Despite extensive research efforts to characterize peripheral regulatory T (Treg) cells expressing transcription factor Foxp3, their subset complexity, phenotypic characteristics, TCR repertoire and Ag specificities remain ambiguous. In this study, we identify and define two subsets of peripheral Treg cells differing in Foxp3 expression level and TCR repertoires. Treg cells expressing a high level of Foxp3 and TCRs not used by naive CD4+ T cells present a stable suppressor phenotype and dominate the peripheral Treg population in unmanipulated mice. The second Treg subset, expressing a lower level of Foxp3 and using TCRs shared with naive CD4+ T cells constitutes a small fraction of all Treg cells in unmanipulated mice and enriches Treg population with the same Ag specificities as expressed by activated/effector T cells. This Treg subset undergoes extensive expansion during response to Ag when it becomes a major population of Ag-specific Treg cells. Thus, Treg cells expressing TCRs shared with naive CD4+ T cells have a flexible phenotype and may down-regulate Foxp3 expression which may restore immune balance at the conclusion of immune response or convert these cells to effector T cells producing inflammatory cytokines.

The presence of Foxp3+ regulatory CD4+ T cells in tumor lesions is considered one of the major causes of ineffective immune response in cancer. It is not clear whether intratumoral Treg cells represent Treg cells pre-existing in healthy mice, or arise from tumor-specific effector CD4+ T cells and thus representing adaptive Treg cells. The generation of Treg population in tumors could be further complicated by recent evidence showing that both in humans and mice the peripheral population of Treg cells is heterogenous and consists of subsets which may differentially respond to tumor-derived antigens. We have studied Treg cells in cancer in experimental mice that express naturally selected, polyclonal repertoire of CD4+ T cells and which preserve the heterogeneity of the Treg population. The majority of Treg cells present in healthy mice maintained a stable suppressor phenotype, expressed high level of Foxp3 and an exclusive set of TCRs not used by naive CD4+ T cells. A small Treg subset, utilized TCRs shared with effector T cells and expressed a lower level of Foxp3. We show that response to tumor-derived antigens induced efficient clonal recruitment and expansion of antigen-specific effector and Treg cells. However, the population of Treg cells in tumors was dominated by cells expressing TCRs shared with effector CD4+ T cells. In contrast, Treg cells expressing an exclusive set of TCRs, that dominate in healthy mice, accounted for only a small fraction of all Treg cells in tumor lesions. Our results suggest that the Treg repertoire in tumors is generated by conversion of effector CD4+ T cells or expansion of a minor subset of Treg cells. In conclusion, successful cancer immunotherapy may depend on the ability to block upregulation of Foxp3 in effector CD4+ T cells and/or selectively inhibiting the expansion of a minor Treg subset.