CD45RA Distinguishes CD4+CD25+CD127-/low TSDR Demethylated Regulatory T Cell Subpopulations With Differential Stability and Susceptibility to Tacrolimus-Mediated Inhibition of Suppression.

BACKGROUND: Adoptive transfer of forkhead box protein (FOX)3 regulatory T (Treg) cells offers a promising strategy to reduce damage to an allograft by the recipient's immune system. Identification of cell surface markers sufficient to purify Treg cells expanded ex vivo to remove cellular contaminants requires optimization. Furthermore, the expanded Treg must be able to survive, expand, and suppress in allograft recipients exposed to immunosuppressants, such as tacrolimus (TAC). Reduced CD127 expression enhances identification of Treg in the human CD4CD25 population. CD45RA expression identifies naive CD4CD25 Treg with an enhanced stability of Treg phenotype. METHODS: We combine an analysis of CD45RA, CD25, and CD127 expression to identify subpopulations of CD4CD127CD25 cells. Regulatory T cells were sorted according to expression of CD25 and CD45RA and expanded in the presence of a physiological relevant concentration of TAC. Regulatory T cell-specific demethylation region (TSDR) demethylation, FOXP3 expression, and suppression were analyzed. RESULTS: CD4CD127CD25CD45RA Treg cells had a stable TSDR demethylated FOXP3 phenotype after expansion whereas CD4CD127CD25CD45RA Treg cell lost the TSDR demethylated phenotype. CD45RA Treg had a greater capacity to suppress after expansion with TAC. CONCLUSIONS: Although CD45RA Treg retained a greater suppressive capacity when expanded with TAC, the marked loss of the TSDR demethylated status highlights the potential for loss of stability of these cells in transplant recipients treated with TAC based immunosuppression. We show that a population of CD4CD127CD45RA Regulatory T cell may offer the best compromise between susceptibility to loss of suppression when exposed to TAC and maintenance of a TSDR demethylated phenotype following in vitro expansion.

Assessing the prognostic value of preoperative carcinoembryonic antigen-specific T-cell responses in colorectal cancer.

Current dogma suggests that tumor-reactive IFN-γ-producing (TH1-type) T-cells are beneficial to patient outcome; however, the clinical consequence of these responses with respect to long-term prognosis in colorectal cancer (CRC) is not understood. Here, we compared the utility of preoperative, peripheral blood-derived IFN-γ(+) T-cell responses specific to carcinoembryonic antigen (CEA), 5T4, or control antigens (n = 64) with tumor staging and clinical details (n = 87) in predicting five-year outcome of CRC patients who underwent resection with curative intent. Although disease recurrence was more likely in patients with stage III tumors, the presence of preoperative, CEA-specific IFN-γ-producing T-cells identified patients at a statistically significantly greater risk of tumor recurrence following surgical resection, irrespective of tumor stage (odds ratio = 5.00, 95% confidence interval = 1.96 to 12.77, two-sided P <.001). Responses to other antigens, including 5T4, did not reflect outcome. Whilst these results initially appear surprising, they could improve prognostication and help redirect adjuvant treatments.

Cholera toxin enhances vaccine-induced protection against Mycobacterium tuberculosis challenge in mice.

Interleukin (IL)-17 is emerging as an important cytokine in vaccine-induced protection against tuberculosis disease in animal models. Here we show that compared to parenteral delivery, BCG delivered mucosally enhances cytokine production, including interferon gamma and IL-17, in the lungs. Furthermore, we find that cholera toxin, delivered mucosally along with BCG, further enhances IL-17 production by CD4(+) T cells over mucosal BCG alone both in the lungs and systemically. This boosting effect of CT is also observed using a vaccine regimen of BCG followed by the candidate vaccine MVA85A. Using a murine Mycobacterium tuberculosis (M.tb) aerosol challenge model, we demonstrate the ability of cholera toxin delivered at the time of a priming BCG vaccination to improve protection against tuberculosis disease in a manner at least partially dependent on the observed increase in IL-17. This observed increase in IL-17 in the lungs has no adverse effect on lung pathology following M.tb challenge, indicating that IL-17 can safely be boosted in murine lungs in a vaccine/M.tb challenge setting.

Analysis of the T-cell receptor repertoires of tumor-infiltrating conventional and regulatory T cells reveals no evidence for conversion in carcinogen-induced tumors.

A significant enrichment of CD4(+)Foxp3(+) T cells (regulatory T cells, Treg) is frequently observed in murine and human carcinomas. As Tregs can limit effective antitumor immune responses, thereby promoting tumor progression, it is important that the mechanisms underpinning intratumoral accumulation of Tregs are identified. Because of evidence gathered mostly in vitro, the conversion of conventional T cells (Tconv) into Tregs has been proposed as one such mechanism. We assessed the contribution of conversion in vivo by analyzing the TCR (T-cell receptor) repertoires of Tconvs and Tregs in carcinogen-induced tumors in mice. Our results indicate that the TCR repertoires of Tregs and Tconvs within tumor-infiltrating lymphocytes (TIL) are largely distinct. Indeed, the cell population with the greatest degree of repertoire similarity with tumor-infiltrating Tregs was the Treg population from the tumor-draining lymph node. These findings demonstrate that conversion of Tconvs does not contribute significantly to the accumulation of tumor-infiltrating Tregs; rather, Tconvs and Tregs arise from different populations with unique TCR repertoires. Enrichment of Tregs within TILs most likely, therefore, reflects differences in the way that Tregs and Tconvs are influenced by the tumor microenvironment. Elucidating the nature of these influences may indicate how the balance between tumor-infiltrating Tregs and Tconvs can be manipulated for therapeutic purposes.

CD59 blockade enhances antigen-specific CD4+ T cell responses in humans: a new target for cancer immunotherapy?

CD59, a broadly expressed GPI-anchored molecule, regulates formation of the membrane attack complex of the complement cascade. We previously demonstrated that mouse CD59 also down-modulates CD4(+) T cell activity in vivo. In this study, we explored the role of CD59 on human CD4(+) T cells. Our data demonstrate that CD59 is up-regulated on activated CD4(+) T cells and serves to down-modulate their activity in response to polyclonal and Ag-specific stimulation. The therapeutic potential of this finding was explored using T cells isolated from colorectal cancer patients. The findings were striking and indicated that blockade of CD59 significantly enhanced the CD4(+) T cell response to two different tumor Ags. These data highlight the potential for manipulating CD59 expression on T cells for boosting weak immune responses, such as those found in individuals with cancer.

Regulating the immune response to tumours.

Naturally occurring regulatory T cells (Tregs) have been shown to suppress immune responses to self-antigens, thereby limiting autoimmunity. In the case of tumours, where immune responses to self-antigens are beneficial and lead to elimination of the tumour, such suppressive activity is actually detrimental to the host. Manipulation of Tregs holds great promise for the immunotherapy of cancer. Several studies performed using rodent models and indicate that Tregs cells inhibit effective anti-tumour immune responses and that their removal promotes tumour rejection. The increasing number of studies of Tregs in patients with cancer also point to a role for these cells in promoting disease progression. This review summarises the findings of these studies and addresses the advantages and potential pitfalls of manipulating Treg activity for the treatment of cancer.

CD4+CD25+FOXP3+ regulatory T cells suppress anti-tumor immune responses in patients with colorectal cancer.

BACKGROUND: A wealth of evidence obtained using mouse models indicates that CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) maintain peripheral tolerance to self-antigens and also inhibit anti-tumor immune responses. To date there is limited information about CD4(+) T cell responses in patients with colorectal cancer (CRC). We set out to measure T cell responses to a tumor-associated antigen and examine whether Treg impinge on those anti-tumor immune responses in CRC patients. METHODOLOGY AND PRINCIPAL FINDINGS: Treg were identified and characterized as CD4(+)CD25(+)FOXP3(+) using flow cytometry. An increased frequency of Treg was demonstrated in both peripheral blood and mesenteric lymph nodes of patients with colorectal cancer (CRC) compared with either healthy controls or patients with inflammatory bowel disease (IBD). Depletion of Treg from peripheral blood mononuclear cells (PBMC) of CRC patients unmasked CD4(+) T cell responses, as observed by IFNgamma release, to the tumor associated antigen 5T4, whereas no effect was observed in a healthy age-matched control group. CONCLUSIONS/SIGNIFICANCE: Collectively, these data demonstrate that Treg capable of inhibiting tumor associated antigen-specific immune responses are enriched in patients with CRC. These results support a rationale for manipulating Treg to enhance cancer immunotherapy.