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.

Publisher Correction: Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment.

Epithelial ovarian cancer (EOC) often presents with metastases and ascites. Granulocytic myeloid-derived suppressor cells are an immature population that impairs antitumor immunity. Since suppressive granulocytes in the ascites of patients with newly diagnosed EOC were morphologically mature, we hypothesized that PMN were rendered suppressive in the tumor microenvironment (TME). Circulating PMN from patients were not suppressive but acquired a suppressor phenotype (defined as ≥1 log10 reduction of anti-CD3/CD28-stimulated T cell proliferation) after ascites supernatant exposure. Ascites supernatants (20 of 31 supernatants) recapitulated the suppressor phenotype in PMN from healthy donors. T cell proliferation was restored with ascites removal and restimulation. PMN suppressors also inhibited T cell activation and cytokine production. PMN suppressors completely suppressed proliferation in naive, central memory, and effector memory T cells and in engineered tumor antigen-specific cytotoxic T lymphocytes, while antigen-specific cell lysis was unaffected. Inhibition of complement C3 activation and PMN effector functions, including CR3 signaling, protein synthesis, and vesicular trafficking, abrogated the PMN suppressor phenotype. Moreover, malignant effusions from patients with various metastatic cancers also induced the C3-dependent PMN suppressor phenotype. These results point to PMN impairing T cell expansion and activation in the TME and the potential for complement inhibition to abrogate this barrier to antitumor immunity.

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.

Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment.

Local acidification of stroma is proposed to favour pre-metastatic niche formation but the mechanism of initiation is unclear. We investigated whether Human Melanoma-derived exosomes (HMEX) could reprogram human adult dermal fibroblasts (HADF) and cause extracellular acidification. HMEX were isolated from supernatants of six melanoma cell lines (3 BRAF V600E mutant cell lines and 3 BRAF wild-type cell lines) using ultracentrifugation or Size Exclusion Chromatography (SEC). Rapid uptake of exosomes by HADF was demonstrated following 18 hours co-incubation. Exposure of HDAF to HMEX leads to an increase in aerobic glycolysis and decrease in oxidative phosphorylation (OXPHOS) in HADF, consequently increasing extracellular acidification. Using a novel immuno-biochip, exosomal miR-155 and miR-210 were detected in HMEX. These miRNAs were present in HMEX from all six melanoma cell lines and were instrumental in promoting glycolysis and inhibiting OXPHOS in tumour cells. Inhibition of miR-155 and miR-210 activity by transfection of miRNA inhibitors into HMEX reversed the exosome-induced metabolic reprogramming of HADF. The data indicate that melanoma-derived exosomes modulate stromal cell metabolism and may contribute to the creation of a pre-metastatic niche that promotes the development of metastasis.

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.

Immunogenicity of subcutaneously administered therapeutic proteins--a mechanistic perspective.

The administration of therapeutic proteins via the subcutaneous route (sc) is desired for compliance and convenience, but could be challenging due to perceived immunogenic potential or unwanted immune responses. There are clinical and preclinical data supporting as well as refuting the generalized notion that sc is more immunogenic. We provide a mechanistic perspective of immunogenicity of therapeutic proteins administered via the sc route and discuss strategies and opportunities for novel therapeutic approaches to mitigate immunogenicity.

Human ovarian tumor ascites fluids rapidly and reversibly inhibit T cell receptor-induced NF-κB and NFAT signaling in tumor-associated T cells.

Human memory T cells present in ovarian tumor ascites fluids fail to respond normally to stimulation via the T cell receptor (TCR). This immunosuppression is manifested by decreases in NF-κB and NFAT activation, IFN-γ production, and cell proliferation in response to TCR stimulation with immobilized antibodies to CD3 and CD28. The anergy of the tumor-associated T cells (TATs) is mediated by soluble factors present in ovarian tumor ascites fluids. The non-responsiveness of the T cells is quickly reversed when the cells are assayed in the absence of the ascites fluid, and is rapidly reestablished when a cell-free ascites fluid is added back to the T cells. Based upon the observed normal phosphorylation patterns of the TCR proximal signaling molecules, the inhibition of NF-κB, and NFAT activation in response to TCR stimulation, as well as the ability of the diacylglycerol analog PMA and the ionophore ionomycin to bypass the ascites fluid-induced TCR signaling arrest, the site of the arrest in the activation cascade appears to be at or just upstream of PLC-γ. An identical TCR signaling arrest pattern was observed when T cells derived from normal donor peripheral blood were incubated with either malignant or nonmalignant (cirrhotic) ascites fluids. The immunosuppressive activity of ascites fluids reported here suggests that soluble factors acting directly or indirectly upon T cells present within tumors contribute to the anergy that has previously been observed in T cells derived from malignant and nonmalignant inflammatory microenvironments. The soluble immunosuppressive factors represent potential therapeutic targets for ovarian cancer.

Changes in ovarian tumor cell number, tumor vasculature, and T cell function monitored in vivo using a novel xenograft model.

Despite an initial response to chemotherapy, most patients with ovarian cancer eventually progress and succumb to their disease. Understanding why effector T cells that are known to infiltrate the tumor do not eradicate the disease after cytoreduction is critically important to the development of novel therapeutic strategies to augment tumor immunity and improve patient outcomes. Such studies have been hampered by the lack of a suitable in vivo model. We report here a simple and reliable model system in which ovarian tumor cell aggregates implanted intraperitoneally into severely immunodeficient NSG mice establish tumor microenvironments within the omentum. The rapid establishment of tumor xenografts within this small anatomically well-defined site enables the recovery, characterization, and quantification of tumor and tumor-associated T cells. We validate here the ability of the omental tumor xenograft (OTX) model to quantify changes in tumor cell number in response to therapy, to quantify changes in the tumor vasculature, and to demonstrate and study the immunosuppressive effects of the tumor microenvironment. Using the OTX model, we show that the tumor-associated T cells originally present within the tumor tissues are anergic and that fully functional autologous T cells injected into tumor-bearing mice localize within the tumor xenograft. The transferred T cells remain functional for up to 3 days within the tumor microenvironment but become unresponsive to activation after 7 days. The OTX model provides for the first time the opportunity to study in vivo the cellular and molecular events contributing to the arrest in T cell function in human ovarian tumors.

Memory T cells in the chronic inflammatory microenvironment of nasal polyposis are hyporesponsive to signaling through the T cell receptor.

A majority of T cells from chronic inflammatory tissues derived from patients with nasal polyposis were found to express an effector memory phenotype. We report here that these memory T cells failed to activate NF-κB in response to TCR stimulation but responded normally when the proximal TCR signaling molecules were bypassed with PMA and ionomycin. The dysfunction of these cells was associated with a decrease in the phosphorylation of several TCR proximal signaling molecules including ZAP70, Lck and SLP-76. In addition to the disruption in the TCR signaling pathway, the nasal polyp-associated T cells were shown to have a defect in their ability to translocate LAMP-1 to the cell surface. The results presented here establish that the phenotype and anergy of the T cells in the nasal polyp are similar to those which is seen in memory T cells derived from human tumors and other sites of chronic inflammation.

Downregulation of CD40 signal and induction of TGF-ß by phosphatidylinositol mediates reduction in immunogenicity against recombinant human Factor VIII.

Factor VIII (FVIII) is an important coagulation cofactor and its deficiency causes Hemophilia A, a bleeding disorder. Replacement therapy using recombinant FVIII is currently the first line of therapy for Hemophilia A, but the development of neutralizing antibody is a major clinical complication for this therapy. Recently, it has been shown that FVIII associated with phosphatidylinositol (PI)-containing lipidic nanoparticles reduced development of neutralizing antibodies in Hemophilia A mice (Peng A, Straubinger RM, Balu-Iyer SV. 2010. AAPS J 12(3):473-481). Here, we investigated the underlying mechanism of this reduction in antibody response in culturing conditions. In vitro, PI interfered with the processing of FVIII by cultured dendritic cells (DC), resulting in a reduction in the upregulation of phenotypic costimulatory signal CD40. Furthermore, PI increased secretion of regulatory cytokines Transforming Growth Factor ß1 and Interleukin 10 (IL-10) but reduced the secretion of proinflammatory cytokines IL-6 and IL-17. The data suggest that PI reduces immunogenicity of FVIII by modulating DC maturation and inducing secretion of regulatory cytokines.

Humanized mouse model of ovarian cancer recapitulates patient solid tumor progression, ascites formation, and metastasis.

Ovarian cancer is the most common cause of death from gynecological cancer. Understanding the biology of this disease, particularly how tumor-associated lymphocytes and fibroblasts contribute to the progression and metastasis of the tumor, has been impeded by the lack of a suitable tumor xenograft model. We report a simple and reproducible system in which the tumor and tumor stroma are successfully engrafted into NOD-scid IL2Rγ(null) (NSG) mice. This is achieved by injecting tumor cell aggregates derived from fresh ovarian tumor biopsy tissues (including tumor cells, and tumor-associated lymphocytes and fibroblasts) i.p. into NSG mice. Tumor progression in these mice closely parallels many of the events that are observed in ovarian cancer patients. Tumors establish in the omentum, ovaries, liver, spleen, uterus, and pancreas. Tumor growth is initially very slow and progressive within the peritoneal cavity with an ultimate development of tumor ascites, spontaneous metastasis to the lung, increasing serum and ascites levels of CA125, and the retention of tumor-associated human fibroblasts and lymphocytes that remain functional and responsive to cytokines for prolonged periods. With this model one will be able to determine how fibroblasts and lymphocytes within the tumor microenvironment may contribute to tumor growth and metastasis, and will make it possible to evaluate the efficacy of therapies that are designed to target these cells in the tumor stroma.

Phosphatidylserine reduces immune response against human recombinant Factor VIII in Hemophilia A mice by regulation of dendritic cell function.

A major clinical complication in the treatment of Hemophilia A using exogenously administered recombinant Factor VIII (FVIII) is the development of neutralizing antibodies. It has been shown previously that FVIII complexed with phosphatidylserine (PS) reduces the development of total and neutralizing antibody titers in hemophilic mice. The effect of complexation of FVIII with PS upon dendritic cell (DC) uptake, maturation and processing, T-cell proliferation and cytokine secretion profiles was investigated. Flow cytometric studies of DC showed that PS inhibited the up-regulation of cell surface co-stimulatory markers (CD86 and CD40). PS reduced T-cell proliferation and significantly increased levels of TGF-ß and IL-10 but reduced secretion of IL-6 and IL-17 compared to controls. The data suggest that PS reduces immunogenicity of FVIII by regulating dendritic cell maturation and subsequent T-lymphocyte activity through modulation of cytokine secretion. A possible mechanism for PS-mediated induction of FVIII tolerance is discussed.

Reciprocal functional modulation of the activation of T lymphocytes and fibroblasts derived from human solid tumors.

Fibroblasts are a dominant cell type in most human solid tumors. The possibility that fibroblasts have the capacity to interact with and modulate the function of tumor-associated T lymphocytes makes them a potential therapeutic target. To address this question, primary cultures of fibroblasts derived from human lung tumors were established and cultured with T cells derived from the same tumor. The tumor fibroblasts significantly enhance the production of IFN-gamma and IL-17A by the tumor-associated T cells following a CD3/CD28-induced activation of the T cells. This enhancement was fibroblast cell dose-dependent and did not require direct contact between the two cell types. Tumor-associated fibroblast-conditioned media similarly enhanced both IFN-gamma and IL-17A in activated T cells, and this enhancement was significantly reduced by Abs to IL-6. Conditioned media derived from activated lymphocyte cultures significantly enhanced IL-6 production by tumor fibroblasts. A similar enhancement of IFN-gamma and IL-17A was observed when activated T cells from a normal donor were cultivated with skin fibroblasts derived from the same donor. These results establish that fibroblasts and autologous lymphocytes, whether derived from the tumor microenvironment or from nonmalignant tissues, have the capacity to reciprocally interact and modulate function. In contrast to other reports, fibroblasts are shown to have an immunostimulatory effect upon activated T lymphocytes. The ability of fibroblasts to enhance two T cell cytokines known to have an impact upon tumor progression suggests that fibroblasts play an important role in tumor pathogenesis that could be exploited therapeutically.

T cells and stromal fibroblasts in human tumor microenvironments represent potential therapeutic targets.

The immune system of cancer patients recognizes tumor-associated antigens expressed on solid tumors and these antigens are able to induce tumor-specific humoral and cellular immune responses. Diverse immunotherapeutic strategies have been used in an attempt to enhance both antibody and T cell responses to tumors. While several tumor vaccination strategies significantly increase the number of tumor-specific lymphocytes in the blood of cancer patients, most vaccinated patients ultimately experience tumor progression. CD4+ and CD8+ T cells with an effector memory phenotype infiltrate human tumor microenvironments, but most are hyporesponsive to stimulation via the T cell receptor (TCR) and CD28 under conditions that activate memory T cells derived from the peripheral blood of the cancer patients or normal donors. Attempts to identify cells and molecules responsible for the TCR signaling arrest of tumor-infiltrating T cells have focused largely upon the immunosuppressive effects of tumor cells, tolerogenic dendritic cells and regulatory T cells. Here we review potential mechanisms by which human T cell function is arrested in the tumor microenvironment with a focus on the immunomodulatory effects of stromal fibroblasts. Determining in vivo which cells and molecules are responsible for the TCR arrest in human tumor-infiltrating T cells will be necessary to formulate and test strategies to prevent or reverse the signaling arrest of the human T cells in situ for a more effective design of tumor vaccines. These questions are now addressable using novel human xenograft models of tumor microenvironments.