Differential expression of CCR7 defines two distinct subsets of human memory CD4+CD25+ Tregs.

Natural Tregs play an essential role in controlling self-tolerance but the in vivo sites of Treg-mediated suppression remain controversial. We have previously reported the identification of distinct naïve and memory Treg populations in human circulating lymphocytes. Here we show that memory Tregs contain high proportions of inflammatory chemokine-expressing cells and comprise two populations that differ in the expression of the lymphoid chemokine receptor CCR7 and represent the counterparts of conventional CCR7(+) central memory (CM) and CCR7(-) effector memory (EM) T cells. CM and EM Tregs exert comparable ex vivo suppressor functions but the EM population is more prominent among Tregs as compared to conventional CD4(+) T cells, and is the main Treg subset found in ovarian tumors. Our data suggest that a division of labor between CM and EM Tregs ensures tolerance at lymphoid and peripheral locations including tumor sites.

Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming.

The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8(+) T cell responses, through in vivo cross-priming, has remained unclear. In this article, we report that repeated vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG ODN 7909, a potent stimulator of B cells and T helper type 1 (Th1)-type immunity, resulted in the early induction of specific integrated CD4(+) Th cells and antibody responses in most vaccinated patients, followed by the development of later CD8(+) T cell responses in a fraction of them. The correlation between antibody and T cell responses, together with the ability of vaccine-induced antibodies to promote in vitro cross-presentation of NY-ESO-1 by dendritic cells to vaccine-induced CD8(+) T cells, indicated that elicitation of NY-ESO-1-specific CD8(+) T cell responses by cross-priming in vivo was associated with the induction of adequate levels of specific antibodies. Together, our data provide clear evidence of in vivo cross-priming of specific cytotoxic T lymphocytes by a recombinant tumor antigen vaccine, underline the importance of specific antibody induction for the cross-priming to occur, and support the use of this type of formulation for the further development of efficient cancer vaccines.

Assessment of CD4+ T cells specific for the tumor antigen SSX-1 in cancer-free individuals.

Proteins encoded by genes of the SSX family are specifically expressed in tumors and are therefore relevant targets for cancer immunotherapy. One of the first identified family members, SSX-1, is expressed in a large fraction of synovial sarcomas as a fusion protein together with the product of the SYT gene. In addition, the full-length SSX-1 antigen is frequently expressed in tumors of several other histological types such as sarcoma, melanoma, hepatocellular carcinoma, ovarian cancer and myeloma. To date, however, SSX-1 specific T cell responses have not been investigated and no SSX-1 derived T cell epitopes have been described. Here, we have assessed the presence of CD4(+) T cells directed against the SSX-1 antigen in circulating lymphocytes of cancer-free individuals. After a single in vitro stimulation with a pool of peptides spanning the entire SSX-1 protein we could detect and isolate SSX-1-specific CD4(+) T cells from 5/5 donors analyzed. SSX-1-specific polyclonal populations isolated from these cultures recognized peptides located in three distinct regions of the protein containing clusters of sequences with significant predicted binding to frequently expressed MHC class II alleles. Characterization of specific clonal CD4(+) T cell populations derived from one donor allowed the identification of several naturally processed epitopes recognized in association with HLA-DR. These data document the existence of a significant repertoire of CD4(+) T cells specific for SSX-1 derived sequences in circulating lymphocytes of any individual that can be exploited for the development of both passive and active immunotherapeutic approaches to control disease evolution in cancer patients.

Epitope clustering in regions undergoing efficient proteasomal processing defines immunodominant CTL regions of a tumor antigen.

Identification of immunodominant CD8(+) T cell responses to frequently expressed tumor antigens across MHC class I polymorphism is essential for the implementation of cancer immunotherapy. However, the key factors that determine immunodominance are not fully understood. Because of its frequent expression in tumors and its spontaneous immunogenicity, NY-ESO-1 is a prime target of cancer vaccines and an ideal model antigen for elucidating the molecular basis of immunodominant tumor-specific CD8(+) T cell responses. Here, we have assessed CD8(+)T cell responses to full-length NY-ESO-1 in cancer patients. We identified 3 immunodominant regions of the protein located within 3 distinct clusters of MHC class I binding sequences that co-localize with previously defined clusters of MHC class II binding sequences, are predicted to be hydrophobic and undergo efficient proteasomal processing. Our results support the concept that epitope clustering within defined protein regions identifies tumor antigen immunodominant regions and suggest a general strategy for their identification.

Identification of two Melan-A CD4+ T cell epitopes presented by frequently expressed MHC class II alleles.

Because of its expression pattern restricted to cells of the melanocytic lineage and to melanoma cells, Melan-A is an important target of immunotherapeutic approaches for the treatment of melanoma. Identification of Melan-A derived sequences recognized by specific T cells is therefore of great interest for the development of these therapeutic strategies. Using circulating CD4(+) T cells from healthy donors, we identified two Melan-A-derived CD4(+) T cell epitopes mapping to the 1-20 and 91-110 regions of the protein and restricted by HLA-DR11 and HLA-DR52 molecules, respectively. CD4(+) T cells specific for the identified epitopes were able to recognize the native antigen when endogenously expressed by antigen presenting cells and tumor cells. In addition, CD4(+) T cells specific for Melan-A 91-110 recognized the epitope after exogenous processing and presentation of Melan-A recombinant protein. Identification of these epitopes will be instrumental for the evaluation of the immune response to Melan-A in cancer patients.

Rapamycin-mediated enrichment of T cells with regulatory activity in stimulated CD4+ T cell cultures is not due to the selective expansion of naturally occurring regulatory T cells but to the induction of regulatory functions in conventional CD4+ T cells.

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.

A phenotype based approach for the immune monitoring of NY-ESO-1-specific CD4+ T cell responses in cancer patients.

Because of its frequent expression in tumors and spontaneous immunogenicity in advanced cancer patients, NY-ESO-1 is presently viewed as a prototype tumor antigen for the development of cancer vaccines. A prerequisite for the analysis of NY-ESO-1-specific T cell responses in vaccinated patients is the assessment of the complete T cell repertoire available for the antigen. Here, we have assessed frequency and fine specificity of CD4+ T cells reactive against NY-ESO-1-derived sequences in circulating lymphocytes from cancer patients with spontaneous responses to the antigen. We found that, relative to healthy donors, this frequency was only moderately increased in cancer patients. The reactivity of these cells, however, was directed against the same immunodominant regions previously identified for healthy donors. On account of these data, we developed an approach for the immune monitoring of NY-ESO-1-specific CD4+ T cell responses based on the assessment of CD4+ T cell populations of defined phenotype. Using this approach, a similar frequency of NY-ESO-1-specific CD4+ T cells was found among naive T cells of healthy donors and cancer patients. In contrast, among antigen-experienced T cells, NY-ESO-1-specific CD4+ T cells were exclusively detectable in cancer patients. We anticipate that this phenotype-based approach will be useful for the immune monitoring of vaccine-induced responses in vaccination trials using NY-ESO-1 as well as other tumor antigens.

Quantitative and qualitative assessment of circulating NY-ESO-1 specific CD4+ T cells in cancer-free individuals.

The germ cell antigen NY-ESO-1 is characterized by its frequent expression in patients bearing cancers of various histological types, that positively correlates with stage of disease, together with its frequent spontaneous immunogenicity in patients with advanced cancer. Because of these features, NY-ESO-1 is presently viewed as a prototype antigen for the development of cancer vaccines aimed at preventing disease progression. To gain a global view of the CD4+ T cell repertoire available for NY-ESO-1 in individuals of different genetic background, in this study, we have addressed the presence, frequency, and fine specificity of CD4+ T cells reactive against NY-ESO-1-derived sequences among circulating lymphocytes from healthy donors. NY-ESO-1 specific CD4+ T cells were present among circulating lymphocytes at a frequency between 0.5 and 5 precursors per million CD4+ T cells. In the majority of the cases, the reactivity of NY-ESO-1 specific CD4+ T cells was directed towards immunodominant regions located in the carboxyl-terminal half of the protein. Interestingly, immunodominant regions were confined to parts of the NY-ESO-1 protein containing hotspot sequences with predicted high binding for multiple frequently expressed MHC class II molecules. In contrast, no reactivity was found against the amino-terminal part of the protein, which was concomitant with the paucity, in this region, of sequences with predicted high binding to MHC class II molecules.

Identification of B cell epitopes recognized by antibodies specific for the tumor antigen NY-ESO-1 in cancer patients with spontaneous immune responses.

Expression of the germ line antigen NY-ESO-1 in adult somatic tissues other than testis is strictly found in association with cancer. Patients bearing NY-ESO-1 expressing tumors often develop integrated specific immune responses to the antigen, encompassing T cell and antibody responses. Hence, detection of NY-ESO-1 specific antibody responses can be considered as a cancer biomarker of great interest. Here, we used synthetic peptides spanning the sequence of the NY-ESO-1 protein to assess antibody responses in cancer patients. This approach allowed the identification of peptides containing linear B cell epitopes. Some peptides were recognized by the majority of seropositive patients thus identifying several distinct regions of the protein containing frequently recognized B cell epitopes. The results of this study provide the first appraisal of the diversity of naturally-occurring NY-ESO-1 specific antibodies and could be instrumental in the monitoring of therapy-induced antibody responses in cancer patients receiving NY-ESO-1-based vaccines.

A peripheral circulating compartment of natural naive CD4 Tregs.

CD4CD25 Tregs play a central role in the maintenance of peripheral self tolerance by keeping autoreactive T cells in check. Whereas the thymic origin of CD4CD25 Tregs, as a distinct lineage, has been inferred, understanding of their developmental pathways has remained elusive. In both mice and humans, peripheral CD4CD25 Treg populations have been described as composed of antigen-experienced T cells that fail to significantly proliferate following TCR stimulation but suppress proliferation and effector functions of CD25 T cells. Here we show that analysis of CD25 expression in human circulating CD4 T lymphocytes with respect to their in vivo differentiation stages identifies a distinct subset of CD25CCR7CD62LCTLA-4FOXP3 cells contained in the CD45RA/RO naive fraction. The subset, which we have named natural naive Tregs (NnTregs), is prominent in young adults and decreases with age together with the total naive CD4 population. NnTregs are anergic following stimulation in the absence of IL-2 and exert ex vivo cell-cell contact-mediated suppressor functions. In addition, they proliferate in response to stimulation with autologous APCs, which indicates a high enrichment in T cells bearing self-reactive TCRs. The definition of this subset has important implications for the analysis of human naturally occurring Tregs and for their targeting in therapeutic immune interventions.