Tumor Antigen–Specific FOXP3+ CD4 T Cells Identified in Human Metastatic Melanoma: Peptide Vaccination Results in Selective Expansion of Th1-like Counterparts

Abstract

We have previously shown that vaccination of HLA-A2 metastatic melanoma patients with the analogue Melan-A 26-35(A27L) peptide emulsified in a mineral oil induces ex vivo detectable specific CD8 T cells. These are further enhanced when a TLR9 agonist is codelivered in the same vaccine formulation. Interestingly, the same peptide can be efficiently recognized by HLA-DQ6-restricted CD4 T cells. We used HLA-DQ6 multimers to assess the specific CD4 T-cell response in both healthy individuals and melanoma patients. We report that the majority of melanoma patients carry high frequencies of naturally circulating HLA-DQ6-restricted Melan-A-specific CD4 T cells, a high proportion of which express FOXP3 and proliferate poorly in response to the cognate peptide. Upon vaccination, the relative frequency of multimer+ CD4 T cells did not change significantly. In contrast, we found a marked shift to FOXP3-negative CD4 T cells, accompanied by robust CD4 T-cell proliferation upon in vitro stimulation with cognate peptide. A concomitant reduction in TCR diversity was also observed. This is the first report on direct ex vivo identification of antigen-specific FOXP3+ T cells by multimer labeling in cancer patients and on the direct assessment of the impact of peptide vaccination on immunoregulatory T cells.

The Tumor Antigen Cyclin B1 Hosts Multiple CD4 T Cell Epitopes Differently Recognized by Pre-Existing Naive and Memory Cells in Both Healthy and Cancer Donors

“…They also did not generate a huge number of T cell lines when pools of peptides were used as immunogens, suggesting that either these T cells are not able to be expanded or that our culture conditions are not appropriate for their expansion. Spontaneous CD4 T cell responses to the cancer Ag OCT4 or MUC-1 have already been observed in healthy donors (43,44), but remained unusual as compared with other tumor Ags (8,11,12,45,46). For the first time, to our knowledge, we demonstrated that the spontaneous CD4 T cell response to CCNB1 persists in cancer patients and is not therefore dampened by the tumors.…”

Adjuvants That Improve the Ratio of Antigen-Specific Effector to Regulatory T Cells Enhance Tumor Immunity

“…After 7 days, antigen-specific Teffs and Tregs were examined by flow cytometry in inguinal dLNs and spleen. We initially vaccinated mice with OVA peptide alone, OVAþCpG AEIFA to determine whether this system could faithfully reproduce the effect observed in the blood of patients with melanoma vaccinated with MelanAþCpGþIFA (21). This was indeed the case, with CpG AEIFA significantly increasing the OVA-specific Teff:Treg ratio in vaccine-dLNs and spleen (Supplementary Fig.…”

“…In a clinical study of patients with melanoma immunized with NY-ESO-1 protein in ISCOMATRIX, vaccination increased the frequency of NY-ESO-1-specific Tregs in peripheral blood mononuclear cells (PBMC) and tumor tissue (20). We have shown that therapeutic vaccination of patients with melanoma with Melan-A peptide plus CpG-ODN in Montanide results in robust expansion of Melan-A-specific CD8 þ T cells within PBMCs with a concomitant decrease in Melan-A-specific Tregs (1,2,21). Importantly, although we observed a decrease in vaccine-specific Tregs, the total polyclonal Treg population remained unchanged.…”

High-throughput monitoring of human tumor-specific T-cell responses with large peptide pools

“…In contrast to CD8 C T cells, CD4 C T cells recognize epitopes presented by MHC class II molecules and libraries of longer (20-mer) overlapping peptides are generally used to screen for antigen-specific responses. 19 We thus measured the response of Melan-A/ MART-1 [25][26][27][28][29][30][31][32][33][34][35][36] -specific CD4 C T-cell clones 20 spiked into PBMCs of HLA-DQ6 C donors in the presence of 20-mers overlapping peptide pools (1 mM each). As shown in Fig.…”