Tumor-Associated Exosomes: A Potential Therapeutic Target for Restoring Anti-Tumor T Cell Responses in Human Tumor Microenvironments.

Exosomes are a subset of extracellular vesicles (EVs) that are released by cells and play a variety of physiological roles including regulation of the immune system. Exosomes are heterogeneous and present in vast numbers in tumor microenvironments. A large subset of these vesicles has been demonstrated to be immunosuppressive. In this review, we focus on the suppression of T cell function by exosomes in human tumor microenvironments. We start with a brief introduction to exosomes, with emphasis on their biogenesis, isolation and characterization. Next, we discuss the immunosuppressive effect of exosomes on T cells, reviewing in vitro studies demonstrating the role of different proteins, nucleic acids and lipids known to be associated with exosome-mediated suppression of T cell function. Here, we also discuss initial proof-of-principle studies that established the potential for rescuing T cell function by blocking or targeting exosomes. In the final section, we review different in vivo models that were utilized to study as well as target exosome-mediated immunosuppression, highlighting the Xenomimetic mouse (X-mouse) model and the Omental Tumor Xenograft (OTX) model that were featured in a recent study to evaluate the efficacy of a novel phosphatidylserine-binding molecule for targeting immunosuppressive tumor-associated exosomes.

Novel phosphatidylserine-binding molecule enhances antitumor T-cell responses by targeting immunosuppressive exosomes in human tumor microenvironments.

BACKGROUND: The human tumor microenvironment (TME) is a complex and dynamic milieu of diverse acellular and cellular components, creating an immunosuppressive environment, which contributes to tumor progression. We have previously shown that phosphatidylserine (PS) expressed on the surface of exosomes isolated from human TMEs is causally linked to T-cell immunosuppression, representing a potential immunotherapeutic target. In this study, we investigated the effect of ExoBlock, a novel PS-binding molecule, on T-cell responses in the TME. METHODS: We designed and synthesized a new compound, (ZnDPA)6-DP-15K, a multivalent PS binder named ExoBlock. The PS-binding avidity of ExoBlock was tested using an in vitro competition assay. The ability of this molecule to reverse exosome-mediated immunosuppression in vitro was tested using human T-cell activation assays. The in vivo therapeutic efficacy of ExoBlock was then tested in two different human tumor xenograft models, the melanoma-based xenomimetic (X-)mouse model, and the ovarian tumor-based omental tumor xenograft (OTX) model. RESULTS: ExoBlock was able to bind PS with high avidity and was found to consistently and significantly block the immunosuppressive activity of human ovarian tumor and melanoma-associated exosomes in vitro. ExoBlock was also able to significantly enhance T cell-mediated tumor suppression in vivo in both the X-mouse and the OTX model. In the X-mouse model, ExoBlock suppressed tumor recurrence in a T cell-dependent manner. In the OTX model, ExoBlock treatment resulted in an increase in the number as well as function of CD4 and CD8 T cells in the TME, which was associated with a reduction in tumor burden and metastasis, as well as in the number of circulating PS+ exosomes in tumor-bearing mice. CONCLUSION: Our results establish that targeting exosomal PS in TMEs with ExoBlock represents a promising strategy to enhance antitumor T-cell responses.

Preclinical evaluation of cancer immune therapy using patient-derived tumor antigen-specific T cells in a novel xenograft platform.

OBJECTIVES: With a rapidly growing list of candidate immune-based cancer therapeutics, there is a critical need to generate highly reliable animal models to preclinically evaluate the efficacy of emerging immune-based therapies, facilitating successful clinical translation. Our aim was to design and validate a novel in vivo model (called Xenomimetic or 'X' mouse) that allows monitoring of the ability of human tumor-specific T cells to suppress tumor growth following their entry into the tumor. METHODS: Tumor xenografts are established rapidly in the greater omentum of globally immunodeficient NOD-scid IL2Rγnull (NSG) mice following an intraperitoneal injection of melanoma target cells expressing tumor neoantigen peptides, as well as green fluorescent protein and/or luciferase. Changes in tumor burden, as well as in the number and phenotype of adoptively transferred patient-derived tumor neoantigen-specific T cells in response to immunotherapy, are measured by imaging to detect fluorescence/luminescence and flow cytometry, respectively. RESULTS: The tumors progress rapidly and disseminate in the mice unless patient-derived tumor-specific T cells are introduced. An initial T cell-mediated tumor arrest is later followed by a tumor escape, which correlates with the upregulation of the checkpoint molecules programmed cell death-1 (PD-1) and lymphocyte-activation gene 3 (LAG3) on T cells. Treatment with immune-based therapies that target these checkpoints, such as anti-PD-1 antibody (nivolumab) or interleukin-12 (IL-12), prevented or delayed the tumor escape. Furthermore, IL-12 treatment suppressed PD-1 and LAG3 upregulation on T cells. CONCLUSION: Together, these results validate the X-mouse model and establish its potential to preclinically evaluate the therapeutic efficacy of immune-based therapies.

Exosomes Represent an Immune Suppressive T Cell Checkpoint in Human Chronic Inflammatory Microenvironments.

Background: T cells present in chronic inflammatory tissues such as nasal polyps (from chronic rhinosinusitis patients) have been demonstrated to be hypo-responsive to activation via the TCR, similar to tumor-specific T cells in multiple different human tumor microenvironments. While immunosuppressive exosomes have been known to contribute to the failure of the tumor-associated T cells to respond optimally to activation stimuli, it is not known whether they play a similar role in chronic inflammatory microenvironments. In the current study, we investigate whether exosomes derived from chronic inflammatory microenvironments contribute to the immune suppression of T cells. Methods: Exosomes were isolated by ultracentrifugation and characterized by size and composition using nanoparticle tracking analysis, scanning electron microscopy, antibody arrays and flow exometry. Immunosuppressive ability of the exosomes was measured by quantifying its effect on activation of T cells, using nuclear translocation of NFκB as an activation endpoint. Results: Exosomes were isolated and characterized from two different types of chronic inflammatory tissues - nasal polyps from chronic rhinosinusitis patients and synovial fluid from rheumatoid arthritis patients. These exosomes arrest the activation of T cells stimulated via the TCR. This immune suppression, like that which is seen in tumor microenvironments, is dependent in part upon a lipid, ganglioside GD3, which is expressed on the exosomal surface. Conclusion: Immunosuppressive exosomes present in non-malignant chronic inflammatory tissues represent a new T cell checkpoint, and potentially represent a novel therapeutic target to enhance the response to current therapies and prevent disease recurrences.

Sialic Acid-Dependent Inhibition of T Cells by Exosomal Ganglioside GD3 in Ovarian Tumor Microenvironments.

The tumor microenvironment is rendered immunosuppressive by a variety of cellular and acellular factors that represent potential cancer therapeutic targets. Although 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. In this study, 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 TCR. This arrest is inhibited by Ab 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, because their enzymatic removal from exosomes or liposomes results in a loss of inhibitory capacity. Collectively, these data define GD3 as a potential immunotherapeutic target.

Exosomes Associated with Human Ovarian Tumors Harbor a Reversible Checkpoint of T-cell Responses.

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. In addition, 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 T cells. Cancer Immunol Res; 6(2); 236-47. ©2018 AACR.

Patient-derived xenografts of low-grade B-cell lymphomas demonstrate roles of the tumor microenvironment.

To discern features of non-Hodgkin lymphomas (NHL) that are autonomous from those that are shaped by the tumor environment (TE), we used patient-derived xenografts (PDX) to probe the effects on neoplastic cells of manipulating the TE. Properties of neoplastic cells that are often considered to be autonomous include their relative independence from stromal support, their relative survival and/or proliferation advantages compared with nonneoplastic cells, and their state of differentiation. Prior approaches to creation of PDX models likely select for neoplasms, which are the most capable of engraftment, potentially masking the effects of the TE. To overcome this bias, we developed a robust protocol that rapidly produced xenografts with more than 85% of unselected, cryo-preserved, B-cell NHL specimens, including low-grade tumors such as follicular and marginal zone lymphoma. To discern features that are shaped by the TE, we extensively studied 4 low-grade lymphoma specimens. B-cell engraftment required components of the native TE; specifically, CD4+ cells. The relative survival of neoplastic compared with nonneoplastic B cells was not autonomous in 2 specimens; specifically, neoplastic B cells from 2 specimens showed a greater dependence on the TE than normal B cells for engraftment. Furthermore, the differentiation of neoplastic B cells was dependent on the TE; mature B-cell neoplasms converted to plasmacytoma-like lesions in the grafts. These results highlight the central and patient-specific roles of the TE in maintaining the relative survival of neoplastic cells compared with normal cells and in controlling the differentiation of neoplastic cells.

Extracellular Vesicles Present in Human Ovarian Tumor Microenvironments Induce a Phosphatidylserine-Dependent Arrest in the T-cell Signaling Cascade.

The identification of immunosuppressive factors within human tumor microenvironments, and the ability to block these factors, would be expected to enhance patients' antitumor 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 80 nm. 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 immunosuppressive 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.

Recruitment of memory B cells to lymph nodes remote from the site of immunization requires an inflammatory stimulus.

Successful recall Ab responses require recruitment of quiescent memory B cells to secondary lymphoid organs. However, the cellular dynamics of memory cells responding to local antigenic challenge at lymphoid sites distal from the initial Ag encounter are not well understood. We show in this study that memory B cells generated following s.c. immunization in one footpad generate secondary responses to soluble Ag given i.p. but not to Ag given s.c. in the contralateral footpad unless LPS is coadministered. Memory B cells do not express CD62L, and CD62L(-ve) cells cannot enter lymph nodes unless LPS-mediated inflammation is induced there. Functional TLR4 is required on the B cells, as well as on non-B cells, in the lymph node to achieve full recruitment. Furthermore, splenectomized mice fail to respond to such inflammatory s.c. challenge in contralateral footpads, unlike lymphadenectomized mice lacking the original draining lymph nodes. Splenectomized mice also fail to respond to i.p. challenge with soluble Ag. Together, these data indicate that, unlike the central memory pool of T cells, which circulates through resting lymph nodes, the majority of long-lived memory B cells are spleen resident and require inflammatory signals for mounting recall responses at distal challenge sites.

Inhibition of terminal differentiation of B cells mediated by CD27 and CD40 involves signaling through JNK.

B cells responding to cognate Ag in vivo undergo clonal expansion that is followed by differentiation into Ab-secreting plasma cells or into quiescent restimulable memory. Both these events occur in the germinal center and require that cells exit from proliferation, but the signals that lead to one or the other of these mutually exclusive differentiation pathways have not been definitively characterized. Previous experiments have shown that signals transduced through the TNFRs CD27 and CD40 at the time of B cell stimulation in vitro or in vivo can influence this cell fate decision by inhibiting terminal differentiation and promoting memory. In this study, we show that the PIQED domain of the cytoplasmic tail of murine CD27 and the adapter molecule TNFR-associated factor 2 are involved in this effect. Using pharmacological inhibitors of signaling intermediates, we identify JNK as being necessary and sufficient for the observed inhibition of terminal differentiation. While JNK is involved downstream of CD40, inhibition of the MEK pathway can also partially restore plasma cell generation, indicating that both signaling intermediates may be involved. We also show that inhibition of induction of IFN regulatory factor 4 and B lymphocyte induced maturation protein 1 are downstream events common to both receptors.