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The involvement of γδ TCR-bearing lymphocytes in immunological memory has gained increasing interest due to their functional duality between adaptive and innate immunity. γδ T effector memory (TEM) and central memory (TCM) subsets have been identified, but their respective roles in memory responses are poorly understood. In the present study, we used subsequent mouse cytomegalovirus (MCMV) infections of αβ T cell deficient mice in order to analyze the memory potential of γδ T cells. As for CMV-specific αβ T cells, MCMV induced the accumulation of cytolytic, KLRG1+CX3CR1+ γδ TEM that principally localized in infected organ vasculature. Typifying T cell memory, γδ T cell expansion/proliferation in organs and blood was higher and more efficient after secondary viral challenge than after primary infection. Viral control upon MCMV reinfection involved the T-cell receptor, and was associated with a preferential amplification of private and unfocused TCR δ chain repertoire as evidenced by next generation sequencing. The γδ T cell secondary response to MCMV was composed by a combination of clonotypes expanded post-primary infection and, more unexpectedly, of novel expanded clonotypes. Finally, Long-term-primed γδ TCM cells, but not γδ TEM cells, protected T cell-deficient hosts against MCMV-induced death upon adoptive transfer, probably through their ability to survive and to generate TEM in the recipient host. Overall, our study uncovered memory properties of long-lived TCM γδ T cells that confer protection in a chronic infection, highlighting the interest of this T cell subset in vaccination approaches.AUTHOR SUMMARYCytomegalovirus (CMV) is a widespread, latent virus that can cause severe organ disease in immune-compromised patients. Anti-CMV memory immune responses are essential to control viral reactivation and/or reinfection events that commonly take place in solid organ transplantation. The role of γδ T-cell receptor bearing lymphocytes could be crucial in this context where immunosuppressive/ablative treatments cause suboptimal and/or delayed αβ T cell responses. Here we asked whether γδ T cells could compensate for the absence of αβ T cells in the long-term control of mouse CMV infection. Three months post-primary viral challenge in αβ-T cell deficient mice, γδ T cells displayed similar features as cytolytic, CMV-specific αβ CD8 T cells. We showed that previous priming with CMV endowed γδ T cells with an enhanced antiviral potential and that long-term maintenance of γδ-mediated antiviral protection was dependent on γδ central memory T cells (TCM). The γδ T cell response to a secondary CMV challenge was dependent on γδ TCR-signaling and generated a private TCR δ repertoire as observed in human. Our results sustain the adaptive-like properties of these unconventional T cells and reveal the interest of targeting γδ TCM subset in novel antiviral vaccination approaches.

International audience; Background The reported association of mTOR-inhibitor (mTORi) treatment with a lower incidence of cytomegalovirus (CMV) infection in kidney transplant recipients (KTR) who are CMV seropositive (R+) remains unexplained. Methods The incidence of CMV infection and T-cell profile was compared between KTRs treated with mTORis and mycophenolic acid (MPA), and in vitro mTORi effects on T-cell phenotype and functions were analyzed. Results In KTRs who were R+ and treated with MPA, both αβ and γδ T cells displayed a more dysfunctional phenotype (PD-1+, CD85j+) at day 0 of transplantation in the 16 KTRs with severe CMV infection, as compared with the 17 KTRs without or with spontaneously resolving CMV infection. In patients treated with mTORis ( n =27), the proportion of PD-1+ and CD85j+ αβ and γδ T cells decreased, when compared with patients treated with MPA ( n =44), as did the frequency and severity of CMV infections. mTORi treatment also led to higher proportions of late-differentiated and cytotoxic γδ T cells and IFN γ -producing and cytotoxic αβ T cells. In vitro , mTORis increased proliferation, viability, and CMV-induced IFN γ production of T cells and decreased PD-1 and CD85j expression in T cells, which shifted the T cells to a more efficient EOMES low Hobit high profile. In γδ T cells, the mTORi effect was related to increased TCR signaling. Conclusion Severe CMV replication is associated with a dysfunctional T-cell profile and mTORis improve T-cell fitness along with better control of CMV. A dysfunctional T-cell phenotype could serve as a new biomarker to predict post-transplantation infection and to stratify patients who should benefit from mTORi treatment. Clinical Trial registry name and registration number: Proportion of CMV Seropositive Kidney Transplant Recipients Who Will Develop a CMV Infection When Treated With an Immunosuppressive Regimen Including Everolimus and Reduced Dose of Cyclosporine Versus an Immunosuppressive Regimen With Mycophenolic Acid and Standard Dose of Cyclosporine A (EVERCMV), NCT02328963

γδ T lymphocytes are unconventional immune cells, which have both innate- and adaptive-like features allowing them to respond to a wide spectrum of pathogens. For many years, we and others have reported on the role of these cells in the immune response to human cytomegalovirus (HCMV) in transplant patients, pregnant women, neonates, immunodeficient children, and healthy people. Indeed, and as described for CD8+ T cells, CMV infection leaves a specific imprint on the γδ T cell compartment: (i.) driving a long-lasting expansion of oligoclonal γδ T cells in the blood of seropositive individuals, (ii.) inducing their differentiation into effector/memory cells expressing a TEMRA phenotype, and (iii.) enhancing their antiviral effector functions (i.e. cytotoxicity and IFNγ production). Recently, two studies using murine CMV (MCMV) have corroborated and extended these observations. In particular, they have illustrated the ability of adoptively transferred MCMV-induced γδ T cells to protect immune deficient mice against virus-induced death. In vivo, expansion of γδ T cells is associated with the clearance of CMV infection as well as with reduced cancer occurrence or leukemia relapse risk in kidney transplant patients and allogeneic stem cell recipients, respectively. Taken together, all these studies show that γδ T cells are important immune effectors against CMV and cancer, which are life-threatening diseases affecting transplant recipients. The ability of CMV-induced γδ T cells to act independently of other immune cells opens the door to the development of novel cellular immunotherapies that could be particularly beneficial for immunocompromised transplant recipients.

Human  T cells are usually considered to contribute to fast-acting local immune responses. Their somewhat limited T cell receptor (TCR) diversity implies that large subsets of  T cells share the capacity to respond to the same restricted set of antigens, rather than showing the fine specificity toward extremely diverse antigens, as is characteristic of  T cells. This has been well demonstrated for V 9V 2 T cells, particularly in non-human primate models. However, much less is known about the other subsets of  T cells, herein collectively called V 2 neg  T cells. Most of these cells express the V 1 chain, some express the V 3 chain, and very few express the four remaining V chains (V 4 to V 8). All these V chains can be associated with any of the six V chains (V 2, 3, 4, 5, 8, 9). V 2 neg  T cells are mainly located in epithelial tissues and the spleen, and are barely found in the circulation in normal physiological conditions. This tissue localization has limited their analysis. Establishment of murine models is difficult since murine and human  T cell populations vary greatly. For example, the equivalent of murine dendritic epithelial  T cells (DETC) does not exist in humans, and conversely, the equivalent of human V 9V 2 T cells is present only in primates. Therefore, human V 2 neg  T cells have mostly been examined during pathological situations where their circulating levels are increased. Like V 9V 2 T cells, V 1 and V 3 T cells have been shown to be involved in widely diverse pathological contexts, such as infection, cancer, auto-immunity, and inflammation. This suggests that  T cells respond to a variety of altered microenvironments induced by these situations. It is acknowledged that  T cells can recognize ubiquitous stress-induced conserved antigens in their native form, and altered-self or foreign ligands presented on non-polymorphic molecules in total independence of classical MHC molecules. Since V 2 neg  T cells can recognize broadly distributed antigens and are localized at the interface with the outer environment within epithelial tissues, V 2 neg  T cells can act as a first line of defense in the surveillance of body integrity and microorganism infections. Nevertheless, V 2 neg  T cells can also display effector/memory phenotypes similar to conventional MHC-restricted  T cells. This suggests an ability to mount long-lasting anamnestic immunity similar to conventional  T cells. Here, we will review what is currently known about V 2 neg  T cells highlighting the pathological situations where they expand. We will also discuss what is known concerning the cellular and molecular mechanisms of their activation and their effector functions.

gammadelta T cells recognize stress-induced autoantigens and contribute to immunity against infections and cancer. Our previous study revealed that Vdelta2-negative ((neg)) gammadelta T lymphocytes isolated from transplant recipients infected by cytomegalovirus (CMV) killed both CMV-infected cells and HT29 colon cancer cells in vitro. To investigate the antitumor effects of Vdelta2(neg) clones in vivo, we generated hypodermal HT29 tumors in immunodeficient mice. Concomitant injections of Vdelta2(neg)clones, in contrast to Vdelta2(+) cells, prevented the development of HT29 tumors. Vdelta2(neg) clones expressed chemokine C-C motif receptor 3 (CCR3) and migrated in vitro in response to chemokines secreted by HT29 cells, among which were the CCR3 ligands macrophage inflammatory protein-1delta and monocyte chemoattractant protein-4. More importantly, a systemic i.p. treatment with Vdelta2(neg) clones delayed the growth of HT29 s.c. tumors. The effect of in vivo gammadelta T-cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-CCR3 antibody. gammadelta T-cell passive immunotherapy was dependent on the cytotoxic activity of the gammadelta effectors toward their targets because Vdelta2(neg) clones were not able to inhibit the growth of A431 hypodermal tumors. Our findings suggest that CMV-specific Vdelta2(neg) cells could target in vivo cancer cells, making them an attractive candidate for antitumor immunotherapy.

International audience; Neonatal thymectomy in BALB/c mice has been described as a model of gastric mucosa-associated lymphoid tissue (MALT) lymphoma (GML). By using this experimental system, we screened, for the first time to our knowledge, Helicobacter pylori GML-associated strains for their capacity to promote disease. A cohort of BALB/c mice underwent thymectomy at day 3 after birth (d3Tx). Successful thymic ablation was evaluated by the degree of lymphopenia in blood samples collected at 4 weeks of age. d3Tx and non-thymectomized controls were infected with either GML strains (B38 or B47) or control strains (SS1 or TN2GF4). Gastric samples collected at 6, 12, and 18 months after infection were studied for bacteria content, and submitted to histological, immunochemical, molecular, and immunological analyses. Severe gastric inflammation was only observed in d3Tx mice. In these animals, the gastric lamina propria was infiltrated with lymphoid cells organized in follicles composed of B cells with few infiltrating T cells. PCR of D/J IgH gene segments proved the monoclonality of infiltrating B cells, which strongly correlated with the presence of lymphoepithelial lesions. B-cell infiltrates were particularly prominent in mice infected with the B47-GML strain. No pathological changes were detected in noninfected d3Tx mice. We identified new H. pylori isolates adapted to the mouse stomach with high potential of GML development, which is only revealed in hosts rendered lymphopenic by neonatal thymic ablation.

Natural killer T (NKT) cells are a subset of mature alpha beta TCR(+) cells that co-express NK lineage markers. Whereas most NKT cells express a canonical Valpha14/Vbeta8.2 TCR and are selected by CD1d, a minority of NKT cells express a diverse TCR repertoire and develop independently of CD1d. Little is known about the selection requirements of CD1d-independent NKT cells. We show here that NKT cells develop in RAG-deficient mice expressing an MHC class II-restricted transgenic TCR (Valpha2/Vbeta8.1) but only under conditions that lead to negative selection of conventional T cells. Moreover development of NKT cells in these mice is absolutely dependent upon an intact TCR alpha-chain connecting peptide domain, which is required for positive selection of conventional T cells via recruitment of the ERK signaling pathway. Collectively our data demonstrate that NKT cells can develop as a result of high avidity TCR/MHC class II interactions and suggest that common signaling pathways are involved in the positive selection of CD1d-independent NKT cells and conventional T cells.

The present study describes three subsets of bone marrow-derived Lin CD43+B220(low) (B220(low)) progenitor cells which represent distinct stages in hematopoietic development. These populations differ in their expression of CD24 and ckit and the occurrence of IgH gene rearrangement. B220(low) CD24-ckit(hi) progenitors have their IgH loci in germ-line configuration and are multipotent since they can give rise to B cells, T cells and myeloid cells. B220lowckit(hi) cells which have acquired CD24 expression have retained IgH loci in germ-line configuration and the ability to generate B cells, however, they have lost the ability to generate T cells and myeloid cells. Thus acquisition of CD24 by B220(low) cells occurs concurrently with the transition from a multipotent to a lineage-restricted progenitor population. B220(low)CD24+ cells expressing low levels of ckit are also lineage restricted, giving rise to only B cells and have begun D-J(H) rearrangement, implying that initiation of IgH rearrangement coincides with the down-regulation of ckit expression.

We performed a comprehensive analysis of T cell receptor (TCR) γ rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that TCRγ rearrangements are present in CD44+CD25+Pro-T thymocytes much earlier than expected. TCRγ rearrangements increase significantly from the Pro-T to the CD44−CD25+Pre-T cell transition, and follow different patterns depending on each Vγ gene segment, suggesting that ordered waves of TCRγ rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of TCRγ genes occur concurrently with TCRδ and D-Jβ rearrangements, but before Vβ gene assembly. Productive TCRγ rearrangements do not increase significantly before the Pre-T cell stage and are depleted in CD4+CD8+double-positive cells from normal mice. In contrast, double-positive thymocytes from TCRδ−/− mice display random proportions of TCRγ rearranged alleles, supporting a role for functional TCRγ/δ rearrangements in the γδ divergence process.

International audience; Binding of ligand FasL to its receptor Fas triggers apoptosis via the caspase cascade. FasL itself is homotrimeric, and a productive apoptotic signal requires that FasL be oligomerized beyond the homotrimeric state. We generated a series of FasL chimeras by fusing FasL to domains of the Leukemia Inhibitory Factor receptor gp190 which confer homotypic oligomerization, and analyzed the capacity of these soluble chimeras to trigger cell death. We observed that the most efficient FasL chimera, called pFasL, was also the most polymeric, as it reached the size of a dodecamer. Using a cellular model, we investigated the structure-function relationships of the FasL/Fas interactions for our chimeras, and we demonstrated that the Fas-mediated apoptotic signal did not solely rely on ligand-mediated receptor aggregation, but also required a conformational adaptation of the Fas receptor. When injected into mice, pFasL did not trigger liver injury at a dose which displayed anti-tumor activity in a model of human tumor transplanted to immunodeficient animals, suggesting a potential therapeutic use. Therefore, the optimization of the FasL conformation has to be considered for the development of efficient FasL-derived anti-cancer drugs targeting Fas.

The role of human intraepithelial Vδ1(+) γδ T cell cytotoxic effectors in the immune surveillance against metastatic colon cancer has never been addressed, despite their reported capacity to infiltrate colon carcinomas and to kill colonic cancer cells in vitro. We previously showed that Vδ1(+) γδ T cells are enriched in blood in response to cytomegalovirus (CMV) infection, and that such increase may be protective against epithelial cancers. The objective of the present study was to investigate whether CMV-induced Vδ1(+) γδ T lymphocytes could inhibit the propagation of human colon tumors in vivo, in order to evaluate their immunotherapeutic potential in this context. Even though metastases are an important cause of death in various cancers including colorectal cancer (CRC), the anti-metastatic effect of immune effectors has been poorly analyzed. To this purpose, we set up a reliable model of metastatic colon cancer through orthotopic implantation of luciferase-expressing human HT29 cells in immunodeficient mice. Using bioluminescence imaging to follow the outcome of colonic cancer cells, we showed that a systemic treatment with CMV-induced Vδ1(+) γδ T cells could not only inhibit primary colon tumor growth but also the emergence of secondary tumor foci in the lungs and liver. Finally, our data lead to propose that Vδ1(+) γδ T lymphocytes may directly influence the appearance of metastases independently from their control of primary tumor size. These findings, which extend our previous work, pave the road for the potential manipulation of Vδ1(+) γδ T lymphocytes in novel anti-CRC immunotherapeutic protocols.

Whereas interactions between the TCRalpha beta and self MHC:peptide complexes are clearly required for positive selection of mature CD4(+) and CD8(+) T cells during intrathymic development, the role of self or foreign ligands in maintaining the peripheral T cell repertoire is still controversial. In this report we have utilized keratin 14-beta2-microglobulin (K14-beta2m)-transgenic mice expressing beta2m-associated ligands exclusively on thymic cortical epithelial cells to address the possible influence of TCR:ligand interactions in peripheral CD8(+) T cell homeostasis. Our data indicate that CD8(+) T cells in peripheral lymphoid tissues are present in normal numbers in the absence of self MHC class I:peptide ligands. Surprisingly, however, steady state homeostasis of CD8(+) T cells in the intestinal epithelium is severely affected by the absence of beta2m-associated ligands. Indeed TCRalpha beta(+) IEL subsets expressing CD8alpha beta or CD8alpha alpha are both dramatically reduced in K14-beta2m mice, suggesting that the development, survival or expansion of CD8(+) IEL depends upon interaction of the TCR with MHC class I:peptide or other beta2m-associated ligands elsewhere than on thymic cortical epithelium. Collectively, our data reveal an unexpected difference in the regulation of CD8(+) T cell homeostasis by beta2m-associated ligands in the intestine as compared to peripheral lymphoid organs.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.

The history of T-cell development is highly connected to the physiology of the thymus. Following the pioneering observations of J. F. A. P. Miller on the role of the thymus in T lymphocyte production, many laboratories have studied the events that take place in this gland. As shall be reviewed in this chapter and elsewhere in this book, the events known to take place in the thymus have remained fundamentally unchanged and include: colonization of the thymus by bone-marrow derived precursors; commitment to the T-cell lineage; rearrangement of the TCR genes; selection of T-cells according to their MHC and antigenic specificity, and finally, export from the thymus to the periphery. However, new molecular insights obtained more recently have reaffirmed these events. During the 1980s, the main interest of most T-cell development labs focused on the selection mechanisms that resulted in the T-cell repertoire. This was prompted by the molecular characterization of the T-cell receptor, as well as the availability of reagents (monoclonal antibodies, and so forth), which allowed the demonstration of positive and negative selection. Only a few labs focused instead on the earlier events of T-cell development, before positive and negative selection take place. More recently, this situation has changed with more labs studying the early events of T-cell development in the thymus. We have really only begun to understand the basic events that take place before the CD4+CD8+ (double positive [DP]) stage. This situation means that many basic and important developments await to be discovered and understood in early T-cell development.

The origin and specificity of alphabeta TCR(+) T cells that express CD8alphaalpha have been controversial issues. Here we provide direct evidence that precursors of functional CD8alphaalpha T cells are positively selected in the thymus in the presence of agonist self-peptides. Like conventional positive selection, this agonist selection process requires functional TCR alpha-CPM, whereas it is independent of CD8beta expression. Furthermore, CD8alphaalpha expression on mature, agonist-selected T cells does not imply selection by MHC class I, and CD8alphaalpha(+) T cells can be either class I or class II restricted. Our data define a distinct agonist-dependent, positive selection process in the thymus, and they suggest a function for CD8alphaalpha distinct from the conventional TCR coreceptor function of CD8alphabeta or CD4.

Thymic positive and negative selection of developing T lymphocytes confronts us with a paradox: How can a T-cell antigen receptor (TCR)-major histocompatibility complex (MHC)/peptide interaction in the former process lead to transduction of signals allowing for cell survival and in the latter induce programmed cell death or a hyporesponsive state known as anergy? One of the hypotheses put forward states that the outcome of a TCR-MHC/peptide interaction depends on the cell type presenting the selecting ligand to the developing thymocyte. Here we describe the development and lack of self-tolerance of CD8+ T lymphocytes in transgenic mice expressing MHC class I molecules in the thymus exclusively on cortical epithelial cells. Despite the absence of MHC class I expression on professional antigen-presenting cells, normal numbers of CD8+ cells were observed in the periphery. Upon specific activation, transgenic CD8+ T cells efficiently lysed syngeneic MHC class I+ targets in vitro and in vivo, indicating that thymic cortical epithelium (in contrast to medullary epithelium and antigen-presenting cells of hematopoietic origin) is incapable of tolerance induction. Thus, compartmentalization of the antigen-presenting cells involved in thymic positive selection and tolerance induction can (at least in part) explain the positive/negative selection paradox.

Cytomegalovirus causes serious pathologies in immune-compromised hosts. γδ T cells increase in the peripheral blood of renal transplant recipients concomitantly to a decrease of CMV viral antigenemia, indicating that they participate to the immune response against CMV. Moreover, γδ T cell amplification is associated with a reduced risk of skin cancer in these patients. We chose to use the mouse model of CMV infection to study the capacity of γδ T cells to protect mice against CMVinfection and cancer.We showed that in the absence of αβ T cells in TCRα-/- mice (αβ-γδ+), different γδ T cell subsets are increased in CMV target organs. A concomitant decrease of viral load was observed in TCRα-/- mice which survived CMV infection, in contrast to CD3ε-/- mice which died and displayed damage to the lungs and liver. γδ T cell antiviral protective effect was also observed in the absence of NK, αβ T and B cells, showing the crucial role that these cells could play in immunodeficient contexts where other immune players are compromised.We showed the ability of CMV to inhibit the growth of subcutaneous colonic tumors (MC38) and melanomas (B16F10) in immunodeficient mice, thus revealing an anti-tumor role of CMV independently of immunity and γδ T cells. CMV was able to infect these tumor cell lines in accordance with a direct anti-tumor effect of the virus, through apoptosis (B16F10) or by means of a still unresolved mechanism. Finally, CMV also inhibits the growth of human colonic tumors, leading to the hypothesis that a viral-mediated indirect anti-tumor effect could also operate.; L’infection à cytomégalovirus (CMV) cause des pathologies graves en absence d’immunité. Les lymphocytes T (LT) γδ participent à la réponse anti-CMV puisqu’ils s’amplifient dans le sang de patients transplantés rénaux concomitamment à une diminution de la charge virale. D’autre part, l’amplification T γδ est associée à un risque moindre de cancers cutanés chez ces patients. Nous avons choisi d’utiliser le modèle murin de l’infection à CMV afin d’étudier la capacité des LTγδ à protéger les souris contre l’infection et le cancer.Nous avons montré qu’en absence de LTαβ dans des souris TCRα-/- (αβ-γδ+), différentes sous populations de LTγδ s’amplifient dans les organes cibles du CMV. Le contrôle de la charge virale observé in situ suite à leur amplification protège les souris TCRα-/- des dommages hépatiques/pulmonaires et de la mort, alors que les souris CD3ε-/- (αβ-γδ-) succombent à l’infection. Enfin, l’effet protecteur des LTγδ est également observé en absence de NK, de LTαβ et de LB, montrant l’importance que peuvent avoir ces cellules dans un contexte d’immunodéficience touchant les autres acteurs immunitaires.Nous avons montré la capacité du CMV à inhiber la croissance de tumeurs coliques (MC38) et de mélanomes (B16F10) implantés en sous-cutané dans des souris immunodéficientes, révélant un rôle anti-tumoral du CMV indépendant de l’immunité et des LTγδ. La permissivité au CMV de ces lignées tumorales suggère un effet direct du virus, par apoptose (B16F10) ou par un mécanisme encore indéterminé (MC38). Enfin, une inhibition comparable est observée pour une lignée carcinomateuse humaine, présupposant un effet indirect du virus sur le microenvironnement tumoral.

Cytomegalovirus (CMV), a Beta Herpes virus, is considered as a paradigm for viral evasion.It is an important opportunistic pathogen in immunocompromised patients and a major cause of congenital birth defects when acquired in utero. CMV encodes molecules to prevent antigen presentation to αβ T cells through inhibition of MHC Class I expression and to suppress NK cell functions by mimicking or down-regulating ligands of NK receptors (NKR). These evasion mechanisms are not expected to affect γδ T cells and, as a matter of fact, their response to CMV has been widely reported in many different physiopathological contexts as well as in CMV-seropositive healthy donors (Dechanet et al, 1999)( Scheper, 2013). Our aim was to understand how CMV induce γδ T cell response. We used recombinant adenoviruses expressing each of the four US genes, and a mutant HCMV deleted for these 4 genes (CMV-DUS). We observed an induction of HLA-I expression by the control adenovirus, and an inhibition by US2, US3 and US11. When using CMV-DUS, infected cells expressed much more native HLA-I than CMV-WT infected cells. Interestingly and in sharp contrast to αβ T cells, γδ T cell were activated to produce IFNg when cultured with fibroblasts infected with CMV-WT, but not when fibroblasts were infected with CMV-DUS. These results indicate that HLA-I molecules regulate γδ T cells through mechanisms that are under investigation in our team. The immune escape processes developed by CMV could thus promote γδ over αβ T cell response and explain the important response of γδ T cells to the virus in immunosuppressed individuals.; Le cytomégalovirus (CMV), un β-herpes virus, est considéré comme un modèle d'immuno-évasion virale. Il s'agit d'un agent pathogène opportuniste fréquent chez les patients immunodéprimés et une cause majeure de malformations congénitales lors de l'acquisition in utero. Le CMV code pour des protéines (i) qui empêchent la présentation de l'antigène aux lymphocytes T αβ notamment par l'inhibition de l'expression des molécules HLA-I et (ii) qui suppriment les fonctions des cellules NK en imitant ou en régulant à la baisse les ligands des récepteurs NK (NKR). Ces mécanismes d'évasion ne devraient pas affecter les lymphocytes T γδ dont la reconnaissance antigénique est indépendante du HLA-I, et d’ailleurs leur réponse au CMV a été largement rapportée dans de nombreux contextes physiopathologiques. Notre objectif était de comprendre comment les mécanismes d’immuno-évasion du CMV affectent la réponse des lymphocytes T γδ. Nous avons utilisé des adénovirus recombinants exprimant chacun des quatre gènes du CMV impliqués dans l’inhibition de l’expression du HLA-I, et un mutant du HCMV déficient pour ces 4 gènes (CMV-∆US). Nous avons observé une induction de l'expression de HLA-I par l'adénovirus control, et une inhibition par US2, US3 et US11. Lors de l'utilisation de CMV-∆US, les cellules infectées exprimaient beaucoup plus de HLA-I que les cellules infectées par CMV-WT. De façon intéressante et à l’opposé des lymphocytes T αβ, les lymphocytes T γδ produisent plus d'IFNy en présence de fibroblastes infectés par le CMV-WT, qu’avec des fibroblastes infectés par CMV-∆US. Ces résultats indiquent que les molécules HLA-I régulent les lymphocytes T γδ grâce à des mécanismes qui sont en cours d'investigation dans notre équipe. Les processus d'échappement immunitaire développés par le CMV pourraient ainsi favoriser la réponse des lymphocytes T γδ par rapport à celle des lymphocytes T αβ et expliquer le rôle important des cellules T γδ dans le contrôle du virus chez les individus immunodéprimés. D'autre part, les acides nucléiques et les protéines du HCMV ont été trouvés dans les tissus tumoraux, mais la relation précise entre le HCMV et le cancer reste un sujet de débat. La plupart du temps, HCMV est décrit comme un virus oncomodulateur avec un rôle pro-tumoral. Notre objectif était d'utiliser le modèle de la souris pour tester in vivo l'impact de CMV de souris (MCMV) sur la croissance des cellules tumorales. Nous avons observé que MCMV pourrait inhiber la croissance de tumeurs sous-cutanées de côlon MC38 chez les souris immunodéficientes. Encore plus surprenant lorsque l'on considère la spécificité d’espèce des CMV, l'infection par le MCMV inhibe de la même façon la croissance des cellules cancéreuses du côlon humain HT29, qui n’est pas affectée par le HCMV. In vitro, les protéines MCMV précoces (IE-1) sont détectées dans des cellules cancéreuses humaines et murines après l'infection. Cependant, peu de cellules cancéreuses sont retrouvées positives pour le MCMV dans les tumeurs HT29 prélevées sur des souris infectées par le MCMV. De manière surprenante, le MCMV inhibe la prolifération des cellules cancéreuses de côlon humain contrairement au HCMV. De plus, la transcription de l'interféron β humain est induite après une infection par le MCMV. Cette induction n'a pas été observée après l'infection par le HCMV. En conclusion, nos données suggèrent un potentiel effet anti-tumoral de MCMV sur les cellules cancéreuses du côlon humain (HT29), qui pourrait être au moins partiellement médiée par l'interféron β. Ces résultats ouvrent la voie à l'utilisation potentielle du MCMV en tant que traitement du cancer du côlon humain.