Consecutive pancreatic sections were analysed by triple immunofluorescence to simultaneously evaluate the expression of CD8 or FOXP3 along with insulin and glucagon. After stepwise deparaffinisation and rehydration (xylene-I and xylene II, 20 min/each; ethanol 100%, 95%, 80%, 75%, 5 min/each), sections were subjected to heat-induced antigen retrieval using Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, 0.05% Tween-20, pH 9.0) for 20 min at 100°C. After cooling and incubation in 5% goat serum (Sigma) to reduce non-specific reactions, sections were stained in 5% goat serum overnight at 4°C with antibodies against CD8 (RRID:AB_2075537; 1:50, final concentration 3.2 µg/ml) or FOXP3 (clone 236A/E7, RRID:AB_467556; 1:40, 12.5 µg/ml). After 3 washes in PBS, sections were incubated with pre-diluted guinea pig anti-insulin (RRID:AB_2800361) for 20 min, followed by rabbit anti-glucagon (RRID:AB_10013726; 1:100, final concentration 352 µg/ml) in 5% goat serum for 1 h. Secondary antibodies were subsequently added at 1:500 in PBS for 1 h: Alexa-Fluor594-labelled goat anti-guinea pig IgG (RRID:AB_2535856), Alexa-Fluor647-labelled goat anti-rabbit IgG (RRID:AB_2535813), Alexa-Fluor488-labelled goat anti-mouse IgG (RRID:AB_2534088). Sections were counter-stained with DAPI (Sigma; 1:3,000) and mounted with Vectashield antifade (RRID:AB_2336789) or fluorescence mounting medium (Dako/Agilent) and analysed immediately or stored at 4°C prior to acquisition on a Leica TCS SP5 confocal laser scanning microscope with LAS software and manual counting of CD8 + and FOXP3 + cells. CD8 + insulitis was defined based on ≥15 CD8 + cells/islet. FOXP3 + insulitis was defined based on ≥1 FOXP3 + cells/islet.

T-cell assays

Frozen-thawed PBMCs were processed as described [START_REF] Martinuzzi | acDCs enhance human antigenspecific T-cell responses[END_REF][START_REF] Mallone | Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society[END_REF], plated at 10 6 cells/well in 96-well flatbottom plates and stimulated for 48 h, as described [START_REF] Martinuzzi | acDCs enhance human antigenspecific T-cell responses[END_REF], in the presence of 10 µg/ml of the following antigens: proinsulin (kindly provided by Lilly), insulin (Actrapid®, Novo Nordisk), C peptide (SynPeptide), preproinsulin signal peptide (GL Biochem), GAD65 (Diamyd), intracellular IA-2 (amino acids 214-591; kindly provided by J.F. Elliott, University of Alberta, Canada), adenoviral lysate (produced in-house) and phytohemagglutinin (PHA, 1 µg/ml; Sigma). All antigens were >95% pure and with an endotoxin concentration <5 EU/mg. At the end of the 48 h culture, nonadherent cells were washed and plated at 2x10 5 cells/well in triplicate wells of 96-well PVDF enzyme-linked immunospot (ELISpot) plates coated with either an anti-interferon (IFN)-γ or antiinterleukin (IL)-10 antibody (U-CyTech). After 6 h of incubation, plates were developed as described [START_REF] Martinuzzi | acDCs enhance human antigenspecific T-cell responses[END_REF], counted on a BioSys 5000 Pro-SF Bioreader and results expressed as mean spotforming cells/10 6 PBMCs normalised across samples. T-cell responses were scored as positive for counts >2SD above spontaneous background responses in the absence of antigen, as determined by receiver-operator characteristic analysis.

RESULTS

Decreased beta-cell area and mixed CD8/FOXP3 + insulitis at the time of surgery

Quantitative immunochemistry was performed on four blocks from the head of the pancreas, distant from the tumour and from areas of chronic pancreatitis (Fig. 2a). On slides stained for insulin (section area 56-250 mm², total 513 mm²), a total of 119 islets were identified. On slides stained for glucagon (section area 44-219 mm², total 416 mm²) a total of 320 islets were identified. Representative images are shown in Fig. 2b-c. The total fractional islet areas were 0.54% and 1.31% of the pancreas area on slides stained for insulin and glucagon respectively, ranging from 0.19% to 3.6% in different sections (Fig. 2d). When considering the 4 blocks altogether, the betacell/islet area and alpha-cell/islet area were 50% (range 0-97%) and 65% (range 18-100%), respectively (Fig. 2d). Sixty-five islets (55%) were insulin-positive (referred to as insulincontaining islets, ICIs, from hereon), with wide heterogeneity across blocks (range 0-95%); 263 islets (82%) were glucagon-positive (range 36-100%) (Fig. 2e).

Representative islets are shown that display peri-insulitis (Fig. 2f-h) and invasive (intra-islet) insulitis (Fig. 2i-k). Overall, CD8 + , CD4 + and regulatory (FOXP3 + ) peri-insulitis was observed in 28%, 16% and 8% of the islets, respectively. CD8 + , CD4 + and FOXP3 + intra-islet insulitis was observed in 8%, 5% and 16% of the islets, respectively (Figure 2e).

In type 1 diabetes, insulitis is preferentially localized in ICIs as compared to insulin-deficient islets (IDIs, i.e. insulin-negative and glucagon-positive) [START_REF] Carré | Presumption of guilt for T cells in type 1 diabetes: lead culprits or partners in crime depending on age of onset?[END_REF]. We therefore analysed adjacent sections by triple immunofluorescence to verify whether this was the case also in our patient and to quantify the number of CD8 + vs. FOXP3 + cells. First, the specificity of FOXP3 staining was confirmed on tonsil tissue sections and on sorted, in-vitro expanded Tregs and Teffs (ESM Fig. 1). Representative images of CD8/insulin/glucagon and FOXP3/insulin/glucagon staining are shown in Fig. 3. Results were remarkably similar between sections stained for CD8 and FOXP3 (Fig. 4a-b). ICIs accounted for 58-63% of islets. CD8 + and FOXP3 + insulitis (defined as ≥15 CD8 + cells/islet and ≥1 FOXP3 + cells/islet, respectively) were present in 90% and 85% of ICIs vs. 32% and 35% of IDIs, respectively (p≤0.001). Insulitis displayed a predominant peri-islet pattern for both CD8 + and FOXP3 + cells (85% and 73% in ICIs vs. 100% and 100% in IDIs, respectively). Also when analysing the number of CD8 + cells and FOXP3 + cells per islet (Fig. 4c-d), both were more abundant in ICIs than in IDIs. Overall, the number of CD8 + cells was approximately 10-fold higher than for FOXP3 + cells in peri-islet infiltrates, while it was similar in the less common intra-islet infiltrates. As reported [START_REF] Rodriguez-Calvo | Increased immune cell infiltration of the exocrine pancreas: a possible contribution to the pathogenesis of type 1 diabetes[END_REF], some infiltration was also noted in the exocrine tissue for CD8 + cells; and, to a lesser extent, for FOXP3 + cells.

Collectively, these findings document a heterogeneous pattern of beta-cell loss and ICI-polarised insulitis, as commonly observed in type 1 diabetic patients [START_REF] Coppieters | Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients[END_REF]. This insulitis displayed a preferential peri-islet pattern and an unusual FOXP3 + infiltrate suggestive of an ongoing regulatory process.

Regulatory polarisation of circulating beta-cell-reactive T-cell responses waning over time

Given the hallmarks of heterogeneous beta-cell loss and preservation along with the regulatory features of insulitis observed in pancreatic sections, we analysed the responses against beta-cell antigens of circulating T cells obtained 4 and 9 years after surgery (Fig. 5). We employed an assay that stimulates unfractionated PBMCs with protein/polypeptide antigens to detect CD4 + and, to a lesser extent, CD8 + T-cell responses, without biasing towards IFN-γ or IL-10 responses [START_REF] Martinuzzi | acDCs enhance human antigenspecific T-cell responses[END_REF]. Weak IFN-γ responses were initially present, mainly against proinsulin and IA-2, accompanied by a dominant IL-10 response against proinsulin and, to a lesser extent, C-peptide, GAD and insulin. In the follow-up sample taken 5 years later (i.e. after anti-ZnT8 seroconversion and the rise in insulin needs), IL-10 responses remained dominant but did not significantly increase. Conversely, IFN-γ responses were more robust against all the beta-cell antigens tested, and significantly increased overall (p=0.03). Both IFN-γ and IL-10 responses remained stable against the adenoviral lysate and PHA positive controls. The dominant IFN-γ responses detected in 2 age-matched type 1 diabetic patients are shown for comparison.

Collectively, these results suggest a pattern of T-cell autoimmunity with an initial regulatory polarisation that waned over time.

DISCUSSION

Several lines of evidence support the diagnosis of type 1 diabetes in this patient: positive and increasing GADA titres; the late appearance of anti-ZnT8 aAbs; the associated thyroid autoimmunity and positivity for anti-smooth muscle cell aAbs; and the histopathological finding of insulitis and decreased beta-cell area 6 years after diabetes diagnosis. However, this patient maintained some endogenous insulin secretion for up to 8 years post-surgery, suggesting a slowly evolving form of type 1 diabetes. A plausible histopathological correlate of this slow clinical progression was an insulitis pattern enriched in FOXP3 + T cells. Also in peripheral blood, T-cell responses against islet antigens were skewed towards secretion of the regulatory cytokine IL-10, while IFN-γ is usually found in type 1 diabetic patients [START_REF] Arif | Autoreactive T cell responses show proinflammatory polarization in diabetes but a regulatory phenotype in health[END_REF].

Given the age of this patient and the higher degree of beta-cell loss and insulitis observed in children [START_REF]Insulitis in human type 1 diabetes: The quest for an elusive lesion[END_REF][START_REF] Campbell-Thompson | Insulitis and beta-Cell Mass in the Natural History of Type 1 Diabetes[END_REF][START_REF] Arif | Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes[END_REF][START_REF] Leete | The Effect of Age on the Progression and Severity of Type 1 Diabetes: Potential Effects on Disease Mechanisms[END_REF], the most informative comparison is with published case series of adult recentonset type 1 diabetic and at-risk single-aAb + cases. Moreover, quantification of insulitis in the pancreas of living type 1 diabetic patients is seldom available [START_REF] Imagawa | Pancreatic biopsy as a procedure for detecting in situ autoimmune phenomena in type 1 diabetes: close correlation between serological markers and histological evidence of cellular autoimmunity[END_REF][START_REF] Krogvold | Insulitis and characterisation of infiltrating T cells in surgical pancreatic tail resections from patients at onset of type 1 diabetes[END_REF], and the best-matched case series may be the one reported by the DiViD study on 6 living patients aged 24-35 years with recent-onset type 1 diabetes [START_REF] Krogvold | Insulitis and characterisation of infiltrating T cells in surgical pancreatic tail resections from patients at onset of type 1 diabetes[END_REF].

First, in the present case, 55% of the islets were insulin-positive, which is in the 18-66% range observed in the DiViD study. The proportion of ICIs and insulitis-positive islets was very variable across DiViD patients and, like in our case, from one section to another [START_REF] Krogvold | Insulitis and characterisation of infiltrating T cells in surgical pancreatic tail resections from patients at onset of type 1 diabetes[END_REF]. It should however be noted that the sections analysed herein were probably more distant between each other than the ones in the DiViD study. The beta-cell area in our patient was 0.27% of the total pancreas surface, which is even lower than the 0.44-1.2% observed in DiViD patients [START_REF] Krogvold | Insulitis and characterisation of infiltrating T cells in surgical pancreatic tail resections from patients at onset of type 1 diabetes[END_REF], and much lower than the average 0.8% observed in single-aAb + non-diabetic adult donors [START_REF] Diedisheim | beta-cell Mass in Nondiabetic Autoantibody-Positive Subjects: An Analysis Based on the Network for Pancreatic Organ Donors Database[END_REF]. Moreover, the beta-cell/islet area ratio was lower than for alpha cells, whereas this ratio, although highly variable, is higher for beta cells in non-diabetic subjects [START_REF] Cabrera | The unique cytoarchitecture of human pancreatic islets has implications for islet cell function[END_REF][START_REF] Brissova | Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy[END_REF]. Thus, several lines of evidence suggest that significant beta-cell loss took place before surgery. Moreover, this beta-cell loss may have been underestimated because we only had access to tissue from the head of the pancreas, which is the less affected region in type 1 diabetic patients [START_REF] Poudel | Beta-cell destruction and preservation in childhood and adult onset type 1 diabetes[END_REF].

Second, CD8 + lymphocytic infiltrates were detected in 36% of islets (28% peri-islet plus 8% intraislet) as compared to 5-58% of CD3 + ICIs in the DiViD study (i.e. 5%, 12%, 23%, 33%, 52% and 58% for the 6 DiViD patients) [START_REF] Krogvold | Insulitis and characterisation of infiltrating T cells in surgical pancreatic tail resections from patients at onset of type 1 diabetes[END_REF]. Also in other type 1 diabetes case series, the fraction of infiltrated islets is typically low (<10%) [START_REF] Carré | Presumption of guilt for T cells in type 1 diabetes: lead culprits or partners in crime depending on age of onset?[END_REF][START_REF]Insulitis in human type 1 diabetes: The quest for an elusive lesion[END_REF][START_REF] Campbell-Thompson | Insulitis and beta-Cell Mass in the Natural History of Type 1 Diabetes[END_REF]. At earlier disease stages, little or no insulitis is observed (1-9% of islets) in single-and multiple-aAb + donors, even in those with predisposing HLA haplotypes [START_REF] Carré | Presumption of guilt for T cells in type 1 diabetes: lead culprits or partners in crime depending on age of onset?[END_REF][START_REF] Campbell-Thompson | Insulitis and beta-Cell Mass in the Natural History of Type 1 Diabetes[END_REF][START_REF] Veld | Screening for insulitis in adult autoantibodypositive organ donors[END_REF][START_REF] Rodriguez-Calvo | Pancreas Pathology During the Natural History of Type 1 Diabetes[END_REF]. Thus, substantial insulitis lesions were present in this patient, more extensive than those observed in most type 1 diabetic cases.

Third, the striking histopathological hallmark of the present case was the abundance of infiltrating FOXP3 + cells, which are virtually absent in the pancreas of type 1 diabetic donors, and even in non-diabetic cases [START_REF] Carré | Presumption of guilt for T cells in type 1 diabetes: lead culprits or partners in crime depending on age of onset?[END_REF]. In a series of 16 recent-onset type 1 diabetic cases, scattered FOXP3 + cells were detected in two islets of one single patient and not in non-diabetic controls, using the same antibody employed herein [START_REF] Willcox | Analysis of islet inflammation in human type 1 diabetes[END_REF]. Similarly, FOXP3 gene expression in the DiViD series was modest [START_REF] Krogvold | Insulitis and characterisation of infiltrating T cells in surgical pancreatic tail resections from patients at onset of type 1 diabetes[END_REF]. Moreover, the insulitis of type 1 diabetic patients mostly displays a peri-islet localization, with very little islet invasion [START_REF] Carré | Presumption of guilt for T cells in type 1 diabetes: lead culprits or partners in crime depending on age of onset?[END_REF][START_REF] Rodriguez-Calvo | Pancreas Pathology During the Natural History of Type 1 Diabetes[END_REF]. This was also the case of this patient, as all T cells were predominantly observed around the islets. However, FOXP3 + cells were also found inside the islets. This FOXP3-enriched insulitis was mirrored in the blood by the presence of islet-reactive T cells that were skewed towards a regulatory, IL-10-secreting phenotype, as reported in slow-progressing type 1 diabetic and healthy adults [START_REF] Arif | Autoreactive T cell responses show proinflammatory polarization in diabetes but a regulatory phenotype in health[END_REF][START_REF] Tree | Naturally arising human CD4 T cells that recognize islet autoantigens and secrete IL-10 regulate pro-inflammatory T cell responses via linked suppression[END_REF] and in 'partially regulated', IL-10 + , pauci-aAb + , pauciimmune type 1 diabetic children and adolescents [START_REF] Arif | Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes[END_REF].

Despite significant beta-cell loss and islet infiltration, insulin requirements remained modest over time, suggesting a slowly evolving islet destruction. Although the weight loss after surgery likely contributed to these reduced insulin needs, this lack of progression is striking when considering the additional beta-cell deprivation imposed by duodenopancreatectomy. Collectively, these histopathological, immunological and clinical findings suggest that, after having destroyed a significant beta-cell fraction, the autoimmune reaction remained efficiently regulated over several years. We may speculate that the autoimmune process in the islets may have been hindered by exposure to the tumour microenvironment, which is often enriched in suppressive cytokines such as transforming growth factor (TGF)-β that drive Treg differentiation [START_REF] Oleinika | Suppression, subversion and escape: the role of regulatory T cells in cancer progression[END_REF]. Indeed, TGF-β mRNA was reportedly expressed in two pancreatic serous cystadenomas [START_REF] Van Laethem | Different expression of transforming growth factor beta 1 in pancreatic ductal adenocarcinoma and cystic neoplasms[END_REF]. The CD8 + T-cell infiltration may have been another driver for FOXP3 + T cells, as suggested in the NOD mouse [START_REF] Grinberg-Bleyer | Pathogenic T cells have a paradoxical protective effect in murine autoimmune diabetes by boosting Tregs[END_REF].

Finally, the late raise of GADA titres, anti-ZnT8 seroconversion and increase in circulating IFN-γ T-cell responses against islet antigens concomitant with increased insulin needs suggest that immune regulation was eventually lost. This progression may be in favour of a relapsing-remitting model of type 1 diabetes [START_REF] Von Herrath | Type 1 diabetes as a relapsing-remitting disease?[END_REF]. Several autoimmune diseases, e.g. vitiligo, multiple sclerosis and rheumatoid arthritis, follow a course marked by flares alternating with remission periods. In the present case, the early histopathological findings and late clinical and autoimmune progression are consistent with an initial autoimmune flare followed by several years of remission before eventual relapse. Nonetheless, some endogenous insulin secretion was maintained. Also in other autoimmune conditions, end-stage organ failure is not invariably attained even after significant tissue loss. For instance, subclinical hypothyroidism, i.e. positivity of anti-thyreoperoxydase aAbs and increased thyroid-stimulating hormone levels with normal thyroxine levels, is reported to progress to overt hypothyroidism at a rate of only 4% per year [START_REF] Biondi | Subclinical Hyperthyroidism[END_REF]. Indeed, the prevalence of subclinical aAb + conditions is largely higher than that of overt functional failure for most organspecific autoimmune diseases [START_REF] Van Den Driessche | Type 1 diabetes and autoimmune polyglandular syndrome: a clinical review[END_REF][START_REF] Merbl | Newborn humans manifest autoantibodies to defined self molecules detected by antigen microarray informatics[END_REF]. In the case of type 1 diabetes, most slow-onset forms are associated with isolated GADA, as initially observed in this patient. While the majority eventually requires insulin treatment, one third remains insulin-free [START_REF] Sorgjerd | Time dynamics of autoantibodies are coupled to phenotypes and add to the heterogeneity of autoimmune diabetes in adults: the HUNT study, Norway[END_REF]. However, in the context of pancreas transplantation, anti-ZnT8 seroconversion has been shown to predict loss of graft function [START_REF] Occhipinti | Zinc transporter 8 autoantibodies increase the predictive value of islet autoantibodies for function loss of technically successful solitary pancreas transplant[END_REF]. A similar phenomenon may have occurred in our patient.

In conclusion, we here describe the case of a type 1 diabetic patient who, despite positivity for GADA and other aAbs, significant insulitis and beta-cell loss, and partial pancreatectomy, remained well-controlled with low-dose insulin till anti-ZnT8 seroconversion 14 years after diabetes onset. Both islet infiltrates and circulating islet-reactive T cells were skewed towards a regulatory phenotype. These observations suggest an islet autoimmune process that remained spontaneously regulated for several years after substantial beta-cell destruction. The definition of the mechanisms behind these indolent forms of more or less "benign" autoimmunity may lead to better understanding of type 1 diabetes physiopathology and to the definition of novel therapeutic targets [START_REF] Ehlers | Who let the dogs out? The ever-present threat of autoreactive T cells[END_REF][START_REF] Mallone | Presumption of innocence for beta cells: why are they vulnerable autoimmune targets in type 1 diabetes?[END_REF].

Figure 1 .

 1 Figure 1. Schematic timeline of the evolution of aAbs, clinical parameters, treatments and related pancreas histopathology and blood T-cell analyses. The patient's clinical history before and after surgery (2011) is depicted. The time of sampling for pancreas tissue (2011) and PBMCs (2015, 2020) is indicated.

Figure 2 .

 2 Figure 2. Tissue macroscopy, immunohistochemistry and quantification of endocrine areas and insulitis at surgery. a. Macroscopy of the tumour and surrounding pancreas. The tumour (red arrow), the duct of Wirsung (white arrow) and the head of the pancreas where tissue sections were taken (black arrow) are indicated. Scale bar, 10 mm. b-c. Representative tissue sections stained for insulin (INS, b) and glucagon (GCG, c). d. Percent tissue section area for islets, beta (β) cells and alpha (α) cells (d, left); and percent islet tissue area for beta and alpha cells (d, right). e. Percent positive islets for the indicated markers. Each symbol represents one of the 4 tissue blocks analysed; black symbols display values for the 4 blocks considered altogether. f-h. Representative peri-insulitis stained for CD8 (f), CD4 (g) and FOXP3 (h). i-k. Representative invasive insulitis (intra-islet) stained for CD8 (i), CD4 (j) and FOXP3 (k). Scale bar, 100 µm.

Figure 3 .

 3 Figure 3. Immunofluorescence images of pancreas sections at surgery. a-c. Representative staining for CD8 (green), insulin (INS, red) and glucagon (GCG, blue) of one ICI (a), one IDI (b) and of exocrine tissue (c). d-f. Representative staining for FOXP3 (green), INS (red) and GCG (blue) of one ICI (d), one IDI (e) and of exocrine tissue (f). Scale bars, 50 µm.

Figure 4 .

 4 Figure 4. Immunofluorescence quantification. a-b. Fraction of ICIs and IDIs (top rows), insulitis-positive ICIs and IDIs (middle rows) and of peri-islet insulitis in insulitis-positive ICIs and IDIs in CD8-stained sections (a) and FOXP3-stained sections (b). CD8 + and FOXP3 + insulitis was scored as positive in islets with ≥15 CD8 + cells and ≥1 FOXP3 + cells, respectively. p≤0.001 for the comparison between insulitis-positive ICIs and IDIs by Fisher exact test (for both CD8 and FOXP3 staining). c-d. Number of CD8 + cells (c) and FOXP3 + cells (d) in ICIs (red symbols), IDIs (blue symbols) and exocrine tissue (gray symbols). For ICIs and IDIs, cell numbers are displayed for total islets (circles) and for those displaying a peri-islet infiltrate (squares) and an intra-islet infiltrate (diamonds). ***p≤0.0004, *p=0.02 by Mann-Whitney test.

Figure 5 .

 5 Figure 5. IFN-γ and IL-10 T-cell responses against beta-cell autoantigens 4 and 9 years after surgery (2015, 2019). Frozen-thawed PBMCs were analysed by ELISpot following a 48 h culture in the presence of the indicated beta-cell antigens, an adenoviral (AdV) lysate and PHA positive controls or no antigen. Each symbol represents the mean number of spot-forming cells/10 6 PBMCs from triplicate wells. The dotted line indicates the positive cut-off, corresponding to 2SD above mean values from wells stimulated in the absence of antigen. The responses detected in 2 agematched type 1 diabetic (T1D) patients are shown for comparison. *p=0.03 by Wilcoxon signed rank test.

Fig. 2 a

 2 Fig.2

Fig. 4

 4 Fig.4

Fig. 5

 5 Fig.5

Acknowledgments

. We thank Dr. Raphaël Scharfmann (INSERM U1016 Cochin Institute, Paris) for reviewing the manuscript. Data availability. Data are available from the authors on request. Funding. This work was performed within the Département Hospitalo-Universitaire (DHU) AutHorS and supported by grants from the JDRF (2-SRA-2016-164-Q-R), the Fondation pour la Recherche Medicale (EQU20193007831), the Agence Nationale de la Recherche (ANR-19-CE15-0014-01) and the Innovative Medicines Initiative 2 Joint Undertaking under grant agreements 115797 and 945268 (INNODIA and INNODIA HARVEST), which receive support from the EU Horizon 2020 program, the European Federation of Pharmaceutical Industries and Associations, JDRF, and The Leona M. & Harry B. Helmsley Charitable Trust. ZZ is supported by JDRF Postdoctoral Fellowship 3-PDF-2020-942-A-N. The funding sources had no involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the manuscript.

Authors' relationships and activities. The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their work. Contribution statement. PF performed histology quantifications; FB, DF, GS performed histopathology studies; GA, ZZ performed T-cell assays; BD performed surgery; CB, FD provided intellectual input; RM, EL designed the study, interpreted the data and wrote the manuscript; EL is the diabetologist in charge of the patient. All authors approved the version to be published. The corresponding author confirms that he had full access to all the data in the study and had final responsibility for the decision to submit for publication.

ELECTRONIC SUPPLEMENTARY MATERIALS (ESM)

Immuno-regulated insulitis and slow-progressing type 1 diabetes after duodenopancreatectomy

Pauline Faucher, Frédéric Beuvon, Daniela Fignani, Guido Sebastiani, Georgia Afonso, Zhicheng Zhou, Bertrand Dousset, Christian Boitard, Francesco Dotta, Roberto Mallone and Etienne Larger.

ESM


Bernard Mignotte
email: mignotte@genetique.uvsq.fr

Jessie Colin

Sylvain Brun

Isabelle Guenal

Apoptosis: the fly point of view

It is now established that genes involved in the execution of programmed cell death by apoptosis are relatively well conserved throughout evolution. However, the control of commitment to apoptosis exhibits some differences between organisms. In C. elegans, genetic studies have led to the identification of the ced genes (cell death) ced-3 and ced-4 that are essential to trigger cell death, and ced-9 that antagonizes the activities of ced-3 and ced-4. CED-3 is a caspase (cysteinyl aspartase) and CED-9 is homologous to proteins of the Bcl-2 family. In mammals, two main pathways of apoptosis have been identified. The intrinsic pathway is regulated by the bcl-2 family genes (the homologs of CED-9) but is more complex than in C. elegans and most data suggest that this family controls caspase activation by regulating mitochondrial membrane permeability and the release in the cytoplasm of proapoptotic factors including cytochrome c, which participate in caspases activation. An extrinsic pathway, which is activated by the binding of ligands on death receptors, activates in some cases caspases in a Bcl-2 independent fashion.

In Drosophila the way of action of of Bcl-2 homologs is probably different. No release of mitochondrial proteins during Drosophila cell death has been demonstrated. Instead, numerous actors involved in the cell death process migrate from cytoplasm to mitochondria. Furthermore, although numerous homologs of actors of the extrinsic pathway exist in Drosophila, the signaling cascade seems quite different.

On the other hand, the genetic approach performed in Drosophila has resulted in the discovery of new apoptosis regulators encoded by genes located on the 75C region of the third chromosome. The products of these genes (RPR, HID, GRIM and SICKLE) act through binding to IAPs (Inhibitor of Apoptosis Proteins), disrupting inhibition of caspases managed by IAP. Their action on IAPs is similar to that of mammalian Smac/Diablo and Omi/HtrA2 but, unlike these, RPR, HID, GRIM and SICKLE act upstream or independently of mitochondria. More recently, genetic and genomic tools available in Drosophila have also allowed discovering micro RNA (Bantam and mir-14) that regulates apoptosis.

Finally, homologs of various oncogenes and tumor suppressor genes which control apoptosis have also been identified in Drosophila. In this paper, we review the regulation of apoptosis in Drosophila, and we discuss the extent to which the data obtained in this organism can be helpful to understand apoptosis in other species.

I n t ro d u c t i o n

The rapid increase of knowledge about apoptosis mechanisms directly results from the diversity of both approaches and organisms used for this purpose. Genetic studies in Caenorhabditis elegans have led to the identification of genes involved in programmed cell death (PCD) [1]. Afterward, it has been found that the mammalian genes of the bcl-2 family are the homologs of ced-9 and egl-1 that regulate PCD in the nematode (Table 1). Furthermore, genes encoding caspases (cysteinyl aspartases) and caspase activators are respectively the homologs of ced-3 and ced-4 [2,3]. In contrast to the situation found in C. elegans, in mammals, apoptosis control is mediated by multigenic families containing numerous members such as bcl-2 and caspase families. Three families of proteins involved in apoptosis are found in nematodes, Drosophila and mammals: CED-3, -4, -9 are homologous to caspases, caspases activators (like Apoptosis activating factor 1, Apaf-1) and proteins of the Bcl-2 (B Cell Lymphoma 2) family. In Drosophila and mammals, caspase inhibitors (IAP, Inhibitor of Apoptosis Proteins) and proteins able to abrogate this inhibition also participate in the control of caspase activity (Smac/Diablo, RPR, HID, GRIM…).

Nematode

Drosophila is one of the most powerful models to study apoptosis. Many genetic tools are available including collections of Drosophila lines containing single gene disruptions, gene targeting or the ability to drive gene or RNAi expression in a temporally and spatially regulated manner [4]. Furthermore, the complete annotated sequence of its genome is now available. The aim of the present paper is to focus on the data obtained with Drosophila as a model organism.

C a s p a s e -d e p e n d e n t c e l l d e a t h o c c u r s i n D ro s o p h i l a

Caspases have been implicated in the execution of apoptosis in Drosophila melanogaster by the observation that expression of baculovirus p35 protein, a caspase inhibitor, blocks cell death in vivo in Drosophila [5]. Furthermore, recently, Drosophila caspases have also been involved in autophagic cell death [6,7], a type of developmental cell death that we will not discuss in this review.

Data from the Drosophila genome sequence have permitted to identify seven genes encoding caspases [review: 8]. Among them, 3 procaspases contain a long prodomain and have been classified as apical caspases. STRICA contains a serine and threonine rich prodomain of unknown function. Like the mammalian caspase-8, DREDD harbor a DED domain but is rather involved in the immune response [9,10]. Like the mammalian caspases -9 and -2, DRONC contains a CARD domain. It is the only fly caspase known to be regulated by the molting hormone ecdysone [START_REF] Dorstyn | Proc. Natl. Acad. Sci[END_REF] and RNA interference experiments have suggested that DRONC is important for programmed cell death during embryogenesis [START_REF] Quinn | [END_REF]. Although in some tissues, effector caspase activation and cell death can occur independently of DRONC [13], it appears as a regulator of cell number in certain developmental cell death pathways and during stress-induced apoptosis [14]. Thus, DRONC could be the functional homologue of mammalian caspase-9 but has also other functions not related to cell death [15].

Four caspases without prodomain and classified as effector caspases have been identified in Drosophila: DCP-1, drICE, DAMM and DECAY.

DCP-1 has been shown to be essential to some but not all developmental cell death [16] and to nurse cells apoptosis [17,18]. Furthermore, studies of DCP-1 expression in the eye tissue show that some apoptotic inducers can activate DCP-1 in vivo [19].

DAMM and DECAY show a similar pattern of expression [20,21]. They are ubiquitously expressed in Drosophila embryos during early stages of development, in larval salivary glands and midgut, two tissues which undergo histolysis during larval/pupal metamorphosis, and in adult egg chambers, suggesting that they play a role in developmental programmed cell death.

drICE is expressed at all stages of Drosophila development at which PCD can be induced [22] and immunodepletion of drICE from S2 cell lysates have shown that drICE is an essential effector of apoptosis. [23]. Furthermore, drICE cleaves both the Drosophila homolog of the caspase-activated DNase (CAD) and its inhibitor (ICAD) permitting the activation of CAD [24]. Thus, involvement of drICE as an effector caspase seems well established.

However, in Drosophila as well as in other organisms [25] apoptosis is not the sole type of programmed cell death. Furthermore, caspase activation does not always lead to cell death and, instead, like other components of the apoptotic machinery have also functions [26], and important to other biological process. Indeed, as mentioned above, in addition to its function in cell death, DRONC, the initiator cell death caspase in Drosophila, may play a direct or indirect role in cell migration [27], sperm differentiation [28], and compensatory cell proliferation [29]. Elsewhere, DREDD is clearly involved in NFκB-Relish transcription factor activation leading to induction of antibacterial peptide genes.

A p a t h wa y o f c a s p a s e s reg u l a t io n i d e n t i f i e d i n D ro s o p h i l a

In Drosophila, the study of genes involved in apoptosis signaling has begun in the 90's with the discovery of the essential role of the 75C chromosomal region in developmental or irradiation-induced programmed cell death [30]. This region contains four genes without known homologs in mammals or nematodes. rpr (reaper), hid (head involution defective) and grim can induce apoptosis alone whereas the more recently identified skl (sickle) seems to act in synergy with grim or rpr [31][32][33]. All four proteins display a homology in their N-terminal domain that is involved in their PCD promoting activity and has been called RHG (Reaper Hid Grim). Cell death induced by these proteins can be blocked by caspase inhibitors like the baculovirus p35 protein or IAPs (Inhibitor of Apoptosis Protein: DIAP1, DIAP2, dBRUCE and Deterin in Drosophila [34] or mammalian c-IAP1 and c-IAP2 [35]). DIAP1 inhibits caspases by binding of BIR1 or BIR2 (Baculovirus IAP Repeat) domain to caspases. Since RPR, HID and GRIM are able to alleviate this inhibition by their binding to the BIR domains of DIAP1 [36], it has been proposed that RPR, HID and GRIM act mainly by releasing caspases from the inhibitory action of IAPs and by promoting IAP degradation (Figure 1).

More recent studies have given a precise description of the molecular details involved in DIAP1/caspases interactions. DRONC processing occurs in two steps. The first step appeared to occur continuously even in the absence of an apoptotic signal. The second step was observed only in apoptotic cells indicating that the initial step in DRONC processing occurs continuously in Drosophila cells and that DIAP1 is required to prevent excess accumulation of this first form of processed DRONC [37]. BIR2 domain of DIAP1 recognizes a 12-residues sequence in DRONC. Furthermore, the DRONC-binding surface on BIR2 coincides with that required for binding to the N-terminal of RHG proteins [38].

A similar situation has recently been described for the effector caspase drICE. Inhibition of drICE by DIAP1 occurs only after the cleavage of its N-terminal and involves a conserved surface groove on BIR1. RHG proteins use their N-terminal IAP-binding motifs to bind to the same surface groove, hence relieving DIAP-mediated inhibition of drICE [39].

All these data point out that the main way of controlling proapoptotic caspases activation in Drosophila is not the same than in C. elegans or mammals. Indeed, caspase activation by activators/adaptators is a crucial control point of caspase-dependent apoptosis signaling in nematodes and mammals (CED-4 in nematodes, FADD or Apaf-1 in mammals). In contrast, in Drosophila, the continuous inhibition of DRONC activity seems necessary to maintain cell survival [40,41].

Another IAP-interacting protein that harbors a conserved N-terminal IAP-binding motif has been identified in Drosophila. This protein, called Jafrac2, is a thioredoxin peroxidase that resides in the endoplasmic reticulum but is rapidly released into the cytosol following induction of apoptosis. Similarly to RHG proteins, Jafrac2 displaces DRONC from DIAP1 by competing with DRONC for the binding of DIAP1 suggesting that Jafrac2 triggers cell death by liberating DRONC from DIAP1-mediated inhibition [37].

These results have led to the conclusion that DIAP1 is the principal blockade to abolish in order to activate caspase-mediated cell death in Drosophila (Figure 1).

Figure 1. Schematic representation of apoptosis signaling pathways identified in Drosophila.

To survive, Drosophila cells must counteract the continuous DARK-mediated processing of DRONC. DIAP1 appears as the major brake able to neutralize this proapoptotic constitutive signal. Thus, it is the key factor to target in order to activate caspase-mediated cell death in Drosophila. This survival effect of DIAP1 can be alleviated by several apoptotic signaling. Binding of RHG proteins such as RPR, HID, GRIM or SKL can disrupt the caspase inhibitory action of DIAP1. The endoplasmic reticulum released thioredoxin Jaffrac2 can provide the same effect. Debcl, a proapoptotic member of the Bcl-2 family can stimulate, in a Buffy inhibitable manner, the activation of caspases in a way that could involve mitochondria. RHG proteins could also participate in this mitochondrial pathway. Bantam miRNA inhibits apoptosis by downregulating hid expression. Tumor suppressor proteins such as p53, Salvador, Warts and Hippo can inhibit cell death by inducing RHG proteins and/or regulating the turnover of DIAP1. Ras/MAPK pathway controls survival by inhibiting HID. An extrinsic pathway, involving homologs of TNFR, that does not involve a direct activation of caspases, but merely the JNK pathway leads also to cell death in a way that could be inhibited by DIAP1.

Ras/MAPK pathway

I A P p ro t e i n s a re E 3 u b i q u i t i n l i g a s e s i n v o l v e d i n t h e p ro t e a s o m e p a t h wa y

Recently the ubiquitin/proteasome pathway has been implicated in the regulation of apoptosis in mammals and in Drosophila. This is one of the main pathways of intracellular proteolysis concerning polyubiquitinated proteins that are degraded by the 26S proteasome.

IAPs carry on their C-terminal end a RING (Really Interesting New Gene) finger domain responsible for a E3 ubiquitin ligase activity. This activity enables IAPs to ubiquitinate caspases bound on their BIR domain [42] and then directs them to the 26S proteasome. Furthermore, as shown in Figure 2, DIAP1 can be auto-ubiquitinated in a UbcD1 E2 conjugase-dependent way under stimulation of apoptosis inducers of the RHG group [43]. Moreover, a genetic screen of modifiers of a chimeric RPR/GRIM protein-induced cell death has enabled the discovery of Morgue (Modifier of Rpr Grim, Ubiquitously Expressed), an F-box protein with a non-functional E2 conjugase domain. F-box proteins are adapter proteins able to recruit a specific substrate protein in a multimeric E3 called SCF (SkpA-Cul1-F-box protein) complex. In the presence of the death inducer RPR, Morgue stimulates DIAP1 ubiquitination. Indeed, DIAP1 has a short half-life and morgue mutants present high levels of DIAP1. Thus, Morgue is also implicated in regulating the turn-over of DIAP1. These studies have also pointed out the roles of Uba1, an E1 ubiquitin activating enzyme, SkpA, a component of the SCF complex and Fat Facets, a deubiquitinating enzyme, in Morgue-induced targeting of DIAP1 to the proteasome [44,45]. Thus, ubiquitin/proteasome-mediated destruction of DIAP1 is a key event during the initiation of caspase-mediated apoptosis in Drosophila [ review : 46].

In addition, flies also possess an apoptosis signaling pathway related to the Jun kinase pathway of mammals. Wengen is a Drosophila type III membrane protein with conserved cysteine-rich residues in the extracellular domain, a hallmark of the TNFR superfamily (Figure 1). Wengen can bind to the Drosophila homolog of TRAF2 (TNF Receptor-Associated Factor 2) [47] and act as a receptor of Eiger [48], a type II transmembrane protein with a C-terminal TNF homology domain which induces cell death through a process which completely depends on its ability to activate the JNK pathway but may not require new transcripts controlled by Jun [49]. Although this cell death process is blocked by DIAP1, it does not require the activity of the caspase-8 homolog DREDD [50]. Indeed, DIAP1 can also modulate cell death by inhibiting JNK activation through the ubiquitin/proteasome pathway and the binding of RHG proteins to DIAP1 can also activate this pathway [51].

M i t o c h o n d r i a a n d a p o p t o s i s i n D ro s o p h i l a

In C. elegans, mitochondria seem to play a minor role in apoptosis. In contrast, in mammals, proapoptotic members of the Bcl-2 family like Bax trigger the release of various key proapoptotic factors from the inter-membrane space of mitochondria (review: [52] and Vandenabeele, this book). These factors include cytochrome c, Apoptosis Inducing Factor (AIF), Endonuclease G, Smac/DIABLO (Second mitochondria-derived activator of caspase/direct IAP-binding protein with low PI), the serine protease Omi/HtrA2 and several caspases. Once released in the cytoplasm, cytochrome c binds to the WD40 domain of Apaf-1 (that is missing in C. elegans) and leads to the formation of a cytochrome c/Apaf-1/caspase 9 complex called "apoptosome" in which caspase-9 is autoactivated and then initiates a caspases cascade. Antiapoptotic members like Bcl-2 inhibit this release and apoptosis induced by Bax or many other death stimuli [53].

The role of mitochondria in Drosophila programmed cell death remains elusive [review : 54]. Most data show that cytochrome c is not released from mitochondria during apoptosis in vivo. However, some studies suggest that cytochrome c might regulate apoptosis without being released in cytoplasm [55][56][57]. Interestingly, the Drosophila proapoptotic Bcl-2 homolog Debcl (Death Executioner Bcl-2 Homologue), which localizes to mitochondria, triggers cytochrome c release and apoptosis when expressed in mammalian cells, while in Drosophila cells, it triggers the translocation of DRONC to mitochondria and caspase activation without cytochrome c release, suggesting that cytochrome c might participate in apoptosis without being released from mitochondria. However, a very recent study shows in a cell-free system that recombinant Drosophila cytochrome c DC3 or DC4 (the products of the two cytochrome c genes found in Drosophila) failed to activate caspases in Drosophila cell lysates, but induced caspase activation in human cell extracts [58]. Furthermore, silencing expression of either or both DC3 and DC4 had no effect on apoptosis or activation of DRONC and drICE in Drosophila cells suggesting that caspase activation occurs independently of cytochrome c in these cells [58].

However, mammalian Bcl-2 inhibits both developmental and radiation-induced cell death in vivo [59]. Furthermore, rpr-induced apoptosis in Drosophila is inhibited by Bcl-2 and, as observed in mammals, this inhibition is associated with the preservation of mitochondrial homeostasis [60]. Interestingly, mitochondria seem to be targeted by RHG domain proteins. Although the three genes rpr, hid and grim are unknown in mammals, they are functional and their action is associated with their mitochondrial localization in cellulo and/or cytochrome c release in a vertebrate acellular essays [61][START_REF] Haining | Proc. Natl. Acad. Sci[END_REF][START_REF] Evans | [END_REF].

Furthermore, the study of RHG proteins has revealed a functional role for these proteins at the mitochondrial level in Drosophila. In addition to their N-terminal RHG domain, RPR, GRIM and SKL contain an internal domain able to target them to the mitochondria. First identified in the third helix of GRIM, this domain has been called GH3 (Grim Helix 3) [64]. It is predicted to be structured in amphipathic alpha-helixes with one hydrophobic and one hydrophilic face and has been found also in RPR and SKL.

During apoptosis induction, Grim relocalizes to mitochondria in a GH3-dependent but N-terminal-and caspase activity-independent manner. In vivo, the RHG and the GH3 domains cooperate for apoptosis induction. Indeed, RHG and GH3 domains of GRIM can activate independently apoptosis and synergize to induce programmed cell death efficiently [64]. The role of the GH3 domain has also been studied in mammalian cells. In this system, the GH3 domain is essential for Grim proapoptotic activity, whereas the RHG domain is dispensable. The GH3 domain is required and sufficient for Grim targeting to mitochondria and for cytochrome c release in a Bax, Bak, caspase-and IAP-insensitive manner [65].

The C-terminal mitochondrial targeting sequence of Bcl-xL can alleviate GH3 deficiency [66]. Furthermore, the C-terminal domain of HID, although not containing a GH3, is somehow similar to the C-terminal mitochondrial targeting sequence of Bcl-xL and can substitute to the GH3 of RPR. Studies of RPR also revealed that its GH3 domain is involved in its mitochondrial localization, the induction of IAP degradation and cell killing [66]. These data suggest that IAP destabilization by RPR and probably by HID requires their localization to mitochondria.

All these data strongly suggest that mitochondria play an important role in apoptosis signaling in Drosophila even if further investigations are needed to understand their exact contribution.

D ro s o p h i l a d e a t h a d a p t o r p ro t e i n s : D a r k a n d d FA D D , t h e h o m o l o g s o f A p a f -1 / C e d -4 a n d FAD D

An homolog of the Apaf-1/Ced-4 protein, called DARK, HAC-1 or Dapaf-1, has been identified [67][68][69]. Study of Dark mutants and gene silencing by RNAi showed that this gene is required for normal developmental apoptosis [67,68]. Furthermore, Dark cooperates with RHG proteins to induce cell death in the eye [68,69]. Numerous data suggest that Dark is responsible for the basal level of DRONC activation [41]. However, the molecular details of caspase activation by Dark remain elusive [70]. Some data suggest that two isoforms of Dark can be produced by alternative splicing [67]. A short isoform, structurally related to CED-4 could induce caspase activation and cell death autonomously as CED-4 does in C. elegans [START_REF] Kanuka | Proc. Natl. Acad. Sci. USA[END_REF]. However, the most abundant isoform contains an additional C-terminal region comprising multiple WD repeats (WD-40 domain) that could mediate protein-protein interactions. This long isoform is in this way similar to mammalian Apaf-1. Indeed, Dark and cytochrome c seem to cooperate with DRONC and drICE in an apoptosome-like complex localized at mitochondria and responsible for the processing of both caspases DRONC and drICE in Drosophila cells [57,[67][68][69][START_REF] Li | [END_REF].

A Drosophila homolog of FADD, called dFADD, that contains a death domain homologous to the mammalian FADD death domain has also been identified [73]. dFADD acts downstream of IMD in the pathway that controls the expression of the antibacterial peptide genes [74,75]. dFADD contains a death domain homologous to the FADD death domain and binds to DREDD thus enhancing the cell death activity and proteolytic processing of DREDD suggesting the existence of a FADD-like apoptosis pathway in Drosophila [73]. However, dFADD is probably not involved in Eiger (the Drosophila homolog of TNF)-induced apoptosis. Rather, as mentioned above Eiger seems to induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of DRONC [50] (Figure 1).

Two D ro s o p h i l a h o m o l o g s o f t h e B c l -2 / C e d -9 f a m i l y

Surprisingly, no fly homolog of the bcl-2/ced-9 gene family has been found in genetic screens. Only the analysis of the genome permitted to identify two Drosophila members of the bcl-2 family [76]. Debcl (also called dborg-1, dbok or drob-1) is the first studied member and it has been shown to be proapoptotic [77][78][79][START_REF] Igaki | Proc. Natl. Acad. Sci. USA[END_REF][START_REF] Zhang | [END_REF]. More recently, the second one, called Buffy (or dBorg-2), has been shown to be merely antiapoptotic [82].

The Debcl protein is closer to Bok than to any other mammalian member of the Bcl-2 family. Like Bok, it contains BH1, BH2 and BH3 domains and a putative domain of membrane anchoring. Using RNAi methods, this protein has been shown to be necessary to developmental cell death [79]. Like most members of the family, Debcl is located at intracellular membranes and mostly at the outer mitochondrial membrane both in mammalian and Drosophila cells [START_REF] Igaki | Proc. Natl. Acad. Sci. USA[END_REF][START_REF] Zhang | [END_REF]. In mammalian cells [START_REF] Zhang | [END_REF], but not in Drosophila [57], Debcl triggers cytochrome c release. Experiments with mammalian cells has also allowed to show that Debcl-induced cell death is inhibited by Bcl-2 and Bcl-x L [79] and that Debcl specifically binds to the antiapoptotic members of the Bcl-2 family. However we failed to detect any protection offered by Bcl-2 against Debcl in vivo in Drosophila (Colin et al., unpublished results). Genetic interactions between debcl and other apoptosis regulators suggest that debcl and dark act in the same apoptotic pathway whereas, RHG proteins would act independently of debcl [79]. However, whether or not apoptosis induced by debcl is caspase-dependent merits further investigations. It is possible that Debcl, like Bax in mammals, can induce both caspase-dependent and -independent cell death. In this respect, it is noteworthy that mammalian Bax induces caspase-independent apoptosis in Drosophila [60].

On the contrary, although Buffy does not contain evident BH4 domain, and is structurally related to Debcl [83], ablation of Buffy by RNA interference leads to ectopic apoptosis, whereas overexpression of Buffy results in the inhibition of developmental and gamma irradiation-induced apoptosis thus exhibiting antiapoptotic properties [82]. Furthermore, Buffy interacts genetically and physically with Debcl to suppress Debclinduced cell death. Studies of genetic interactions suggest that Buffy acts downstream of RPR, GRIM and HID to block caspase-dependent cell death. In addition, similarly to Bcl-2, Buffy inhibits cell cycle progression [82].

G e n e t i c c o n t ro l o f c e l l d e a t h b y m i c ro R N A s

Recently, a new way of apoptosis regulation involving the so called micro RNAs (miRNAs) has been pointed out using Drosophila. miRNAs are small RNAs encoded by the genome. Transcripted but not translated, they adopt a hairpin structure which undergoes a maturation process that consists in a cleavage by a type III RNAse called Dicer (also involved in RNAi maturation). Maturation produces RNAs of about twenty nucleotides which, once charged in a complex similar to the RISC (RNA-Induced Silencing Complex, involved in RNAi), are able to inhibit translation of target mRNAs. In animals, this process seems to occur through a mechanism that preserves the stability of the mRNA target [84][85][86]. Recognition is made by partial complementarities with a sequence of the target mRNA 3'UTR which allows one miRNA to inhibit several genes. Hundreds of miRNA genes have been found in diverse animals, and many of these are phylogenetically conserved [85].

Two loci encoding miRNAs involved in apoptosis regulation have been identified in Drosophila: bantam/clochette and mir-14 (reviewed in [87]).

bantam has been found in a screen of genes involved in growth regulation [88,89]. Deletion of this locus gives a dwarf phenotype (bantam was the name of a dwarf chicken) due to both a cell proliferation and an apoptosis regulation role of bantam. bantam stimulates proliferation by driving both G1/S and G2/M transitions and inhibiting apoptosis induced by this overproliferation or by overexpression of E2F/DP [90]. Indeed, five target sequences of bantam are in the 3'UTR of the hid mRNA and the miRNA of bantam can abolish hid translation without affecting its mRNA level [90]. The target(s) responsible for the proliferative effect of bantam, that should be an inhibitor(s) of proliferation, remains to be identified. Interestingly, bantam miRNA expression is controlled by developmental signals.

mir-14 has been identified as a modifier of rpr-induced apoptosis in Drosophila eye [91]. Indeed, mutated mir-14 increases rpr-induced apoptosis. What is (are) the target(s) of mir-14? In agreement with an antiapoptotic effect of mir-14, its overexpression inhibits rpr-, hid-, grim-and dronc-induced apoptosis. However, target sequences of mir-14 have not been found in the mRNA of these genes. On the other hand, mir-14 does not affect apoptosis induced by Strica or Tramtrack (an inhibitor of Ras). Thus, one possibility is that mir-14 inhibits the drice gene that is an effector common to rpr, hid, grim and dronc. In agreement with this possibility, mir-14 mutants exhibit a high level of drICE. However, a direct action of mir-14 on drice remains to be demonstrated.

A p o p t o s i s c o n t ro l b y o n c o p ro t e i n s a n d t u m o r s u p p re s s o r p ro t e i n s i n D ro s o p h i la

Numerous genes involved in tumorigenesis act on cell proliferation and/or apoptosis. Some of them, as myc and e2f activate both process. As a consequence, proliferating cell are more susceptible to death signals [92], and, in many cases, inactivation of antiapoptotic tumor suppressors like p53 cooperate with oncogene activation in tumorigenic transformation. In Drosophila, genes that control both proliferation and apoptosis have also been found. Some of them are homologs of known mammalian oncogenes (e2f [93], myb [94], Src [95] etc…) or tumor suppressor genes (for example l(2)Tid [96]). Noteworthy, hid is downregulated by a Ras-dependent survival signaling [97,98]. Others cancer related genes have been identified through genetic screens performed in the fly. We will focus on two examples of tumor suppressor genes for which links with the apoptotic machinery have been established: DmP53 and Salvador/Hippo/Warts.

A single homolog of the p53 protein, called Dmp53, has been identified in 2000 by three groups [99][100][101]. The DNA-binding and transactivation domains of this protein are similar to that of its mammalian counterpart. However, it contains a specific N-terminal domain and is notably different in the MDM2 binding motif (a negative regulator of p53 that has not been found in Drosophila). As mammalian p53, Dmp53 is not required for developmental apoptosis but is involved in radiation [102, 103] and reactive oxygen species [104] -induced apoptosis. However, may be in relation to a role of Dmp53 in DNA repair, in some circumstances Dmp53 can also protect from DNA damage-induced cell death [105].

The rpr and skl genes are targets of Dmp53 [99,102] but apoptosis induced by Dmp53 does not require RPR [106] and is not inhibited by p35 [99,100], suggesting that, like its C. elegans homolog [107] and like the mammalian p53 in some cases [108], Dmp53 could be able to induce an effector caspase-independent cell death pathway. Furthermore, Dmp53, unlike its mammalian counterpart but like C. elegans p53 is not involved in the cell cycle arrest observed after irradiation [109]. These observations suggest that cell cycle inhibition by the proteins of the p53 family has appeared after the arthropod/vertebrate dichotomy. The study of Dmp53-induced apoptosis could then provide a model p53-induced apoptosis disconnected from the cell cycle.

Screens for Drosophila loss of function mutations that result in tissue overgrowth has allowed to discover new tumor suppressor genes in Drosophila. Three interesting genes that promote both cell cycle exit and cell death have been identified, salvador (Sav, also called Shar-pei), Warts (also called LATS) and Hippo (also called dMST) [110,111]. Interestingly, the human orthologs of these genes have been found mutated in some cancer cell lines. These three genes negatively regulate expression of Cyclin E, downregulate the Drosophila inhibitor of apoptosis protein DIAP1, and induce hid expression. Sav binds to the Warts serine/threonine kinase of the myotonic dystrophy protein kinase family [111]. Sav also binds to a regulatory domain of Hippo that is essential for its function. Since DIAP1 is phosphorylated in a Hippo-dependent manner in Drosophila S2 cells and Hippo can phosphorylate DIAP1 in vitro, Hippo could promote apoptosis by reducing cellular amounts of DIAP1. In addition, Sav is an unstable protein that is stabilized by Hippo [112]. These results indicate that Hippo, Warts and Sav function together to restrict tissue growth in vivo both by favoring cell cycle exit and by inducing cell death. This control of PCD is done through a reduction of cellular amounts of DIAP1 and induction of hid expression [113][114][115][116].

C o n c l u d i n g rem a r ks

Mechanisms involved in the control and execution of apoptosis in Drosophila exhibit numerous common traits with what exists in mammals. However, conservation during evolution of most of the genes involved in cell death is not associated with the maintenance of the mechanisms involved. This is true for both the intrinsic and the extrinsic pathway. In mammals, the release from mitochondria of proapoptotic factors including cytochrome c, AIF and Endo G (see the chapter "Proteins released from mitochondria during apoptosis" by P. Vandenabeele) is a decisive event of the intrinsic pathway of cell death, controlled by the Bcl-2 protein family. In contrast, although homologs of Bcl-2 proteins are found in Drosophila, no such protein release occurs during Drosophila cell death. Rather, numerous actors involved in the cell death process migrate from cytoplasm to mitochondria. Since Debcl can trigger cytochrome c release from mammalian mitochondria, it could be that the ability of some mitochondrial proteins to cooperate with Debcl to permeabilize outer mitochondrial membrane has been acquired during subsequent stage of evolution.

Concerning the extrinsic pathway, although Drosophila contains homologs of TNFα, FADD and caspase-8, an evolutionary conserved extrinsic pathway of apoptosis does not seem to function. On the one hand, the homolog of TNF activates the JNK pathway, which in turn activates caspases. On the other hand, the homolog of FADD can function as an activator of the caspase-8 homolog (DREED) but it does not seem to be involved in apoptosis regulation. Therefore, direct activation of effector caspases by the FADD/Caspase-8 pathway is also an evolutionary recent event.

In spite of these dissimilarities, Drosophila has been shown to be a powerful modelsystem to identify cell death regulators. Indeed, identification of proapoptotic regulators acting as disruptors of IAP inhibition of caspases has been possible first in Drosophila and functional analogs of these proteins (Smac/Diablo and Omi/HtrA2), although displaying different cellular localization (and relecolization) have subsequently been found in mammals. The power of the genetic approach with this organism let us glimpse that it will provide us much more other advances to our knowledge of apoptosis regulation among the life kingdom.

Figure 2 .

 2 Figure 2. IAP proteins are E3 ubiquitin ligases controlling caspase activation. In a living cell, DRONC is continuously inhibited and targeted to proteasome by DIAP1 which has a E3 ubiquitin ligase activity, thus mediating DRONC degradation. Upon a death signal, RHG proteins can cooperate with Morgue and a SCF complex to ubiquitinate DIAP1 and the RHG protein. This releases DRONC and promotes caspase activation.

Table I .

 I Conservation during evolution of proteins involved in caspase-dependent cell death.

				Mammals	Drosophila
		Caspases	CED-3	Casp-9, -3…	DREDD, DRONC,
					DECAY, drICE…
		Adaptators	CED-4	Apaf-1, FADD…	Dark, DFADD
		Antiapoptotic	CED-9	Bcl-2, Bcl-x L ...	Buffy
		proteins
	Bcl-2 family	Proapoptotic proteins		Bax, Bak, Bok...	Debcl
		BH3 only	EGL-1	Bik, Bad…	?
		proteins
		Inhibitors of		c-IAP1, c-IAP2…	DIAP1, DIAP2,
		caspases			Deterin
		Disruptors of		Smac/Diablo,	RPR, HID, GRIM,
		the anti-caspase		Omi/HtrA2	SICKLE…
		activity of IAPs

A c k n o w l e d g e m e n t s

We thank S. Gaumer for his critical reading of the manuscript. Our work was supported in part by grants from the Association pour la Recherche contre le Cancer (#3367) and from the Ligue Contre le Cancer (Comité des Yvelines).