The influence of cyclosporin alone, or cyclosporin and methotrexate, on the incidence of mixed haematopoietic chimaerism following allogeneic sibling bone marrow transplantation for severe aplastic anaemia

We previously reported a randomized trial comparing Cyclosporin-A (CsA) and short-term methotrexate versus CsA alone for graft-versus-host disease (GvHD) prophylaxis in 71 patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) from a human leucocyte antigen-identical sibling for severe aplastic anaemia (SAA). We found a better survival in the group receiving the two-drug prophylaxis regimen with no significant difference in the probability of developing GvHD between the two groups. The present study details chimaeric analysis and its influence on survival and GvHD occurrence in 45 of the original 71 patients in whom serial samples were available. Analysis was carried out in a blinded prospective manner. Seventy-two per cent achieved complete donor chimaerism (DC), 11% stable mixed chimaerism (SMC) and 17% progressive mixed chimaerism (PMC). The overall 5-year survival probability was 82% (+/-11%) with a significant survival advantage (P = 0.0009) in DC or SMC compared to those with PMC. Chronic GvHD was more frequent in DC patients, whereas no patient with SMC developed chronic GvHD. Graft failure occurred in 50% of the PMC group. This study demonstrates the relevance of chimaerism analysis in patients receiving HSCT for SAA and confirms the occurrence of mixed chimaerism in a significant proportion of recipients.

Leukaemic transformation of donor cells in a patient receiving a second allogeneic bone marrow transplant for severe aplastic anaemia

Allogeneic blood or bone marrow transplantation is a successful treatment for leukaemia and severe aplastic anaemia (SAA). Graft rejection following transplantation for leukaemia is a rare event but leukaemic relapse may occur at varying rates, depending upon the stage of leukaemia at which the transplant was undertaken and the type of leukaemia. Relapse is generally assumed to occur in residual host cells, which are refractory to, or escape from the myeloablative conditioning therapy. Rare cases have been described, however, in which the leukaemia recurs in cells of donor origin. Lack of a successful outcome of blood or bone marrow transplantation for severe aplastic anaemia (SAA), however, is due to late graft rejection or graft-versus-host disease. Leukaemia in cells of donor origin has rarely been reported in patients following allogeneic bone marrow transplantation for SAA. This report describes leukaemic transformation in donor cells following a second allogeneic BMT for severe aplastic anaemia. PCR of short tandem repeats in bone marrow aspirates and in colonies derived from BFUE and CFU-GM indicated the donor origin of leukaemia. Donor leukaemia is a rare event following transplantation for severe aplastic anaemia but may represent the persistence or perturbation of a stromal defect in these patients inducing leukaemic change in donor haemopoietic stem cells.

Treatment of relapse after allogeneic bone marrow transplantation with reduced intensity conditioning (FLAG +/- Ida) and second allogeneic stem cell transplant

Acute leukaemias in relapse after allogeneic stem cell transplantation (SCT) respond poorly to donor leucocyte infusions (DLI) compared with chronic myeloid leukaemia (CML), at least in part because of faster disease kinetics. Fludarabine-containing 'non-myeloablative' chemotherapy followed by further allo SCT may offer more rapid and effective disease control. We report 14 patients with relapse after allo SCT for acute leukaemia [seven acute myeloid leukaemia (AML), five acute lymphoblastic leukaemia (ALL)] or refractory anaemia with excess blasts in transformation (RAEB-t, n = 2) treated with fludarabine, high-dose cytosine arabinoside (ara-C) and granulocyte colony-simulating factor (G-CSF) with (n = 10) or without (n = 2) idarubicin (FLAG +/- Ida) or DaunoXome (FLAG-X) (n = 2) and second allo SCT from the original donor. Donors were fully human leucocyte antigen (HLA) -matched in 13 cases with a single class A mismatch in one. Actuarial overall survival was 60% and disease-free survival was 26% at 58 months. Remissions after the second SCT were longer than those after the first bone marrow transplantation (BMT) in eight of the 13 assessable patients to date. Haematopoietic recovery was rapid. Transplants were well tolerated with no treatment-related deaths. The major complication was graft-versus-host disease (GvHD, acute >/= grade II-2 cases, chronic - eight cases, two limited, six extensive) although there have been no deaths attributable to this. FLAG +/- Ida and second allo SCT is a safe and useful approach and may be more effective than DLI in the treatment of acute leukaemias relapsing after conventional allo SCT.

Short Report:Engraftment of T-cell-depleted allogeneic haematopoietic stem cells using a reduced intensity conditioning regimen

Graft-versus-host disease (GVHD) remains a significant complication in patients undergoing allogeneic stem cell transplantation (SCT) using a reduced intensity conditioning regimen. Although T-cell depletion (TCD) reduces the risk of GVHD after a myeloablative conditioning regimen, it is associated with an increased risk of graft failure. We have therefore examined whether TCD compromises engraftment using a fludarabine-based conditioning regimen. Fifteen patients have been transplanted using such a regimen of whom 13 underwent ex vivo TCD. All but one patient demonstrated durable engraftment and no patient receiving a TCD product developed severe GVHD. Thus, TCD may play a role in GvHD prophylaxis using such regimens.

Monitoring of lineage-specific chimaerism allows early prediction of response following donor lymphocyte infusions for relapsed chronic myeloid leukaemia

Donor lymphocyte infusions (DLI) have been shown to enhance the graft-versus-leukaemia (GVL) effect and induce haematological and molecular remission in patients with relapsed CML following allogeneic bone marrow transplantation (BMT). The potent donor cell-mediated cytolysis following DLI may lead to a short period of aplasia before the re-establishment of donor haematopoiesis. The absence of detectable donor cells in patients prior to DLI infusion may result in permanent aplasia in certain patients. We report on four patients who relapsed 1, 3, 6.5 and 7 years post-BMT for chronic phase CML and were treated with DLI from their original BMT donor. Polymorphic short tandem repeats (STRs) were used to assess haematological chimaerism both prior to and following DLI. At the time of relapse, STR-PCR indicated the presence of donor cells in all four patients, at levels ranging from 1-40%. A clinical and molecular response was seen in 4/4 patients following a short period of cytopenia and all patients remain in clinical remission with a follow-up of 2 months-3 years post-DLI. STR-PCR indicated that a response was occurring during the period of pancytopenia when metaphase analysis was unsuccessful. Lineage-specific analysis of the cellular response to DLI was monitored using STR-PCR of peripheral blood (PB) and bone marrow (BM) lymphocyte-enriched fractions and CD2-positive and -negative T cell fractions. In one patient BM and PB CD34-positive and -negative fractions were also assessed. A change in the ratio of donor:recipient cells in the PB lymphocyte fraction was the earliest molecular indication of an anti-leukaemic response. Subsequent conversion to donor chimaerism occurred in the other lineages and the granulocyte fraction was the last lineage to convert. In conclusion, lineage-specific STR-PCR permits detailed monitoring of subtle changes in donor/recipient cell dynamics in specific lineages following DLI during the crucial pancytopenic phase and may be a useful predictor of haematological response to DLI therapy.

Effect of ultraviolet-B light on lymphocyte activity at doses at which normal bone marrow stem cells are preserved

Ultraviolet B (UVB) light is known to be immunosuppressive, but, probably because of a small UVC component in the emission spectra of some of the UVB lamps used, reports vary on effective dose levels. To prevent potentially lethal graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation, alloreactive donor T-cell activity must be suppressed. In this study, a narrow wavelength UVB lamp (TL01, 312 nm peak emission) was used to determine what doses of UVB were required to abolish rat lymphocyte proliferation while simultaneously preserving rat bone marrow progenitor cell and primitive hematopoietic stem cell viability. Lymphocyte proliferation, as measured by 3H-Thymidine incorporation, in response to lectin stimulation was abolished below detection at doses greater than 3,500 J/m2. When T-cell clonogenicity was measured in a limiting dilution assay, a small fraction (0.6%) was maintained at doses up to 4,000 J/m2. Cytotoxic T-lymphocyte (CTL) activity was reduced after treatment with 4,000 J/m2, but a significant level of cytotoxicity was still maintained. Natural killer cell cytolytic activity was not affected by doses up to 4,000 J/m2. At 4,000 J+m2 there was a 10% survival of colony-forming units-granulocyte-macrophage; a 1% and 4% survival of day-8 and day-12 colony-forming units-spleen, respectively; and 11% survival of marrow repopulating ability cells. Up to 25% of late cobblestone area forming cells (4 to 5 weeks), reflecting the more immature hematopoietic stem cells, were preserved in bone marrow treated with 4,000 J/m2, indicating that early stem cells are less sensitive to UVB damage than are more committed progenitor cells. Thus, a potential therapeutic window was established at approximately 4,000 J/m2 using this light source, whereby the potentially GVHD-inducing T cells were suppressed, but a sufficient proportion of the cells responsible for engraftment was maintained.

PCR amplification of short tandem repeat sequences allows serial studies of chimaerism/engraftment following BMT in rodents

Animal models of bone marrow transplantation (BMT) allow evaluation of new experimental treatment strategies. One potential strategy involves the treatment of donor marrow with ultra-violet B light to allow transplantation across histocompatibility boundaries without an increase in graft rejection or graft-versus-host disease. A major requirement for a new experimental protocol, particularly if it involves manipulation of the donor marrow, is that the manipulated marrow gives rise to long-term multilineage engraftment. DNA based methodologies are now routinely used by many centres to evaluate engraftment and degree of chimaerism post-BMT in humans. We report the adaptation of this methodology to the serial study of engraftment in rodents. Conditions have been defined which allow analysis of serial tail vein samples using PCR of short tandem repeat sequences (STR-PCR). These markers have been used to evaluate the contribution of ultraviolet B treated marrow to engraftment following BMT in rodents without compromising the health of the animals under study. Chimaerism data from sequential tail vein samples and bone marrow from selected sacrificed animals showed excellent correlation, thus confirming the validity of this approach in analysing haemopoietic tissue. Thus the use of this assay may facilitate experimental studies in animal BMT.

Polymerase chain reaction based methods for assessing chimerism following allogeneic bone marrow transplantation

It is important to be able to assess the contribution of donor cells to the graft followmg bone marrow transplantation (BMT), as complete engraftment of marrow progenitors that can give rise to long term donor derived hemopoiesis may be important in long-term disease-free survival. The contribution of the donor marrow, both in terms of filling the marrow "space" created by the intense conditioning regimen and in its ability to mediate a graft versus leukemia effect may be assessed by studying the kinetics of the engraftment process. As BMT involves repopulation of the host hemopoietic system with donor cells, recipients of allogeneic marrow are referred to as hemopoietic chimeras. A donor chimera is an individual who exhibits complete donor hemopoiesis and we would imagine that donor chimertsm carries the best long-term prognosis. A patient who has both donor and recipient cells coexistmg in a stable fashion post-BMT without hematological evidence of relapse or graft rejection is referred to as a mixed chimera. Mixed chimerism may be a prelude to graft rejection or leukemic relapse; therefore, it is important to be able to monitor the presence of these cells in a precise manner.

A rapid dot-blot assay to assess chimerism following sex-mismatched bone marrow transplantation

Mixed chimerism may occur more frequently than previously thought following allogeneic bone marrow transplantation and may have implications in terms of relapse, graft-versus-host disease and immune reconstitution. DNA analysis using single or multilocus polymorphic probes cannot reliably discriminate between donor and recipient cells below a level of 10%. We used probe pHY2.1, a cloned segment of tandemly repeated DNA (2000 copies) on the long arm of chromosome Y. A dot blot procedure allowed us to immobilize DNA directly from 50 microliter of peripheral blood or bone marrow. Cross-reactivity was eliminated by hybridization at conditions of extreme stringency (65 degrees C, 50% formamide). Mixing experiments detected male DNA at a level of 0.1% after 10 h exposure. Five patients were studied serially post-bone marrow transplantation. One patient showed mixed chimerism for 12 months, one had complete autologous recovery and the remaining three showed complete engraftment. All results were verified by standard karyotyping on bone marrow cells. This technique is a simple, rapid and sensitive assay for chimerism following sex mismatched bone marrow transplantation.

role of SLO in aGVHD : alterando o paradigma de allo-activação das células T, The

A indução da doença do transplante contra o hospedeiro (GVHD) depende da activação das células dadoras T pelas células do hospedeiro que apresentamantigenio (APCs). A teoria prevalecente descreve que estas interacções ocorrem nos órgãos linfáticos secundários (SLO), tais como os nóduloslinfáticos (LN), as placas de Peyer’s (PP) e o baço (SP). Esta hipótese foi testada usando ratinhos homozigóticos aly/aly (alinfoplasia) que não têm LN nem PP, usando como controlo os ratinhos heterozigóticos (aly/+) da mesma ninhada. Os dois grupos foram irradiados com dose letal após a remoção do baço aos ratinhos aly/aly (LN/PP/SP-/-), enquanto nos ratinhos aly/+ o baço foi deslocado e recolocado. Ambos receberam transplante de medula óssea (BMT) de ratinhos dadores singénicos (aly/aly, H-2b) ou de ratinhos alogénicos, com diferente complexo principal de histocompatibilidade (MHC) (BALB/c, H-2dou B10.BR, H-2k). A severidade de GVHD foi medida pela sobrevivência,e pelo sistema de pontuação, bem estabelecido, quer de doença clínica quer de doença dos órgãos alvo. Surpreendentemente, todos os ratinhos LN/PP/SP-/-sobreviveram, desenvolvendo GVHD clinicamente significativo, comparável,em severidade, com o observado nos ratinhos LN/PP/SP+/+. Além disso, asanálises histopatológicas demonstraram que os ratinhos LN/PP/SP-/-receptores de BMTdesenvolveram significativamente mais GVHD no fígado,no intestino, e na pele quando comparados com os animais singénicos decontrolo. Os ratinhos LN/PP/SP-/-desenvolveram também GVHD hepático mais severo quando comparados com os ratinhos de controlo LN/PP/SP+/+. Diferenças semelhantes foram ainda observadas, logo ao 7º dia, para o GVHDhepático entre os grupos alogénicos. Para identificar quais os órgãos extra-linfáticos do receptor que poderão servir como sítios iniciais de exposição a antigenios alogénicos, na ausência de SLO, foi examinada a expansão das células T (CD3+), a sua activação (CD69+), e a sua proliferação (CFSE) na medula óssea, 3 dias depois do BMT. Em cada caso, os ratinhos LN/PP/SP-/-transplantados com medula de dadores alogénicos apresentaram númerosabsolutos significativamente maiores quer de células, quer de divisõescelulares, se comparados com os LN/PP/SP+/+. Para garantir que as diferenças experimentais observadas nos animais aly/aly, no sistema díspar do MHC, não são apenas um fenómeno dependente da estirpe de ratinho, foramtransplantados ratinhos sem baço FucT dko (LN/PP/SP-/-), previamente tratados com o anticorpo monoclonal (mAb) anti-MadCAM-1. Após o BMT estes ratinhos apresentaram elevada pontuação clínica de GVHD, mostrando que os SLO não são necessários para a indução de GVHD. Em estudos de transplante-versus-leucemia usando hospedeiros homozigóticos (LN/PP/SP-/-) estes ratinhos morreram devido a expansão tumoral e não devido a GVHD.Estudos in vitro mostraram que a capacidade das APCs, quer das célulasdendríticas (DCs) esplénicas, quer das DCs derivadas da medula óssea, dosratinhos aly/aly e aly/+ eramcomparável. Colectivamente, estes resultados são consistentes com a noção de que os SLO não são necessários para a activação alogénica das celulas T, sugerindo que a medula óssea pode ser umlocal alternativo, embora menos eficiente, para o reconhecimento alogénico deantígenos e consequente activação das células dadoras T. Estas observações desafiam o paradigma de que os tecidos linfáticos secundários sãonecessários para a indução de GVHD.

From T cell “exhaustion” to anti-cancer immunity

The immune system has the potential to protect from malignant diseases for extended periods of time. Unfortunately, spontaneous immune responses are often inefficient. Significant effort is required to develop reliable, broadly applicable immunotherapies for cancer patients. A major innovation was transplantation with hematopoietic stem cells from genetically distinct donors for patients with hematologic malignancies. In this setting, donor T cells induce long-term remission by keeping cancer cells in check through powerful allogeneic graft-versus-leukemia effects. More recently, a long awaited breakthrough for patients with solid tissue cancers was achieved, by means of therapeutic blockade of T cell inhibitory receptors. In untreated cancer patients, T cells are dysfunctional and remain in a state of T cell "exhaustion". Nonetheless, they often retain a high potential for successful defense against cancer, indicating that many T cells are not entirely and irreversibly exhausted but can be mobilized to become highly functional. Novel antibody therapies that block inhibitory receptors can lead to strong activation of anti-tumor T cells, mediating clinically significant anti-cancer immunity for many years. Here we review these new treatments and the current knowledge on tumor antigen-specific T cells.

L'étude du rôle immunomodulateur des IVIG dans un modèle murin humanisé de réaction du greffon contre l'hôte

La maladie du greffon contre l’hôte (GvHD) est une complication majeure des greffes de cellules souches hématopoïétiques (HSCT) qui survient dans 30 à 70% des cas et peut causer la mort, malgré un traitement prophylactique bien conduit. Il existe donc une réelle demande clinique pour améliorer ces traitements prophylactiques. Parce que ces traitements prophylactiques reposent en général sur des agents immunosuppresseurs, ceux-ci contribuent à diminuer la reconstitution immunitaire du patient, ce qui a un impact défavorable sur les infections et les taux de rechute d’hémopathie maligne, et donc limite leur utilisation. Les immunoglobulines (IVIG) pourraient représenter une alternative intéressante puisqu’elles ont des propriétés immunomodulatrices et qu’elles sont de plus couramment utilisées en clinique pour traiter des patients ayant un déficit immunitaire. Leur capacité à réduire l’apparition et la sévérité de la GvHD, sans toutefois inhiber ou nuire à la reconstitution immunitaire chez le patient n’a néanmoins jamais été clairement démontrée. Les objectifs de ce projet sont donc d’évaluer l’efficacité des IVIG à réduire l’incidence et la sévérité de la GvHD dans un modèle murin humanisé de GvHD, ainsi que de déterminer le mécanisme d’action des IVIG. Ce modèle consiste à injecter des huPBMCs à des souris immunodéprimées ne pouvant les rejeter. Les résultats obtenus suggèrent que les IVIG possèdent un effet immunomodulateur permettant de réduire les signes cliniques et de retarder l’apparition de la GvHD, tout en permettant l’apparition de cellules NK. Les IVIG agiraient de façon indirecte sur les huPBMCs afin d’induire l’apparition des cellules NK.

Modulation des réactions alloimmunitaires par les cytokines maîtresses IFN-γ et TGF-β

L’injection de cellules immunologiquement compétentes à un hôte histo-incompatible amène une réaction qui peut se traduire par la maladie du greffon-contre-l’hôte (GVHD). La GVHD demeure une barrière importante à une utilisation plus répandue de la greffe allogénique de cellules hématopoïétiques (AHCT), pourtant un traitement efficace pour traiter de nombreuses maladies. Une meilleure compréhension des mécanismes qui sous-tendent cette pathologie pourrait en faciliter le traitement et la prévention. L’Interféron-gamma (IFN-γ) et le Transforming Growth Factor-béta (TGF-β) sont deux cytokines maîtresses de l’immunité impliquées dans la fonction et l’homéostasie des cellules greffées. Nous démontrons chez la souris que l’IFN-γ limite la reconstitution lympho-hématopoïétique de façon dose-dépendante en mobilisant des mécanismes d’apoptose et en inhibant la prolifération cellulaire. Le TGF-β est quant à lui généralement connu comme un immunosuppresseur qui contrôle l’immunité en utilisant plusieurs voies de signalisation. Le rôle relatif de ces voies en AHCT est inconnu. Nous avons étudié une de ces voies en greffant des cellules provenant de donneurs déficients pour le gène SMAD3 (SMAD3-KO), un médiateur central de la voie canonique du TGF-β, à des souris histo-incompatibles. Bien que l’absence de SMAD3 ne cause aucune maladie chez nos souris donneuses, l’injection de cellules SMAD3-KO amène une GVHD du colon sévère chez le receveur. Cette atteinte est caractérisée par une différenciation Th1 et une infiltration massive de granulocytes témoignant d’un rôle central de SMAD3 dans la physiologie des lymphocytes T CD4 et des cellules myéloïdes. Nous avons focalisé ensuite nos efforts sur le rôle de SMAD3 chez les lymphocytes T CD4 en sachant que SMAD3 était actif chez les lymphocytes T CD4 tolérants. Nous avons découvert que SMAD3 était rapidement inactivé après une activation des cellules T, suggérant que l’inactivation de SMAD3 était fonctionnellement importante pour briser l’état de tolérance. Des études de micro-puces d’ADNc nous ont montré que SMAD3 contrôlait en effet l’expression de nombreux transcrits de gènes connus comme étant reliés à la tolérance et/ou à des processus biologiques dont les rôles dans le maintien de la tolérance sont plausibles.

Étude du rôle des lymphocytes T chez les receveurs pédiatriques de greffe de sang de cordon ombilical

La transplantation de sang de cordon ombilical (TSCO) est utilisée pour traiter les enfants atteints de maladies hématologiques en l’absence de donneurs apparentés compatibles. Elle est associée avec des risques plus élevés d’échec de greffe et d’infections opportunistes dans les premiers mois qui suivent la transplantation en comparaison avec une greffe de moelle osseuse. Par contre, la TSCO comporte un risque plus faible de maladie du greffon contre l’hôte et une incidence comparable de rechute de leucémie. Ces quatre complications impliquent directement les lymphocytes T. Dans le but de mieux comprendre le schéma particulier des évènements qui suivent la TSCO et d’améliorer le pronostic des patients, nous avons étudié le potentiel fonctionnel, la persistance et la reconstitution antivirale des lymphocytes T au sein d’un groupe d’enfants transplantés de sang de cordon ombilical (SCO). Étant donné que le SCO contient une majorité de lymphocytes T naïfs, nous avons étudié les lymphocytes T spécifiques au HLA-A2:Melan-A26-35 A27L; seul répertoire naïf et abondant caractérisé chez l’homme. Nous avons observé que les lymphocytes T du SCO se différencient en populations effectrices, s’oligoclonalisent, produisent de l’IFN-γ et lysent spécifiquement leur cible suite à la stimulation. Néanmoins, ces cellules produisent moins d’IFN-γ et sont moins bifonctionnelles que leurs homologues issus du sang périphérique d’adultes. Chez les patients, les lymphocytes T du SCO s’épuisent après la TSCO : ils s’oligoclonalisent dramatiquement, sont principalement en différenciation terminale, et une importante fréquence exprime PD-1 (« programmed death-1 ») dans les 3 à 6 premiers mois post-greffe. Très peu de patients sont capables de développer des réponses antivirales durant cette période et la fréquence de lymphocytes T qui expriment PD-1 semble aussi avoir un impact sur le risque subséquent de faire une rechute de leucémie. La deuxième vague de lymphocytes T émergeant à 6 mois post-TSCO mène à une population fonctionnelle et diversifiée. En conclusion, la fonctionnalité des lymphocytes T présents dans les 3 à 6 premiers mois post-TSCO doit être rétablie pour améliorer les risques d’infections opportunistes et de rechute de leucémie.

Étude des fonctions immunomodulatrices des lymphocytes T « Doubles-Négatifs »

La réaction du greffon contre l’hôte (GvH) est responsable d’un grand taux de morbidité et de mortalité chez les patients recevant des greffes de cellules souches (GCSH) allogéniques. Dans ce contexte, les cellules T régulatrices sont largement étudiées et semblent avoir un grand potentiel d’utilisation dans le domaine de la thérapie cellulaire de la GvH. Parmi les populations cellulaires T régulatrices, les lymphocytes T CD4-CD8- TCRαβ+ « Doubles-Négatifs » (DN), qui ne représentent que 1-3% des lymphocytes T, ont été décrits. Ces cellules ont des propriétés inhibitrices de la réponse immunitaire qui s’avèrent spécifiques aux antigènes auxquels elles ont préalablement été exposées. La répression de la réponse immunitaire par les cellules T DN régulatrices semble être un mécanisme important impliqué dans l’induction de la tolérance aux allo-antigènes. De plus, ces cellules confèrent une tolérance immunitaire dans des modèles de greffes allogéniques et xénogéniques. En effet, ces cellules ont la capacité d’inhiber la réaction contre un allo-antigène auquel elles ont été exposées, sans inhiber la réaction contre un allo-antigène inconnu. Les cellules T DN ont été isolées et caractérisées chez l’homme où elles ont la capacité d’interagir avec des cellules présentatrices d’antigènes (APCs) par un contact cellulaire, comme chez la souris. Cependant, leur capacité immunomodulatrice reste inconnue chez l’humain. Notre objectif consistait donc principalement à étudier le rôle et le mécanisme d’action des cellules T DN régulatrices humaines in vitro, en étudiant leur capacité à inhiber une réaction lymphocytaire mixte (MLR). Nous avons montré que les cellules T DN stimulées par un allo-antigène donné inhibent des cellules syngéniques effectrices dirigées contre ce même alloantigène mais n’inhibent pas des cellules syngéniques effectrices dirigées contre un autre alloantigène, démontrant ainsi la spécificité aux antigènes de ces cellules. De plus, les T DN non stimulées par un allo-antigène n’ont pas de rôle inhibiteur. Cependant, durant cette inhibition, nous n’observons pas de modulation de l’expression des marqueurs d’activation et d’induction de l’apoptose. Afin d’étudier le mécanisme d’action des cellules T DN, nous avons mesuré l’expression intracellulaire de la granzyme B. Les résultats démontrent que les cellules T DN stimulées expriment un niveau significativement plus élevé de granzyme B que les cellules T DN non-stimulées par l’allo-antigène. Ceci suggère que l’immunosuppression induite par les cellules T DN stimulées pourrait passer par la voie granzyme B. Le mécanisme utilisé par ces cellules reste à être confirmé par nos futures expériences.