Purpose Recent advances in immunotherapy highlight the antitumor effects of immune- checkpoint inhibition despite a relatively limited subset of patients receiving clinical benefit. The selective class I histone deacetylase inhibitor (HDACi) entinostat has been reported to have immunomodulatory activity including targeting of immune suppressor cells in the tumor microenvironment. Thus, we decided to assess whether entinostat could enhance anti-PD-1 treatment and investigate those alterations in the immunosuppressive tumor microenvironment that contribute to the combined anti-tumor activity. Experimental design We utilized syngeneic mouse models of lung (LLC) and renal cell (RENCA) carcinoma, and assessed immune correlates, tumor growth and survival following treatment with entinostat (5 or 10 mg/kg, P.O.) and a PD-1 inhibitor (10 and 20 mg/kg, s.c.). Results Entinostat enhanced the antitumor effect of PD-1 inhibition in two syngeneic mouse tumor models by reducing tumor growth and increasing survival. Entinostat inhibited the immunosuppressive function of both PMN- and M-MDSC populations. Analysis of MDSC response to entinostat revealed significantly reduced arginase-1, iNOS and COX-2 levels, suggesting potential mechanisms for the altered function. We also observed significant alterations in cytokine/chemokine release in vivo with a shift from an immunosuppressive to a tumor suppressive microenvironment. Conclusions Our results demonstrate that entinostat enhances the antitumor effect of PD-1 targeting through functional inhibition of MDSCs, and a transition away from an immune suppressive tumor microenvironment. These data provide a mechanistic rationale for the clinical testing and potential markers of response of this novel combination in solid tumor patients.

The induction of immunologic unresponsiveness by i.v. administration of Ag-coupled lymphoid cells has been studied extensively, but the mechanisms remain unclear. We have further explored this model by examining the role of Fas/Fas ligand (FasL)-mediated apoptosis. Using i.v. injection of trinitrophenyl-coupled splenocytes (TNP-spl) as tolerogen, we found that Fas signaling for apoptosis in the spleen cells delivered by FasL in the recipient is the critical event. The requirement for Fas and FasL was overcome by prior induction of apoptosis in TNP-spl, making the tolerogen 100 times more potent. Prevention of apoptosis by a caspase inhibitor blocks tolerance. Interestingly, while blocking CD40/CD40 ligand interaction does not prevent tolerance induction, an agonist anti-CD40 Ab turns tolerogenic TNP-spl into an immunizing Ag. Studies further showed that tolerance is induced through cross-presentation of Ag in a class I MHC-dependent manner by CD8+CD11c+ lymphoid-derived dendritic cells to regulatory T cells. The results provide a mechanism for a well-established method of inducing immunologic unresponsiveness.

Although mounting evidence indicates that platelets participate in the modulation of both innate and adaptive immunity, the mechanisms by which platelets exert these effects have not been clearly defined. The study reported herein uses a previously documented adoptive transfer model to investigate the ability of platelet-derived membrane vesicles to communicate activation signals to the B-cell compartment. The findings demonstrate for the first time that platelet-derived membrane vesicles are sufficient to deliver CD154 to stimulate antigen-specific IgG production and modulate germinal center formation through cooperation with responses elicited by CD4(+) T cells. The data are consistent with the hypothesis that platelets modulate inflammation and adaptive immunity at sites distant from the location of activation and that platelet-derived membrane vesicles are sufficient to mediate the effect.