Maha Ayyoub

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.

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.

Human memory FOXP3+ Tregs secrete IL-17 ex vivo and constitutively express the TH17 lineage-specific transcription factor RORγt

Recent studies have suggested a close relationship between CD4+FOXP3+ regulatory T cells (Tregs) and proinflammatory IL-17-producing T helper cells (TH17) expressing the lineage-specific transcription factor RORγt. We report here the unexpected finding that human memory Tregs secrete IL-17 ex vivo and constitutively express RORγt. IL-17-secreting Tregs share some phenotypic and functional features with conventional TH17 cells, expressing high levels of CCR4 and CCR6 and low levels of CXCR3. However, unlike conventional TH17 cells, they express low levels of CD161 and mostly fail to cosecrete IL-22 and TNF-α ex vivo. Ex vivo secretion of IL-17 and constitutive expression of RORγt by human memory Tregs suggest that, in addition to their well-known suppressive functions, these cells likely play additional, as yet undescribed, proinflammatory functions.

Recombinant vaccinia/fowlpox NY-ESO-1 vaccines induce both humoral and cellular NY-ESO-1-specific immune responses in cancer patients

NY-ESO-1 is a cancer/testis antigen expressed in a range of human malignancies, and a number of vaccine strategies targeting NY-ESO-1 are being developed. In the present study, the safety and immunogenicity of recombinant vaccinia-NY-ESO-1 and recombinant fowlpox-NY-ESO-1 were analyzed in a series of 36 patients with a range of different tumor types. Each construct was first tested individually at two different dose levels and then in a prime-boost setting with recombinant vaccinia-NY-ESO-1 followed by recombinant fowlpox-NY-ESO-1. The vaccines were well tolerated either individually or together. NY-ESO-1-specific antibody responses and/or specific CD8 and CD4 T cell responses directed against a broad range of NY-ESO-1 epitopes were induced by a course of at least four vaccinations at monthly intervals in a high proportion of patients. CD8 T cell clones derived from five vaccinated patients were shown to lyse NY-ESO-1-expressing melanoma target cells. In several patients with melanoma, there was a strong impression that the natural course of the disease was favorably influenced by vaccination.

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.

Antigenicity and immunogenicity of Melan-A/MART-1 derived peptides as targets for tumor reactive CTL in human melanoma.

Summary: Some cancer patients mount spontaneous T‐ and B‐cell responses against their tumor cells. Autologous tumor reactive CD8 cytolytic T lymphocyte (CTL) and CD4 T‐cell clones as well as antibodies from these patients have been used for the identification of genes encoding the target antigens. This knowledge opened the way for new approaches to the immunotherapy of cancer. In this review, we describe the characterization of the structure‐function properties of the melanocyte/melanoma tumor antigen Melan‐A/MART‐1, the assessment of the T‐cell repertoire available against this antigen in healthy individuals, and the analysis of naturally acquired and/or vaccine‐induced CTL responses to this antigen in patients with metastatic melanoma.

IL-1β and IL-2 convert human Treg into TH17 cells

Natural CD4(+)CD25(+) regulatory T cells (Treg) and interleukin 17 (IL-17)-producing T helper cells (T(H)17) carry out opposite functions, the former maintaining self-tolerance and the latter being involved in inflammation and autoimmunity. We report here that stimulation of human Natural Treg under T(H)17 polarizing conditions in the presence of IL-2 converts them into T(H)17 cells. Conversion of Tregs into T(H)17 cells occurs both from natural naïve Tregs (NnTregs) and, to a higher extent, from memory Tregs (MTregs). Among antigen presenting cells, fresh monocytes activated by microbial stimuli were the most efficient inducers of T(H)17 cells from Tregs. Conversion of Treg into T(H)17 cells was induced by IL-1beta and involved down-regulation of the Treg lineage transcription factor FOXP3 and suppressive functions. Our results indicate that, under inflammatory conditions, in the presence of IL-2, Treg can be converted into pro-inflammatory T(H)17 cells and establish a functional link between inflammation and autoimmunity.

Proteasome-Assisted Identification of a SSX-2-Derived Epitope Recognized by Tumor-Reactive CTL Infiltrating Metastatic Melanoma

The tumor Ag SSX-2 (HOM-MEL-40) was found by serological identification of Ags by recombinant expression cloning and was shown to be a cancer/testis Ag expressed in a wide variety of tumors. It may therefore represent a source of CD8+ T cell epitopes useful for specific immunotherapy of cancer. To identify potential SSX-2-derived epitopes that can be recognized by CD8+ T cells, we used an approach that combined: 1) the in vitro proteasomal digestion of precursor peptides overlapping the complete SSX-2 sequence; 2) the prediction of SSX-2-derived peptides with an appropriate HLA-A2 binding score; and 3) the analysis of a tumor-infiltrated lymph node cell population from an HLA-A2+ melanoma patient with detectable anti-SSX-2 serum Abs. This strategy allowed us to identify peptide SSX-241–49 as an HLA-A2-restricted epitope. SSX241–49-specific CD8+ T cells were readily detectable in the tumor-infiltrated lymph node population by multimer staining, and CTL clones isolated by multimer-guided cell sorting were able to lyse HLA-A2+ tumor cells expressing SSX-2.

Human RORγt+ TH17 cells preferentially differentiate from naive FOXP3+Treg in the presence of lineage-specific polarizing factors

RORγt+ TH17 cells are a proinflammatory CD4+ T-cell population associated with autoimmune tissue injury. In mice, priming of TH17 requires TGF-β, which alone directs the priming of FOXP3+ regulatory T cells (Treg), in association with inflammatory cytokines. Priming of human TH17 cells from conventional naive CD4+ T cells under similar conditions, however, has proved difficult to achieve. Here, we report that differentiation of human TH17 cells preferentially occurs from FOXP3+ naive Treg (NTreg) in the presence of IL-2 and IL-1β and is increased by IL-23 and TGF-β. IL-1β–mediated differentiation correlated with IL-1RI expression in stimulated NTreg and was accompanied by induction of RORγt along with down-regulation of FOXP3. IL-17–secreting cells in NTreg cultures cosecreted TNF-α and IL-2 and contained distinct subpopulations cosecreting or not cosecreting IFN-γ and other TH17-associated cytokines. Polarized NTreg contained significant subpopulations of CCR6-expressing cells that were highly enriched in IL-17–secreting cells. Finally, analysis of CCR6 expression with respect to that of IL-1RI identified distinct IL-17–secreting subpopulations that had maintained or lost their suppressive functions. Together our results support the concept that priming of human TH17 from naive CD4+ T cells preferentially takes place from FOXP3+ Treg precursors in the presence of lineage-specific polarizing factors.

Lack of tumor recognition by hTERT peptide 540–548-specific CD8+ T cells from melanoma patients reveals inefficient antigen processing

Telomerase is a ribonucleoprotein complex responsible for the maintenance of the length of the telomeres during cell division, which is active in germ‐line cells as well as in the vast majority of tumors but not in most normal tissues. The wide expression of the human telomerase catalytic subunit (hTERT) in tumors makes it an interesting candidate vaccine for cancer. hTERT‐derived peptide 540–548 (hTERT540) has been recently shown to be recognized in an HLA‐A*0201‐restricted fashion by T cell lines derived from peptide‐stimulated peripheral blood mononuclear cells (PBMC) from healthy donors. As a first step to the inclusion of this peptide in immunotherapy clinical trials, it is crucial to assess hTERT540‐specific T cell reactivity in cancer patients as wellas the ability of hTERT‐specific CD8+ T lymphocytes to recognize and lyse hTERT‐expressing target cells. Here, we have analyzed the CD8+ T cell response to peptide hTERT540 in HLA‐A*0201 melanoma patients by using fluorescent HLA‐A*0201/hTERT540 peptide tetramers. HLA‐A*0201/hTERT540 tetramer+ CD8+ T cells were readily detected in peptide‐stimulated PBMC from a significant proportion of patients and could be isolated by tetramer‐guided cell sorting. hTERT540‐specific CD8+ T cells were able to specifically recognize HLA‐A*0201 cells either pulsed with peptide or transiently transfected with a minigene encoding the minimal epitope. In contrast, they failed to recognize hTERT‐expressing HLA‐A*0201+ target cells. Furthermore, in vitro proteasome digestion studies revealed inadequate hTERT processing. Altogether, these results raise questions on the use of hTERT540 peptide for cancer immunotherapy.