Firouzeh Korangy

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.

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.

Myeloid Derived Suppressor Cells Inhibit Natural Killer Cells in Patients with Hepatocellular Carcinoma via the NKp30 Receptor

Several immune suppressive mechanisms that evade the host immune response have been described in patients with hepatocellular carcinoma (HCC); one of these mechanisms is expansion of myeloid‐derived suppressor cells (MDSCs). MDSCs have been shown to inhibit T cell responses in tumor‐bearing mice, but little is known about these cells in humans. Here, we have analyzed and characterized the effect of MDSCs on the innate immune system, in particular, their interaction with natural killer (NK) cells in patients with HCC. MDSCs from patients with HCC inhibited autologous NK cell cytotoxicity and cytokine secretion when cultured together in vitro. This suppression was dependent on cell contact, but did not rely on the arginase activity of MDSCs, which is a hallmark function of these cells. However, MDSC‐mediated inhibition of NK cell function was dependent mainly on the NKp30 on NK cells. Conclusion: Our study suggests a new role for MDSCs in patients with HCC in disarming the innate immune system and further contributing to the immune suppressor network in these patients. These findings have important implications when designing immunotherapy protocols. (HEPATOLOGY 2009.)

Myeloid derived suppressor cells in human diseases

Myeloid derived suppressor cells (MDSC) have been described as a heterogeneous cell population with potent immune suppressor function in mice. Limited data are available on MDSC in human diseases. Interpretation of these data is complicated by the fact that different markers have been used to analyze human MDSC subtypes in various clinical settings. Human MDSC are CD11b+, CD33+, HLA-DR(neg/low) and can be divided into granulocytic CD14⁻ and monocytic CD14+ subtypes. Interleukin 4Rα, VEGFR, CD15 and CD66b have been suggested to be more specific markers for human MDSC, however these markers can only be found on some MDSC subsets. Until today the best marker for human MDSC remains their suppressor function, which can be either direct or indirect through the induction of regulatory T cells. Immune suppressor activity has been associated with high arginase 1 and iNOS activity as well as ROS production by MDSC. Not only in murine models, but even more importantly in patients with cancer, different drugs have been shown to either reverse the immune suppressor function of MDSC or directly target these cells. Systemic treatment with all-trans-retinoic acid has been shown to mature human MDSC and reverse their immune suppressor function. Alternatively, MDSC can be targeted by treatment with the multi-targeted receptor tyrosine kinase inhibitor sunitinib. This review will provide a comprehensive summary of the recent literature on human MDSC.

Myeloid-Derived Suppressor Cells in Inflammatory Bowel Disease: A New Immunoregulatory Pathway

BACKGROUND & AIMS CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) have been shown to cause T-cell tolerance in tumor-bearing mice; however, little is known about the role of MDSCs in chronic inflammation. Here, for the first time, we have identified and analyzed their role in inflammatory bowel disease (IBD). METHODS Repetitive adoptive transfer of clone 4/T-cell receptor (CL4-TCR) transgenic CD8(+) T cells into VILLIN-hemagglutinin (HA) transgenic mice was performed on days 1, 12, and 27. Recipient mice were analyzed for immunopathology, HA-specific CD8(+) T-cell responses, and CD11b(+)Gr-1(+) MDSCs (frequency, phenotype, expression analysis, and in vitro as well as in vivo function). In addition, peripheral blood from patients with active Crohns disease and ulcerative colitis was examined for the presence and function of human MDSCs denoted as CD14(+)HLA-DR(-/low) cells. RESULTS Repetitive transfer of HA-specific CD8(+) T cells prevented VILLIN-HA recipient mice from development of severe enterocolitis, which is seen after a single transfer of T cells. Repeated transfer of antigen-specific T cells led to an increase in the frequency of nitric oxide synthase 2 and arginase-expressing CD11b(+)Gr-1(+) MDSCs in spleen and intestine of VILLIN-HA mice with immunosuppressive function. Cotransfer of MDSCs with HA-specific CD8(+) T cells into naive VILLIN-HA mice ameliorated enterocolitis, indicating a direct immune regulatory effect of MDSCs on induction of IBD by antigen-specific T cells. Finally, an increase in the frequency of human MDSCs with suppressor function was observed in peripheral blood from patients with IBD. CONCLUSIONS These results identify MDSCs as a new immune regulatory pathway in IBD.

Plasticity of human Th17 cells and iTregs is orchestrated by different subsets of myeloid cells

CD4+ T helper cell differentiation is essential for mounting robust immune responses without compromising unresponsiveness toward self-tissue. Here, we show that different subsets of myeloid cells isolated from human peripheral blood modulate TGF-β-dependent CD4+ T-cell developmental programs ex vivo. Human CD14+HLA-DR(-/low) myeloid-derived suppressor cells (MDSCs) induce Foxp3+ regulatory T cells, whereas CD14+HLA-DR+ monocytes promote generation of IL-17-secreting RORc+ Th17 cells when cocultured with naive CD4+ T cells. More importantly, not only do these 2 subsets modulate the de novo induction of Tregs and Th17 cells from CD4+ T cells, but MDSCs also catalyze the transdifferentiation of Foxp3+ regulatory T cells from monocyte-induced Th17 cells. The mechanism of such Th17 plasticity is dependent on MDSC-derived TGF-β and retinoic acid. Our results identify a previously unknown feature of the different subsets of CD14+ myeloid cells namely their pivotal role in immune response regulation and plasticity of CD4+ T helper cells. We propose that different subsets of myeloid cells in humans can orchestrate the differentiation of naive CD4+ T cells into effector/regulatory T-cell subsets. The balance between these 2 subsets can impact the outcome of immune reaction from inflammation to tolerance.

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

BackgroundThe 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.Methods40 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.ResultsNone 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.ConclusionsLow 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 registrationNCT00444782

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.

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, CD4+CD25+Foxp+ 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 &agr;-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/m2 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/m2) induced a decrease in the absolute and relative frequency of CD4+CD25+Foxp+ 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/m2 on days 8 and 21. Finally, &agr;-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 CD4+CD25+Foxp+ regulatory T cells in peripheral blood and could be used in combination with immunotherapeutic approaches in HCC.

CD49d Is a New Marker for Distinct Myeloid-Derived Suppressor Cell Subpopulations in Mice

Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of cells that negatively regulate the immune response during tumor progression, inflammation, and infection. In this study, through gene-expression analysis, we have identified a new marker, CD49d, which is expressed exclusively on CD11b+Gr-1dull/int. MDSCs. We have characterized two subpopulations of MDSCs based on CD49d expression in two different settings, a mouse model of inflammatory bowel disease and tumor-bearing mice. The CD49d+ subset of MDSCs was mainly monocytic and strongly suppressed Ag-specific T cell proliferation in an NO-dependent mechanism similar to Gr-1dull/int. MDSCs. Alternatively, CD49d− cells were granulocytic and poorly inhibited T cell proliferation compared with CD11b+Gr-1high cells. Both mouse models showed preferential expansion of the granulocytic CD49d− subset. We suggest that CD49d can be used as an alternative marker for Gr-1 to differentiate between the subpopulations of MDSCs together with CD11b, which will ultimately help in understanding the mechanisms of immune suppression by MDSCs.