PHENOTYPIC AND FUNCTIONAL MODULATION OF REGULATORY T CELLS IN MELANOMA PATIENTS

Recent studies indicated that regulatory T cells (Tregs) are implicated in the suppression of the immune response against tumors. Accumulation of Tregs in peripheral blood and in tumor microenvironment has been described for patients with various types of cancer. The balance between T effector cells and Tregs, both at tumor site or at local draining lymph nodes is subverted thus limiting the immune response against cancer and restraining the effect of immunotherapy. As concern melanoma, data till now available confirm the presence of Tregs at tumor site, however no in deep analysis on phenotypic or functional characterization of these cells have been provided. This thesis is aimed at evaluating the role of CD4+ Tregs in the immune response against melanoma either by in vivo or in vitro approaches. Among molecules found in mice as expressed by Tregs, lymphocytes activation gene-3 (LAG-3) has been described to be expressed by activated Tregs and more in general as a molecule involved in the control of T cell expansion and homeostasis. In this thesis I explored the expression of LAG-3 in human CD4+ T cells and found that LAG-3 identifies a discrete subset of CD4+CD25highFoxp3+ T cells. This CD4+CD25highFoxp3+LAG-3+ population is preferentially expanded in lymphocytes of tumor-invaded lymph nodes and in lymphocytes infiltrating visceral and sub cute metastasis of melanoma patients. Ex-vivo analysis showed that CD4+CD25highFoxp3+LAG-3+ T cells are functionally active cells that release the immunosuppressive cytokines interleukin-10 (IL-10) and transforming growth factor beta (TGF-1). An in vitro suppression assay using CD4+CD25highLAG-3+ T cells sorted from in vitro expanded CD4+CD25high Tregs showed that this subset of cells is endowed with potent suppressor activity that requires cell-to-cell contact. All together, our data show that LAG-3 defines an active CD4+CD25high Tregs subset in melanoma patients whose frequency is expanded at tumor sites. My data showed that Tregs are accumulated in different immunological districts of patients with melanoma and they also stress the notion that in melanoma patients these Tregs are preferentially in an activation status. However, the real impact of these cells on tumor progression has not been totally clarified. To get insights on the relevance of Tregs in the immunological response to tumor, a phase II randomized trial of multipeptide vaccination in stage IIB-C/III melanoma patients has been designed to include the administration of low dose cyclophosphamide (CTX; 300mg/m2) and low dose interleukin-2 (IL-2; 3x106). CTX has been described as limiting the expansion of Tregs, while IL-2 was given with the aim of expanding tumor-specific responses. The modulation that these drugs exert on different T cell compartment, namely Tregs and conventional T cells was evaluated for its impact on patients’ immunological response. Careful ex-vivo immunological monitoring has been performed aimed at assessing the status of vaccine-induced immune response and the levels of Tregs. Importantly, Treg frequency was defined combining physical and functional markers trying to take into account the plasticity of this population. We observed that CTX has a limited efficacy and a transient effect on Tregs modulation; frequency of Tregs identified by multiparametric fluorescence-activated cell sorting (FACS) analysis as CD4+CD25highFoxp3+ dropped in peripheral blood mononuclear cells (PBMCs) collected 4-7 days after CTX administration, with 6 out of 13 patients displaying a reduction ranging from 20 to 65 %. IL-2 showed higher immunomodulatory effects, expanding both circulating conventional activated CD4+ T cells and Tregs; interestingly, a fraction of these Tregs displayed a Th-1 like phenotype, expressing ex-vivo T-bet (Th1 specific T-box transcription factor) and interferon-INF-. Importantly, this enhanced frequency of Tregs does not significantly affect patients’ immunization assessed ex-vivo by human leukocyte antigen (HLA)-A*0201/peptide multimer staining and IFN- ELISpot assays.