The identification of immunosuppressive factors within human tumor microenvironments, and the ability to block these factors, would be expected to enhance patients’ anti-tumor immune responses. We previously established that an unidentified factor, or factors, present in ovarian tumor ascites fluids reversibly inhibited the activation of T cells by arresting the T cell signaling cascade. Ultracentrifugation of the tumor ascites fluid has now revealed a pellet that contains small extracellular vesicles (EV) with an average diameter of 80nm. The T cell arrest was determined to be causally linked to phosphatidylserine (PS) that is present on the outer leaflet of the vesicle bilayer, as a depletion of PS expressing EV or a blockade of PS with anti-PS antibody significantly inhibits the vesicle induced signaling arrest. The inhibitory EV were also isolated from solid tumor tissues. The presence of immune suppressive vesicles in the microenvironments of ovarian tumors and our ability to block their inhibition of T cell function represent a potential therapeutic target for patients with ovarian cancer.

The tumor microenvironment is rendered immunosuppressive by a variety of cellular and acellular factors, which represent potential cancer therapeutic targets. While exosomes isolated from ovarian tumor ascites fluids have been previously reported to induce a rapid and reversible T cell arrest, the factors present on or within exosomes that contribute to immunosuppression have not been fully defined. Here, we establish that GD3, a ganglioside expressed on the surface of exosomes isolated from human ovarian tumor ascites fluids, is causally linked to the functional arrest of T cells activated through their T cell receptor. This arrest is inhibited by antibody blockade of exosomal GD3 or by the removal of GD3+ exosomes. Empty liposomes expressing GD3 on the surface also inhibit the activation of T cells, establishing that GD3 contributes to the functional arrest of T cells, independent of factors present in exosomes. Finally, we demonstrate that the GD3-mediated arrest of the TCR activation is dependent upon sialic acid groups, since their enzymatic removal from exosomes or liposomes results in a loss of inhibitory capacity. Collectively, these data define GD3 as a potential immunotherapeutic target.

Nano-sized membrane-encapsulated extracellular vesicles isolated from the ascites fluids of ovarian cancer patients are identified as exosomes based on their biophysical and compositional characteristics. We report here that T cells pulsed with these tumor-associated exosomes during TCR-dependent activation inhibit various activation endpoints including translocation of NFκB and NFAT into the nucleus, upregulation of CD69 and CD107a, production of cytokines and cell proliferation. Additionally, the activation of virus-specific CD8+ T cells that are stimulated with the cognate viral peptides presented in the context of class I MHC is also suppressed by the exosomes. The inhibition occurs without loss of cell viability, and coincidentally with the binding and internalization of these exosomes. This exosome-mediated inhibition of T cells was transient and reversible: T cells exposed to exosomes can be reactivated once exosomes are removed. We conclude that tumor-associated exosomes are immunosuppressive, and represent a therapeutic target blockade of which would enhance the antitumor response of quiescent tumor-associated T cells and prevent the functional arrest of adoptively transferred tumor-specific T cells or chimeric antigen receptor (CAR) T cells.

While a number of extrinsic factors are known to promote the survival of plasma cells (PCs), the signaling intermediates involved remain poorly characterized. Here we identified inducible nitric oxide synthase (iNOS) as an intermediate that supported the survival of PCs. PCs deficient in iNOS (Nos2(-/-) PCs) showed enhanced death in vitro, after transfer into congenic adoptive hosts, and in chimeras made with wild-type and Nos2(-/-) bone marrow. The iNOS-mediated protection involved activation of protein kinase G and modulation of endoplasmic reticulum stress components. Activation of caspases was also diminished. We found that iNOS was required for PCs to respond to some prosurvival mediators associated with bone marrow stromal cells and that at least one mediator, interleukin 6, fed directly into this pathway by inducing iNOS.