Primary sterile necrotic cells fail to cross-prime CD8(+) T cells.

Necrotic cells are known to activate the innate immune system and trigger inflammation by releasing damage associated molecular patterns (DAMPs). However, how necrotic cells influence the induction of antigen-specific CD8(+) T cell-mediated adaptive immune responses under sterile conditions, in the absence of pathogen associated molecular patterns (PAMPs), remains poorly understood. Here, we examined antigen-specific CD8(+) T-cell responses to primary sterile necrotic tumor cells both in vitro and in vivo. We found that primary necrotic cells alone fail to generate CD8(+) T cell-dependent immune responses toward cell-associated antigens. We show that necrotic cells trigger CD8(+) T-cell immunity only in the presence of PAMPs or analogs, such as p(dI-dC) and/or unmethylated CpG DNA. The electroporation of tumor cells with these PAMPs prior to necrosis induction triggered antigen-specific CD8(+) T-cell responses through a TLR9/MyD88-dependent pathway. In addition, we found that necrotic cells contain factors that can block the cross-priming of CD8(+) T cells even under non-sterile conditions and can serve as a possible mechanism of immunosuppression. These results suggest that antigen-specific CD8(+) T-cell responses to primary necrotic tumor cells can be induced in the presence of PAMPs and thus have a substantial impact on the development of antitumor vaccination strategies.

Human CCR4+ CCR6+ Th17 cells suppress autologous CD8+ T cell responses.

The role of Th17 cells in cancer patients remains unclear and controversial. In this study, we have analyzed the phenotype of in vitro primed Th17 cells and further characterized their function on the basis of CCR4 and CCR6 expression. We show a novel function for a subset of IL-17-secreting CD4(+) T cells, namely, CCR4(+)CCR6(+)Th17 cells. When cultured together, CCR4(+)CCR6(+)Th17 cells suppressed the lytic function, proliferation, and cytokine secretion of both Ag-specific and CD3/CD28/CD2-stimulated autologous CD8(+) T cells. In contrast, CCR4(-)CCR6(+) CD4(+) T cells, which also secrete IL-17, did not affect the CD8(+) T cells. Suppression of CD8(+) T cells by CCR4(+)CCR6(+)Th17 cells was partially dependent on TGF-ß, because neutralization of TGF-ß in cocultures reversed their suppressor function. In addition, we also found an increase in the frequency of CCR4(+)CCR6(+), but not CCR4(-)CCR6(+) Th17 cells in peripheral blood of hepatocellular carcinoma patients. Our study not only underlies the importance of analysis of subsets within Th17 cells to understand their function, but also suggests Th17 cells as yet another immune evasion mechanism in hepatocellular carcinoma. This has important implications when studying the mechanisms of carcinogenesis, as well as designing effective immunotherapy protocols for patients with cancer.

A phase II open label trial evaluating safety and efficacy of a telomerase peptide vaccination in patients with advanced hepatocellular carcinoma.

BACKGROUND: The sole effective option for patients with advanced HCC is sorafenib and there is an urgent need to develop new therapeutic approaches. Immunotherapy is a promising option that deserves major investigation. In this open label, single arm clinical trial, we analyzed the effect of a low dose cyclophosphamide treatment in combination with a telomerase peptide (GV1001) vaccination in patients with advanced HCC. METHODS: 40 patients with advanced HCC were treated with 300 mg/m2 cyclophosphamide on day -3 followed by GM-CSF + GV1001 vaccinations on days 1, 3, 5, 8, 15, 22, 36 followed by 4-weekly injections. Primary endpoint of this phase II trial was tumor response; secondary endpoints evaluated were TTP, TTSP, PFS, OS, safety and immune responses. RESULTS: None of the patients had a complete or partial response to treatment, 17 patients (45.9%) demonstrated a stable disease six months after initiation of treatment. The median TTP was 57.0 days; the median TTSP was estimated to be 358.0 days. Cyclophosphamide, GV1001 and GM-CSF treatment were well tolerated and most adverse events, which were of grade 1 or 2, were generally related to the injection procedure and injection site reactions. GV1001 treatment resulted in a decrease in CD4+CD25+Foxp3+ regulatory T cells; however, no GV1001 specific immune responses were detected after vaccination. CONCLUSIONS: Low dose cyclophosphamide treatment followed by GV1001 vaccinations did not show antitumor efficacy as per tumor response and time to progression. Further studies are needed to analyze the effect of a combined chemo-immunotherapy to treat patients with HCC. TRIAL REGISTRATION: NCT00444782.

Low-dose cyclophosphamide treatment impairs regulatory T cells and unmasks AFP-specific CD4+ T-cell responses in patients with advanced HCC.

Immunotherapy represents a potential therapeutic option for patients with hepatocellular carcinoma (HCC). However, CD4CD25Foxp regulatory T cells, which suppress potential antigen-specific T-cell responses, are increased in patients with HCC and might impair the effect of an immune-based therapeutic approach. In this study, we demonstrate that depletion of regulatory T cells in vitro unmasks alpha-fetoprotein-specific T-cell responses in HCC patients. On the basis of these results, we performed a clinical trial, in which 13 patients with advanced HCC ineligible for any other type of treatment were treated with 150, 250, or 350 mg/m cyclophosphamide on day 1 and 29 to suppress regulatory T cells in these patients (NCT00396682). The primary end point of this trial was regulatory T-cell number and function. Low-dose cyclophosphamide treatment (150 and 250 mg/m) induced a decrease in the absolute and relative frequency of CD4CD25Foxp regulatory T cells in peripheral blood on days 8 and 29. Suppressor function of regulatory T cells was impaired after treatment of patients with 250 mg/m on days 8 and 21. Finally, alpha-fetoprotein-specific T-cell responses were unmasked in 6/13 treated patients. In summary, systemic treatment of HCC patients with low-dose cyclophosphamide decreases the frequency and suppressor function of circulating CD4CD25Foxp regulatory T cells in peripheral blood and could be used in combination with immunotherapeutic approaches in HCC.

A new population of myeloid-derived suppressor cells in hepatocellular carcinoma patients induces CD4(+)CD25(+)Foxp3(+) T cells.

BACKGROUND & AIMS: Several studies have shown that development of hepatocellular carcinoma (HCC) generates a number of immune suppressive mechanisms in these patients. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that have been shown to inhibit T-cell responses in tumor-bearing mice, but little is known about these cells in humans owing to a lack of specific markers. In this study, we have investigated the frequency and function of a new population of MDSC denoted here as CD14(+)HLA-DR(-/low) in HCC patients. We have also identified a novel, MDSC-mediated immune regulatory pathway in these patients. METHODS: We have directly isolated and characterized MDSCs for phenotype and function from peripheral blood (n = 111) and tumor (n = 12) of patients with HCC. RESULTS: The frequency of CD14(+)HLA-DR(-/low) cells in peripheral blood mononuclear cells (PBMC) from HCC patients was significantly increased in comparison with healthy controls. CD14(+) HLA-DR(-/low) cells were unable to stimulate an allogeneic T-cell response, suppressed autologous T-cell proliferation, and had high arginase activity, a hallmark characteristic of MDSC. Most important, CD14(+)HLA-DR(-/low) cells from HCC patients induced a CD4(+)CD25(+)Foxp3(+) regulatory T-cell population when cocultured with autologous T cells. CONCLUSION: CD14(+)HLA-DR(-/low) cells are a new population of MDSC increased in blood and tumor of HCC patients. We propose a new mechanism by which MDSC exert their immunosuppressive function, through the induction of CD4(+)CD25(+)Foxp3(+) regulatory T cells in cocultured CD4(+) T cells. Understanding the mechanism of action of MDSC in HCC patients is important in the design of effective immunotherapeutic protocols.

Direct ex vivo analysis of dendritic cells in patients with hepatocellular carcinoma.

AIM: To analyze the phenotype and function of dendritic cells (DC) from patients with hepatocellular carcinoma (HCC) in order to understand their role in this disease. METHODS: Myeloid dendritic cells were enumerated in peripheral blood of HCC patients. CD80, CD83, CD86 and HLA-DR expression on naive and stimulated myeloid dendritic cells from peripheral blood were analyzed. Myeloid dendritic cells were isolated from peripheral blood and their function was tested. Phagocytosis was analyzed using FITC-dextran beads, peptide specific stimulation, the capacity to stimulate allogeneic T cells and secretion of cytokines upon poly dI:dC was tested. RESULTS: Myeloid dendritic cells were reduced in patients with HCC. No differences in CD80, CD83, CD86 and HLA-DR expression were found on naive and stimulated myeloid dendritic cells from HCC patients and healthy controls. Normal phagocytosis or stimulation of peptide specific T cells was observed in contrast to an impaired allo-stimulatory capacity and a reduced IL-12 secretion. CONCLUSION: Impaired IL-12 production of mDCs in patients could lead to an impaired stimulatory capacity of naive T cells suggesting that IL-12 directed therapies may enhance tumor specific immune responses in HCC patients.

Increased populations of regulatory T cells in peripheral blood of patients with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a poor prognosis and one for which immunotherapy remains a viable option. Experimental tumor models have shown that regulatory T cells, a functionally unique subset of T cells, can suppress effective antitumor immune responses. This suppression might explain the poor outcome of some of the immunotherapy protocols currently being used. A better understanding of the role of regulatory T cells in HCC is important for design of future immunotherapy-based clinical protocols. We have studied regulatory T cells from 84 patients with HCC and 74 controls, including healthy donors, patients with chronic hepatitis B virus and hepatitis C virus infection and nonviral liver cirrhosis. Regulatory T cells were identified by fluorescence-activated cell sorting using a panel of antibodies and by real-time PCR analysis for Foxp3 expression. Functional studies were done to analyze their inhibitory role. Finally, regulatory T cells were analyzed in tumors and ascites from patients with HCC. Patients with HCC have increased numbers of CD4+CD25+ regulatory T cells in their peripheral blood, which express high levels of HLA-DR, GITR, and low or no CD45RA. These cells were anergic toward T-cell receptor stimulation and, when cocultured with activated CD4+CD25- cells, potently suppressed their proliferation and cytokine secretion. There were also high numbers of regulatory T cells in tumor-infiltrating lymphocytes of HCC patients comparable with the increase in their peripheral blood. Our data suggest that the increase in frequency of regulatory T cells might play a role in modulation of the immune response against HCC and could be important in design of immunotherapeutic approaches.

Spontaneous tumor-specific humoral and cellular immune responses to NY-ESO-1 in hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is the fifth most common cancer around the world. Although several therapeutic approaches for treatment of HCC are available, survival rates for HCC patients are still very poor because of inefficient treatment options. For HCC, as well as other tumors, antigen-specific immunotherapy remains a viable approach that is dependent on the definition of tumor-associated antigens. NY-ESO-1, a member of the cancer testis antigen family, is one possible candidate for a tumor-specific antigen in HCC. The aim of this study was to show the relevance of NY-ESO-1 in hepatocellular carcinoma. EXPERIMENTAL DESIGN: Sera samples from 189 HCC patients were analyzed for NY-ESO-1-specific antibodies. Forty-nine HCC patients were screened for NY-ESO-1 mRNA expression in HCC tissue. Selected patients were followed for up to 3 years to correlate their immune response with their clinical course of events. NY-ESO-1-specific CD4+ and CD8+ T-cell responses from NY-ESO-1 seropositive patients were analyzed and a NY-ESO-1+ specific cytotoxic T-cell line was generated. RESULTS: Twelve of 49 analyzed tumor samples expressed NY-ESO-1 mRNA and 23 of 189 patients showed NY-ESO-1-specific antibody responses. These humoral immune responses were accompanied by NY-ESO-1-specific functional CD4+ and CD8+ T-cell responses. Finally, NY-ESO-1 humoral responses were dependent on the presence of NY-ESO-1-expressing tumors. CONCLUSIONS: This is the first report of a spontaneous immune response in HCC patients to a known tumor-specific antigen, NY-ESO-1 protein. Our data favor the possibility of immunotherapeutic strategies for the treatment of HCC.