Monitoring of human CD4 T cell responses in metastatic melanoma and arthritic patients

SUMMARY : Detailed knowledge of the different components of the immune system is required for the development of new immunotherapeutic strategies. CD4 T lymphocytes represent a highly heterogeneous group of cells characterized by various profiles of cytokine production and effector vs. regulatory functions. They are central players in orchestrating adaptive immune responses: unbalances between the different subtypes can lead either to aggressive autoimmune disorders or can favour the uncontrolled growth of malignancies. In this study we focused on the characterization of human CD4 T cells in advanced stage melanoma patients as well as in patients affected by various forms of autoimmune inflammatory spondyloarthropathies. In melanoma patients we report that a population of FOXP3 CD4 T cells, known as regulatory T cells, is overrepresented in peripheral blood, and even more in tumor-infitrated lymph nodes as well as at tumor sites, as compared to healthy donors. In tumor-infiltrated lymph nodes, but not in normal lymph nodes or in peripheral blood, FOXP3 CD4 T cells feature a highly differentiated phenotype (CD45RA-CCR7+/-), which suggests for a recent encounter with their cognate antigen. FOXP3 CD4 T cells have been described to be an important component of the several known immune escape mechanisms. We demonstrated that FOXP3 CD4 T cells isolated from melanoma patients exert an in vitro suppressive action on autologous CD4 T cells, thus possibly inhibiting an efficient anti-tumor response. Next, we aimed to analyse CD4 T cells at antigen-specific level. In advanced stage melanoma patients, we identified for the first time, using pMHCII multimers, circulating CD4 T cells specific for the melanoma antigen Melan-A, presented by HLA-DQB1 *0602. Interestingly, in a cohort of melanoma patients enrolled in an immunotherapy trails consisting of injection of a Melan-A derived peptide, we did not observe signif cant variations in the ex vivo frequencies of Melan-A specific CD4 T cells, but important differences in the quality of the specific CD4 T cells. In fact, up to 50% of the ex vivo Melan-A/DQ6 specific CD4 T cells displayed a regulatory phenotype and were hypoproliferative before vaccination, while more effector, cytokine-secreting Melan-A/DQ6 specific CD4 T cells were observed after immunization. These observations suggest that peptide vaccination may favourably modify the balance between regulatory and effector tumor-specific CD4 T cells. Finally, we identified another subset of CD4 T cells as possible mediator of pathology in a group of human autoimmune spondyloarthropathies, namely Th17 cells. These cells were recently described to play a critical role in the pathogenesis of some marine models of autommunity. We document an elevated presence of circulating Th17 cells in two members of seronegative spondyloarthropathies, e.g. psoriatic arthritis and ankylosing spondylitis, while we do not observe increased frequencies of Th17 cells in peripheral blood of rheumatoid arthritic patients. In addition, Th17 cells with a more advanced differentiation state (CD45RA-CCR7-CD27-) and polyfunctionality (concomitant secretion of IL-17, IL-2 and TNFα) were observed exclusively in patients with seronegative spondylarthropathies. Together, our observations emphasize the importance of CD4 T cells in various diseases and suggest that immunotherapeutic approaches considering CD4 T cells as targets should be evaluated in the future.

HCV infection after liver transplantation is associated with lower levels of alloreactive CD4+CD25+CD45RO+IL-7R+high+ T cells

Aim: Expression of IL-7R discriminates alloreactive CD4 T cells (Foxp3 negative), from IL-7Rlow regulatory CD4 T cells (Foxp3 positive). Chronic hepatitis C virus infection (HCV) reduces expression of IL-7R on T cells thus promoting persistence of infection. The aim of this study was to analyze the effect of HCV infection on the expression of IL-7R of activated CD4+ T cells in liver transplant patients. Patients and methods: We analyzed PBMC from liver transplant recipients for the expression of CD4, CD25, FoxP3, IL-7R (24 HCV negative and 29 HCV-chronically infected). We compared these data with non-transplanted individuals (52 HCV-chronically infected patients and 38 healthy donors). Results: In HCV-infected liver transplant recipients, levels of CD4+CD25+CD45RO+IL-7R+ T cells were significantly reduced (10.5+/-0.9%) when compared to non-HCV-infected liver transplant recipients (17.6+/-1.4%) (P<0.001), while both groups (HCV-infected and negative transplant recipients) had significantly higher levels than healthy individuals (6.6+/-0.9%) (P<0.0001). After successful antiviral therapy (sustained antiviral response), 6 HCV-infected transplant recipients showed an increase of CD4+CD25+CD45RO+IL-7R+ T cells, reaching levels similar to that of non-HCVinfected recipients (10.73+/-2.63% prior therapy versus 21.7+/-6.3% after clearance of HCV). (P<0.05) In 4 non-responders (i.e. HCVRNA remaining present in serum), levels of CD4+CD25+CD45RO+IL-7R+ T cells remained unmodified during and after antiviral treatment (11.8+/- 3.3% versus 11.3+/-3.3% respectively). Conclusions: Overall, these data indicate that CD4+CD25+CD45RO+IL-7R+ T cells appear to be modulated by chronic HCV infection after liver transplantation. Whether lower levels of alloreactive T cells in HCV-infected liver transplant recipients are associated with a tolerogenic profile remains to be studied.