Inhibition Of Antigen Presentation Research Articles

Abstract 4988: Reprogramming the immunologic microenvironment through radiation and Axl targeting

Abstract There is increasing evidence that hypofractionated high dose ionizing radiation (RT) can enhance antitumor immune responses in many cancers. In some cases the combination of RT and checkpoint immunotherapy can enhance antitumor immune responses. Here, we developed a model to study the genetic, microenvironmental, and immunologic factors of immune mediated antitumor responses after radiation. Two different tumor clones from the same parental transgenic PyMT mammary carcinoma model reveal similar growth characteristics but different responses to RT primarily due to an antitumor immune response. The responsive Py117 tumors increase expression of PD-L1 after RT and the antitumor response is enhanced with immune checkpoint antibodies (Ab) targeting PD-1 and CTLA-4. However the unresponsive Py8119 tumors do not respond to radiation or combination immunotherapy. Key differences in the microenvironment include greater infiltration of suppressive macrophages, lower MHCI on tumor cells, and low T cell infiltrates. Upon profiling differences in the tumors we found that Axl a receptor tyrosine kinase of the TAM family associated with invasion and metastasis in a broad range of tumors was overexpressed on the surface of Py8119 cells but not Py117. We genetically knocked out Axl in Py8119 cells using CRISPR/Cas technology and found greater tumor latency and enhanced radiosensitivity in the mouse but not in culture. The effects are largely immune mediated as the RT response was diminished in nude mice. On analysis of the tumor cells we found that MHCI is significantly increased and secretion of a number of chemokines is diminished after Axl targeting. In the tumor microenvironment there was significant decrease in macrophages, increase in myeloid dendritic cells, and resulting influx of T cells by 10 days after RT. These data suggest that Axl may not only mediate invasion and metastasis but can influence immunoserveilance and response to therapy through suppression of antigen presentation and supporting a tumor promoting immune microenvironment through chemokine signaling. Citation Format: Todd A. Aguilera, Marjan R. Rafat, Laura Castellini, Mihalis S. Kariolis, Rie vonEbyen, Edward E. Graves, Amato J. Giaccia. Reprogramming the immunologic microenvironment through radiation and Axl targeting. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4988.

Mycobacterium tuberculosis: Strategies of offense and defense

Strategies of macrophage attack by Mycobacterium tuberculosis and defense against immune response are considered in detail. The mechanisms of cell invasion, strategies of survival within phagosomes, methods for the suppression of antigen presentation, and pathways of M. tuberculosis transition into the persisting latent state are discussed.

A VP22-Null HSV-1 Is Impaired in Inhibiting CD1d-Mediated Antigen Presentation

CD1d-restricted T (natural killer T [NKT]) cells are important for controlling a herpes simplex virus (HSV) infection. One of the mechanisms of immune evasion by HSV is to downregulate CD1d-mediated activation of NKT cells. VP22 is an HSV-1-encoded protein responsible for reorganizing the host cell's cytoskeletal network and viral spreading. We have previously shown that modification of the cytoskeleton can alter CD1d-mediated antigen presentation. In this study, we found that an HSV-1 lacking VP22 (ΔUL49) was impaired in its ability to inhibit CD1d-mediated antigen presentation compared with the wild-type (WT) virus; this was reversed by a repair virus (UL49R) in CD1d-expressing cells. We further demonstrated that CD1d recycling was inhibited by infection with WT and UL49R, but not the ΔUL49 virus. Ectopic expression of VP22 in CD1d-expressing cells complemented the VP22-deficient virus in inhibiting antigen presentation. Moreover, inhibiting viral protein synthesis rescued VP22-dependent inhibition of CD1d antigen presentation. In conclusion, our findings suggest that VP22 is required (but not sufficient) for the inhibition of CD1d-mediated antigen presentation by an HSV-1 infection.

The Epstein-Barr Virus Glycoprotein gp150 Forms an Immune-Evasive Glycan Shield at the Surface of Infected Cells

Cell-mediated immunity plays a key role in host control of viral infection. This is exemplified by life-threatening reactivations of e.g. herpesviruses in individuals with impaired T-cell and/or iNKT cell responses. To allow lifelong persistence and virus production in the face of primed immunity, herpesviruses exploit immune evasion strategies. These include a reduction in viral antigen expression during latency and a number of escape mechanisms that target antigen presentation pathways. Given the plethora of foreign antigens expressed in virus-producing cells, herpesviruses are conceivably most vulnerable to elimination by cell-mediated immunity during the replicative phase of infection. Here, we show that a prototypic herpesvirus, Epstein-Barr virus (EBV), encodes a novel, broadly acting immunoevasin, gp150, that is expressed during the late phase of viral replication. In particular, EBV gp150 inhibits antigen presentation by HLA class I, HLA class II, and the non-classical, lipid-presenting CD1d molecules. The mechanism of gp150-mediated T-cell escape does not depend on degradation of the antigen-presenting molecules nor does it require gp150’s cytoplasmic tail. Through its abundant glycosylation, gp150 creates a shield that impedes surface presentation of antigen. This is an unprecedented immune evasion mechanism for herpesviruses. In view of its likely broader target range, gp150 could additionally have an impact beyond escape of T cell activation. Importantly, B cells infected with a gp150-null mutant EBV displayed rescued levels of surface antigen presentation by HLA class I, HLA class II, and CD1d, supporting an important role for iNKT cells next to classical T cells in fighting EBV infection. At the same time, our results indicate that EBV gp150 prolongs the timespan for producing viral offspring at the most vulnerable stage of the viral life cycle.

Risk of graft-versus-host disease with rituximab-containing conditioning regimens in allogeneic hematopoietic stem cell transplant.

Graft-versus-host disease represents a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant patients. There is growing evidence that B lymphocytes may play a role in the pathogenesis of acute graft-versus-host disease. The purpose of this retrospective cohort study was to evaluate the efficacy of rituximab-containing conditioning regimens in decreasing graft-versus-host disease in allogeneic hematopoietic stem cell transplant patients who received standardized tacrolimus-based graft-versus-host disease prophylaxis regimens. Patients were divided into two cohorts, based on the presence (RTX, n = 54) or absence (No-RTX, n = 105) of rituximab in the conditioning regimen and were matched 1:2 for major graft-versus-host disease risk factors. The incidence of grade II-IV acute graft-versus-host disease was not different between the two groups (37% vs. 26%, p = 0.147). When restricting the analysis to recipients of peripheral blood hematopoietic stem cell transplants, the RTX group had a higher incidence of grade II-IV acute graft-versus-host disease, relapse, or death prior to day 100 (55% vs. 36%, p = 0.037). The median time to the onset of acute graft-versus-host disease was no different between the RTX and No-RTX groups (67 vs. 74 days, respectively, p = 0.141). Inhibition of antigen presentation by B cells with rituximab-based conditioning regimens does not appear to reduce the incidence of acute graft-versus-host disease in allogeneic hematopoietic stem cell transplant recipients.

Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.

Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.

The inhibition of CD1d-mediated antigen presentation upon an HSV-1 infection is VP22-dependent (VIR1P.1134)

Abstract CD1d-restricted T (NKT) cells are important for controlling a herpes simplex virus (HSV) infection. One of the mechanisms of immune evasion by HSV is to down-regulate CD1d-mediated activation of NKT cells. VP22 is an HSV-1-encoded protein responsible for reorganizing the host cell’s cytoskeletal network and viral spreading. We have shown that modification of the cytoskeleton can alter CD1d-mediated antigen presentation. In this study, we found an HSV-1 lacking VP22 (ΔUL49) was impaired in its ability to inhibit CD1d-mediated antigen presentation compared to wild-type (WT) virus; this was reversed by a repair virus (UL49R) in CD1d-expressing cells. Of importance, the effects of VP22 were shown to be exerted on the T322/S323 dyad, critical residues for the virus to reduced antigen presentation. We further demonstrated that CD1d recycling was inhibited by infection with WT and UL49R, but not ΔUL49 virus. Rather than inhibiting antigen presentation by CD1d, ectopic expression of VP22 in CD1d-expressing cells complemented the VP22-deficient virus in inhibiting antigen presentation. Therefore, VP22 is required (but not sufficient) for the inhibition of CD1d-mediated antigen presentation by an HSV-1 infection.

CTLA4-Ig treatment improves RBP4-induced adipose tissue inflammation and insulin resistance triggered by MyD88, JNK, ERK and p38 pathways (IRC8P.443)

Abstract Adipose tissue (AT) inflammation and impaired insulin action is a major cause of type 2 diabetes. RBP4 is an adipocyte- and liver-derived protein that has an important role in insulin resistance, metabolic syndrome and AT inflammation. RBP4 elevation causes AT inflammation by activating innate immunity that elicits an adaptive immune response. Our aims are to determine the signaling pathways involved in RBP4-induced macrophage activation and the resulting antigen presentation and Th1 polarization and whether the blockade of antigen presentation improves AT inflammation and insulin resistance. RBP4-overexpressing mice (RBP4-Ox) are insulin resistant and glucose intolerant and have increased AT macrophage and Th1 cell infiltration. In RBP4-Ox, AT macrophages display enhanced JNK, ERK and p38 phosphorylation, and in vitro inhibition of these pathways reduces macrophage activation and macrophage-induced CD4 T cell proliferation and Th1 polarization. Moreover, macrophages obtained from MyD88 knockout mice and activated with RBP4 do not secrete TNF, IL12 and IL-1b and fail to induce CD4 T cell proliferation and Th1 polarization. Treatment of RBP4-Ox mice with CLTA4-Ig reduces AT inflammation and improves insulin resistance. Thus, RBP4 causes insulin resistance, at least partly, through MyD88 pathway and downstream by activating JNK, ERK and p38 pathways. These pathways induce macrophage activation and Th1 polarization, which can be blocked by inhibiting antigen presentation.

Ethyl pyruvate, a modulator of dendritic cell activation and survival (INM1P.437)

Abstract Danger Signals induce dendritic cells (DCs) to mature and present antigen in a pro-inflammatory context. When remaining immature however, DCs confer tolerance. Hence the need for new protocols inducing tolerogenic DCs. We assessed the effects of ethyl pyruvate (EP), ester derivative of pyruvate with anti-inflammatory properties, on DC responses. We found that EP (10mM) abolished pro-inflammatory cytokine induction (IL-12, TNFa, IL-6) by the TLR ligand and strong DC activator LPS in bone marrow-derived dendritic cells (BMDCs) at 8, 24, 48 and 72h post-stimulation. Furthermore, EP inhibited the IFN-I response and the anti-inflammatory cytokine IL-10 induced by LPS stimulation. Determining the effects of EP on BMDC viability with a dose titration response showed that EP (10mM) strongly inhibited in vitro DC activation without inducing death. Moreover, EP could prevent LPS-induced killing that normally occurs 72h post-stimulation. EP also decreased the up-regulation of BMDC surface MHC-II and co-stimulatory molecules, suggesting a role in inhibiting antigen presentation. Investigating the mechanism of action, EP decreased Erk and JNK phosphorylation but not p38 phosphorylation. Our results indicate that EP inhibits most of the biological responses of DCs to LPS. We are currently examining further the molecular mechanisms of EP’s action in BMDCs to highlight novel targets to inhibit DC activation.

MECHANISMS OF VITAMIN D ACTION ON THE IMMUNE SYSTEM

Besides the well-known effects upon bone metabolism, vitamin D (VD) plays important roles in many other processes in the body, including immune regulation. VD action is carried out through its cellular membrane receptor, which is expressed in a variety of human organs and tissues, e.g., most cells of immune system, as well as epithelial cells lining the mucous membranes. The cell-membrane bound VD receptor is transferred to the cytoplasm, to form a functional complex with vitamin A and its receptor. This complex provides either inhibiting, or enhancing effect upon transcription of hundreds genes in the nuclear DNA, including those that regulate cell growth, differentiation, apoptosis, thus preventing malignancy and angiogenesis. The following effects of VD are supposed with respect to immune system: VD inhibits antigen presentation by dendritic cells, supresses Th1-cell differentiation and the production of Th1-cytokines, shifts the balance of Th1/Th2 cell responses towards the Th2 response, exerts inhibitory effect upon Th17 cells, promotes Treg cell development, and increases their activity. In addition, VD boosts production of «endogenous antibiotics» that may provide powerful effects upon Gram-positive and Gram-negative bacteria, fungi and viruses. Therefore, VD seems quite important for prevention of autoimmune and atopic diseases: multiple sclerosis, rheumatoid arthritis, type 1 diabetes, Crohn’s disease, ulcerative colitis, development of asthma in children and chronic obstructive pulmonary disease. VD protects from a wide range of infections, including tuberculosis, leprosy and respiratory infections, and prevents the development of several tumors. Almost half the population of different countries has a VD hypovitaminosis, often hidden and undiagnosed, and this can be a leading cause of weakened immunity and increased morbidity. The diagnostics of VD hypovitaminosis, prevention and treatment of hypovitaminosis should be among the most important healthcare tasks in Russia.

IL-6/STAT3 signaling impaired induction of cancer-antigen specific T cells via down-regulation of dendritic cells in tumor microenvironment.

590 Background: Immunosuppression in tumor microenvironments is a critical issue for cancer immunotherapy. Recently, the effectiveness of immuno-check point therapy has been reported on various types of solid tumors. But, the effectiveness in colorectal cancer was poor compared to other cancers, such as melanoma and renal cell carcinoma. Correct regulation of dendritic cell (DC) function in tumors is important for inducing anti-tumor immunity. We have been demonstrated that IL-6 inhibits antigen presentation by DCs through STAT3 activation in tumor-bearing mice. In this study, we focused on the role of the IL-6/STAT3 signaling cascade in human DCs. Methods: Both IL-6 and pSTAT3 expressions in tumor sites of colorectal cancers was verified by immunohistostaining. CD11b+CD11c+DCs in cancer tissues and PBMCs were isolated by fluorescence-activated cell sorting system and investigated about the surface molecules such as HLA-DR, T cell stimulating ability, and effector gene expression levels. Moreover, we investigated influence of IL-6/STAT3 signaling in human DCs in vitro. Results: The results of IHC revealed that IL-6 was preferentially produced by cancer-associated fibroblasts and immune cells in the tissues of colorectal cancers. In addition, it was confirmed that STAT3 was activated in tumor-infiltrating immune cells. Tumor infiltrating CD11b+CD11c+ DCs highly induced IL-6 gene, down-regulated surface expression of HLA-DR, and attenuated T cell stimulating ability. In vitro experiments showed that IL-6-mediated STAT3 activation reduced surface expression of HLA-DR. COX2, cathepsin L (CTSL), and arginase activity were are involved in the IL-6-mediated down-regulation of surface expression levels of HLA-DR expression levels on DCs. Gene expressions of CTSL, ARG1 as well as IL6 in tumor infiltrating CD11b+CD11c+DCs were much higher than those of PBMCs. Conclusions: IL-6-mediated STAT3 activation inhibits functional maturation of DCs, causing suppression of anti-tumor immunity in colorectal cancer. Therefore, inhibition of the IL-6/STAT3 signaling pathway could be a promising strategy for improving immunotherapies for colorectal cancer patients.

The Role of the Cytoplasmic RNA Sensor Retinoic Acid Inducible Gene- I (RIG-I) in Allogeneic Stem Cell Transplantation

The success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by acute graft-versus-host disease (GVHD). Improving the procedure depends on identifying the mechanisms that contribute to this damaging T cell reactivity, while preserving graft-versus-leukemia (GVL) activity against hematopoietic malignancies."Tonic" type I IFN signaling in BMT recipients and therapeutic application of recombinant IFN-α have been shown to play an important role in defining the balance between GVHD and GVL responses, but the molecular mechanisms inducing this protective response remains unknown. In this regard, pattern recognition receptors (PRRs) that detect cytosolic nucleic acids and lead to the production of large amounts of type I interferons (IFN-α/β) such as the family of RIG-I-like receptors (RLRs) are of particular interest. RLRs, a subfamily of the cytoplasmic DExD/H- box family of helicases, consist of three members: retinoic acid inducible gene I (RIG-I), melanoma differentiation factor 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). RIG-I senses viral and bacterial RNA to induce the production of type I interferons, proinflammatory cytokines and inflammasome activation. Double-stranded RNA (dsRNA) carrying a 5′-triphosphate (3pRNA) has been identified as the natural ligand for RIG-I and serves as a selective trigger for RIG-I signaling. Although initially characterized as a main regulator for antiviral host defense, mice deficient in components of the RLR and type I IFN signaling pathway develop inflammatory bowel disease (IBD)-related pathologies. Furthermore, patients suffering from IBD show a highly significant downregulation of RIG-I in ileal epithelial cells and in a recent meta-analysis of genome-wide association studies (GWAS) data, IFNAR1 and MDA-5 were identified as primary candidate genes in susceptible loci for IBD. Together, these results indicate that RLRs and type I IFN signaling have important functions in the suppression of IBD by yet ill defined mechanisms. Given that the pathophysiology of GVHD shares several features with inflammatory bowel disease (IBD), we tested the role of the RIG-I pathway in the context of allo-HSCT.We utilized MAVS-deficient mice, which lack a common adapter for RIG-I signaling, as recipients in an MHC-disparate (BALB/c into B6) model of allo-HSCT. Compared to wild-type (WT) B6 mice, MAVS-KO mice receiving allogeneic BM + T cells displayed significantly worse GVHD mortality (Fig.1 A). Given the enhanced GVHD observed in the absence of RIG-I signaling, we hypothesized that selective engagement of RIG-I by 3pRNA (RIG-I ligand) in vivo would protect recipients from GVHD. Using a B6 into BALB/c model we observed that i.v. administration of a selective RIG-I ligand on d-1 significantly reduced mortality, weight loss and intestinal GVHD histopathology (Fig. 1B and data not shown). In addition, the translocation of LPS and microorganisms from the bowel lumen through the damaged intestinal mucosa to the systemic circulation during pre-transplant conditioning represents a crucial step in GVHD pathophysiology. We observed that administration of RIG-I ligand prior to allo-HSCT augmented intestinal barrier function measured by less fluorescence in the serum after FITC-dextran gavage compared to untreated WT recipients (Fig. 1C). Moreover, RIG-I stimulation augmented epithelial regeneration as determined by organoid formation from freshly isolated crypts (Fig. 1D) and inhibited activation of dendritic cells (DCs) during pre-transplant conditioning (Fig. 1E). To investigate the impact of 3pRNA administration during GVT, we used luciferase+ A20 bioluminescence in B6 into BALB/c tumor challenge recipients demonstrating that RIG-I ligands do not limit GVL (data not shown). Taken together, our results (i) uncover a previously unknown role of the RIG-I-MAVS signaling pathway in GVHD and (ii) offer a novel strategy to foster epithelial regeneration and inhibit antigen presentation during pre-transplant conditioning, while maintaining GVL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interleukin-10: a pleiotropic regulator in pregnancy.

Pregnancy is a unique and well-choreographed physiological process that involves intricate interplay of inflammatory and anti-inflammatory milieu, hormonal changes, and cellular and molecular events at the maternal-fetal interface. IL-10 is a pregnancy compatible cytokine that plays a vital role in maintaining immune tolerance. A wide array of cell types including both immune and non-immune cells secret IL-10 in an autocrine and paracrine manner. IL-10 binds to a specific receptor complex and activates JAK-STAT and PI3K-Akt signaling pathways while inhibiting NF-κB signaling pathway. IL-10 exerts its anti-inflammatory effects mainly by decreasing pro-inflammatory cytokines such as IL-1, IL-6, IL-12, and TNF-α, by inducing heme oxygenase-1, and by inhibiting antigen presentation via blocking major histocompatibility complex (MHC) class II expression. Prior studies from our group and others have shown that IL-10 also functions as a potent protector against vascular dysfunction, and enhancement of IL-10 may serve as an immunotherapeutic intervention to treat adverse pregnancy outcomes. This review seeks to critically evaluate the archetypal functions of IL-10 as an immune suppressive factor as well as its novel functions as a vascular protector and modulator of endoplasmic reticulum (ER) stress and autophagy in the context of normal and adverse pregnancy outcomes.

Cooperation between Epstein-Barr virus immune evasion proteins spreads protection from CD8+ T cell recognition across all three phases of the lytic cycle.

CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IE<E<<L). Moreover, double-knockdown experiments showed that BILF1 and BNLF2a co-operate to further inhibit antigen presentation of L epitopes. Together, these data firstly indicate which potential immune-evasion functions are actually relevant in the context of lytic virus replication, and secondly identify lytic-cycle phase-specific effects that provide mechanistic insight into the immunodominance pattern seen for CD8+ T cell responses to EBV lytic antigens.

Cloning, expression and characterisation of a type II cystatin from Schistosoma japonicum, which could regulate macrophage activation.

Cystatin play an important role in parasite immune evasion. It is involved in many immune responses processes regulations such as inhibiting antigen presentation, modifying cytokines production and macrophage polarization. In recent years, more and more cystatins were used in treating some inflammatory diseases such as asthma and inflammation bowel diseases; however, cystatins from Schistosoma japonicum were rarely studied. In the present study, we have cloned a cystatin from the adult stage of Schistosoma japonicum, named as SjCystatin, and its sequence shares conserved domains with other type II family cystatins. It was further verified by enzyme inhibition assays. SjCystatin retained its inhibitory activity under a wide range of pH values and temperatures, can maintain its inhibitory activity at pH 6.5-7.5 and 37°C, respectively. Then, we investigated the effects of SjCystatin on the lipopolysaccharide (LPS)-induced activated RAW264.7. Results showed that SjCystatin inhibit LPS-induced nitric oxide production in a dose-dependent manner. LPS-induced TNF-α and IL-6 production began to be inhibited at least 6h after SjCystatin stimulation. SjCystatin significantly increased IL-10 production at 6h after stimulation and its effect on IL-10 production diminished quickly. These results imply that SjCystatin can induce M2 macrophage polarization and can be expected to serve as a potential drug source for the medication of inflammatory disorders like other cystatins.

Ethyl pyruvate, an inhibitor of high-mobility group box 1 (HMGB1) release, modulates dendritic cell activation and survival (TRAN3P.897)

Abstract Dendritic cells (DCs) activated by stimulatory danger signals initiate the detrimental anti-graft immune responses that lead to transplant rejection. High-mobility group box1 (HMGB1) is a nuclear DNA-binding protein that signals danger if released, contributing to DC activation and acute allograft rejection. Preventing HMGB1 release is a way to block its effects, promoting less active DCs. Here we assessed the effects of ethyl pyruvate (EP), proposed to potentially prevent HMGB1 release from cells, on DC responses. We found that EP (10mM) abolished pro-inflammatory cytokine (IL-12, IL-6 and TNFa) and IL-10 induction by the strong DC stimulus LPS in myeloid bone marrow-derived dendritic cells (BMDCs). EP also significantly decreased BMDC surface MHC expression, suggesting a possible role in inhibiting antigen presentation. We also determined EP effects on BMDC viability and found that EP strongly inhibited in vitro DC activation without inducing death. Furthermore, EP could prevent LPS-induced apoptosis that normally occurs 72h post-stimulation. Moreover, EP suppressed DC responses to other TLR stimuli including CpGs and R848. Our results indicate that EP inhibits most of the biological responses of DCs to LPS and other TLR stimuli. We are investigating the link between EP and HMGB1 release prevention from BMDCs. Testing the effect of HMGB1 blockade on DC activation will highlight a novel target to induce tolerogenic DCs and prolong solid organ graft survival.

Blocking oncogenic RAS enhances tumour cell surface MHC class I expression but does not alter susceptibility to cytotoxic lymphocytes

Mutations in the RAS family of oncogenes are highly prevalent in human cancer and, amongst its manifold effects, oncogenic RAS impairs the expression of components of the antigen presentation pathway. This allows evasion of cytotoxic T lymphocytes (CTL). CTL and natural killer (NK) cells are reciprocally regulated by MHC class I molecules and any gain in CTL recognition obtained by therapeutic inactivation of oncogenic RAS may be offset by reduced NK cell activation. We have investigated the consequences of targeted inactivation of oncogenic RAS on the recognition by both CTL and NK cells. Inactivation of oncogenic RAS, either by genetic deletion or inactivation with an inducible intracellular domain antibody (iDAb), increased MHC class I expression in human colorectal cell lines. The common RAS mutations, at codons 12, 13 and 61, all inhibited antigen presentation. Although MHC class I modulates the activity of both CTL and NK cells, the enhanced MHC class I expression resulting from inactivation of mutant KRAS did not significantly affect the in vitro recognition of these cell lines by either class of cytotoxic lymphocyte. These results show that oncogenic RAS and its downstream signalling pathways modulate the antigen presentation pathway and that this inhibition is reversible. However, the magnitude of these effects was not sufficient to alter the in vitro recognition of tumour cell lines by either CTL or NK cells.

E. coli Heat-labile Enterotoxin B Subunit as a Platform for the Delivery of HIV Gag p24 Antigen.

Multiple vaccination strategies have been devised against HIV-1 including delivery of HIV moieties in attenuated or replication defective recombinant microbial agents alone or in combination with priming agents in form of soluble proteins or naked DNA. For the priming agents to be effective, adjuvants might be essential in directing the immune response to a desired outcome. E. coli enterotoxin B subunit (LTB) is an effective adjuvant and carrier for other proteins and epitopes. Here we show that conjugation of HIV gag p24 to LTB enhances the T cell response to gag p24 by increasing rate of T cell division compared to other treatments. Because HIV vaccines are likely to be multivalent, we further investigated whether gag p24 inhibits antigen presentation of an unrelated antigen, OVA. Addition of gag p24 to OVA-responsive DO.11.10 cell culture did not have adverse effects on antigen presentation. Interestingly, the presence of LTB in these cultures significantly increased proliferation of DO.11.10 cells. In all, the results suggest the use of LTB to boost immune responses against HIV gag p24 in systemic priming regimens with oral recombinant HIV vaccines.

Simvastatin inhibits secretion of Th17‐polarizing cytokines and antigen presentation by DCs in patients with relapsing remitting multiple sclerosis

Statins, widely used cholesterol-lowering agents, have also been demonstrated to have antiinflammatory effects. Here, we characterize the capacity of simvastatin to target DCs and modulate T-cell priming and Th17-cell differentiation, in a cohort of patients with relapsing remitting multiple sclerosis (RRMS). We report that simvastatin inhibits IL-1β, IL-23, TGF-β, IL-21, IL-12p70, and induces IL-27 secretion from DCs in RRMS patients, providing an inhibitory cytokine milieu for Th17 and Th1-cell differentiation. The effect on DCs is mediated via induction of SOCS1, SOCS3, and SOCS7 gene expression, which are associated with the inhibition of STAT1, STAT3, and ERK1/2 phosphorylation. A geranylgeranyl transferase inhibitor replicated simvastatin's effects on DC cytokine secretion, implicating that simvastatin-induced depletion of isoprenoids mediates this effect. Simvastatin inhibited antigen presentation by DCs via suppression of the MHC class I expression, costimulatory molecules CD80 and CD40, and by inducing a dramatic loss of dendritic processes. The changes in DC morphology were also mediated via inhibition of geranylgeranylation. The therapeutic use of geranylgeranylation inhibitors may provide selective inhibition of key pathogenic cytokines that drive the autoimmune response in MS; their use represents a promising therapeutic approach that requires further clinical testing.

Transcriptome characterization of immune suppression from battlefield-like stress

Transcriptome alterations of leukocytes from soldiers who underwent 8 weeks of Army Ranger training (RASP, Ranger Assessment and Selection Program) were analyzed to evaluate impacts of battlefield-like stress on the immune response. About 1400 transcripts were differentially expressed between pre- and post-RASP leukocytes. Upon functional analysis, immune response was the most enriched biological process, and most of the transcripts associated with the immune response were downregulated. Microbial pattern recognition, chemotaxis, antigen presentation and T-cell activation were among the most downregulated immune processes. Transcription factors predicted to be stress-inhibited (IRF7, RELA, NFκB1, CREB1, IRF1 and HMGB) regulated genes involved in inflammation, maturation of dendritic cells and glucocorticoid receptor signaling. Many altered transcripts were predicted to be targets of stress-regulated microRNAs. Post-RASP leukocytes exposed ex vivo to Staphylococcal enterotoxin B showed a markedly impaired immune response to this superantigen compared with pre-RASP leukocytes, consistent with the suppression of the immune response revealed by transcriptome analyses. Our results suggest that suppression of antigen presentation and lymphocyte activation pathways, in the setting of normal blood cell counts, most likely contribute to the poor vaccine response, impaired wound healing and infection susceptibility associated with chronic intense stress.