The extent to which T cell-mediated immune surveillance is impaired in human cancer remains a question of major importance, given its potential impact on the development of generalized treatments for advanced disease where the highest degree of heterogeneity exists. Here we report the first global analysis of immune dysfunction in patients with advanced hepatocellular carcinoma (HCC). Using multi-parameter FACS analysis, we quantified the cumulative frequency of T regulatory cells (Tregs), exhausted CD4+ helper T cells, and myeloid-derived suppressor cells (MDSC) to gain concurrent views on the overall level of immune dysfunction in these inoperable patients. We documented augmented numbers of Tregs, MDSC, PD-1+ exhausted T cells and increased levels of immunosuppressive cytokines in HCC patients, compared to normal controls, revealing a network of potential mechanisms of immune dysregulation in HCC patients. In dampening T cell-mediated anti-tumor immunity, we hypothesized that these processes may facilitate HCC progression and thwart the efficacy of immunotherapeutic interventions. In testing this hypothesis, we demonstrated that combined regimens to deplete Tregs, MDSC, and PD-1+ T cells in advanced HCC patients restored production of granzyme B by CD8+ T cells, reaching levels observed in normal controls, and also modestly increased the number of IFN-γ producing CD4+ T cells. These clinical findings encourage efforts to restore T cell function in patients with advanced stage disease, by highlighting combined approaches to deplete endogenous suppressor cell populations that can also expand effector T cell populations.

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Rationale: Previous studies from our laboratory have shown that peripheral blood mononuclear cells (PBMCs) from patients with chronic obstructive pulmonary disease (COPD) prone to exacerbations with nontypeable Haemophilus influenzae have impaired responses to lipoprotein P6. We hypothesized that an underlying immunosuppressive network could be responsible for the defective antibacterial immunity observed in these patients. We Objectives: We performed an in-depth characterization of Tregs, T effector cells, and MDSC in COPD and correlated their levels and function with disease severity.Methods: Treg, effector T cell, and MDSC frequency from patients with COPD and healthy subjects' PBMCs were analyzed by flow cytometry. Treg immunosuppressive capacity was measured by in vitro suppression assay. The frequency of interferon-g producing T cells and T-cell proliferation were measured after blocking CTLA-4 and PD-1. Plasma proinflammatory and immunosuppressive cytokine levels were measured.Measurements and Main Results: Significantly increased levels of Tregs, MDSC, and PD-1 1 exhausted effector T cells were present in patients with COPD compared with healthy subjects. Tregs from patients with COPD suppressed P6-specific T-cell proliferation to a greater extent than Tregs from healthy subjects. Plasma levels of Treg-generated cytokines, IL-10, and transforming growth factor-b were elevated. Blockade of CTLA-4 resulted in significant augmentation of T-cell IFN-g production in patients with COPD.Conclusions: Functionally suppressive Tregs, MDSCs, and exhausted PD-1 1 T cells contribute to effector T-cell dysfunction in COPD.

BACKGROUND Sorafenib is an oral antiangiogenic agent administered in advanced-stage hepatocellular carcinoma (HCC). Based on preclinical and human studies, we hypothesized that, in addition to its antiangiogenic properties, sorafenib may beneficially reduce the extent of the immunosuppressive network in HCC patients. To test this hypothesis, we examined whether alterations in the immunosuppressive burden of advanced-stage HCC patients correlated with clinical outcome. METHODS In before and after sorafenib treatment, blood samples collected from 19 patients with advanced HCC, the frequency of PD-1+ T cells, Tregs, and myeloid derived suppressor cells (MDSC) were quantified by multiparameter FACS. Cytokine levels in plasma were determined by ELISA. RESULTS Overall survival (OS) was significantly impacted by the reduction in the absolute number of both CD4+PD-1+ T cells and CD8+PD-1+ T cells following sorafenib treatment. Significant decreases in the frequency and absolute number of Foxp3+ Tregs were also observed, and a statistically significant improvement in OS was noted in patients exhibiting a greater decrease in the number of Foxp3+ Tregs. The ratio of CD4+CD127+PD-1− T effector cells to CD4+Foxp3+PD-1+ Tregs was significantly increased following treatment with sorafenib. Increased frequency of CD4+CD127+ T effector cells in the posttreatment samples significantly correlated with OS. CONCLUSION This study is the first to our knowledge to demonstrate the potent immunomodulatory effects of sorafenib therapy on PD-1+ T cells and Tregs and the ensuing correlation with survival. These phenotypes could serve as predictive biomarkers to identify HCC patients who are likely to benefit from sorafenib treatment. TRIAL REGISTRATION Registration is not required for observational studies. FUNDING This study was supported by NCI Core Grant to RPCI (NIH P30 CA016056) and discretionary funds to Y. Thanavala.

A bottleneck for immunotherapy of cancer is the immunosuppressive microenvironment in which the tumor cells are located. Regardless of the fact that large numbers of tumor-specific T cells can be generated in patients by active immunization or adoptive transfer, these T cells do not readily translate to tumor cell killing in vivo. The immune regulatory mechanism which prevents autoimmunity may be harnessed by tumor cells for the evasion of immune destruction. Regulatory T cells, myeloid-derived suppressor cells, inhibitory cytokines and immune checkpoint receptors are the major components of the immune system acting in concert to cause the subversion of anti-tumor immunity in the tumor microenvironment. This redundant immunosuppressive network may pose an impediment to efficacious immunotherapy, thus facilitating tumor progression. Cancer progression clearly documents the failure of immune control over relentless growth of tumor cells. Detailed knowledge of each of these factors responsible for creating an immunosuppressive shield to protect tumor cells from immune destruction is essential for the development of novel immune based therapeutic interventions of cancer. Multi-pronged targeted depletion of these suppressor cells may restore production of granzyme B by CD8+ T cells and increase the number of IFN-γ producing CD4+ T cells.