Professeur Marie-Thérèse Rubio

Docteur Arnaud Campidelli

Cécile Docteur

Pochon

Yingying Wang

L I Zhen

Yihua Liu

Yun Luo

Yang Xu

Duan Jie

W U Xianglei

X U Song

Yueying Li

Xie Zhe

PANG Xiaomeng, PENG Zhang Lin

X U Lu

Dan Yong

Zhang Pan

Ganggang

Deng

Chongsheng QIAN ° A Campidelli °

Caroline Laroye

Véronique Decot

Loïc Reppel

Yu Xiong

Manon Mouginot

Loic Reppel

Jean-Francois Stoltz

Chongsheng Qian

Daniele Bensoussan

Veronique Decot

Véronique Decot

Loïc Reppel

Adenovirus-specific T-cell Subsets in Human Peripheral Blood and After IFN-γ Immunomagnetic selection

Curative or pre-emptive Adenovirus specific T-cell transfer from matched unrelated or third party haploidentical donors after HSCT, including UCB transplantations: A successful phase I/II multicenter clinical trial

UCB and recipient are better tolerated. Thus, as a lower HLA compatibility is required, grafts can be found nearly for each patient.

Moreover, a lower incidence of GVHD is observed compared to Bone Marrow (BM) and PBSC transplantations. However, due to the low HSC doses available in UCB grafts, poor immune reconstitution and delayed engraftment become the major limit [START_REF] Rocha | Umbilical cord blood cells from unrelated donor as an alternative source of hematopoietic stem cells for transplantation in children and adults[END_REF].

Although up to now HSCT is the only curative treatment for most of hematological malignancies, post-HSCT complications occur frequently and lead to a high incidence of transplant-related morbidity and mortality.

Among the different complications, we will mention: graft failure,

GVHD and infections.

Graft failure is a life-threatening complication. Conditioning regimen plays an important role in preventing graft failure secondary to alloreactive immune responses mediated by persisting residual host immunity [START_REF] Masouridi-Levrat | Immunological Basis of Bone Marrow Failure after Allogeneic Hematopoietic Stem Cell Transplantation[END_REF]. There are two major types of conditioning regimens:

Myeloablative (MA) and Reduced Intensity Conditioning (RIC)/Non-MA (NMA) regimens. Graft failure is more common in RIC/NMA Allogeneic HSCT (Allo-HSCT) patients compared to MA patients [START_REF] Baron | Graft-versus-tumor effects after allogeneic hematopoietic cell transplantation with nonmyeloablative conditioning[END_REF]. The other risk factors also include donor-recipient HLA mismatch, insufficient number of transplanted stem cells, and viral infections such as Cytomegalovirus (CMV) infection [START_REF] Wolff | Second hematopoietic stem cell transplantation for the treatment of graft failure, graft rejection or relapse after allogeneic transplantation[END_REF][START_REF] Spellman | The detection of donor-directed, HLA-specific alloantibodies in recipients of unrelated hematopoietic cell transplantation is predictive of graft failure[END_REF]. A second transplantation with pre-infusion conditioning may be successful, but transplant-related mortality remains high [START_REF] Guardiola | Second early allogeneic stem cell transplantations for graft failure in acute leukaemia, chronic myeloid leukaemia and aplastic anaemia. French Society of Bone Marrow Transplantation[END_REF].

Graft-versus-host disease (GVHD) remains one of the major causes of morbidity following allogeneic HSCT. GVHD is categorized as acute or chronic according to time of presentation. Acute GVHD (aGVHD) was defined historically to be limited to the first 100 days following HSCT, while chronic GVHD (cGVHD) was defined historically to be out of 100 days after HSCT. However, the distinction is not always clear as aGVHD often evolves into cGVHD. Moreover, with the implementation of NMA conditioning regimen, the timing of GVHD occurrence changed. This led the National Institutes of Health (NIH) to standardize the diagnostic criteria for GVHD helping in the identification of acute and chronic GVHD. The decision-making diagram is given below (Figure 1). It is also important to exclude other possible causes such as infections, drug toxicity or other pathology. A biopsy or laboratory test will confirm the diagnosis [START_REF] Jagasia | National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: I. The 2014 Diagnosis and Staging Working Group report[END_REF][START_REF] Filipovich | National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging working group report[END_REF].

Figure 1 Clinical signs of acute and chronic GVHD

It was demonstrated that the disparity in the minor and major HLA antigens between donor and recipient presented a positive correlation with the incidence and the severity of aGVHD [START_REF] Lee | High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation[END_REF][START_REF] Loiseau | HLA Association with hematopoietic stem cell transplantation outcome: the number of mismatches at HLA-A, -B, -C, -DRB1, or -DQB1 is strongly associated with overall survival[END_REF]. While some investigators demonstrated that the risk factors of aGVHD also included age of both the recipient and the donor, gender disparity (with multiparous female donors carrying particularly a higher risk), ineffective GVHD prophylaxis, intensity of the transplant conditioning regimen and viral infections [START_REF] Hahn | Risk factors for acute graft-versus-host disease after human leukocyte antigen-identical sibling transplants for adults with leukemia[END_REF][START_REF] Nash | Acute graft-versus-host disease: analysis of risk factors after allogeneic marrow transplantation and prophylaxis with cyclosporine and methotrexate[END_REF][START_REF] Cantoni | Evidence for a bidirectional relationship between cytomegalovirus replication and acute graft-versus-host disease[END_REF]. Ferrara and Reddy proposed three phases for the mechanism of aGVHD [START_REF] Ferrara | Pathophysiology of graft-versus-host disease[END_REF][START_REF] Harris | Advances in predicting acute GVHD[END_REF]:

- Acute GVHD predominately affects skin, upper and lower gastrointestinal (GI) tract and liver. Some nonspecific signs occur occasionally in eye and oral mucosa. Acute GVHD grading scheme is based on the site and severity of the manifestation (Table 1) [START_REF] Goyal | Grading acute graft-versus-host disease: time to reconsider[END_REF].

A commonly used prophylactic regimen for GVHD includes a combination of a calcineurin inhibitor (cyclosporine or tacrolimus) with mycophenolate mofetil (MMF) [START_REF] Arnaout | Complications of allogeneic hematopoietic stem cell transplantation[END_REF]. Some investigators reported that in vivo T-cell depletion significantly reduced the incidence and severity of GVHD [START_REF] Finke | Standard graft-versus-host disease prophylaxis with or without anti-T-cell globulin in haematopoietic cell transplantation from matched unrelated donors: a randomised, open-label, multicentre phase 3 trial[END_REF], but it also increased risk of relapse of leukemia and incidence of infections, especially viral infections [START_REF] Farhadfar | Overview of the progress on haploidentical hematopoietic transplantation[END_REF][START_REF] Watcharananan | Cytomegalovirus, adenovirus, and polyomavirus co-infection among pediatric recipients of allogeneic stem cell transplantation: characteristics and outcome[END_REF]. The treatment of isolated grade II cutaneous aGVHD includes corticosteroids or tacrolimus. For acute grade III-IV GVHD, treatment includes high dose systemic steroids, cyclosporine, and antithymocyte globulin [START_REF] Arnaout | Complications of allogeneic hematopoietic stem cell transplantation[END_REF]. In case patients are dependent or refractory to corticosteroids, other immunosuppressive drugs can be proposed as a second line treatment.

The risk factors of cGVHD are similar to aGVHD. Limited cGVHD may not need systemic therapy. However, extensive cGVHD needs immunosuppressive therapy, alternating prednisone and cyclosporine is the standard first line therapy [START_REF] Farhadfar | Overview of the progress on haploidentical hematopoietic transplantation[END_REF].

Infections are also one of the major causes of morbidity and mortality after HSCT. Independent risk factors of post-HSCT infections include presence of myeloablation, delay of immune reconstitution, use of immunosuppressive drugs and GVHD [START_REF] Sahin | An overview of infectious complications after allogeneic hematopoietic stem cell transplantation[END_REF]. There are three different phases for post-HSCT infections [START_REF] Wingard | Hematopoietic stem cell transplantation: an overview of infection risks and epidemiology[END_REF]:

- Viral infections are a common cause of morbidity and mortality after HSCT which can be seen in all post-transplant phases. The most common viral pathogen is CMV which reactivates in 60-70% of pre-transplant seropositive patients and primary infections occur in about 30% of seronegative patients with seropositive donors [START_REF] Boeckh | How we treat cytomegalovirus in hematopoietic cell transplant recipients[END_REF][START_REF] Ljungman | Cytomegalovirus in hematopoietic stem cell transplant recipients[END_REF]. Despite effective antiviral drugs like ganciclovir and foscarnet implemented as a pre-emptive treatment, life threatening CMV disease still occurs in 10% of post-HSCT patients [START_REF] Blyth | Donor-derived CMV-specific T cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation[END_REF]. Epstein -Barr virus (EBV) also reactivates, usually from the donor graft, very frequently after HSCT. EBV related Post-transplant lymphoproliferative disease (EBV-PTLD) is a complication with a high mortality rate up to 80% before anti-EBV therapy [START_REF] Styczynski | Outcome of treatment of Epstein-Barr virus-related post-transplant lymphoproliferative disorder in hematopoietic stem cell recipients: a comprehensive review of reported cases[END_REF]. Rituximab (anti-CD20 antibody) is effective as a preemptive therapy for EBV infection or treatment for established PTLD.

However, only 50%-60% of patients with PTLD achieve durable remissions after treatment with Rituximab [START_REF] Choquet | Efficacy and safety of rituximab in B-cell post-transplantation lymphoproliferative disorders: results of a prospective multicenter phase 2 study[END_REF]. Adenovirus (ADV) is also a common pathogen in viral infections. It is seen more frequently in children compared to in adult after HSCT (6%-28% vs 0%-6%) [START_REF] Matthes-Martin | European guidelines for diagnosis and treatment of adenovirus infection in leukemia and stem cell transplantation: summary of ECIL-4 (2011)[END_REF][START_REF] Lion | Molecular monitoring of adenovirus in peripheral blood after allogeneic bone marrow transplantation permits early diagnosis of disseminated disease[END_REF][START_REF] Van Tol | Adenovirus infection in paediatric stem cell transplant recipients: increased risk in young children with a delayed immune recovery[END_REF][START_REF] Walls | Adenovirus: an increasingly important pathogen in paediatric bone marrow transplant patients[END_REF].

An antiviral drug, cidofovir, seems to be effective for ADV infection [START_REF] Bordigoni | Treatment of adenovirus infections in patients undergoing allogeneic hematopoietic stem cell transplantation[END_REF][START_REF] Ljungman | Cidofovir for adenovirus infections after allogeneic hematopoietic stem cell transplantation: a survey by the Infectious Diseases Working Party of the European Group for Blood and Marrow Transplantation[END_REF], but this treatment has not received authorization from French regulatory agencies for this indication. A lipid-ester oral form of cidofovir, brincidofovir, is currently being evaluated for refractory ADV infection and disease in immunocompromised pediatric and adult patients [START_REF] Paolino | Eradication of disseminated adenovirus infection in a pediatric hematopoietic stem cell transplantation recipient using the novel antiviral agent CMX001[END_REF][START_REF] Florescu | Safety and efficacy of CMX001 as salvage therapy for severe adenovirus infections in immunocompromised patients[END_REF][START_REF] Grimley | Brincidofovir for Asymptomatic Adenovirus Viremia in Pediatric and Adult Allogeneic Hematopoietic Cell Transplant Recipients: A Randomized Placebo-Controlled Phase II Trial[END_REF]. A randomized placebo-controlled phase II study recently reported a clearance of ADV viral load using Brincidofovir (2 mg/kg if <50kg, twice weekly) as a pre-emptive strategy in 67% patients compared to 33% in the placebo. However, results were impaired by gastrointestinal toxicity leading to early treatment discontinuation and more frequent incidence of acute GVHD (50% vs 17%, respectively) [START_REF] Grimley | Brincidofovir for Asymptomatic Adenovirus Viremia in Pediatric and Adult Allogeneic Hematopoietic Cell Transplant Recipients: A Randomized Placebo-Controlled Phase II Trial[END_REF]. Other viral pathogens are also observed after HSCT, such as human herpesvirus type 6 (HHV-6), BK-virus (BKV), respiratory syncytial virus (RSV), varicella zoster virus (VZV), herpes simplex virus (HSV) for the most frequent (Table 2). As previously reported, the 2 main complications encountered after HSCT that escape conventional drugs are GVHD and viral infections. We were interested in this background part of our work to report the reciprocal interaction between GVHD and viral infections and the impact of their respective treatment on each other. We reported on figure 4 the different types of interactions we have conceptualized.

Figure 4 Interactions between GVHD and viral infections

In the first part of this background section, I will focus on the interaction between post-transplant viral infections and GVHD. Many evidences proved that viral infections after HSCT enable increase the incidence of GVHD. On the other hand, GVHD and the immunosuppressive drugs used for GVHD are a risk of viral infection.

In the second part of the background section, we will present an exhaustive review of all the published results concerning the viral specific T cell (VST) transfer for the treatment of viral infections or diseases post-HSCT. We present these data as cumulative results focusing both on efficacy and GVHD incidence. This subpart is the subject of a review.

We complete these data with an overview of the clinical trial currently underway.

In the third part of the background section, we tried to understand if immunosuppressive drugs administered for prophylaxis or for treatment of GVHD could have a negative impact on VST, as most of post-HSCT patients candidate to VST infusions receive immunosuppressive drugs for GVHD occurrence.

Part I: GVHD and Viral Infection Interaction

Only few studies focused on an effect of GVHD on viral infection incidence. Indeed, the immunosuppression related to GVHD itself and the immunosuppressive drugs enable the emergence of viral infections.

Nathan Cantoni et al [START_REF] Cantoni | Evidence for a bidirectional relationship between cytomegalovirus replication and acute graft-versus-host disease[END_REF] analyzed the evidence for a bidirectional relationship between CMV replication and aGVHD. Patients were at a significantly increased risk of developing aGVHD during CMV replication, the authors also confirmed that GVHD and its therapy could induce CMV replication. Ustun et al evaluated 5178 pediatric and adult patients who underwent HSCT between 1974 and 2010 [START_REF] Ustun | Human parainfluenza virus infection after hematopoietic stem cell transplantation: risk factors, management, mortality, and changes over time[END_REF]. They reported that GVHD was one of the risk factors for Human Parainfluenzae Virus (HPIV) infections. Some investigators reported that GVHD usually preceded CMV infection [START_REF] Miller | Cytomegalovirus infection after bone marrow transplantation: an association with acute graft-v-host disease[END_REF][START_REF] Appleton | Human herpes virus-6 infection in marrow graft recipients: role in pathogenesis of graft-versus-host disease. Newcastle upon Tyne Bone Marrow Transport Group[END_REF]. Some studies indicated that treatment of GVHD, such as steroid therapy, led to an increased risk of viral reactivation and other opportunistic infections [START_REF] Paczesny | Acute graft-versus-host disease: new treatment strategies[END_REF][START_REF] Bartee | Virotherapy using myxoma virus prevents lethal graft-versus-host disease following xeno-transplantation with primary human hematopoietic stem cells[END_REF].

Watcharananan et al [START_REF] Watcharananan | Cytomegalovirus, adenovirus, and polyomavirus co-infection among pediatric recipients of allogeneic stem cell transplantation: characteristics and outcome[END_REF] patients. The authors found that HHV-6 reactivation was associated with a higher incidence of grades II-IV aGVHD in the MAC group (P =0.01).

However, no significant impact was observed in the RIC group (P=0.97).

In multivariate analysis, HHV-6 reactivation had no significant effect on the relapse rate [START_REF] Aoki | Impact of Human Herpesvirus-6 Reactivation on Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation[END_REF]. Verhoeven et al detected HHV-6 by PCR in a cohort of 106 pediatric HSCT recipients. No significant difference was reported concerning incidence of aGvHD (all grades) between children with and without HHV-6 viremia (28% and 22%, P=0.6). However, a significantly higher incidence of grade II-IV aGVHD (90%) was observed in patients with HHV-6 reactivation before GVHD (P = 0.006) [START_REF] Verhoeven | Reactivation of Human Herpes Virus-6 After Pediatric Stem Cell Transplantation: Risk Factors, Onset, Clinical Symptoms and Association With Severity of Acute Graft-Versus-Host Disease[END_REF]. Admiraal et al reported a retrospective study for viral infections in 273 HCT patients.

They found that HHV6 reactivations were predictive for a higher incidence on grade II-IV GVHD (P<0.0001) and that both HHV6 and EBV were predictive on grade III-IV GVHD (P=0.015 and p=0.018, respectively) [START_REF] Admiraal | Viral Reactivations and Associated Outcomes in Context of Immune Reconstitution after Pediatric Hematopoietic Cell Transplantation[END_REF]. Willemse et al reported that 5 of 130 patients post allo-HSCT had Hepatitis E virus (HEV) infection. All of them developed suspected (n=4) or confirmed (n=1) hepatic GVHD. The authors hypothesized that HEV can provoke or sustain hepatic GVHD [START_REF] Willemse | Hepatitis E virus infection and hepatic GvHD in allogeneic hematopoietic stem cell transplantation recipients[END_REF].

Finally, Peterson et al reported that incidence of aGVHD (all grades) had no significant difference between the BKV-positive and BKV-negative cohort. However, a significantly higher proportion of BKV-positive patients than patients in the BKV-negative group developed severe GVHD (grades≥II) (80% vs 50 %, p = 0.013) [START_REF] Peterson | Reactivation of polyomavirus in the genitourinary tract is significantly associated with severe GvHD and oral mucositis following allogeneic stem cell transplantation[END_REF]. selection based on multimers [START_REF] Cobbold | Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers[END_REF] or IFNγ-capture system [START_REF] Feuchtinger | Safe adoptive transfer of virus-specific T-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation[END_REF] in less than 48 hours (Figure 1). In this review, we report cumulative results of a large number of published studies both in terms of efficacy and of GVHD incidence.

Infusion of ex vivo expanded virus-specific T cells

The recovery of virus-specific T-cell immunity is crucial for patients after HSCT to avoid viral infections or reactivations. At the beginning, donor lymphocyte infusions (DLI) were used to treat serious viral infections or Epstein-Barr virus (EBV)-associated lymphoproliferation after HSCT [START_REF] Heslop | Donor T cells to treat EBV-associated lymphoma[END_REF][START_REF] Hromas | Donor leukocyte infusion as therapy of life-threatening adenoviral infections after T-cell-depleted bone marrow transplantation[END_REF][START_REF] Papadopoulos | Infusions of donor leukocytes to treat Epstein-Barr virus-associated lymphoproliferative disorders after allogeneic bone marrow transplantation[END_REF]. Unfortunately, this method was not implemented broadly because of low frequency of VSTs compared to the much higher frequency of alloreactive T cells which can cause GVHD. Some interesting experiments reported in the 1990s and at the beginning of the 2000s were at the origin of the development of specific adoptive immunotherapies [START_REF] Heslop | Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients[END_REF][START_REF] Feuchtinger | Safe adoptive transfer of virus-specific T-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation[END_REF][START_REF] Walter | Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor[END_REF]. Therefore, ex vivo expanded virus-specific T cells were developed reducing drastically the number of alloreactive T cells.

CMV-VSTs

From 1995 to 2017, different groups generated CMV specific T cells using peripheral-blood mononuclear cells (PBMCs) from the HSCT-donors or from third party donors co-cultured with different CMV related stimulators such as autologous fibroblasts infected with the CMV AD169 strain [START_REF] Walter | Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor[END_REF], PBMCs loaded with CMV lysate [START_REF] Einsele | Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy[END_REF][START_REF] Perruccio | Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation[END_REF], dendritic cells (DCs) transfected with Ad5f35pp65 or pulsed with CMV antigen [START_REF] Blyth | Donor-derived CMV-specific T cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation[END_REF][START_REF] Peggs | Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines[END_REF][START_REF] Micklethwaite | Ex vivo expansion and prophylactic infusion of CMV-pp65 peptide-specific cytotoxic T-lymphocytes following allogeneic hematopoietic stem cell transplantation[END_REF][START_REF] Micklethwaite | Prophylactic infusion of cytomegalovirus-specific cytotoxic T lymphocytes stimulated with Ad5f35pp65 gene-modified dendritic cells after allogeneic hemopoietic stem cell transplantation[END_REF][START_REF] Mackinnon | Adoptive cellular therapy for cytomegalovirus infection following allogeneic stem cell transplantation using virus-specific T cells[END_REF][START_REF] Koehne | Immunotherapy with Donor T Cells Sensitized with Overlapping Pentadecapeptides for Treatment of Persistent Cytomegalovirus Infection or Viremia[END_REF] or CMV peptide mixes derived from full length pp65 and/or IE1 [START_REF] Bao | Adoptive immunotherapy with CMV-specific cytotoxic T lymphocytes for stem cell transplant patients with refractory CMV infections[END_REF]. Altogether, 47 post-HSCT patients received adoptive CMV specific T cell (CMV-VST) infusions (range from 10 5 to 3×10 6 /kg or 0.06 to 100×10 7 /m 2 ) to treat CMV infections or diseases refractory to antiviral drugs (acyclovir, foscarnet, cidofovir or ganciclovir) 4 to 479 days following HSCT (Table 1). Forty (85.1%) patients presented a complete clearance of CMV load in peripheral blood after one or two CMV-VST infusions, including 20 patients who did not receive any antiviral drug after CMV-VST infusion. Three of forty seven patients (6.4%) presented grade I cutaneous GVHD post CMV-VST infusion, including 2 patients (4.2%) with de novo GVHD. Moreover, 110 post-HSCT patients received CMV-VST (range 10 5 to 3×10 6 /kg or 0.06 to 100×10 7 /m 2 ) as a prophylaxis 28 to 115 days following HSCT [START_REF] Blyth | Donor-derived CMV-specific T cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation[END_REF][START_REF] Walter | Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor[END_REF][START_REF] Perruccio | Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation[END_REF][START_REF] Micklethwaite | Ex vivo expansion and prophylactic infusion of CMV-pp65 peptide-specific cytotoxic T-lymphocytes following allogeneic hematopoietic stem cell transplantation[END_REF][START_REF] Micklethwaite | Prophylactic infusion of cytomegalovirus-specific cytotoxic T lymphocytes stimulated with Ad5f35pp65 gene-modified dendritic cells after allogeneic hemopoietic stem cell transplantation[END_REF].

Eighteen patients (16%) presented CMV reactivations post CMV-VST infusion including 5 patients (4.5%) with CMV diseases. Graft versus Host Disease I to IV was observed in eighteen patients (16%) post CMV-VST infusion. Interestingly, two groups compared the incidence of CMV reactivations or diseases in the patients who received prophylactic CMV-VST and in control cohorts [START_REF] Blyth | Donor-derived CMV-specific T cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation[END_REF][START_REF] Perruccio | Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation[END_REF]. Incidence of CMV reactivations was of 16% (12/75 patients) in the prophylactic group and of 66% in the control cohorts (107/161 patients). Moreover, one of the two groups compared the incidence of aGVHD and cGVHD between the patients receiving prophylactic CMV-VST (n=50) and control cohorts (n=128) and no significant difference was observed [START_REF] Blyth | Donor-derived CMV-specific T cells reduce the requirement for CMV-directed pharmacotherapy after allogeneic stem cell transplantation[END_REF].

EBV-VSTs

We reviewed 10 published studies [START_REF] Heslop | Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients[END_REF][START_REF] Gustafsson | Epstein-Barr virus (EBV) load in bone marrow transplant recipients at risk to develop posttransplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells[END_REF][START_REF] Imashuku | Unsuccessful CTL transfusion in a case of post-BMT Epstein-Barr virus-associated lymphoproliferative disorder (EBV-LPD)[END_REF][START_REF] Comoli | T cell therapy of Epstein-Barr virus and adenovirus infections after hemopoietic stem cell transplant[END_REF][START_REF] Doubrovina | Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation[END_REF][START_REF] Haque | Treatment of Epstein-Barr-virus-positive post-transplantation lymphoproliferative disease with partly HLA-matched allogeneic cytotoxic T cells[END_REF][START_REF] Haque | Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial[END_REF][START_REF] Vickers | Establishment and operation of a Good Manufacturing Practice-compliant allogeneic Epstein-Barr virus (EBV)-specific cytotoxic cell bank for the treatment of EBV-associated lymphoproliferative disease[END_REF][START_REF] Gallot | T-cell therapy using a bank of EBV-specific cytotoxic T cells: lessons from a phase I/II feasibility and safety study[END_REF][START_REF] Barker | Successful treatment of EBV-associated posttransplantation lymphoma after cord blood transplantation using third-party EBV-specific cytotoxic T lymphocytes[END_REF] from 1991-2017 about EBV-VSTs generated from PBMC from HSCT donors or third party donors stimulated with the autologous EBV-lymphoblastoid cell line (LCL). Altogether, 166 patients received EBV-VST after HSCT (range from 0.5 to 5×10 6 /kg or 1 to 5×10 7 /m 2 ). One hundred and one of 164 patients received EBV-VST to prevent EBV-related lymphoproliferative disease as prophylaxis [START_REF] Heslop | Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients[END_REF] (Table 1). No patient developed lymphoma up

Infusion of freshly and rapidly isolated VSTs

As traditional ex vivo expanded VSTs require a long generation time (between 10 days to 1 month), patients with acute viral reactions cannot receive VST unless they were prepared for each patient before HSCT, which is cost-intensive and time-consuming and is a brake to the diffusion of this technique. In addition, technically demanding protocols also limit the broad clinical implementation of this method. Although short culture protocols were implemented, it still spends around 10 days to generate VSTs. VST banking should be a good way to rapidly obtain off-the-shelf VST ready for clinical infusion, but the culture process remains time-consuming and cost-intensive mainly to establish a large number of VST lines covering most of HLA types including the rarer

[105].
An alternative method has also been developed to freshly isolate VSTs from a leukapheresis by an immunomagnetic strategy using HLA-peptide multimers [START_REF] Cobbold | Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers[END_REF] or IFNγ-capture system [START_REF] Feuchtinger | Safe adoptive transfer of virus-specific T-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation[END_REF]. This method is faster (24-48 h), easier and less expensive for broad clinical implementation.

Virus-specific T cells by HLA-peptide multimers

CMV-VSTs

To our knowledge, three investigators [START_REF] Cobbold | Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers[END_REF][106][107][108] reported the treatment of post-HSCT CMV infection using adoptive transfer of fresh CMV-specific CD8 + T cells generated by an immunomagnetic strategy based on HLA-peptide multimers from HSCT donors or third-party haploidentical donors. Thirty three patients received CMV-specific CD8 + T cells (dose range from 0.78 to 872×10 3 /kg) for treatment of CMV infections refractory to antiviral drugs. Twenty eight of 32 evaluable patients (87%) achieved complete or partial viral load response (Table 2).

Four patients had aGHVD I-III (n=3) or cGVHD (n=1) within one month of CMV-VST infusion. However, three of them had developed previous GVHD before infusion, the remaining one had received a natural killer cell transfusion and an unselected DLI at 21 and 8 days before CMV-VST infusion, respectively.

EBV-VSTs and ADV-VSTs

Uhlin and colleagues only reported one successful experience in the treatment of EBV-associated lymphoma after HSCT [109] and one failure in the treatment of an ADV infection resistant to cidofovir [107] (Table 2).

No GVHD associated with VST infusions was observed.

Multimers are most readily made with class I HLA and can only select CD8+ T cells but not CD4+ T-cell subset restricted by class II HLA [110].

In some viral infections, for example ADV infection, immune response is mainly supported by CD4+T cells [START_REF] Feuchtinger | Safe adoptive transfer of virus-specific T-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation[END_REF]111]. Moreover CD4+ T-cells are necessary to support in vivo expansion and survival of CD8+ T cells [START_REF] Heslop | Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients[END_REF]112] to maintain virus-specific immune response following adoptive transfer for a long time. In contrast, IFNγ-capture approach can select both CD4+ and CD8+ antigen-specific T cells in an HLA unrestricted manner.

Virus-specific T cells IFNγ-capture system

Recovered peripheral blood mononuclear cells (PBMCs) are stimulated for 6 to 16 hours with a viral antigen [START_REF] Feuchtinger | Safe adoptive transfer of virus-specific T-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation[END_REF][113][114][115][116]. These cells are subsequently processed using the Cytokine Capture System (CCS, Miltenyi Biotec) based on an IFN-γ immunomagnetic technology on the CliniMACS device (Miltenyi Biotec), as previously described [117, 118] .

This technique can rapidly isolate multi-clone VSTs including CD4+ and CD8+ cells.

CMV-VSTs

Three investigators generated CMV-VSTs using immunomagnetic isolation based on IFNγ-capture system from PBMC of HSCT donors or

EBV-VSTs

Two studies reported the generation of EBV-VST using immunomagnetic isolation based on IFNγ-capture system from PBMC of HSCT donors stimulated ex vivo with one or a pool of EBV peptides.

BKV-VSTs

Recently, Pello et al generated BKV-VST by immunomagnetic isolation based on IFNγ-capture system using PepTivators BKV-LT and BKV-VP1 as stimulators. One post-HSCT patient received BKV-VST infusion for treatment of refractory severe BKV haemorrhagic cystitis, resolution of both the symptoms and viraemia was achieved after BKV-VST transfer without GVHD [126] (Table 2).

Discussion and Conclusion

In vitro studies of Immunosuppressive drug effects on T cells or VSTs

Andersson We reviewed the publications for the VST infusion after HSCT from 1995 to 2017 (Qian et al, 2017, under submission). In most of the studies, the influence of immunosuppressive drugs on the effect of VST in vivo was usually not analyzed. However, we observed that, altogether, 330 patients received VSTs for the treatment of viral infections or diseases.

In vivo studies of Immunosuppressive drug effects on T cells or VST

Among them, 239 patients (72%) achieved a complete clinical response (n=209, 63%) or a partial response (n=30, 9%) to VST infusion, although almost all the patients received ≥1 immunosuppressive drugs including ATG/ alemtuzumab as prophylaxis or treatment of GVDH. These results are in favor of a low influence of immunosuppressive drugs onto the effect of VSTs in vivo, as previously reported [START_REF] Doubrovina | Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation[END_REF]113].

Objectives

As we have just reported, viral infections and GVHD influence each other as well as immunosuppressive treatment of GVHD may have an impact on VSTs efficacy and VSTs may promote the reactivation of GVHD.

In this context, where patients who really need VST infusion may suffer from GVHD and are concomitantly treated by immunosuppressive drugs for GVHD prophylaxis or treatment, we wondered which parameters could influence VST efficacy. Indeed, only a very low dose of VST isolated by an IFN based immunomagnetic technique can be infused into the patients. It is well known now that the efficacy of VST is directly related to their in vivo expansion when they encounter the blood-circulating virus. We focused on two main parameters that can influence VST expansion:

-First, we analyzed the different T-cell subsets among isolated ADV-VSTs, as the expansion capacity varies between the subsets, the most mature expanding less than the immature ones.

-Second, we were interested in whether corticosteroids

Results

ADV-VST subpopulations

The T cell compartment was classically divided into four subpopulations:

-Naïve T lymphocytes (TN) which are mature cells that have never yet encountered an antigen.

-Effector T lymphocytes (TEFF) which rapidly eliminate pathogens or tumors according to different effector functions.

-Memory T cells which, once produced, can respond rapidly to subsequent antigenic restimulation including central memory T cells (TCM) and effector memory T cells (TEM) [136].

In the recent years, Gattinoni team identified another memory T subpopulation with stem cell properties so-called T memory stem cells (TSCM) (Figure 5) [137, 138]

Results

Methylprednisolone had a inhibitive effect on ADV-VST proliferation in the first 2 to 4 days but not after 10 to 19 days. Surprisingly, ADV-VST with MP 1 µg/mL and 2 µg/mL even presented a slight increase in IFN-γ secretion after restimulation with ADV-peptide pool compared to ADV-VST without MP, although there was no significant difference. The As patients who receive VST infusion often also receive corticosteroids as a first line immunosuppressive treatment for acute GVHD, we were interested in whether corticosteroids presented an impact on ADV-VSTs.

Introduction

Based on our previous experience of ADV-VST [117, 118], we focused on the impact of Methylprednisolone (MP) 1 μg/mL or 2 μg/mL on in vitro expanded ADV-VSTs after the IFN-γbased immunomagnetic selection. Functional properties of ADV-VSTs were investigated in three culture conditions either without MP, with MP 1 μg/mL or MP 2 μg/mL.

Methods

Generation of ADV-specific T Cells

Leukapheresis collections were performed in 8 healthy donors.

Recovered peripheral blood mononuclear cells (PBMCs) were stimulated for 6 hours with an adenovirus peptide pool: PepTivator-AdV5 Hexon (Miltenyi Biotec, Bergisch Gladbach, Germany). These cells were subsequently processed using the Cytokine Capture System (CCS, (experimental release -spontaneous release)/(maximum releasespontaneous release) x 100.

Mixed Lymphocyte Reaction

Peripheral blood mononuclear cells (PBMCs) from 3 allogeneic blood donors were irradiated (25 Gy) and used as stimulating cells. In vitro expanded ADV-specific T cells were used as responder cells. Stimulating cells (1×10 5 cells/well) and responder cells (1×10 5 cells/well) were mixed in a 96-well round-bottom culture plate and incubated for 72 hours at 37°C, 5% CO 2 . In the same way as proliferative assay, Cell proliferation kit and fluorometer Victor 4® (PerkinElmer, Massachusetts, USA) were used to detect and measure allogeneic proliferation.

Statistical analysis

after a 2 to 4 day expansion (1.04±0.5 and 1.05±0.31 vs 1.41±0.63, respectively), MP 1 μg/ml group presented significant decrease of growth rate compared to the group without MP (p<0.05, Dunn's Multiple

Comparison Test) (Figure 2 a). However both ADV-VST with MP 1 μg/mL and 2 μg/mL presented higher growth rate than the ADV-VST without MP after a 10 to 19 day expansion (4.79±6.04 and 3.68±2.40 vs 3.24±3.18), but no significant difference was observed between the three groups (Figure 2 b).

Specific immune reaction

Specific IFN-γ secretion after antigen restimulation IFN-γ secretion after restimulation with the same ADV peptide pool was detected in 7 evaluable ADV-VST lines by Elispot assay. Surprisingly, ADV-VST with MP 1 µg/mL and MP 2 µg/mL presented a slight increase in IFN-γ secretion compared to ADV-VST without MP (19654±12586 SFC/10 6 and 22303±9123 SFC/10 6 vs 19544±15514 SFC/10 6 , respectively) but no significant difference was reached (Figure 3).

Specific proliferation after antigen restimulation

In the five evaluable ADV-VST lines, all of the ADV-VST with MP 1 µg/mL (n=5) and MP 2 µg/mL (n=5) presented a notable decrease in specific proliferation after restimulation with ADV peptide compared to ADV-VST without MP (54088±30211 and 51364±24894 vs 111572±51578 respectively), a significant difference was observed between MP 2μg/mL and without MP group. (Figure 4). Moreover, in the positive control stimulated with PHA, a remarkable decrease inproliferation was also observed in all ADV-VST lines with MP 1 µg /ml and MP 2 µg /ml (Data not shown).

Specific cytotoxicity

A cytotoxicity essay was performed in three evaluable ADV-VST lines.

The specific lysis of ADV-VST with MP 1 μg/mL and MP 2 μg/mL was relatively decreased compared to ADV-VST without MP at an effector-to-target cell ratio of 40:1, 20:1, 10:1 and 5:1 (Figure 5), but no significant difference was observed between the three groups at all different effector-to-target cell ratio.

Alloreactivity

ADV-VST alloreactivity against 2 different irradiated PBMCs of healthy donors was detected in five evaluable ADV-VST lines. As expected, alloreactivity of ADV-VST with MP 1 μg/mL and MP 2 μg/mL significantly decreased compared to ADV-VST without MP (p<0.01 and p<0.05, respectively) (Figure 6). Moreover both MP 1 μg/ml and 2 μg/ml showed a significant inhibition of alloreactivity in the positive control (PBMCs 1 against irradiated PBMCs 2 or PBMCs 2 against irradiated PBMCs 1) (Data not shown).

Discussion

Adoptive anti-viral cellular immunotherapy by infusion of virus-specific T cells (VSTs) has been reported feasible and effective for the SFCs/10 6 PBMC). In comparison, ADV-specific IFN-γ secretion in the 5 evaluable (M)MUD was lower (234 ± 178, p=0.10).

General immune recovery of after ADV-VST infusion

Lymphocyte, CD3+, CD4+ and CD8+ T cell recovery was detected after ADV-VST infusion. A slight increase in general immune recovery was observed at D14 and was amplified after D30 (Figure S1).

Outcome:

During follow-up, we observed 30 infectious episodes (other than ADV) in 10 patients with 7 early infectious complications (within the first month post ADV-VST infusion). Bacterial infections were the most frequent (50%) and CMV-and EBV-reactivations post ADV-VST infusion represented 17% of the infections.

Infectious complications evolved favorably, except for patient 02-08.

Legend

Discussion

ADV-VST efficacy relying on their composition

Since expansion is critical for the efficacy of ADV-VSTs, we wondered whether a minimal dose should be infused to observe an anti-viral effect, and/or whether some T cell subsets were critical to support a sustained in vivo expansion.

ADV-VST infused dose

In

-Persistence of ADV-VST

We have just reported that persistence of ADV-VST is related to the expansion and differentiation from the more immature T cell subsets.

Another critical parameter for sustained persistence of ADV-VST could be the donor origin and HLA compatibility between ADV-VST and recipients. Indeed, it was previously reported by Haque et al that there was an association between the number of HLA matches of EBV-VST (from a cell bank) and patient outcome, those with a higher number of HLA matches presented better response at 6 months than those with fewer HLA matches at 6 months (P =0.048) [START_REF] Haque | Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial[END_REF]. However, the response rate was not related to matching or mismatching of any particular HLA According to our clinical and experimental experience, we suggest that durable efficacy of ADV-VSTs relies mainly on the presence of more immature T cell subsets, even in a low frequency, but probably not on VST infused cell dose nor HLA compatibility with the recipient.

ADV-VST efficacy relying on the in vivo environment at the time of infusion.

Patients who need VST immunotherapy often present GVHD or receive immunosuppressive drugs as GVHD prophylaxis or treatment. In order to be sure that ADV-VST will not be the target of immunosuppressive drugs, treatment doses are decreased, but it will be a risk of GVHD occurrence or reactivation. However, are we certain that immunosuppressive drugs have a deleterious effect on VSTs?

In vitro effect

According to in vitro assays, most studies reported that some immunosuppressive drugs, especially cyclosporin A, FK506, MTX and

In vivo effect

Although an in vitro effect is observed, there is no evidence for a deleterious effect of immunosuppressive drugs on VSTs in vivo.

According to the review of nearly all the publications related to VST

Adoptive Cellular Immunotherapy for the treatment of refractory Adenovirus infections after Hematopoietic Stem Cell Transplantation

From bench to bedside Hematopoietic stem cell transplantation (HSCT) is one of the only curative treatments for benign or malignant hematological diseases and primary immune deficiencies. However, viral infections and graft-versus-host disease (GVHD) are among the most frequent complications after HSCT associated with high morbidity and mortality. Viral infections often occur in the absence of specific immune reconstitution in the context of immunosuppression related to GVHD itself or to the prophylaxis or treatment of GVHD. The recommended anti-viral drug treatments have an inconsistent efficacy in this context of immunodeficiency and are not devoid of toxicity. The promising therapeutic alternative is adoptive immunotherapy, in particular the infusion of specific anti-viral T lymphocytes isolated by immunomagnetic technique (VSTs). However, these T lymphocytes may be targeted by immunosuppressive treatments administered for GVHD, but also may be the cause of the onset or reactivation of GVHD. We have shown in this work that the efficacy of VSTs, which is based on their in vivo expansion when they encounter the circulating virus, is mainly allowed by the most immature lymphocyte subpopulations, even in a small proportion. We argue in this work that the efficacy of VSTs and their persistence is mainly based on the presence of the most immature T lymphocyte subpopulations and this regardless of the degree of HLA compatibility between the VSTs and the recipient. Moreover, their moderate sensitivity to corticosteroids, which we have studied in vitro, does not justify the modulation of immunosuppression at the time of infusion of ADV-VSTs, as observed in vivo in the multicenter phase I / II clinical trial we conducted between 2012 and 2015. Indeed, this clinical trial does not report any de novo GVHD after ADV-VSTs infusion. On the other hand, modulation of immunosuppression may potentially be incriminated in the reactivation of GVHD within weeks of ADV-VST infusion. A Phase II comparative trial will bring the evidence of efficacy and will clearly determine the role of VSTs in the reactivation of GVHD.

Keyword: Hematopoietic stem cell transplantation, Adoptive cellular immunotherapy, Adenovirus infection, anti-virus specific T-cell (VST), T cell subpopulation, Corticosteroids

Immunothérapie adoptive pour le traitement des infections à Adénovirus réfractaires après allogreffes de Cellules Souches Hématopoïétiques :

De la recherche fondamentale à la recherche clinique L'allogreffe de cellules souches hématopoïétiques (CSH) est un des seuls traitements curatifs des hémopathies bénignes ou malignes et des déficits immunitaires primitifs. Cependant, les infections notamment virales ainsi que la réaction du greffon contre l'hôte comptent parmi les complications les plus fréquentes des allogreffes associées à une morbidité et une mortalité élevées. Les infections virales surviennent souvent en l'absence de reconstitution immunitaire spécifique dans un contexte d'immunosuppression liée à la GVHD elle-même ou à la prophylaxie ou au traitement de la GVHD. Les traitements médicamenteux anti-viraux préconisés présentent une efficacité inconstante dans ce contexte d'immunodéficience et ne sont pas dénués de toxicité. L'alternative thérapeutique prometteuse est l'immunothérapie adoptive cellulaire notamment celle qui consiste en l'injection de lymphocytes T spécifiques anti-viraux isolés par technique immunomagnétique (VSTs). Cependant, ces lymphocytes T peuvent être la cible des traitements immunosuppresseurs administrés pour la GVHD mais également par eux-mêmes être potentiellement la cause de la survenue ou de la réactivation d'une GVHD. Nous avons montré dans ce travail que l'efficacité des VSTs, qui repose sur leur expansion in vivo lors de la rencontre avec le virus circulant, est principalement permise par les sous-populations lymphocytaires les plus immatures, même si elles ne sont présentes qu'en faible proportion. Nous défendons dans ce travail le fait que l'efficacité des VST ainsi que leur persistance repose prioritairement sur la présence des sous-populations lymphocytaires T les plus immatures et ce quel que soit le degré de compatibilité HLA entre les VSTs et le receveur. De plus, leur sensibilité modérée aux corticoïdes, que nous avons étudiée in vitro, ne justifie pas la modulation de l'immunosuppression lors de l'injection des ADV-VSTs, comme observé in vivo dans le protocole clinique multicentrique de phase I/II que nous avons mené entre 2012 et 2015. En effet, ce protocole clinique ne rapporte aucune GVHD de novo après injection d'ADV-VSTs ; en revanche, la modulation de l'immunosuppression peut potentiellement être incriminée dans la réactivation de GVHD dans les semaines suivant l'injection des ADV-VSTs. La réalisation d'un essai comparatif de phase II permettra de prouver très clairement le rôle des VSTs dans la réactivation de GVHD.

Mot clé : Allogreffe de cellules souches hématopoïétique, Immunothérapie adoptive cellulaire, Adenovirus infection, Lymphocytes T spécifiques anti-virus (VST), Sous-population de lymphocyte T, Corticoïdes

1 IDOFigure 1 :Figure 2 :

 112 Figure 1: Clinical signs of acute and chronic GVHD

Figure 3 :

 3 Figure 3: Post-HSCT infections in different phases

Figure 4 :

 4 Figure 4: Interactions between GVHD and viral infections

Figure 5 :Figure 6 :

 56 Figure 5: Development of T cell sub-populations: phenotypes and

Figure 7 :

 7 Figure 7: Two possible models of T cell differentiation * Figures in articles and reviews are not included

Table 5 :

 5 Recommendations for therapy of EBV-PTLD * Tables in articles and reviews are not included Résumé en français du travail de thèse L'allogreffe de cellules souches hématopoïétiques (CSH) constitue une thérapeutique efficace pour le traitement des nombreuses hémopathies malignes et non malignes telles que les leucémies aiguës ou les lymphomes. Cependant, la transplantation allogénique de cellules souches hématopoïétiques non apparentées ou intra-familiales partiellement incompatibles, entraine un risque important de rejet, de réactions du greffon contre l'hôte (GVH) et un retard de la reconstitution immunitaire. Afin de minimiser le risque de rejet et de GVH, le patient est rendu immunodéprimé. La période d'immunodépression nécessaire à la prévention des effets secondaires de la greffe peut durer 3 à 6 mois voire plus et est propice au développement d'un grand nombre d'infections bactériennes, fongiques et virales parmi lesquelles les infections à Epstein-Barr virus (EBV), cytomégalovirus (CMV) et adénovirus (ADV) sont les plus redoutables. Ces infections représentent une cause majeure de morbidité et de mortalité. L'ADV présente une affinité pour de nombreux tissus et organes et son pouvoir pathogène est très étendu. Asymptomatique dans la majorité des cas pour l'immunocompétent, les infections à ADV se révèlent sévères chez l'immunodéprimé pouvant aboutir à des cystites hémorragiques, pneumonies, hépatites, méningo-encéphalites ou à des atteintes généralisées selon l'âge du patient et le sérotype incriminé. Le taux d'infection à ADV varie de 5 à 21% après greffe de CSH selon l'âge du patient et la méthode de diagnostic, et était associé à un taux de mortalité de plus de 50% avant l'avènement du cidofovir et plus récemment du brincidofovir (forme orale du cidofovir). A l'exception des deux médicaments mentionnés ci-dessus, il n'existe pas de traitement spécifique des infections à ADV. La ribavirine est encore parfois utilisée mais semble inefficace pour les infections disséminées et l'utilisation du cidofovir malgré des résultats très encourageants reste limitée par une importante néphrotoxicité. La faible efficacité ou la toxicité limitante des agents pharmaceutiques et la nécessité d'une reconstitution immunitaire ont donné naissance à une alternative thérapeutique : l'immunothérapie adoptive. Afin de traiter les patients présentant une adénovirose réfractaire aux traitements conventionnels notre équipe a développé une technique rapide et de grade clinique de sélection de lymphocytes T spécifiques anti-virus (VSTs) anti-ADV permettant l'initiation d'un protocole clinique. La production de VSTs anti-ADV est réalisée avec le kit Cytokine Capture System (Mitenyi Biotec) de grade clinique. Après avoir été isolées par gradient de densité ou leukaphérèse, les cellules mononucléées du sang périphérique (PBMC) de donneurs sains sont stimulées par un pool de peptides spécifiques de la protéine Hexon de l'ADV (PepTivator ADV5-Hexon, Miltenyi Biotec) entrainant la production d'IFNγ par les lymphocytes T spécifiques. Tous les leucocytes sont ensuite marqués par un anticorps bivalent anti-CD45 anti-IFNγ mais seuls les lymphocytes T spécifiques anti-virus ayant sécrété de l'IFNγ suite à la stimulation antigénique et qui sont donc spécifiques de l'ADV, seront sélectionnés via un anticorps anti-IFNγ couplé à une bille immunomagnétique. Les cellules marquées par l'anticorps anti-IFNγ couplé à la bille magnétique sont retenues par la colonne magnétique du CliniMACS et constituent les cellules ADV-VSTs, appelées fraction positive. Les cellules non marquées donc non spécifiques de l'ADV et non retenues constituent la fraction négative collectée dans une poche située en dessous de la colonne magnétique. Les ADV-VSTs sélectionnés sont mis en culture durant 3 à 4 semaines dans des flacons de culture contenant du milieu RPMI 1640 + 10% sérum AB (SAB) et de l'interleukine-2 (IL2) à la concentration finale de 1000UI/mL. Des cellules nourricières (fraction négative irradiée à 25 Gy) sont ajoutées à J0 à un ratio de 100 :1 et à J7 à 10 :1. Après expansion, 5 contrôles qualités fonctionnels sont réalisés. Les tests de marquage des cytokines intracellulaires et d'Elispot-IFNγ permettent de mettre en évidence le maintien des fonctions sécrétrices d'IFNγ des ADV-VSTs après une nouvelle stimulation antigénique par le PepTivator-ADV5 Hexon. Le test de cytotoxicité permet de mettre en évidence la capacité des VSTs à détruire leur cible (Hexon). L'alloréactivité résiduelle des ADV-VSTs est mesurée par un test de culture mixte lymphocytaire. Enfin, la réponse proliférative des VSTs est déterminée par le test de transformation lymphocytaire après stimulation antigénique. La maîtrise de cette méthode d'isolement ainsi que des contrôles qualité afférents n'est pas suffisante pour observer l'efficacité de ces VSTs in vivo. En effet, nous avons observé dans la littérature et dans notre expérience que l'efficacité était corrélée au formidable potentiel d'expansion des VSTs in vivo. Les paramètres pouvant influencer cette expansion peuvent être inhérents aux ADV-VSTs eux-mêmes, tels que les sous-populations lymphocytaires T qui constituent les VSTs, ou liés à l'environnement dans lequel se retrouvent les VSTs in vivo, comme par exemple la présence d'immunosuppresseurs. Dans ce travail, nous nous sommes intéressés dans un premier temps aux sous-populations lymphocytaires T naïves et mémoires et dans un second temps nous avons étudié in vitro l'effet des corticoïdes sur les ADV-VSTs. Cependant, l'efficacité ne doit pas être aux dépens de la sécurité. L'effet indésirable le plus redouté reste la GVHD. C'est pourquoi nous rapportons dans la troisième partie de ce travail les résultats d'un essai clinique multicentrique de phase I/II consistant en l'administration d'ADV-VSTs à des patients présentant, après allogreffe de CSH, une adénovirose infection ou maladie réfractaire au traitement anti-viral médicamenteux (Cidofovir). Ainsi, nous avons montré que la présence des sous-populations lymphocytaires mémoires les plus immatures était très importante, même en faible proportion, car ce sont elles qui possèdent le plus fort pouvoir d'expansion et permettront une persistance de la réponse immunitaire anti-ADV. Nous avons d'ailleurs observé dans le protocole clinique que le seul patient pour lequel la charge virale n'a pu être contrôlée avait reçu des ADV-VSTs avec la plus faible proportion de lymphocytes T mémoires immatures. Selon une réflexion argumentée, nous considérons que la composition en sous-populations les plus immatures est le paramètre limitant pour observer une expansion des ADV-VSTs, contrairement à la dose de cellules ou encore la compatibilité HLA entre donneur et receveur. En effet, nous rapportons dans le protocole clinique des résultats comparables lorsque les ADV-VSTs proviennent du donneur non apparenté de CSH ou d'un donneur intra-familial haplo-identique. Nous avons observé un effet transitoire des corticoïdes sur l'expansion in vitro des ADV-VSTs sans effet significatif sur leur fonction. Toutefois, si cet effet transitoire peut effectivement limiter l'expansion des ADV-VSTs in vivo, il est d'autant plus important de ne pas tarder à mettre en oeuvre cette thérapeutique et de traiter les patients le plus tôt possible, tant que la charge virale n'est pas trop élevée. Une telle attitude est probablement préférable à une baisse de l'immunosuppression pour tenter de contrôler la charge virale ADV par l'immunité du patient car cette stratégie risque d'entrainer une réactivation de la GVHD, comme nous l'avons observé dans l'essai clinique. En conclusion, ce travail de thèse a permis de mieux comprendre les conditions qui favorisent l'efficacité des ADV-VSTs et qui limitent leur toxicité. Toutefois, seul un essai clinique comparatif permettra de conclure. Cet essai clinique de phase III à grande échelle est en attente de financement au niveau européen et la France fait partie des pays producteurs (par le biais de notre centre) et investigateurs. Introduction Hematopoietic stem cell transplantation (HSCT) has been used to treat most malignant and non-malignant hematological disorders for about 70 years. In 1959, Thomas and his colleagues first reported that a patient who developed terminal stage leukemia received her identical twin's bone marrow transplantation after total-body irradiation. A three-month remission was observed post transplantation [1]. Thanks to the identification of human leukocyte antigen (HLA) system, the patients can receive allogeneic HSCT from alternative HLA matched donors, while HLA incompatibilities are associated with an increased risk of post-transplant complications. Effective T-cell depletion permitted to reduce incidence of graft versus host disease (GVHD) in allogeneic donor setting. However delay in immune reconstitution and higher risk of infections remained a significant limit [2]. In the recent years, HSCT from haploidentical donors improved significantly using cyclophosphamide post-transplantation and present advantages for patients who lack of a suitable HLA matched donor. Currently, Umbilical cord blood (UCB) became an alternative source of HSCT. Umbilical cord blood present lower alloreactivity compared to conventional bone marrow (BM) or peripheral blood stem cells (PBSC) because UCB contains relative immature lymphocytes. Therefore, disparities of HLA between

  Phase I: Tissue damage and activation of antigen-presenting cells This first phase begins at the time of conditioning. Conditioning regimens, especially MA, damage host tissues and cause a huge secretion of inflammatory cytokines (TNF-α, IL-1 and IL-7) activating recipient antigen presenting cells (APC) which overexpress adhesion, co-stimulatory and HLA molecules. The damage of mucosa induces Lipopolysaccharide (LPS) and bacterial cell wall components release resulting in the activation of macrophages via Toll Like Receptors (TLR) and release of additional inflammatory cytokines. -Phase II: Activation, differentiation and migration of donor T cells Donor mature T lymphocytes, after HSCT, are activated by HLA molecules of host APCs through IL-12 and IL-23 and produce T helper cell type 1 (Th1) cytokines (IL-2, IFN- and IL-6). The synthesis of inflammatory cytokines is partly inhibited by IL-10 from host APCs. Regulatory T cells (Treg) are induced by indoleamine 2,3-dioxygenase (IDO) secretion from host APCs. -Phase III: The proliferation and differentiation of cytotoxic T lymphocytes and Natural Killer (NK) cells Th1 cells promote proliferation and differentiation of activated cytotoxic T lymphocytes (CTLs) and stimulate NK cells, which can induce apoptosis. Inflammatory cytokine production secondary to mucosa damage also causes apoptosis.

Figure 2

 2 Figure 2 Pathophysiology of acute graft-versus-host disease[START_REF] Harris | Advances in predicting acute GVHD[END_REF]

  Phase I. The early pre-engraftment phase involves 2 to 4 weeks after HSCT -Phase II. The early post-engraftment phase involves 2 to 3 months after HSCT -Phase III. The late phase involves beyond the third month after engraftment Neutropenia and mucosal damage lead to infections in the early pre-engraftment phase. Bacteria are the leading responsible pathogens, whereas viral infections are less frequent. Due to impaired cellular and humoral immunity, viral and fungal infections are frequently encountered in the early post-engraftment phase. Bacterial infections are as well as frequently encountered in this phase. In the late phase, in which immune reconstitution continues, recovery of cellular immunity requires a long time. As well as the pathogens seen during the early post-engraftment phase, Varicella zoster virus (VZV) and encapsulated bacterial infections due to impaired opsonization are observed. The infections that are commonly seen in different phases are shown in figure 3.

Figure 3 :

 3 Figure 3: Post-HSCT infections in different phases. (Tomblyn et al 2009) [24]

  Yaghobi et al detected adenovirus (ADV) DNA in 125 HSCT recipients. Adenovirus DNA positive in EDTA-treated blood samples was observed in 16/125 (12.8%) patients post-transplantation [65]. GVHD clinical complications were observed in 6/16 (37.5%) ADV-infected transplant recipients. They stated significant correlations between ADV infections and GVHD-related skin symptoms (P<0.005) and gastrointestinal presentations (P<0.005) at 3 weeks post-HSCT. Olkinuora et al reported a single center retrospective study of 124 pediatric patients who underwent HSCT between 1999 and 2006 [66]. They found that early multiple herpesvirus/ Respiratory viral infections were associated with an increased risk of chronic GVHD (P<0.001). In a large-scale clinical study, Dulery et al monitored weekly HHV6 plasma loads of 235 consecutive patients by quantitative PCR. Patients with an early positive HHV6 PCR assay experienced more frequent grade II-IV aGVHD compared with the patients who had not (p =0.009). This result indicated that HHV6 reactivation after allo-HSCT was associated with the development of aGVHD [67]. Aoki et al conducted a retrospective analysis of the impact of HHV-6 reactivation on transplantation outcomes in 236 allo-HSCT

  Wang et al stated three possibilities of HHV-6 infection on the occurrence of aGVHD[START_REF] Wang | Correlations of human herpesvirus 6B and CMV infection with acute GVHD in recipients of allogeneic haematopoietic stem cell transplantation[END_REF]. First, HHV-6 may increase the GVHD reaction by modifying, directly or indirectly, the regulation of the host immune response. Second, HHV-6 may decrease the threshold of induction of acute GVHD reaction. Third, HHV-6 may increase the expression of key molecules which could lead to the development of GVHD, such as intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1, IL-1, tumor necrosis factor-α (TNF-α) and the CC chemokine RANTES[START_REF] Grivel | Pathogenic effects of human herpesvirus 6 in human lymphoid tissue ex vivo[END_REF].Although controversial studies were reported concerning CMV, viral infections related to HHV6, ADV or BKV seem to be highly correlated with the development of GVHD, especially after MAC conditioning regimen, and with high grades GVHD (II-IV or III-IV), either in pediatric and adult populations. On the other hand, GVHD and immunosuppressive drugs are also important risk factors for the occurrence of viral infections due to impairment of immune response (NK cell and APC functions).AbstractAllogeneic hematopoietic stem cell transplantation (HSCT) is a curative option for treatment of some malignant and nonto viral infections which are a major cause of morbidity and mortality. Although anti-viral agents are now available for most types of viral infections, they are not devoid of side effects and their efficacy is limited when there is no concomitant antiviral immune reconstitution. In recent decades, adoptive transfer of viral specific T-cells (VSTs) became an alternative treatment for viral infection after HSCT. However two major issues are concerned in VSTs transfer: the risk of GVHD and anti-viral efficacy. We report an exhaustive review of the published studies which focus on prophylactic and/or curative therapy by VST transfer for post-HSCT common viral infections. A low incidence of GVHD and a good anti-viral efficacy was observed after adoptive transfer of VSTs. Viral specific T cell transfer is a promising approach for abroad clinical application. However a randomized, controlled study in a large cohort of patients comparing antiviral treatment alone to antiviral treatment combined with VSTs is still needed to demonstrate efficacy and safety. Introduction Viral infections are important causes of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). The early diagnosis of viral infections has been facilitated by the method of polymerase chain reaction (PCR). The incidence of reactivation of latent viruses and of related viral diseases significantly decreased with the prophylactic and preemptive strategies using anti-viral agents. Although anti-viral agents are now available for most types of viral infections, they are not devoid of side effects and their efficacy is sometimes limited in the absence of concomitant anti-viral immune reconstitution [74]. A promising alternative consists in reconstituting the patient`s specific anti-viral immunity with virus-specific T cells (VSTs), either produced by ex vivo expansion[75] within 2 to 8 weeks or isolated by rapid immunomagnetic

  to 15 years of follow-up although they had risk factors that included T-cell depletion or primary immunodeficiency. Sixty five other patients presented proven EBV-PTLD or detectable EBV viral load in peripheral blood and were resistant to conventional treatment like rituximab, chemotherapy or antiviral drugs. Forty six patients (70.8%) achieved complete remission (n=45) and one patient achieved partial remission (n=1). All of the responders presented an increase in EBV-VSTs quantification in blood concomitant with a decrease in EBV-viremia and resolution of EBV-PTLD.In all but one of the 164 patients receiving EBV-VSTs as prophylaxis or treatment of EBV-PTLD or EBV viremia after HSCT, no immediate adverse reaction neither de novo GVHD was observed. One patient presented a possible skin aGVHD five hours after the first infusion, which was resolved by topical steroid and tacrolimus within 24 h. Eight patients (4.9%) had a recurrence of GVHD (6 patients in grade I, 2 patients in grade II) and 13 patients (7.9%) developed a cGVHD resolutive under treatment. However, most of EBV-VSTs donors were HLA mis-matched or even third party donors with minimal HLA matching, and a decrease in steroid therapy dose was performed in some patients waiting for EBV-VSTs. These low cumulative incidences of both recurrent GVHD and chronic GVHD account for the safety of EBV-VST infusion.Multiple-VSTsMore recently, some investigators generated Multiple-VST using PBMCs from the HSCT-donors or third party donors co-cultured with different viral related stimulators, including two-virus-ST[START_REF] Gerdemann | Safety and clinical efficacy of rapidly-generated trivirus-directed T cells as treatment for adenovirus, EBV, and CMV infections after allogeneic hematopoietic stem cell transplant[END_REF] (ADV and EBV), three-virus-ST[99-102] (ADV, CMV and EBV), four-virus-ST[103] (ADV, CMV, EBV and varicella zoster virus (VZV)) or even five-virus-ST[104] (ADV, BKV, CMV, EBV and Human Herpesvirus 6 (HHV6)). Altogether, 96 patients received Multiple-VST (dose range from 0.5 to 10×10 7 /m 2 and 1×10 4 /kg in one study[101]) as a curative treatment of one to four viral infections/diseases (n=83) or as a prophylaxis (n=13). Among the 78 evaluable patients with curative VST-treatment, 64 patients (82%) presented CR (n=43) or PR (n=21) of all the viral infections or diseases including 3 patients with EBV-PTLD (

  third-party donors stimulated ex vivo with pp65 protein[116] or a pool of CMV-pp65 peptides[116, 119, 120].Forty six patients received CMV-VSTs post-HSCT for prophylaxis[116] (n=7), preemptive treatment[116] (n=11) or curative treatment of CMV infection refractory to antiviral drugs [119, 120] (n=28) (Table2). The infused dose of CMV-VST CD4+ cells ranged from 0.28 to 61.4 10 3 cells/kg and CMV-VST CD8+ cells ranged from 0.06 to 20.1 10 3 cells/kg.In the prophylaxis study, no patient required antiviral therapy within the next 6 months and no CMV disease was observed in any patient. In the preemptive study, antiviral treatment was reduced in 9/11 patients and not required in 2 patients. In the cohort of patients treated for CMV infection refractory to antiviral drugs, 15 among 25 evaluable patients (60%) presented a clearance or a significant decrease (>log1) of CMV viremia including 2 cases of CMV encephalitis. Interestingly, it was suggested that efficacy of VST transfer was not related to the T-cell dose as even a very small dose of 360 CD3/kg was shown to generate successful antiviral response[119]. Five patients experienced GVHD I (10.9%) and four, GVHD II-III (8.7%).

  Viral specific T cell transfer for the treatment of post-HSCT viral infections or diseases is associated with a good tolerance and an anti-viral efficacy. Regarding the lower antiviral efficacy reported when VST were infused at late-stage of viral infections or diseases[113, 115], we suggest that preemptive treatment should be considered in the future. Indeed, the Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines recommends that donor or third party EBV-VST should be considered for preemptive therapy of EBV disease. Moreover, EBV-VSTs are now recommended at a first line therapy in EBV-PTLD according to these guidelines[START_REF] Styczynski | Management of Epstein-Barr Virus infections and post-transplant lymphoproliferative disorders in patients after allogeneic hematopoietic stem cell transplantation: Sixth European Conference on Infections in Leukemia (ECIL-6) guidelines[END_REF].Ex vivoexpanded method could significantly enrich VSTs and expand them to a large cell number. However, this time-consuming and cost-intensive technique cannot be broadly implemented. In the recent decade, freshly isolated VST presented safety and efficacy against post HSCT viral infections. The method of HLA-peptide multimers presents a limitation with a class I HLA-restriction allowing isolation of one CD8+ T cell clone with a specificity limited to one peptide presented by one MHC molecule, while the Cytokine Capture System (CCS, Miltenyi Biotec) based on an IFN-γ immunomagnetic technology isolates the functionally active T cells, making it possible to generate polyclonal CD4+ and CD8+ T-cells. Moreover, the polyclonal VSTs are able to target most of viral peptides presented by the patient HLA molecules enhancing anti-virus efficacy, provided that an anti-viral specific immune response is present in the donor. However, if the HSCT donor cannot be recruited again for VST preparation, due to an absence of specific immune response, to a refusal of the donor center in case of MUD donor, or because the graft was MUD UCB, a third party haploidentical donor can be contacted. Indeed, Qian et al reported a series of 6 UCB transplanted patients who received ADV-VSTs from a third party haploidentical donor. No difference regarding intensity or persistence of ADV-specific immune response and ADV viral load clearance was observed between patients who received ADV-VSTs from HSCT original donors and from third party haploidentical donors [125]. This type of donors presents different advantages: rapidly available, a specificimmune response which can be controlled before collection, a full HLA haplo-identity with the recipient and potentially with the HSCT graft, except maybe in case of UCB transplantation. The experience reported in the context of VST banking showed that, although a significantly better immune response is observed at 6 months when HLA matches are higher[START_REF] Haque | Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial[END_REF], efficacy can still be observed even with a single HLA allele matched with the recipient [102].Unlike with the ex vivo expanded method, the number of isolated VSTs with the immunomagnetic technique is very low. However, many investigators reported that even a low dose of infused VSTs was able to induce viral load clearance and in vivo VST expansion[119, 125]. If the dose of infused cells is not critical for expansion, the composition of those cells may play an important role. Indeed, it was previously reported that the presence of a relevant pool of stem memory T cell (TSCM) might be essential for the control of persisting infections, in which effector T cells undergoing functional exhaustion and replicative senescence need to be replenished continuously by less differentiated T cell subsets[127][128][129][130]. We propose that the mature sub-populations of VSTs support the immediate cytotoxicity, while the immature subpopulations play a crucial role in sustaining ADV-VST expansion in vivo and continuous anti-ADV efficacy until patient presents his own immune reconstitution from the graft.Expansion of VSTs also depends on the in vivo environment. Indeed, patients who need VST immunotherapy often present GVHD and/or receive immunosuppressive drugs which may impair VST expansion.Although the impact of immunosuppressive drugs on VST is poorly known, two studies compared anti-virus efficacy of VSTs in patients who received immunosuppressive drugs or not[START_REF] Doubrovina | Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation[END_REF] 113]. They both reported that anti-virus efficacy of VSTs was not significantly different between the two groups. In absence of evidence of a clear impact of immunosuppressive drugs (except T-cell depleting antibodies) on VSTs, and considering that modulation of immunosuppression could secondarily increase the risk of GVHD reactivation, immunosuppression should not be a contraindication to VST infusion. Indeed, discriminating the causes of GVHD, between the accountability of VSTs themselves and the modulation of immunosuppression, still remains difficult. A randomized, controlled phase II or III study in a large cohort of patients comparing antiviral treatment alone to antiviral treatment combined with VSTs is required in order to confirm the safety and efficacy of VST infusion. By providing evidence of the safety of VST infusion, those trials will allow for a wide early implementation of this strategy leading to an improved management of viral infections or diseases. The easy standardized production process of immunomagnetic isolated VSTs will contribute to this dissemination.

Figure 1

 1 Figure 1 Different methods for VST generation. Virus-specific T cells are generated by ex vivo expansion within 2 to 8 weeks or isolated by rapid immunomagnetic selection based on multimers or IFNγ-capture system in less than 48 hours.

[ 30 :

 30 DLI (27: treatment with DLI alone;3: DLI followed by EBV-VST) 19: EBV-VST (17: treatment with EBV-VST alone;2 EBV-VST followed by DLI)] 13/19 EBV-VST: EBV-PTLD CR 17/30 DLI: EBV-PTLD CR EBV-VST: No de novo GVHD DLI: 3 de novo aGVHD II-III ; 1 worsing aGVHD II-III; 3 de novo cGVHD Doubrovina et al., 2012 EBV-VST (From a bank) 8 patients with progressive EBV-PTLD refractory to antiviral drugs, rituximab and chemotherapy Solid organ transfer patient (n=7) HSCT patient (n=1) 3/8 patients: EBV-PTLD CR; 1/1 HSCT patient: no response No GVHD Haque et al., EBV-VST (From a bank) 33 patients with EBV-PTLD refractory to antiviral drugs, rituximab, chemotherapy and radiotherapy Solid organ transfer patients (n=31) HSCT patients (n=2) 14/33 patients : EBV-PTLD CR 3/33 patients : EBV-PTLD PR 2/2 HSCT patients : EBV-PTLD CR No GVHD Haque et al., EBV-VST (From a bank) 10 patients with EBV-PTLD refractory to rituximab or chemotherapy Solid organ transfer patients (n=3) HSCT patients (n=7) 8/10 patients: EBV-PTLD CR 4/7 HSCT patients: EBV-PTLD CR 1 patient (HSCT): possible GVHD Vickers et al, 2014 EBV-VST (From a bank) 11 patients with EBV-PTLD refractory to rituximab and/or chemotherapy Solid organ transfer patients (n=3) HSCT patients (n=8) 3/11 patients: EBV-PTLD CR 1/11 patients: EBV-PTLD PR 3/8 patients HSCT: CR (n=2)+ PR (n=1) No GVHD Gallot et al., EBV-VST (From a bank) 2 patients with EBV-PTLD refractory to rituximab and/or chemotherapy 2/2 patients:EBV-PTLD CR No GVHD Barker et al, CMV-EBV-ADV VST 11 patients with viral infections refractory to antiviral drugs 11/ 11 patients: Decrease i n viral load (one patient had CR of EBV-PTLD) No de novo GVHD Leen et al., 2006 CMV-EBV-ADV VST (From a bank) 50 patients with viral infections refractory to antiviral drugs 18/45 evaluable patients: CR 19/45 evaluable patients: PR 2 de novo GVHD I 4 GVHD I reactivaion 2 GVHD II-III reactivaion Leen et al, 2013 CMV-EBV-ADV or ADV-EBV VST 10 patients with viral infections refractory to antiviral drugs and/or rituximab (CMV=3 ADV=1 EBV=1 EBV-PTLD=1 EBV+ADV=2 CMV+ADV=2) 8/10 patients: CR 4/6 patients with single infection: CR (including 1 EBV-PTLD) 4/4 patients with dual infections:CR 2 non responders: one EBV (viral load fluctuation but no EBV-PTLD development); one CMV (the patient with persistent CMV colitis >4 years before VST infusion ) 1 patient had possible skin GVHD II Gerdemann et al., 2013 CMV-EBV-ADV VST 2 patients with ADV infection refractory to antiviral drugs 1 patient: CR 1 patient: >1.5 log transient reduction of viral load, but died of organ failure associated with ADV disease patients: CR or PR (CMV=1;BKV=1) including one EBV-PTLD resolution. 1/8 patient (HHV6+BKV+EBV): mixed response (EBV and HHV6 were cleared; BKV continued to increase) 3/3 prophylaxis: no relative viral infection > 3 months 1/11 de novo skin GVHD II Papadopoulou et al.,2014 EBV-CMV-ADV-VZV VST 10 patients: prophylaxis 6 patients: CMV reactivation after VST infusion No EBV ADV VZV infection follow-up 12 months 3/10 patients: GVHD II-IV MA et al., 2015

  10 patients :EBV infection (n=2) or PTLD (n=8) refractory to antiviral drugs or rituximab 7/10 patients :EBV load decrease (n=1) or EBV-PTLD CR (n=6) 1/10 patient :GVHD I to II (thought to be related to EBV-VST transfer) Icheva et al, 2013 EBV-VST (IFNγ-capture system) 6 patients with EBV-PTLD refractory to antiviral drugs and rituximab 3 patients (early stage of EBV-PTLD): CR 3 patients (late-stage EBV-PTLD with multiorgan dysfunction)ADV infection refractory to antiviral drugs 5/6 evaluable patients : decrease (n=1) or ADV load clearance (n=4) 1/9 patient: GVHD II Feuchtinger et al, 2006 ADV-VST (IFNγ-capture system) 30 patients with ADV infection refractory to antiviral drugs 14/29 evaluable patients: CR of viremia 3/29 evaluable patients: PR of viremia 5/29 evaluable patients: ADV load clearance at other sites of infection (8/29 patients had no response to ADV-VST: all died) 4/30 patients :GVHD II-III 2/30 patients :GVHD I Feucht et al, 2015 ADV-VST (IFNγ-capture system) 1 patient with severe ADV related respiratory failure refractory to antiviral drug ADV load clearance, ADV specific immune reconstitution and clinical improvement No GVHD Di Nardo et al, 2013 ADV-VST (IFNγ-capture system) 5 patients with ADV infection refractory to antiviral drugs 3/5 patients : ADV load clearance 1/5 patients : ADV load decrease No GVHD Qasim et al, 2013 ADV VST (IFNγ-capture system) 8 patients with ADV disease refractory to antiviral drugs (3 patients had CMV infections) 4/6 evaluable patients: viral load clearance No GVHD Creidy et al, 2016 ADV VST (IFNγ-capture system) 11 patients with ADV infection or disease refractory to antiviral drugs 9/11 patients: CR of viremia and diseases 1/11 patients: ADV load clearance at other sites of infection 3/11 patients: GVHD reactivation (Grade I: n=1; Grade II: n=1; extensive cGVHD: n=1) Qian et al, 2017 BKV VST (IFNγ-capture system) 1 patient with refractory severe BKV haemorrhagic cystitis 1/1: BKV load clearance and resolution of the symptoms No GVHD Pello et al, 2017

  et al tested the effects of FK506 and CsA on cytokine production of peripheral blood mononuclear cell (PBMC) after stimulation with phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore (ionomycin) [131]. They found that both FK506 and CsA significantly inhibited production of IL-2, IL-4, IL-5, IFN- and TNF-. However, the cytokine production was not affected when PBMCs were stimulated with lipopolysaccharide (LPS) and anti-CD28 monoclonal antibody which is a Ca 2 + -independent cell activation process [132]. Strauss et al analyzed seven commonly used immunosuppressive drugs (dexamethasone (DEX), mycophenolic acid (MPA), FK506, CsA, rapamycin (RAP), methotrexate (MTX) and cyclophosphamide (CP)) for apoptosis-induction and effector function in human peripheral T cells and cytotoxic T cell lines (CTL) [133]. They reported that antigen specific proliferation of T cells and cytotoxicity of HLA-A1 antigen specific CTL line were only inhibited by CP and MTX among all the tested drugs. CP and MTX also slightly increased activation-induced cell death (AICD) and CD95-sensitivity. Interestingly, they found that DEX, MPA, CsA, FK506 and RAP only prevented activation of naive T cells, but did not block proliferation of activated T cells nor decrease cytotoxic capacity of CTL lines. Zhan et al tested the effect of CsA onto in vitro expanded EBV-VSTs generated by culture [134]. Cytotoxic function of EBV-VSTs against B-lymphoblastoid cell lines (LCL) was moderately inhibited by CsA greater than or equal to 250 ng/mL during a 4 hour assay. However, the cytotoxic function was significantly reduced when EBV-VSTs were cultured with CsA for 7 days before assay, particularly at a dose between 250 and 1,000 ng/mL of CsA. Fuhrmann et al reported that CsA and FK506 were able to reduce IL2 and TNF-α production of CMV-VSTs in post heart and lung transplantation patient, while IFN-γ production seemed less affected [135].According to in vitro assays, some immunosuppressive drugs, especially CsA, FK506, MTX and Cyclophosphamid were able to decrease T cell proliferation, sometimes targeting mainly naïve T cells, cytokine secretion especially for IL2 and TNF or cytotoxic function of VSTs in case of a long impregnation with CsA. Although an in vitro effect is observed, there is no evidence for a deleterious effect of immunosuppressive drugs on VSTs in vivo. Thus, we report hereafter the

n=27,

  DLIs followed by EBV-VSTs: n=3, and EBV-VSTs followed by DLIs: n=2)[START_REF] Doubrovina | Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation[END_REF]. Fourteen patients received immunosuppressive drugs at the time of cell therapy (steroids alone: n=6, steroids combined with CsA or FK506: n=3, sirolimus alone: n=4, CsA alone: n=1). Among the 14 patients who received immunosuppressive drugs, 64% achieved complete response of EBV-disease compare with 74% of the 35 patients who did not receive immunosuppressive drugs. No significant difference was observed. However, by the time cell infusions began, daily doses of systemic steroids had been reduced to 0.07-0.0375 mg/kg of prednisone (median 0.14 mg/kg) or its equivalent. The authors report that analysis of T-cell responses in these patients did not allow ascribing any clear effect of these agents on the expansion of EBV-CTLs in vivo. Feucht et al treated 30 patients with refractory ADV disease or viremia using ADV-VST infusion [113]. They aimed at a steroid dose of ≤ 1mg/kg prednisolone before ADV-VST treatment. Sixteen patients received steroids at the time of ADV-VST therapy, 12 of 13 (92%) evaluable patients presented in vivo expansion of ADV-VST associated with subsequent virologic response in 12 of 16 (75%) evaluable patients. Moreover, 5 patients still received steroid dose above 1 mg/kg, 3 of the 5 (60%) responded to ADV-VST. The authors stated that in vivo expansion of ADV-VST and virologic response could occur under a low dose of steroids and, in single patients, even under higher doses of steroids. However, only 2 of 7 evaluable patients (29%) who received T-cell-depleting antibodies (antithymocyte globulin (ATG) or alemtuzumab) presented a response to ADV-VST after infusion. The authors considered that treatment with ATG/alemtuzumab seems to decrease the efficacy of ADV-VST. Concerning 2 patients with successful response to ADV-VST despite the use of T-cell-depleting antibodies, they declared that use of ATG/alemtuzumab should not be an absolute contraindication for T-cell therapy. However residual in vivo activity of these antibodies should be detected in future trials.

  presented an impact on ADV-VSTs expansion and function. As corticosteroids are used as a first-line immunosuppressive treatment of GVHD, it is not rare that they are administered to the patient when the question of implementing a treatment by VSTs arises. Proliferative ability and functional properties of ADV-VSTs were investigated in three in vitro culture conditions either without Methylprednisolone (MP), with MP 1 μg/mL or MP 2 μg/mL. However, efficacy must not be at the expense of safety. The most feared toxicity is GVHD when allogeneic T cells are infused. We attempted to confirm the in vivo safety of infused ADV-VST. A phase I/II multicenter pilot study was performed for the treatment of refractory ADV infection or disease after HSCT. As a second objective of this trial we analyzed efficacy on ADV viral load and specific immune reconstitution. Sub-populations of ADV-VST were analyzed in the evaluable patients to attempt to clarify the different in vivo functions of these subsets. The effect of ADV-VSTs in the patients who received immunosuppressive drugs (including steroids) during ADV-VST treatment was studied to understand the impact of immunosuppressive drugs on in vivo ADV-VSTs.

Figure 5

 5 Figure 5 Development of T cell sub-populations: phenotypes and functions. (Gattinoni, 2013)

Figure 6

 6 Figure 6 Hierarchical model of human T cell differentiation. (Gattinoni, 2017)

Figure 7 :

 7 Figure 7: Two possible models of T cell differentiation (Restifo et

  Allogeneic hematopoietic stem cell transplantation (HSCT) is the standard treatment for most malignant and non-malignant hematological disorders and primary immunodeficiencies. Graft versus host disease (GVHD) is one of the major complications after HSCT especially in case of mis-matched or matched unrelated donors ((M)-MUD) or alternative donors. Immunosuppressive treatments required to control GVHD contribute to worsen immunodeficiency. Thereby, GVHD and immunosuppressive treatments increase the risk of infections, especially viral reactivations which are a major cause of morbidity and mortality [144-147]. Effect of anti-viral drugs is limited in the absence of concomitant anti-viral immune reconstitution. Moreover, they are not devoid of severe side effects.Adoptive anti-viral cellular immunotherapy by infusion of virus-specific T cells (VSTs) is a promising alternative treatment for viral infectionsespecially Epstein-Barr virus (EBV)[START_REF] Heslop | Long-term outcome of EBV-specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients[END_REF] cytomegalovirus (CMV)[119] and Adenovirus (ADV)[START_REF] Feuchtinger | Safe adoptive transfer of virus-specific T-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation[END_REF] after HSCT. Interferon(IFN)-γ based immunomagnetic isolation presents the advantage of being a fast Good Manufacturing Practice (GMP)-grade procedure for a wide clinical implementation[113]. Up to now, more than 130 patients received IFN-γ+ VSTs (ADV n=64; EBV n=16; CMV n=53, BKV n=1) with doses ranging from 0.15 to 166 x10 3 IFN-γ+ T cells /kg in eleven clinical studies. About 80.6% patients showed a reduction or complete clearance of viral load concomitantly with in vivo expansion of VST and an acceptable tolerance profile[77, 87, 113-116, 119, 121, 124-126, 148].

  Biotec) based on an IFN-γ immunomagnetic technology, as previously described[117, 118, 149]. Briefly, cells were labeled with a bi-specific antibody: anti-CD45 conjugated to anti-IFN-γ, incubated 45 min in a 37°C, 5% CO 2 incubator to allow IFN-γ secretion. ADV-specific T-cells secreting IFN-γ were then labeled with an anti-IFN-γ antibody conjugated to a magnetic bead. Cells were loaded onto the CliniMACS device (Miltenyi Biotec) and ADV specific T-cells were collected in the positive fraction.In vitro expansion of ADV-specific T-CellADV-specific T cells were in vitro expanded in RPMI-1640 supplemented with 10% human AB serum and 1000 IU/mL of interleukin (IL)-2 (Pepro-Tech, TEBU, Paris, France).Irradiated autologous feeder cells from the negative fraction were added at the beginning and after 7 days at a ratio of 100:1 and 10:1, respectively. Cultures were fed with fresh culture medium every 2 days. All of them were in vitro expanded for 7 to 22 days before applying different culture conditions. Then pre-expanded ADV-VSTs were separated and expansion was proceeded according to 3 different culture conditions (considered as Day 0: D0): without MP (Mylan S.A.S., Saint-Priest, France) (n=8), with a final concentration of MP 1 µg/mL (n=8), or MP 2 µg/mL (n=7) during 28 ± 10 days. Methylprednisolone 1µg/mL and MP 2µg/mL simulate MP 1 mg/kg and MP 2 mg/kg in clinical treatment after calculation according to pharmacokinetics. The absolute cell number of ADV-VST was analyzed at early (Day 2-4) and later time (Day 10-19), growth rate was calculated as: absolute cell number at day 2-4 / day 0 or Day 10-19 / Day 2-4. Then, functional assays were performed after 2-3 weeks of in vitro expansion in presence or not of MP. IFN-γElispot Assay IFN-γ secretion response to PepTivator-ADV5 Hexon was detected by IFN-γ Elispot Assay as previously reported [117]. Briefly, Multiscreen Immobilon-P plates (Millipore, Billerica, MA, USA) were coated with a monoclonal anti-human IFN-γ antibody, 1-D1 K (Mabtech, Stockolm, Sweden). In vitro expanded ADV-specific T cells were added in triplicate wells (100 μL/well), then incubated for 20 hours at 37°C with the PepTivator-ADV5 Hexon. Phytohemagglutinin (PHA) was used as positive control, and culture medium as negative control. The cells were discarded and the plate was washed the next day by Dulbecco's phosphate buffered saline (PBS) (Eurobio, Lille, France) supplemented with 0.1% TWEEN 20 (Sigma-Aldrich, St Louis, USA). A second biotinylated anti-Human IFN-γ antibody (7B 6-1 biotin, Mabtech) and a streptavidin conjugated alkaline phosphatase (Mabtech) were added successively. Then, an alkaline phosphatase conjugate substrate kit (Biorad, Richmond, CA) allowed the visualization of dark spots counted using an automated Elispot reader: Bioreader 4000 PRO-S (BIO-SYS, SERLABO, Entraigues sur la Sorgues, France). The number of specific T-cell responders was calculated after subtracting negative control values. Proliferative Assay Specific proliferation of ADV-VSTs was studied in the presence of PepTivator-ADV5 Hexon in a proliferative assay. Briefly, 1×10 5 in vitro expanded ADV-VSTs were incubated with PepTivator-ADV5 Hexon during 72 hours at 37°C, 5% CO 2 . Phytohemagglutinin (PHA) was used as positive control, and culture medium as negative control. Cell proliferation kit (DELFIA® Cell Proliferation kit) and Fluorometer Victor 4® (PerkinElmer, Massachusetts, USA) were used to detect the proliferation ability. Cytotoxicity Assay Autologous and allogeneic PHA-blasts were generated from 5×10 6 donor PBMCs stimulated with 1 mg/mL PHA and 100 IU/mL IL-2 as previously described [149]. Autologous PHA-blasts pulsed or not with PepTivator-ADV5 Hexon were used as target cells. Target cells were loaded with a fluorescence enhancing ligand (BATDA, bis (acetoxymethyl) 2,2':6',2''-terpyridine-6,6''-dicarboxylate, PerkinElmer, Massachusetts, USA) for 20 min at 37°C, and subsequently washed four times in complete culture medium complemented with 0.1 mmol/L (2mg/L) Sulfinpyrazone (Sigma, Sigma-Aldrich, L'Isle D'Abeau Chesnes, France). Then, target cells were placed in 96-well round-bottom plates at 1×10 4 cells/well. Effector cells were added at an effector-to-target cell ratio of 40:1, 20:1, 10:1 and 5:1 in triplicate, as previously defined in our pre-clinical report [118]. After a 2-hour incubation at 37°C, 5% CO 2 , plates were centrifuged (500g, 5 min) and 20 μL of the supernatant was transferred to a DELFIA® yellow 96 well plate (PerkinElmer, Massachusetts, USA). Two hundred μL of DELFIA® Europium Solution (PerkinElmer, Massachusetts, USA) was added to each well and incubated for 15 minutes at room temperature. Fluorescence was measured by a Fluorometer Victor 4® (PerkinElmer, Massachusetts, USA). The percentage of specific lysis was calculated as follows:

  prophylaxis and treatment of viral infections after HSCT[113]. As patients candidate to ADV-VST infusion often receive steroids as a first line treatment of GVHD, we investigated the effect of methylprednisolone (MP) 1 μg/mL or 2 μg/mL on in vitro expanded ADV-VSTs after IFN-γ based immunomagnetic selection.An obvious inhibitive effect on in vitro expansion of ADV-VSTs was observed in the groups with MP 1 μg/mL and 2 μg/mL at 2 to 4 days. However, although a tendency of decrease was observed in ADV-VST absolute cell number with MP, the growth rate presented a tendency of increase after 10 to 19 days. Our results demonstrated that MP presented a transient inhibitive effect to ADV-VST proliferation in the first 2 to 4 days, while the effect decreased with time. Similarly to our results, some investigators reported a transient decrease in percentage of T lymphocytes after treatment with MP at day 3 in multiple sclerosis patients [150, 151], one of the investigators also demonstrated that the transient decrease was mainly caused by the reduction of CD4+ T cells (P<0.01) especially the naïve compartment (CD4+CD45RA+), while the memory compartment (CD4+CD45RO+) was not reduced and even relatively increased [151]. Concerning the effect of MP on ADV-VST specific immune reaction, it was surprising that MP did not decrease IFN-γ secretion of ADV-VST. Indeed a tendency of an increase in IFN-γ secretion of ADV-VST after restimulation with ADV peptide was observed with MP 1 μg/ml and 2 μg/ml compared to ADV-VST without MP. Then, IFN-γ secretion ability of ADV-VSTs is not impaired by MP. As IFN-γ based immunomagnetic selected ADV-VSTs are mainly composed of mature memory T cells, especially after in vitro expansion [118], we hypothesize they are probably not targeted by MP given that steroids were show to mainly target the naïve T cell compartment [133, 151]. Moreover, the mature memory T cell compartments secrete more IFN-γ than naïve compartments, which can explain a maintained IFN-γ level with ADV-VST with MP 1 μg/mL and MP 2 μg/mL. However, ADV-VST with MP 1 μg/mL and 2 μg/mL presented a decrease in specific proliferation after restimulation with ADV peptide compared to ADV-VST without MP. Specific proliferation of ADV-VSTs was impaired in presence of MP. The specific lysis of ADV-VST with MP 1 μg/mL and MP 2 μg/mL slightly decreased compared to ADV-VST without MP at an effector-to-target cell ratio of 40:1,20:1, 10:1 and 5:1 but it did not reached any significant difference. It showed that MP 1 μg/mL and MP 2 μg/mL presented no or little impact on the specific cytotoxicity of ADV-VSTs. For the alloreactivity of ADV-VSTs against 2 different irradiated PBMCs from healthy donors, an obvious decrease in alloreactivity was observed in ADV-VST with MP 1 μg/mL or 2 μg/mL compared to ADV-VST without MP. MP, usually used as a first line treatment of GVHD, is able to decrease alloreactivity of ADV-VSTs in vitro as expected. According to in vitro assays as previously reported [133-135], some immunosuppressive drugs, especially Cyclosporin A (CsA), FK506, Methotrexate (MTX) and Cyclophosphamid were able to decrease T cell proliferation, sometimes targeting mainly naïve T cells, cytokine secretion especially for IL2 and TNF-γ or cytotoxic function of VSTs in case of a long impregnation with CsA. However, unlike with the observations of in vitro studies, several investigators reported that immunosuppressive treatment (excluding T-cell-depleting antibodies) does not impair the anti-virus in vivo efficacy of VST [93, 113]. In conclusion, MP presented a transient inhibitive effect on in vitro expansion of ADV-VSTs in the first 2 to 4 days. IFN-γ secretion ability of ADV-VSTs after resimulation with ADV peptide and specific cytotoxicity were not or slightly impacted, however MP could decrease the specific proliferation and alloreactivity of ADV-VST. More samples are needed to investigate the impact of other immunosuppressive drugs on VSTs in vitro.

Figure 1 :

 1 Figure1: a: The absolute cell number of ADV-VST with MP 1 μg/mL or 2 μg/mL was lower than ADV-VST without MP after 2 to 4 days expansion, a significant difference was observed between MP 1μg/mL and Without MP group. b: The absolute cell number of ADV-VST with MP 1 μg/mL or 2 μg/mL was lower than ADV-VST without MP after 10 to 19 days expansion but no significant difference.

Figure 2 :

 2 Figure 2: a: The growth rate of ADV-VST with MP 1 μg/mL or 2 μg/mL was lower than ADV-VST without MP after 2 to 4 days expansion, a significant difference was observed between MP 1μg/mL and Without MP group. b: Both ADV-VST with MP 1 μg/mL and 2 μg/mL presented higher growth rate than the ADV-VST without MP after 10 to 19 days expansion but no significant difference. (Growth rate = absolute cell number at day 2-4 / day 0 or Day 10-19 / Day 2-4).

Figure 3 :

 3 Figure 3: ADV-VST with MP 1 µg/mL (n=8) and MP 2 µg/mL (n=7) presented tendency of increase in IFN-γ secretion comparing with ADV-VST without MP, but no significant differences.

Figure 4 :

 4 Figure 4: ADV-VST with MP 1 µg/mL (n=6) and MP 2 µg/mL (n=5) presented a notable decrease in specific proliferation after restimulation with ADV peptide comparing with ADV-VST without MP, a significant difference was observed between MP 2μg/mL and Without MP group.

Figure 5 :

 5 Figure 5:The specific lysis of ADV-VST with MP 1 μg/mL and MP 2 μg/mL was relatively decreased, but no significant difference was observed between the three groups at all different effector-to-target cell ratio.

Figure 6 :

 6 Figure 6: Alloreactivity of ADV-VST with MP 1 μg/mL and MP 2 μg/mL significantly decreased compared with ADV-VST without MP.

FigureFigureFigure

  Figure

Figure S1 :

 S1 Figure S1: General immune recovery of after ADV-VST infusion. A slight increase in general immune recovery was observed at D 14 and was amplified after D 30 .

-

  our clinical study, all of the 11 patients received a very low cell dose of infused ADV-VST (5,83 ± 8,23×10 3 CD3+IFN-γ+ T-cells/kg). Clearance of ADV load with an in vivo expansion of ADV-VST was observed in 10 of 11 patients (91%). In one patient (09-04), only 250 CD3+ IFN-γ+ T-cells/kg were infused and presented a good efficacy of ADV load clearance and ADV-specific immune reconstitution. Similarly, some authors have also reported that a low dose of infused ADV-VST was able to induce ADV load clearance and in vivo ADV-VST expansion [101, 105, 124]. Feucht et al reported that 14 of 21 evaluable patients achieved clearance of ADV load associated with in vivo ADV-VST expansion after infusion at mean T-cell dose of 4.1×10 3 /kg (range from 0.3 to 24×10 3 T-cells/kg). A dose as low as 312 ADV-specific CD3+ T-cells/kg showed effective in vivo expansion with clearance of ADV viremia [113]. Feuchtinger et al also stated that success of adoptive T-cell transfer was not related to the T-cell dose in CMV-VST infusion. Even a very low dose of 360 CD3+IFN+/kg was shown to be sufficient for a successful T-cell transfer [119]. Taking into account all these concordant results, a minimal VST dose may not be requested to observe an anti-viral effect. A low dose of VST is still able to successfully clear ADV load with in vivo ADV-VST expansion. ADV-VST sub-populations If the dose of infused cells is not critical for expansion, the composition of those cells may play an important role. We analyzed the distribution of the main T-cell subsets in ADV-VSTs: TN, TSCM, TCM, TEM and TEFF. All of the different T-cell subsets were observed in the blood sample from healthy donors' ADV-VSTs, both before and after IFN-γ based immunomagnetic selection. As the IFN-γ based immunomagnetic selection system sorts mainly the most differentiated T-cell subsets which secrete IFN with a high intensity, we observed that TEM obviously enriched and were always the major T-cell subset of ADV-specific T cells after immunomagnetic isolation. Although TSCM subset was weakly selected by the IFN-γ based immunomagnetic selection system, it was present in ADV-specific T cells after immunomagnetic isolation. In our clinical study, we analyzed different sub-populations in ADV-VST from four healthy donors. Surprisingly, we observed that the patient 12-11, the only one who died of uncontrolled ADV disease after ADV-VST infusion, received ADV-VST which contained the lowest proportion of immature memory T-cell subsets compared to the other 3 patients (TSCM: 0/kg vs 49 ± 42/kg; TCM: 56/kg vs 145 ± 69/kg). However the infused dose of mature memory T-cell subsets was the highest in patient 12-11 compared with the other 3 patients (TEM: 8.97 x10 3 /kg vs 3.3±3.36 x10 3 /kg, range from 0.19 x10 3 /kg to 8.34 x10 3 /kg).ADV viral load of this patient slightly increased from day 0 to 14 after ADV-VST infusion, while ADV specific immune reconstitution was detected at day 14. However ADV viral load dramatically increased from day 14 to 30 until death due to uncontrolled ADV disease. As previously reported, cytotoxicity is mainly presented by mature T cell sub-populations, while self-renewal and proliferative abilities are mainly observed in immature subsets[137, 138]. It was previously reported that the presence of a relevant pool of TSCM cells might be essential for the control of persisting infections, in which effector T cells undergoing functional exhaustion and replicative senescence need to be replenished continuously by less differentiated T cell subsets[127][128][129][130]. According to this hypothesis, we proposed an explanation for the failure of ADV-VST in controlling ADV viral load between day 14 and 30: the mature sub-populations (TEM and TEFF) played an important role in immediate ADV immune reconstitution and cytotoxicity which contributed to stabilize ADV viral load from day 0 to day 14. Nevertheless, those subsets are not able to expand a lot and die rapidly after cytotoxicity. As TSCM and TCM sub-populations were not or only weakly represented, they were not able to expand and differentiate into TEM and TEFF to continue the cytotoxic effect. In addition, in patient 12-11, the viral load was too high at the time of ADV-VST infusion. As the immature sub-populations had too little time to expand in order to continuously support the ADV specific immune reconstitution, it may have led to the dramatic increase of ADV viral load and the death of the patient.We consider that the mature sub-populations of ADV-VST support the immediate cytotoxicity, while the immature sub-populations play a crucial role to sustain ADV-VST expansion in vivo and continuous anti-ADV efficacy until patient presents own immune reconstitution from the graft.

  antigen locus (A, B, or DR). Leen et al also reported the infusion of third party donors' VSTs. They observed efficacy of VSTs even when only a single HLA allele matched with the recipient, provided that this allele could present relevant virus derived antigens[102]. However, they also described that although these VSTs with such limited HLA matches were able to survive and function in vivo, the immune activity increased far less than that observed in patients who had received VSTs derived from their own (HLA-matched) stem cell donor. More recently, Feucht et al confirmed that T-cell responses observed after infusion of ADV-VSTs were independent of the HLA disparities between donor and recipient.Responses could be observed in 56% of patients with HLA-matched donors (5 of 9 evaluable patients) and in 80% of patients with haploidentical or mismatched donors (16 of 20 patients)[113]. However, no information was given about persistence of ADV-VSTs.In our clinical study, we observed that 5 of 6 post-UCB transplant patients who received ADV-VST from a haploidentical third party donor with a limited HLA matches presented a sustained immune reconstitution at least at day 60 and even at day 90 for most of them. We did not see any difference of intensity and persistence of ADV-specific immune and ADV viral load clearance between the patients who received ADV-VST from HSCT original donors and haploidentical third party donors. The only patient who did not present a sustained efficacy of ADV-VST (patient 12-11) received ADV-VST semi-compatible to the graft. Thus, a lack of viral antigen presentation due to HLA incompatibility cannot be incriminated.

  infusion after HSCT from 1995 to 2017(Qian et al, 2017, under submission), virus specific T cell presented a good anti-virus efficacy although almost all the patients received ≥1 immunosuppressive drug including ATG/ alemtuzumab as prophylaxis or treatment of GVDH at the time of infusion. Two authors compared anti-virus efficacy of ADV-VST in the patients who received immunosuppressive drugs or not[START_REF] Doubrovina | Adoptive immunotherapy with unselected or EBV-specific T cells for biopsy-proven EBV+ lymphomas after allogeneic hematopoietic cell transplantation[END_REF] 113]. They both declared that anti-virus efficacy of ADV-VSTs was not significantly different between the two groups. Similarly, in our clinical study, maintaining immunosuppression (ciclosporin-A or MMF possibly combined with corticosteroid) after ADV-VST infusion did not impair either in vivo expansion of ADV-VST or viral clearance. Feuchtinger's group [113] considered that treatment with ATG/alemtuzumab decreased efficacy of ADV-VST. But they declared that use of ATG/alemtuzumab should not be an absolute contraindication for T-cell therapy since 2 patients achieved successful response to ADV-VST despite the use of ATG/alemtuzumab. Unlike with the observation of Feuchtinger's group [113], ten out of eleven patients received ATG as part of the conditioning regimen with a median time between HSCT and ADV-VST infusion of 117 days (range 23 to 264 days) in our study. Nine patients presented in vivo ADV-VST expansion and clearance of ADV viral load. We suggest that residual in vivo activity of ATG no longer influenced the in vivo expansion of ADV-VST at the time of ADV-VST infusion. However, agreeing with the suggestion of Feuchtinger's group, we consider that residual in vivo activity of these T-cell-depleting antibodies should be detected before VST infusion in future trials according to ATG life-span to confirm no residual ATG in patient serum at the time of VST infusion.GVHD after VST infusionAlthough allo-reactive T cells still persist in the infused VSTs, the purity of VST has improved encouragingly after enrichment by ex vivo expansion or immunomagnetic isolation. Moreover, many patients received a very low dose of VSTs which were mainly generated by immunomagnetic isolation. Such low doses certainly minimize the risk of allo-reactive GVHD after VST infusion. Indeed, many clinical trials have reported low incidence of GVHD after adoptive VST infusion which demonstrated a good tolerance and safety in clinical application. In our study, de novo GVHD was not observed in the first month following ADV-VST infusion. Three of eleven patients (27%) presented GVHD reactivations. One of them presented a grade I acute GVHD which did not require any increase in systemic immunosuppressive drug doses. While the other two patients presented extensive chronic GVHD or grade III acute GVHD at day 7 and day 14 respectively, they stopped immunosuppressive drugs 1 month before or at the day of ADV-VST infusion. Regarding the low dose of infused T cells, we supposed that GVHD reactivations were more likely caused by modulation of immunosuppression in our study. However, it is difficult to discriminate the cases of GVHD between the accountability of ADV-VST themselves and modulation of immunosuppressive drugs. A randomized, controlled study in a large cohort of patients comparing antiviral treatment alone to antiviral treatment combined with ADV-VST will be needed in future studies.In the hypothesis where ADV-VSTs were responsible for GVHD reactivation, maintaining immunosuppressive treatment would be a good way to prevent GVHD reactivation. However, as we observed in our clinical trial, usually immunosuppression is decreased either to allow specific immune reconstitution waiting for ADV-VSTs infusion or to protect ADV-VSTs at the time of infusion. But modulation of immunosuppressive treatment will secondarily increase the risk of GVHD reactivation. In our study, as mentioned above, two patients who stopped immunosuppressive drugs 1 month before or at the day of ADV-VST infusion presented extensive chronic GVHD or grade III acute GVHD at day 7 and day 14 respectively.In conclusion, unlike with the observations of in vitro studies of VSTs with immunosuppressive drugs, it seems that immunosuppressive treatment (excluding T-cell-depleting antibodies) does not impair the anti-virus in vivo efficacy of VST. We suggest that immunosuppressive treatment for GVHD should not be reduced or even discontinued during VST infusion, while residual in vivo activity of T-cell-depleting antibodies should be detected before VST infusion according to their in vivo life-span. Regarding the in vitro impact of immunosuppressive drugs on VSTs, we suggest that post-HSCT patients who develop viremia or viral disease should be infused with VSTs as early as possible, especially for the patients who receive a very low dose of VSTs. Since immunosuppressive drugs mainly impact immature sub-populations, infused VSTs which may present an impaired persistence require more time to in vivo expansion under immunosuppression, potentially requiring a second infusion of VSTs. Nevertheless, this strategy could allow controlling viral infections. However, the results presented with corticosteroids have to be confirmed with other immunosuppressive drugs which often used for GVHD treatment such as CsA, MMF. treatment adoptive transfer of multivirus-specific T cells to clinical routine by performing a multi-national clinical trial to prove efficacy of this life-saving treatment in immunocompromised patients. TRACE will be a blinded placebo-controlled randomized trial to demonstrate efficacy and to monitor acute GVHD and other safety parameters. Around 174 patients are expected to be recruited over 40 months. A new standardized device, CliniMACS Prodigy, has been developed by Miltenyi Biotec, allowing a wide diffusion of the immunomagnetic isolation process according to GMP compliant use. The safety and efficacy of VST transfer will be further better clarified in the future. As mentioned in this document, efficacy of VSTs isolated by immunomagnetic technique relies on their in vivo expansion. In the multicenter clinical trial we performed, we observed that this expansion required around 14 days to be detectable. This delay means that VSTs have to be infused as soon as possible after viral infection diagnosis to let time to VST to expand in vivo and have an anti-viral effect. Following the example of what has recently been published in ECIL guidelines for EBV-VSTs [51] (Table 3-5), prophylactic, pre-emptive or curative use as a first line treatment for viral disease could be considered for the other viruses frequently encountered after HSCT. Moreover, as immunosuppressive drugs do not seem to completely inhibit VST effect, prophylactic or first line curative immunosuppressive treatment for of those therapies will be economically profitable. For example, BK virus (BKV) reactivation and disease can occur in immunosuppressed individuals, such as those who have undergone renal transplantation or HSCT. BKV reactivation after HSCT is frequent and has been associated with severe hemorrhagic cystitis (HC), requiring prolonged hospitalization, altering quality of life and potentially leading to renal failure. Reported cumulative incidence of HC varies from 10 to 70% at 1 year after HSCT [153]. Several risk factors have been identified for the development of BKV disease including myeloablative conditioning, acute graft-versus-host disease, and undergoing HSCT from alternative donor (haplo-identical or umbilical cord blood). There is currently no specific or efficient treatment of severe HC. Responses to cidofovir are unconstant and the drug is difficult to manage because of its renal toxicity. Immune deficiency is a major contributor to the development of BKV-related disease. A proof of concept of immunomagnetic isolation of BKV-ST with a successful clinical benefit has been published recently [126]. We hope that this innovative therapeutic strategy for BKV infection could be transfer in the context of renal transplantation. Indeed, BKV reactivation is a concern in this context, as it results in the development of nephropathy leading to kidney allograft failure in up to 10% of renal transplant recipients. Sharing experience between those two fields of transplantation will help spread this treatment.

Table 1 :

 1 Consensus acute graft-versus-host disease (GVHD) staging and grading with proposed revisions

Table 2 :

 2 Other viral pathogens post HSCT

Table 3 :

 3 Recommendations for prophylaxis against EBV disease

Table 4 :

 4 Recommendations for preemptive therapy of EBV disease

Table 2

 2 Other viral pathogens post-HSCT

		Incidence
	Virus	post HSCT (%)	Traitements	References
			Gancyclovir,
			Acyclovir,
	HHV6	48-72	Cidofovir,	[39]
			Acyclovir,
			Valacyclovir,
	VZV (HHV3)	34.7	Famcyclovir	[40]
	HSV1		Acyclovir,	[41]
		70-80
	HSV2		Valacyclovir	[42]
	BKV	3.6-20	Cidofovir	[43]
	Metapneumovirus (MPV)	2.5	/	[44]
	Respiratory syncytial virus (RSV)	11.6	Ribavirin	[44]
	Human Parainfluenzae Virus (HPIV)	7.1	/	[45]
			oseltamivir,
	Influenza	1.3-44	Inhalation of zanamivir	[46]
	Rhinoviruses and Enteroviruses	3-32	/	[47, 48]
	Hepatitis E virus (HEV)	2.4	/	[49]
	Coronaviruses	8.8	/	[50]
	Although anti-viral drugs are used as prophylaxis or treatment of
	post-transplant viral infections, efficacy can be limited when there is no
	concomitant antiviral immune reconstitution. Moreover, the side effects
	of antiviral drugs are often observed such as tissue toxicity and
	myelosuppression. Adoptive transfer of virus-specific T-cells is becoming

Table 1

 1

	). For

infusion and 6 patients developed CMV reactivation but not the other relative viral infections after infusion during 12 months follow-up.

Table 1 Ex vivo expanded VSTs VST: viral specific T cell; CMV: Cytomegalovirus; EBV: Epstein Barr Virus; ADV: Adenovirus; HHV6: human herpesvirus 6; BKV: BK virus; VZV: varicella zoster virus; GVHD: Graft versus Host disease; PTLD: Post-Transplant Lymphoproliferative disease; CR: Complete remission; PR: Partial remission; HLA: Human Leukocyte Antigen; IFNγ: Interferon-gamma

 1

	VST target	Patients included	Clinical outcome	GVHD post infusion	References
	CMV-VST	9 patients with CMV infection refractory to antiviral	8/9 patients: CMV load clearance	2/9 patients: GVHD I-II	Cobbold et al, 2005
	(HLA-peptide	drugs	1/9 patients: CMV load decrease
	multimers)
	CMV-VST	2 patients with CMV infection refractory to antiviral	2/2 patients: CMV viral load clearance	No de novo GVHD	Schmitt et al, 2011
	(HLA-peptide	drugs
	multimers)
	CMV-VST	6 patients with CMV infection refractory to antiviral	4/6 patients: CMV load clearance	No de novo GVHD	Uhlin et al, 2012
	(HLA-peptide	drugs	1/6 patient: CMV load decrease
	multimers)
	CMV-VST	16 patients with CMV infection refractory to	11/15 evaluable patients: CR or PR on viral load	2/15 evaluable patients: GVHD (aGVHD II-	Neuenhahn et al, 2017
	(HLA-peptide	antiviral drugs (8 CMV-VST from CMV	7/7 patients received CMV-VST from CMV	III: n=1; cGHVD: n=1)
	multimers)	seropositive donors and 8 CMV-VST from	seropositive donors: complete or partial viral load
		CMVseronegative donors)	response
			4/8 patients received CMV-VST from CMV
			seronegative donors: complete viral load response
	CMV-VST	18 patients with CMV infection refractory to	12 /16 evaluable patients: CMV load clearance (n=9)	No de novo GVHD	Feuchtinger et al, 2010
	(IFNγ-capture system)	antiviral drugs	or significant decrease (n=3)
	CMV-VST	11 patients: preemptive treatment	11/11 patients for preemptive treatment: In vivo	5/18 patietnts: GVHD I	Peggs et al, 2011
	(IFNγ-capture system)	7 patients: prophylaxis	expansion of CMV-reactive T cells (2 patients:	2/18 patient: GVHD II
			absence of antiviral drugs; 9 patients: reduction of	1/18 patient: GVHD III
			antiviral drugs)
			7/7 patients for prophylaxis: no CMV disease,
			absence of antiviral drugs
	CMV VST	10 patient with CMV diseases refractory to antiviral	3/9 evaluable patietns: viral load clearance;	1/9 evaluable patient: GVHD III	Creidy et al, 2016
	(IFNγ-capture system)	drugs (3 patients had ADV infections)	stabilization of CMV diseases
	EBV-VST	1 patient: life-threatening EBV-PTLD refractory to	EBV-PTLD CR	No GVHD	Uhlin et al, 2010
	(HLA-peptide	rituximab
	multimers)
	EBV-VST
	(IFNγ-capture system)

Table 2 Immunomagnetic isolated VSTs Part III: Effect of Immunosuppressive drugs in the context of GVHD on Virus Specific T cells

 2

	As previously discussed, adoptive anti-viral cellular immunotherapy
	by infusion of VSTs seems a promising alternative treatment of
	post-HSCT viral infections, presenting efficacy with absence or a low
	associated toxicity. However, VST infusion is often performed during an
	immunosuppressive treatment course, such as corticosteroids, tacrolimus
	(FK506), cyclosporin A (CsA), mycophenolate mofetil (MMF),

Methotrexate and antithymocyte globulin (ATG) which are used to prevent or treat GVHD. We were interested in reviewing the different in vitro or in vivo studies in order to identify if a deleterious effect of immunosuppressive drugs on T cells or VSTs has been demonstrated or, at least, reported.

Acknowledgements

I would like to express my gratitude to all those who helped me during the writing of this thesis.

Methods

Statistical tests were performed using GraphPad Prism 5 software (San Diego, CA, USA). All the data in three groups were analyzed using Friedman test. Dunn's Multiple Comparison Test was used as post-test to compare difference between each group. Statistical significance was fixed a posteriori for a p-value less than 0.05.

Results

ADV-VST Expansion

ADV-VSTs were in vitro expanded either with Methylprednisolone (MP) 1 μg/mL (n=8) or 2 μg/mL (n=7) or without MP (n=8) during 28 ± 10 days. After a 2 to 4 day expansion, the absolute cell number of ADV-VST with MP 1 μg/mL (10.7±6.8×10 6 ) and 2 μg/mL (11.9±6.6×10 6 ) was lower than ADV-VST without MP (14.2±7.3×10 6 ), a significant difference was observed between MP 1μg/mL and without MP group (p<0.05, Dunn's Multiple Comparison Test) (Figure 1a). After a 10 to 19 day expansion, the absolute cell number of ADV-VST with MP 1 μg/mL (33.8±25.3×10 6 ) or 2 μg/ml (34.8±36.0×10 6 ) was also lower than ADV-VST without MP (39.2±33.7×10 6 ), but no significant difference was observed between the three groups (Figure 1b).

Regarding the growth rate of ADV-VST, ADV-VST with MP 1 μg/ml and 2 μg/mL presented slower growth rate than the ADV-VST without MP Kaluza® software (v1.3; Beckman Coulter) was used for analysis of flow cytometry data. The mean number of events acquired in the CD3 IFN-γ gate was 160±133 before isolation and 3345±3214 after isolation.

Results

Production of ADV-VST

Eleven of 13 donors were evaluable for the response to PepTivator-AdV5 Hexon by IFN-γ Elispot assay at the time of immunomagnetic selection (312 ± 188 SFCs/10 6 PBMC, range 31 from 561 SFCs/10 6 PBMC). For UCB transplanted patients, in 5 cases, haploidentical donors could be tested beforehand for ADV-specific IFN-γ response and the best responder was chosen for ADV-VST isolation (447 ± 83

Perspectives

Current antiviral medication is mostly not licenced and does not treat the underlying pathophysiology which is the lack of a specific T-cell immunity. Adoptive transfer of VSTs is a potentially curative, pathogen-specific and non-toxic treatment. As previously reported, VST transfer have already improved patient care. Indeed, although a lot of studies have reported very encouraging results with VSTs as treatment of viral infections or diseases or as prophylaxis with poor adverse events, no efficacy study comparing one arm with conventional anti-viral drug and the other arm conventional anti-viral drug plus VSTs has been published yet. Such a trial is absolutely necessary both for safety and for efficacy evidence. Indeed, comparing adverse event incidence, especially GVHD, between the two arms will help to identify if VST infusion itself promotes GVHD incidence or not. Moreover, frequency of refractory infections after HSCT requires multicentre trials to enrol enough patients. An European Phase III study, TRACE (Transfer of multivirus-specific T cells following transplantation), has been recently built by Tobias Feuchtinger with the support of Miltenyi biotec and with a consortium composed of 6 countries (Germany, Italy, Netherlands, Belgium, United Kingdom and France). A European grant has been sought and decision is pending. In each country, one centre has been chosen for production of VSTs. The overall objective and approach of the TRACE project is to bring the promoting effect on VSTs is not confirmed.

The number of patients concerned by VST infusion is quite low today as this therapy is for HSCT patients who only have a viral infection refractory to conventional anti-viral drugs. However, if infusion is considered at a first line treatment, indications will increase. Moreover, if post-organ-transplant patients with viral infections are also candidate to such infusions, indications will become consistent and the development Annex Article 4