Expression Of MHC Molecules Research Articles

Newcastle Disease Virus Inhibits the Proliferation of T Cells Induced by Dendritic Cells In Vitro and In Vivo.

Newcastle disease virus (NDV) infects poultry and antagonizes host immunity via several mechanisms. Dendritic cells (DCs) are characterized as specialized antigen presenting cells, bridging innate and adaptive immunity and regulating host resistance to viral invasion. However, there is little specific knowledge of the role of DCs in NDV infection. In this study, the representative NDV lentogenic strain LaSota was used to explore whether murine bone marrow derived DCs mature following infection. We examined surface molecule expression and cytokine release from DCs as well as proliferation and activation of T cells in vivo and in vitro in the context of NDV. The results demonstrated that infection with lentogenic strain LaSota induced a phenotypic maturation of immature DCs (imDCs), which actually led to curtailed T cell responses. Upon infection, the phenotypic maturation of DCs was reflected by markedly enhanced MHC and costimulatory molecule expression and secretion of proinflammatory cytokines. Nevertheless, NDV-infected DCs produced the anti-inflammatory cytokine IL-10 and attenuated T cell proliferation, inducing Th2-biased responses. Therefore, our study reveals a novel understanding that DCs are phenotypically mature but dysfunctional in priming T cell responses during NDV infection.

T cells drive negative feedback mechanisms in cancer associated fibroblasts, promoting expression of co-inhibitory ligands, CD73 and IL-27 in non-small cell lung cancer

ABSTRACT The success of immune checkpoint therapy shows tumor-reactive T cells can eliminate cancer cells but are restrained by immunosuppression within the tumor micro-environment (TME). Cancer associated fibroblasts (CAFs) are the dominant stromal cell in the TME and co-localize with T cells in non-small cell lung cancer. We demonstrate the bidirectional nature of CAF/T cell interactions; T cells promote expression of co-inhibitory ligands, MHC molecules and CD73 on CAFs, increasing their production of IL-6 and eliciting production of IL-27. In turn CAFs upregulate co-inhibitory receptors on T cells including the ectonucleotidase CD39 promoting development of an exhausted but highly cytotoxic phenotype. Our results highlight the bidirectional interaction between T cells and CAFs in promoting components of the immunosuppressive CD39, CD73 adenosine pathway and demonstrate IL-27 production can be induced in CAF by activated T cells.

Proof of Concept Study: Mesoporous Silica Nanoparticles, From Synthesis to Active Specific Immunotherapy

Nanomaterials are increasingly valued tools in drug delivery research as they offer enhanced stability, controlled release and more effective drug encapsulation. Though yet to be introduced in clinical trial, mesoporous silica nanoparticles are promising delivery systems, due to their high chemical and mechanical stability while remaining biodegradable. This work provides proof of concept for particle based vaccines as cost-effective alternatives for dendritic cell immunotherapy. Synthesis and surface chemistry of the nanoparticles are optimized for protein conjugation and nanoparticles are characterized for their physicochemical properties and biodegradation. Ovalbumin is used as a model protein to load nanoparticles to produce a nanovaccine. The vaccine is tested in vitro on dendritic cultures to verify particle and vaccine uptake, toxicity, maturation effects and explicitly ovalbumin cross-presentation on MHC class I molecules. The optimized synthesis protocol renders reproducible mesoporous silica nanoparticles, resistant against agglomeration, within the required size range and have carboxylic surface functionalization necessary for protein conjugation. They are biodegradable over a time span of 1 week. This period is adjustable by changing synthesis parameters. UV sterilization of the particles does not induce quality loss, nor does it have toxic effects on cells. Treatment with mesoporous silica nanoparticles increases expression of MHC and costimulatory molecules of dendritic cells, indicating an adjuvant effect of nanoparticles on the adaptive immune system. Nanovaccine uptake and cross-presentation of ovalbumin are observed and the latter is increased when delivered by nanoparticles as compared to control conditions. This confirms the large potential of mesoporous silica nanoparticle based vaccines to replace dendritic-based active specific immunotherapy, offering a more standardized production process and higher efficacy.

Insulin-Reactive T Cells Convert Diabetogenic Insulin-Reactive VH125 B Cells Into Tolerogenic Cells by Reducing Germinal Center T:B Cell Interactions in NOD Mice.

Insulin is a key autoantigen in Type 1 Diabetes (T1D), targeted by both T and B cells. Therefore, understanding insulin-specific T:B cell interactions is important. We have previously reported an insulin-reactive CD4+ T cell, (designated 2H6). Unlike other insulin-reactive T cells, 2H6 cells protect non-obese diabetic (NOD) mice from T1D development, mediated by TGFβ. To investigate insulin-specific T:B cell interactions, we bred 2H6αβ T cell receptor transgenic NOD mice (2H6) with the insulin-reactive B cell receptor transgenic NOD mice (VH125), generating 2H6VH125 NOD mice. Similar to 2H6 mice, 2H6VH125 mice are protected from T1D development. Interestingly, VH125 B cells did not alter the phenotype of 2H6 T cells; however, 2H6 T cells significantly altered the VH125 B cells by reducing the insulin-reactive non-germinal center (GC) and GC B cells, as well as MHC and costimulatory molecule expression on the B cells. Furthermore, the B cells in 2H6VH125 NOD mice exhibited increased non-insulin-specific and a class switched IgG isotype, which can be recapitulated in vivo in Rag-deficient NOD mice by adoptive transfer. In vitro, VH125 B cells from 2H6VH125 mice suppressed the proliferation of 2H6 T cells to insulin antigen but enhanced TGFβ secretion by 2H6 T cells from 2H6VH125 mice compared to 2H6 mice. In summary, our data showed that 2H6 CD4+ T cells alter the phenotype and function of insulin-reactive B cells from pathogenic to tolerogenic cells. In turn, VH125 B cells also modulate the function of the 2H6 T cells. Thus, promoting the interactions between antigen-specific regulatory T cells and B cells may lead to protection from T1D.

Inducing Donor MHC Chimerism with Bone Marrow Derived Exosomes in Non-Human Primates

Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group via hematopoietic mixed chimerism consisting of an allogeneic bone marrow (BM) and kidney co-transplantation from the same donor after non-myeloablative conditioning. In this setting, T cell tolerance is believed to rely on expression of donor MHC molecules in the host's primary and secondary lymphoid organs. Here, we investigated whether donor MHC chimerism could be achieved using donor bone marrow derived extracellular vesicles (exosomes) instead of cells. Exosomes were isolated through serial ultracentrifugation from supernatants of donor BM cells cultured for 48 hours (expressing H38 MHC class I epitope). Two H38- cynomolgus macaque recipients received conditioning consisting of ATGAM and thymic irradiation, with or without total body irradiation (TBI) before a heart and kidney co-transplantation. Both recipients underwent a short course of immunosuppression with a calcineurin inhibitor and co-stimulation blockade with anti-CD154 mAb. A single dose of H38+ donor bone marrow derived exosomes was infused into H38- recipients on post-operative day 2. Image flow cytometry was used to detect the presence of recipient cells displaying donor MHC class I on their surface (cross-dressed) in the blood, thymus, spleen and lymph nodes. Infusion of donor bone marrow derived exosomes resulted in the presence of recipient peripheral blood leukocytes displaying donor MHC class I molecules in both animals. Flow cytometric analysis showed that MHC class I chimerism was restricted to the myeloid cell compartment. Tissues collected from the total body irradiated NHP showed that H38+ cross-dressed cells were also present in the lymph nodes, thymus, and spleen. In a separate study, a NHP recipient treated with an exosome protocol without TBI is currently 54 days post-transplantation with a well-functioning cardiac allograft. Donor MHC chimerism in the form of MHC cross-dressing can be achieved in a NHP model using donor bone marrow derived exosomes. These findings suggest that bone marrow derived exosomes could be used in place of whole bone marrow to induce allograft tolerance. The use of exosomes would allow for the reduction of recipient conditioning and would eliminate the risk of GVHD making this approach clinically safer than traditional BMT.

Dysbiotic oral microbiota and infected salivary glands in Sjögren's syndrome.

Key events in the pathogenesis of Sjӧgren syndrome (SS) include the change of salivary gland epithelial cells into antigen-presenting cell-like phenotypes and focal lymphocytic sialadenitis (FLS). However, what triggers these features in SS is unknown. Dysbiosis of the gut and oral microbiomes is a potential environmental factor in SS, but its connection to the etiopathogenesis of SS remains unclear. This study aimed to characterize the oral microbiota in SS and to investigate its potential role in the pathogenesis of SS. Oral bacterial communities were collected by whole mouthwash from control subjects (14 without oral dryness and 11 with dryness) and primary SS patients (8 without oral dryness and 17 with dryness) and were analyzed by pyrosequencing. The SS oral microbiota was characterized by an increased bacterial load and Shannon diversity. Through comparisons of control and SS in combined samples and then separately in non-dry and dry conditions, SS-associated taxa independent of dryness were identified. Three SS-associated species and 2 control species were selected and used to challenge human submandibular gland tumor (HSG) cells. Among the selected SS-associated bacterial species, Prevotella melaninogenica uniquely upregulated the expression of MHC molecules, CD80, and IFNλ in HSG cells. Concomitantly, P. melaninogenica efficiently invaded HSG cells. Sections of labial salivary gland (LSG) biopsies from 8 non-SS subjects and 15 SS patients were subjected to in situ hybridization using universal and P. melaninogenica-specific probes. Ductal cells and the areas of infiltration were heavily infected with bacteria in the LSGs with FLS. Collectively, dysbiotic oral microbiota may initiate the deregulation of SGECs and the IFN signature through bacterial invasion into ductal cells. These findings may provide new insights into the etiopathogenesis of SS.

Abstract 4976: Ex vivo functional characterization and in vivo efficacy validation of human CD40 agonistic antibodies in the human CD40 knock-in model (CD40 HuGEMM)

Abstract CD40 receptor is a member of TNF-receptor superfamily, broadly expressed on the surface of antigen-presenting cells (APC) including B cells, dendritic cells (DC), macrophages, monocytes, and non-APC like platelets, endothelial cells and even some tumor cells. CD40 is essential for APC activation and signalling via ligation of CD40 ligand located on helper T cells and CD40 on APC to mediate licensing (activating) of APC. Ligation of CD40 on DCs, for example, can induce increased surface expression of costimulatory and MHC molecules, production of pro-inflammatory cytokines, and enhanced T cell activation. CD40 ligation on resting B cells can increase its antigen-presenting function and proliferation. In contrast to its critical role in the induction of effective innate and adaptive immune responses, CD40 signalling on certain malignant cells, particularly B cell lymphomas, triggers tumor apoptosis. The dual function of CD40 signalling provide unique opportunities for the use of agonistic CD40 antibodies in cancer immunotherapy. Indeed, several CD40 monoclonal antibodies are now in different stages of clinical development, some shown promising preliminary results in combination with other cancer immunotherapies. However, we are lacking preclinical models to evaluate efficacy of therapeutic CD40 agonistic antibodies, which usually don’t cross-bind to mouse targets. To fill this gap, we developed a human CD40 knock-in model (CD40 HuGEMM), of which mouse exon 2-5 are replaced by human counterparts. These KI mice express a chimeric CD40 with human extracellular domain & mouse trans-membrane and intracellular sequences. We have characterized the human CD40 expression of CD40 HuGEMM on a broad spectrum of mouse cells, such as lymphocytes, macrophages, DCs, and endothelial cells. In addition, we confirmed that human CD40 antibodies bind to the chimeric m/hCD40 recombinant protein, and lead to increased expression of MHCII on the B cells. Finally, the in vivo efficacy study indicates that human CD40 antibody treatment lead to robust anti-tumor response and contribute to better survival of the mice. Taken together, our CD40 HuGEMM provides a powerful preclinical model to assess the efficacy of the human-specific CD40 agonistic antibodies. It may also serve as a model for proof of concept studies of CD40 agonistic antibodies with other therapies. Citation Format: Daniel Xianfei He, Lei Zheng, Ruilin Sun, Annie Xiaoyu An, Jian Fei, Henry Qixiang Li, Davy Xuesong Ouyang. Ex vivo functional characterization and in vivo efficacy validation of human CD40 agonistic antibodies in the human CD40 knock-in model (CD40 HuGEMM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4976.

Isorhapontigenin shows strong anti-melanoma cell activity, and induces anti-tumor immunity

Abstract Background Melanoma is one of the deadliest forms of skin cancer, and its incidence has continued to rise during the past decade. Isorhapontigenin (ISO) is a new derivative of stilbene compound, isolated from Chinese herb Gnetum Cleistostachyum with capability of antitumor. Purpose The potential anti-melanoma activity, impact on genes’ expression, as well as the influence of immune response against melanoma of ISO were detected in our studies in order to clarify its anti-melanoma effect and the corresponding mechanism. Methods Cytotoxicity of ISO on melanoma cell line B16F10 was studied by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Cell cycle analysis by flow cytometry, annexin V staining, RT-qPCR and Western blot were used to elucidate the mechanism of action of ISO at the cellular level. The in vivo anti-tumor ability of ISO was measured in C57BL/6 mouse syngraft models. Results ISO induced dose-dependent inhibitory effect on the proliferation of B16F10 cell by arresting cells at G0/G1 phase promoting the apoptosis of the cell, which was associated with an increase in the Bax:Bcl-2 ratio and decrease in the proliferative-related genes CYCLIN D1 and SURVIVIN in vitro. In addition, ISO could inhibit the tumor immune escape by promoting the expression of MHC molecules on the membrane of tumor cells, and decreased the infiltration of Tregs and MDSCs which involved in suppressing the anti-tumor effects of immune system. On the other hand, ISO increased the infiltration of T cells, NK cells and NKT cells, which was able to enhance the anti-tumor immune response. Conclusion Taken together, these findings indicate that ISO may be useful as a treatment for Melanoma and thus warrants further investigation.

Expression of MHC-I and II by Microglia and Lymphocytes in the Brain of Diet-Restricted Mice.

Microglia are immunocompetent cells of the central nervous system whose function is to preserve nervous tissue homeostasis; however, under inflammatory conditions, they are associated with tissue damage. Dietary restriction is a nutritional intervention used to delay the onset of chronic disease and inflammation, in addition to improving the functions of the immune system. The aim of this study was to analyze the effect of dietary restriction on microglial expression of MHC molecules. Adult female and male BALB/c mice were fed ad libitum (controls) or kept under dietary restriction (30% reduction in food intake) for 4 wk. Purified brain mononuclear cells were analyzed by flow cytometry staining for CD45, CD11b, MHC-I and MHC-II. Our results show that female animals under dietary restriction had a significant increase in MHC-I expression in microglia (mean fluorescence intensity=7,854 (control) vs. 10,628 (diet-restricted), arbitrary units; p=0.0108), along with increased frequencies of lymphocytes compared to controls (1.39% (control) vs. 7.85% (diet-restricted); p=0.0175), whereas male animals did not show significant differences between groups. Our data suggest a differential effect for dietary restriction on female and male animals, with this nutritional regimen predominantly affecting females. Increased expression of MHC-I by diet-restricted microglia may play a role in maintaining tolerance in the absence of antigenic stimulation.

Impact of tumor-infiltrating LAMP-3 dendritic cells on the prognosis of esophageal squamous cell carcinoma.

Dendritic cells (DCs) are the most potent antigen-presenting cells to induce cytotoxic T lymphocytes in the tumor environment. After acquiring antigens, DCs undergo maturation and their expression of MHC and co-stimulation molecules are enhanced, along with lysosome-associated membrane glycoprotein 3 (LAMP-3), which is a specific marker of mature DCs. In general, mature DCs are usually considered to be immunostimulatory in the cancer microenvironment. In addition, it is known that tumor-infiltrating lymphocytes (TILs) are associated with a good prognosis in esophageal squamous cell carcinoma (ESCC). However, few studies have targeted the interaction between DCs and TILs in the local immunity of ESCC. We investigated the localization of mature DCs within ESCC tissue and their relationship to TILs as well as the clinical outcome. We evaluated 80 ESCC patients who underwent surgical treatment without preoperative treatment, using immunohistochemistry with LAMP-3 and CD8. The results showed that LAMP-3 DCs were predominantly observed in the peritumoral area. Intratumoral CD8 T cells were found to be associated with a favorable prognosis, and the number of infiltrating LAMP-3 DCs was correlated with the number of intratumoral CD8 T cells. At the local tumor site, mature LAMP-3 DCs might be associated with increasing tumor infiltrating CD8 T cells.

A Phase 2 Study to Assess the Immunomodulatory Capacity of a Lecithin-based Delivery System of Curcumin in Endometrial Cancer.

Curcumin is a botanical with anti-tumor and immunomodulatory properties. We hypothesized that curcumin supplementation might influence inflammatory biomarker levels in endometrial carcinoma (EC). In this open-label, non-randomized phase 2 study (NCT02017353), seven EC patients consumed 2 g/day Curcumin Phytosome (CP) orally for 2 weeks. Blood was taken at baseline, days 1, 7, 14, and 21. The following analytes were measured: curcuminoids and metabolites, 56 inflammatory biomarkers, COX-2, frequencies of myeloid-derived suppressor cells, dendritic cells and NK cells, expression of MHC molecules on leukocytes and monocytes and activation/memory status of T cells. Patients completed quality of life (QoL) questionnaires at baseline and end of treatment. Curcumin metabolites were detectable in plasma upon CP intake. CP downregulated MHC expression levels on leukocytes (P = 0.0313), the frequency of monocytes (P = 0.0114) and ICOS expression by CD8+ T cells (P = 0.0002). However, CP upregulated CD69 levels on CD16− NK cells (P = 0.0313). No differences were observed regarding inflammatory biomarkers, frequencies of other immune cell types, T cell activation and COX-2 expression. A non-significant trend to improved QoL was observed. Overall, CP-induced immunomodulatory effects in EC were modest without significant QoL changes. Given the small population and the observed variability in inter-patient biomarker levels, more research is necessary to explore whether benefits of CP can be obtained in EC by different supplementation regimens.Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT02017353; www.clinicaltrialsregister.eu, identifier 2013-001737-40.

Intracellular HSP70L1 inhibits human dendritic cell maturation by promoting suppressive H3K27me3 and H2AK119Ub1 histone modifications.

Epigenetic regulation has been attracting increasing attention due to its role in cell differentiation and behaviors. However, the epigenetic mechanisms that regulate human dendritic cell (DC) differentiation and development remain poorly understood. Our previous studies show that extracellular heat shock protein 70-like protein (HSP70L1) is a potent adjuvant of Th1 responses via stimulating DCs when released from cells; however, the role of intracellular HSP70L1 in DC differentiation and maturation remains unknown. Herein, we demonstrate that intracellular HSP70L1 inhibits human DC maturation by suppressing MHC and costimulatory molecule expression, in contrast to the adjuvant activity of extracellular HSP70L1. The stability of intracellular HSP70L1 is dependent on DNAJC2, a known epigenetic regulator. Mechanistically, intracellular HSP70L1 inhibits the recruitment of Ash1l to and maintains the repressive H3K27me3 and H2AK119Ub1 modifications on the promoter regions of costimulatory, MHC and STAT3 genes. Thus, intracellular HSP70L1 is an inhibitor of human DC maturation. Our results provide new insights into the epigenetic regulation of cell development by intracellular HSP70L1.

Fecal bacteria from ulcerative colitis patients downregulate Tim-3-mediated inhibitory effects on monocytes in vitro

Tim-3 is highly expressed on monocytes and macrophages. Blocking Tim-3 was shown to promote macrophage activation. We previously showed that fecal bacteria from patients with active ulcerative colitis (UC) presented significantly higher capacity to stimulate monocyte activation, resulting in higher expression of MHC molecules, costimulatory molecules, and proinflammatory cytokines, but the underlying mechanism remained unclear. Here, we found that fecal bacteria could significantly downregulate the expression of Tim-3 on CD14+ classical monocytes in vitro. Compared to the monocytes from healthy individuals, the monocytes from UC patients not only presented lower Tim-3 expression directly ex vivo, but also presented lower Tim-3 expression after stimulation. Moreover, the extent of Tim-3 downregulation was higher in UC monocytes than in control monocytes. This effect was, at least in part, attributable to differences in fecal bacterium composition between UC patients and healthy controls, since when tested in unrelated volunteers, the fecal bacteria from UC patients presented higher capacity at mediating Tim-3 downregulation. Fecal bacteria also induced TNF-α and IL-6 secretion from monocytes, which was repressible by the Tim-3 ligand Galectin 9 (Gal-9). Interestingly, we found that monocytes from UC patients presented significantly reduced response to exogenous Gal-9, and the extent of Gal-9-mediated inhibition was directly correlated with the level of Tim-3 expression. Overall, our data suggested that the monocytes from UC patients presented lower Tim-3 expression and reduced response to exogenous Gal-9, and the fecal bacteria from UC patients could potently downregulate Tim-3 expression on monocytes in vitro.

A nanoparticle-incorporated STING activator enhances antitumor immunity in PD-L1-insensitive models of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) has few therapeutic options, and alternative approaches are urgently needed. Stimulator of IFN genes (STING) is becoming an exciting target for therapeutic adjuvants. However, STING resides inside the cell, and the intracellular delivery of CDNs, such as cGAMP, is required for the optimal activation of STING. We show that liposomal nanoparticle-delivered cGAMP (cGAMP-NP) activates STING more effectively than soluble cGAMP. These particles induce innate and adaptive host immune responses to preexisting tumors in both orthotopic and genetically engineered models of basal-like TNBC. cGAMP-NPs also reduce melanoma tumor load, with limited responsivity to anti-PD-L1. Within the tumor microenvironment, cGAMP-NPs direct both mouse and human macrophages (M), reprograming from protumorigenic M2-like phenotype toward M1-like phenotype; enhance MHC and costimulatory molecule expression; reduce M2 biomarkers; increase IFN-γ-producing T cells; augment tumor apoptosis; and increase CD4+ and CD8+ T cell infiltration. Activated T cells are required for tumor suppression, as their depletion reduces antitumor activity. Importantly, cGAMP-NPs prevent the formation of secondary tumors, and a single dose is sufficient to inhibit TNBC. These data suggest that a minimal system comprised of cGAMP-NP alone is sufficient to modulate the tumor microenvironment to effectively control PD-L1-insensitive TNBC.

Connection Between Dysbiotic Oral Microbiota and Deregulation of Salivary Gland Epithelial Cells in Sjogren's Syndrome

Background: Dysbiosis of the gut and oral microbiomes is a potential environmental factor in Sjogren's syndrome (SS), but its connection to the etiopathogenesis of SS remains unclear. This study aimed to characterize the oral microbiota in SS and to investigate its potential role in the pathogenesis of SS. Methods: Oral bacterial communities collected by whole mouthwash from 15 healthy individuals, 10 drug/radiation-induced sicca patients, and 25 primary SS patients were analyzed by pyrosequencing. Four SS-associated species and 2 control species were selected and used to challenge human submandibular gland tumor (HSG) cells. The expression of MHC I, MHC II, CD80, CD86, and IFNλ in HSG cells and the bacterial invasion into HSG cells were examined. Sections of labial salivary gland (LSG) biopsies from 8 non-SS subjects and 15 SS patients were subjected to in situ hybridization using universal and Prevotella melaninogenica-specific probes. Findings: The SS oral microbiota was characterized by an increased bacterial load and Shannon diversity. Although a substantial portion of the changes observed in SS patients overlapped with those in drug/radiation-induced sicca patients, SS-specific changes were identified. Among the selected SS-associated bacterial species, P. melaninogenica uniquely upregulated the expression of MHC molecules, CD80, and IFN λ in HSG cells. Concomitantly, P. melaninogenica efficiently invaded HSG cells. Ductal cells and the areas of infiltration were heavily infected with bacteria in the LSGs with focal lymphocytic sialadenitis. Interpretation: Dysbiotic oral microbiota is associated with deregulation of salivary gland epithelial cells and acquisition of IFN signatures through bacterial invasion into ductal cells. Funding Statement: This study was supported by a grant of the Korean Health Technology R&D Project (HI13C0016) from Ministry of Health & Welfare, Republic of Korea. Labial salivary gland biopsy specimens used in this manuscript are from the Sjogren’s International Collaborative Clinical Alliance [SICCA], funded under contract N01 DE-32636 by the National Institute of Dental and Craniofacial Research, with funding support from the National Eye Institute and Office for Research in Women’s Health. The funders had no role in study design, data collection, analysis, and interpretation, and writing of the report. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: All procedures involving human subjects and materials were performed in accordance with the Helsinki Declaration under approved protocols from the Institutional Review Boards at Seoul St. Mary’s Hospital (KC13ONMI0646) and at Seoul National University, School of Dentistry (S-D20140022, S-D20170004). Informed consent was obtained from all subjects.

Abstract 4722: 4SC-202 increases immunogenicity of tumor cells, induces infiltration of tumor microenvironment with cytotoxic T cells, and primes tumors for combinations with different cancer immunotherapy approaches

Abstract Various histone deacetylases (HDAC) inhibitors were described as beneficially affecting anti-tumoral immune response. Although different HDAC inhibitors were investigated in syngeneic tumor models, their mode of anti-tumoral action (MOA) is not yet fully understood. Here, we analyzed the anti-tumoral MOA of 4SC-202, an orally available clinical stage small molecule inhibitor targeting HDAC class I. To ensure the relevance for the clinical situation we used a clinically equivalent dosage regimen. 4SC-202's effect on expression of tumor-associated antigen (TAA) and MHC molecules was analyzed in vitro and in vivo. Anti-tumoral efficacy and the impact on tumor microenvironment (TME) were analyzed in syngeneic CT26 and C38 models. Transcriptome analysis was performed by RNA-Seq, and the composition of immune cell subpopulations was determined by flow cytometry and immunohistochemistry. 4SC-202 increased expression of TAA and MHC molecules on tumor cells in vitro and in vivo suggesting a beneficial effect on immunogenicity of tumor cells. 4SC-202 significantly inhibited growth of syngeneic tumors at a dose that was inefficacious in immunocompromised mice. IFN-γ and chemokine expression was increased, and pro-inflammatory IL-1β and IL-23 decreased in the TME of CT26 tumors following 4SC-202 treatment. Detailed analysis revealed that 4SC-202 increased the number of cytotoxic CD8+ T cells (CTLs) in the tumor core without affecting their number in blood. Since the abundance of T cells in the tumor is pre-requisite for the efficacy of immune checkpoint blockade as well as the agonistic 4-1BB antibody, combinations of 4SC-202 with anti-PD-1, and anti-4-1BB antibodies were tested in the C38 model. The response rate to the antibodies alone was low in this model reflecting the refractory clinical situation. 4SC-202 was able to control the tumor growth, but did not induce tumor regression, whereas combination therapies resulted in significantly longer survival and durable complete responses in up to 83% of animals for the combination with the anti-PD-1 antibody. 4SC-202 already demonstrated a favorable safety profile with a low rate of > grade 3 treatment-related adverse events (17%) in a phase I study in heavily pretreated hematological cancer patients. With the daily dosing for 14 consecutive days a disease control rate of 83%, 1 partial and 1 complete response could be achieved. Complimentary to these clinical data, 4SC-202's immune priming capacity offers further options for development in combination with various cancer immunotherapy approaches. Combination of 4SC-202 with PD-1 blockade is now under evaluation in a phase Ib/II clinical trial in advanced cutaneous melanoma patients refractory/non-responding to treatment with anti-PD-1 antibodies (‘SENSITIZE', NCT03278665). Citation Format: Svetlana Hamm, Tanja Wulff, Kerstin Kronthaler, Sabine Schrepfer, Ulrike Parnitzke, Anne Catherine Bretz, René Bartz. 4SC-202 increases immunogenicity of tumor cells, induces infiltration of tumor microenvironment with cytotoxic T cells, and primes tumors for combinations with different cancer immunotherapy approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4722.

Abstract LB-126: Nanoparticle-incorporated STING activator as an immunotherapeutic for PD-L1 resistant triple-negative breast cancer

Abstract Current immune checkpoint inhibitors (eg. PD-L1) shows limited efficacy for varies cancer types, especially the difficult-to-treat triple-negative breast cancer. This study uses liposomal nanoparticles (NPs) to deliver the STING agonist, cGAMP, as an alternative approach to amplify innate immune activation and anti-tumor treatment. We studied the impact of cGAMP-NPs on macrophage reprograming and activation by determining M1/M2 biomarkers on polarized M2(+) cells and cytokines from secreted supernatants. The critical role of STING/IFNAR signaling in cGAMP-NP-mediated response was also examined. The efficacy was further evaluated in an orthotopically-transplanted model (C3(1)Tag model) for basal-like triple-negative breast cancer (TNBC) and a spontaneous genetic engineered mouse (GEM) model of basal-like TNBC (C3(1)Tag GEM). Both of these preclinical models that are resistant to PD-L1 checkpoint blockade and most other therapies, which parallels TNBC in humans. cGAMP-NPs were shown to activate STING and induce both innate and adaptive host immune responses. Also, cGAMP-NPs accumulated within macrophages at the tumor site and reversed a pro-tumorigenic microenvironment. Effective tumor suppression was achieved by intravenous delivery in orthotopic and GEM of TNBC with no responsivity to anti-PD-L1 and in B16F10 melanoma with limited responsivity to anti-PD-L1. The anti-tumor immunity was achieved in a STING-dependent fashion that relies on T cells and macrophages but does not require prior knowledge of the tumor antigen. cGAMP-NPs were shown to induce M2-like macrophages to skew towards a M1-like phenotype, cytokine production, MHC and co-stimulatory molecule expression, enhanced CD4+ and CD8+ T cells infiltration, and tumor apoptosis. Moreover, cGAMP-NPs alone induced durable anti-tumor T cell responses and prevented the formation of secondary tumors. We demonstrated the amplified efficacy of liposome formulation relative to soluble cGAMP as an anti-tumor therapeutic that obviates the need of intratumoral injection. Our data suggested that cGAMP-NPs are a potent treatment regimen to modulate the microenvironment of tumors with limited or no responsivity to anti-PD-L1. Citation Format: Ning Cheng, Rebecca Watkins-Schulz, Robert Junkins, Clément David, Brandon M. Johnson, Stephanie A. Montgomery, Kevin J. Peine, David B. Darr, Hong Yuan, Karen P. McKinnon, Qi Liu, Lei Miao, Leaf Huang, Eric M. Bachelder, Kristy M. Ainslie, Jenny P-y Ting. Nanoparticle-incorporated STING activator as an immunotherapeutic for PD-L1 resistant triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-126.

Manipulating the epigenetic framework of T cells with histone deacetylase inhibitors for more robust and durable anti-tumor responses

Abstract Malignant cells often harbor an imbalance in histone acetyltransferase and histone deacetylase (HDAC) activity, which can be disrupted by HDAC inhibitors. We hypothesized that HDAC inhibition can boost anti-tumor T cell responses by modifying tumor cell phenotypes as well as directly altering the transcriptional framework of tumor-specific T cells. To test this, we treated two murine breast cancer cell lines, TS/A and 4T1, with class I (entinostat), or pan-specific (panobinostat), HDAC inhibitors. Culture of tumor cells with either inhibitor increased surface expression of MHC and costimulatory molecules as well as T cell chemoattractants. Tumor growth was significantly impaired by HDAC inhibition in a manner absolutely dependent on adaptive immunity, specifically CD8 T cells and IFNγ production, but not CD4 T cells or B cells. Indeed, CD8 T cell production of effector cytokines was dramatically augmented. This enhanced effector function was paralleled by an increase in T-bet and a decrease in Eomes, consistent with a transcriptional program that is less susceptible to exhaustion. We also found that simply adjusting timing of HDAC inhibition relative to T cell activation could abolish anti-tumor effects or lead to rejection in nearly 50% of mice. These findings corresponded with a significant shift in the responding T cell repertoire. Collectively, our data suggests that HDAC inhibition reprograms the transcriptional profile of T cells in ways that impact priming, improve effector functions, and reduce susceptibility to exhaustion. Thus, appropriately timed HDAC inhibition may synergistically potentiate existing tumor immunotherapies, like T cell reinvigoration strategies.

1185 DNA hypermethylation of MHC class-I genes is associated with reduced expression and survival in melanoma

The aim of this study is to determine the role of DNA methylation in regulating expression of MHC class-I associated genes in melanoma. Reduced MHC class-I expression on the tumor cell surface is an important mechanism of escaping immunosurveillance, and is commonly seen in melanoma. DNA methylation is an epigenetic change that can silence gene expression, and has been implicated in regulating antigen expression and recognition in several cancer types. Abnormal DNA methylation is nearly universal in melanoma, however its role in regulating MHC molecule expression in melanoma is largely unknown. In this study, we investigated the methylation of MHC class-I associated molecules in melanoma tumors from The Cancer Genome Atlas. We uncovered that DNA hypermethylation of MHC class-I genes is a frequent event in melanoma, that hypermethylation is significantly associated with reduced gene expression, and identify the key CpG loci regulating expression. We found that methylation of beta-2 Microglobulin (B-2M) and HLA-B, in particular, may play important roles in melanoma survival. Patients with hypermethylation of B-2M exhibited a twofold decrease in median overall survival (HR =1.73, p=0.0001, Log-rank test). Hypermethylation of HLA-B was associated with significantly decreased 5-year overall survival (48% vs 68%, HR=1.68, p=0.0001, Log-rank test). Univariate and multivariate Cox proportional hazard analysis showed that DNA methylation of B-2M (p=5.8e-8) and HLA-B (p=5.15e-5) are prognostic factors of melanoma overall survival, independent of pathologic stage, age, gender, ulceration or Breslow thickness. Our results suggest that DNA hypermethylation may silence MHC class-I expression, and negatively impact melanoma survival. Based on our findings, targeting DNA methylation of B-2M and HLA-B may be a therapeutic strategy to restore expression of HLA class-I molecules, and potentially achieve improved immune responses in melanoma.

Characterization of a secreted cystatin of the parasitic nematode Haemonchus contortus and its immune-modulatory effect on goat monocytes

BackgroundHaemonchosis is a disease of the small ruminant caused by a nematode parasite Haemonchus contortus, and it is most important and alarming challenges to the small ruminant’s production. The infection of the H. contortus could cause high economic losses worldwide. H. contortus is a blood feeding parasite which penetrates into the abomasal mucosa to feed the blood of the host and causing the anemia and decreased total plasma protein. Modulation and suppression of the immune response of the host by nematode parasites have been reported extensively, and the cysteine protease inhibitor (cystatin) is identified as one of the major immunomodulators.MethodsThe recombinant protein of HCcyst-3 was expressed in a histidine-tagged fusion soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, as well as papain, were identified by fluorogenic substrate analysis. Native HCcyst-3 protein was localized by an Immunohistochemical test. The immunomodulatory effects of HCcyst-3 on cytokine secretion, MHC molecule expression, NO production and phagocytosis were observed by co-incubation of rHCcyst-3 with goat monocytes.ResultsWe cloned and produced recombinant cystatin protein from H. contortus (rHCcyst-3) and investigated its immunomodulatory effects on goat monocyte. The rHCcyst-3 protein is biologically functional as shown by its ability to inhibit the protease activity of cathepsin L, cathepsin B, and papain. The immunohistochemical test demonstrated that the native HCcyst-3 protein was predominantly localized at the body surface and internal surface of the worm’s gut. We demonstrated that rHCcyst-3 could be distinguished by antisera from goat experimentally infected with H. contortus and could uptake by goat monocytes. The results showed that the engagement of rHCcyst-3 decreased the production of TNF-α, IL-1β and IL-12p40. However, it significantly increased the secretion of IL-10 and TGF-β1 in goat monocytes. After rHCcyst-3 exposure, the expression of MHC-II on goat monocytes was restricted. Moreover, rHCcyst-3 could upregulate LPS induced NO production of goat monocytes. Phagocytotic assay by FITC-dextran internalization showed that rHCcyst-3 inhibited the phagocytosis of goat monocytes.ConclusionsOur results suggested that the recombinant cystatin from H. contortus (rHCcyst-3) significantly modulated goat monocyte function in multiple aspects.