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S1: Exosome derived from different ascites fluids show varying degrees of inhibition. S2: Exosomes do not affect T cell death following activation. S3: Exosomes inhibit IL-2 production by T cells. S4: Exosomes inhibit IFN-gamma production by T cells.

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. Cancer Immunol Res; 3(11); 1269–78. ©2015 AACR.

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.

Fig S1. Delipidation of ascites fluid.

List of reagents used in flow cytometry and fluorescence microscopy

Description of additional methods and figure legends for Supplemental Figures

COVID-19, the disease caused by the novel severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), was first detected in December 2019 and has since morphed into a global pandemic claiming over 2.4 million human lives and severely impacting global economy. The race for a safe and efficacious vaccine was thus initiated with government agencies as well as major pharmaceutical companies as frontrunners. An ideal vaccine would activate multiple arms of the adaptive immune system to generate cytotoxic T cell responses as well as neutralizing antibody responses, while avoiding pathological or deleterious immune responses that result in tissue damage or exacerbation of the disease. Developing an effective vaccine requires an inter-disciplinary effort involving virology, protein biology, biotechnology, immunology and pharmaceutical sciences. In this review, we provide a brief overview of the pathology and immune responses to SARS-CoV-2, which are fundamental to vaccine development. We then summarize the rationale for developing COVID-19 vaccines and provide novel insights into vaccine development from a pharmaceutical science perspective, such as selection of different antigens, adjuvants, delivery platforms and formulations. Finally, we review multiple clinical trial outcomes of novel vaccines in terms of safety and efficacy.

The immune system, which encompasses a dynamic network of organs, cells, and molecules, functions to maintain homeostasis as well as to respond to threats. Regulation of the immune system at the ge...

BackgroundThe 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.MethodsWe 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.ResultsExoBlock 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.ConclusionOur results establish that targeting exosomal PS in TMEs with ExoBlock represents a promising strategy to enhance antitumor T-cell responses.

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., We have designed and validated a first‐of‐its‐kind xenograft model, the Xenomimetic Mouse (X‐mouse) model, that has the ability to rapidly and reliably evaluate immune‐based therapies for cancer treatment using patient‐derived tumor‐specific T cells. Our model offers multiple advantages over existing preclinical models including but not limited to easy, accurate and quick measurement of tumor burden, monitoring changes in T‐cell phenotypes at the single cell level and rapid turnaround times. By validating the X‐mouse model using established clinically relevant immune‐based therapies, we demonstrate that it has potential to be a robust preclinical tool that will be of great utility to investigators involved in the development of immunotherapeutic targets and therapeutics for cancer treatment.

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 human 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 nonmalignant 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.

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.

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.

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. Cancer Immunol Res; 3(11); 1269–78. ©2015 AACR.

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.

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.

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.

Nat. Immunol. 15, 275–282 (2014); published online 19 January 2014; corrected after print 21 August 2014 In the version of this article initially published, Figure 6a,b lacked asterisks to indicate P values. The error has been corrected in the HTML and PDF versions of the article.