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FOXP3+ regulatory T cells (Tregs) are critical regulators of self-tolerance and immune homeostasis. In mice and humans, two subsets of FOXP3+ Tregs have been defined based on their differential expression of Helios, a transcription factor of the Ikaros family. Whereas the origin, specificity, and differential function of the two subsets are as yet a source of controversy, their characterization thus far has been limited by the absence of surface markers to distinguish them. In this article, we show that human memory Helios+ and Helios− Tregs are phenotypically distinct and can be separated ex vivo based on their differential expression of IL-1RI, which is restricted to Helios− Tregs, in combination with CCR7. The two populations isolated using this strategy are distinct with respect to the expression of other Ikaros family members. Namely, whereas Eos, which has been reported to mediate FOXP3-dependent gene silencing, is expressed in Helios+ Tregs, Aiolos, which is involved in the differentiation of TH17 and induced Tregs, is instead expressed in Helios− Tregs. In addition, whereas both subsets are suppressive ex vivo, Helios− Tregs display increased suppressive capacity in comparison to Helios+ Tregs, but respond to IL-1β by downregulating their suppressive activity. Together, these data support the concept that human Helios− memory Tregs encompass induced Tregs that can readily respond to changes in the environment by modulating their suppressive capacity.

We read with interest the recent study by Himmel and colleagues ([1][1]) reporting that human circulating FOXP3+ naive T regulatory cells (nTreg), a population that we defined in 2005 ([2][2]), includes Helios+ and Helios− cells, the latter representing, on average, 30% of total nTreg ([1][1]).

Rapamycin is an immunosuppressive drug currently used in different clinical settings. Although the capacity of rapamycin to inhibit the mammalian target of rapamycin serine/threonine protein kinase and therefore T cell cycle progression is well known, its effects are complex and not completely understood. It has been reported recently that TCR-mediated stimulation of murine CD4+ T cells in the presence of rapamycin results in increased proportions of CD4+ T cells with suppressive functions, suggesting that the drug may also exert its immunosuppressive activity by promoting the selective expansion of naturally occurring CD4+ regulatory T cells (Treg). In this study, we show that stimulation of human circulating CD4+ T cells in the presence of rapamycin results indeed in highly increased suppressor activity. By assessing the effect of rapamycin on the growth of nonregulatory and Treg populations of defined differentiation stages purified ex vivo from circulating CD4+ T cells, we could demonstrate that this phenomenon is not due to a selective expansion of naturally occurring Tregs, but to the capacity of rapamycin to induce, upon TCR-mediated stimulation, suppressor functions in conventional CD4+ T cells. This condition, however, is temporary and reversible as it is dependent upon the continuous presence of rapamycin.

Accumulating evidence supports the requirement for both tumor-specific CD8+ and CD4+ T cell responses for efficient tumor rejection to occur. Because of its expression in different tumor types, the cancer/testis Ag encoded by the synovial sarcoma X breakpoint 2 (SSX-2) gene is among the most relevant candidates for the development of generic cancer vaccines. The immunogenicity of SSX-2 has been previously corroborated by detection of specific humoral and CD8+ T cell responses in cancer patients. In this study we report identification of the first CD4+ T cell epitope encoded by SSX-2. The identified epitope mapped to the 19–34 region of the protein and was recognized by CD4+ T cells from an Ag-expressing melanoma patient in association with HLA-DPB1*0101. The absence of detectable response in healthy donors and other patients suggests that SSX-2-specific CD4+ T cells in the responder patient had been previously expanded in vivo in response to the autologous tumor. The epitope did not appear to be presented on the surface of tumor cells at levels sufficient to allow direct recognition. In contrast, it was efficiently presented by autologous dendritic cells, supporting the concept that processing by professional APC is the main pathway through which the CD4+ T cell immunoresponse to tumor Ags occurs in vivo.

The CD8 coreceptor plays a crucial role in both T cell development in the thymus and in the activation of mature T cells in response to Ag-specific stimulation. In this study we used soluble peptides-MHC class I (pMHC) multimeric complexes bearing mutations in the CD8 binding site that impair their binding to the MHC, together with altered peptide ligands, to assess the impact of CD8 on pMHC binding to the TCR. Our data support a model in which CD8 promotes the binding of TCR to pMHC. However, once the pMHC/TCR complex is formed, the TCR dominates the pMHC/TCR dissociation rates. As a consequence of these molecular interactions, under physiologic conditions CD8 plays a key role in complex formation, resulting in the enhancement of CD8 T cell functions whose specificity, however, is determined by the TCR.

The membrane receptor 2B4 is a CD2 family member that is involved in lymphocyte activation. A fraction of human CD8+ αβ T cells up-regulate 2B4 in vivo, and here we demonstrate that this correlates with the acquisition of effector cell properties such as granzyme B and perforin expression, rapid IFN-γ production, and down-regulation of the lymph node homing chemokine receptor CCR7. In PBLs from healthy donors, cytomegalovirus-specific effector T cells were 2B4 positive, whereas naive melanoma Ag (Melan-A/melanoma Ag recognized by T cells-1)-specific T cells were 2B4 negative. In melanoma patients, Melan-A-specific T cells up-regulated 2B4 in parallel with in vivo differentiation. This occurred in PBLs after vaccination with Melan-A peptides and in tumor-infiltrated lymph nodes, likely through disease-associated activation of Melan-A-specific T cells. Thus, 2B4 expression correlates with CD8+ T cell differentiation in vivo.