Surface Expression Of MHC Class Research Articles

Abstract 7479: Targeting the lipid kinase PIKfyve upregulates surface expression of MHC class I to augment cancer immunotherapy

Abstract The widespread success of cancer immunotherapies suggests a promising future - but has been subverted by unresponsiveness and resistance. Current research has furthered the understanding of immunotherapy resistance through the findings of “hot” (responsive) and “cold” (unresponsive) tumors. Here, we focus on the genetic and pharmacological inhibition of PIKfyve, a lipid kinase regulator of autophagic flux and lysosomal biogenesis. We find that inhibition of PIKfyve results in the upregulation of major histocompatibility complex class I (MHC-I) surface expression in cancer cells (through inhibition of autophagic flux), resulting in increased CD8+ T cell-mediated cancer cell death. These findings were corroborated in multiple syngeneic mouse models, which also displayed increased intratumoral functional CD8+ T cells. In addition, the Pikfyve-depletion antitumor responses are CD8+ T cell and MHC-I dependent - as CD8+ T cell and B2m knockout rescued tumor growth - resulting in improved response to immune checkpoint blockable (ICB), adoptive cell therapy, and a therapeutic vaccine. Further, high PIKfyve expression predicts poor ICB response and is prognostic of poor survival in ICB-treated cohorts. Ultimately, these findings collectively suggest PIKfyve as an effective target to enhance immunotherapies through elevation of MHC-I surface expression in cancer cells, and accordingly, PIKfyve inhibitors have novel potential to increase immunotherapy response in cancer patients. Citation Format: Parth Borker, Yi Bao, Yuanyuan Qiao, Arul Chinnaiyan, Jae Eun Choi, Yuping Zhang, Rahul Mannan, Caleb Cheng, Tongchen He, Yang Zheng, Jiali Yu, Mahnoor Gondal, Gabriel Cruz, Sara Sara Grove, Xuhong Cao, Fengyun Su, Rui Wang, Yu Chang, Ilona Kryczek, Marcin Cieslik, Michael D. Green, Weiping Zou. Targeting the lipid kinase PIKfyve upregulates surface expression of MHC class I to augment cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7479.

Targeting the lipid kinase PIKfyve upregulates surface expression of MHC class I to augment cancer immunotherapy.

Despite the remarkable clinical success of immunotherapies in a subset of cancer patients, many fail to respond to treatment and exhibit resistance. Here, we found that genetic or pharmacologic inhibition of the lipid kinase PIKfyve, a regulator of autophagic flux and lysosomal biogenesis, upregulated surface expression of major histocompatibility complex class I (MHC-I) in cancer cells via impairing autophagic flux, resulting in enhanced cancer cell killing mediated by CD8+ T cells. Genetic depletion or pharmacologic inhibition of PIKfyve elevated tumor-specific MHC-I surface expression, increased intratumoral functional CD8+ T cells, and slowed tumor progression in multiple syngeneic mouse models. Importantly, enhanced antitumor responses by Pikfyve-depletion were CD8+ T cell- and MHC-I-dependent, as CD8+ T cell depletion or B2m knockout rescued tumor growth. Furthermore, PIKfyve inhibition improved response to immune checkpoint blockade (ICB), adoptive cell therapy, and a therapeutic vaccine. High expression of PIKFYVE was also predictive of poor response to ICB and prognostic of poor survival in ICB-treated cohorts. Collectively, our findings show that targeting PIKfyve enhances immunotherapies by elevating surface expression of MHC-I in cancer cells, and PIKfyve inhibitors have potential as agents to increase immunotherapy response in cancer patients.

MCK2-mediated MCMV infection of macrophages and virus dissemination to the salivary gland depends on MHC class I molecules

Murine cytomegalovirus (MCMV) infection of macrophages relies on MCMV-encoded chemokine 2 (MCK2), while infection of fibroblasts occurs independently of MCK2. Recently, MCMV infection of both cell types was found to be dependent on cell-expressed neuropilin 1. Using a CRISPR screen, we now identify that MCK2-dependent infection requires MHC class Ia/β-2-microglobulin (B2m) expression. Further analyses reveal that macrophages expressing MHC class Ia haplotypes H-2b and H-2d, but not H-2k, are susceptible to MCK2-dependent infection with MCMV. The importance of MHC class I expression for MCK2-dependent primary infection and viral dissemination is highlighted by experiments with B2m-deficient mice, which lack surface expression of MHC class I molecules. In those mice, intranasally administered MCK2-proficient MCMV mimics infection patterns of MCK2-deficient MCMV in wild-type mice: it does not infect alveolar macrophages and subsequently fails to disseminate into the salivary glands. Together, these data provide essential knowledge for understanding MCMV-induced pathogenesis, tissue targeting, and virus dissemination.

Abstract 6257: Developing HDAC inhibitors as immunomodulators

Abstract Immune checkpoint inhibitors (ICIs) are standard of care for several solid tumor types, including renal cell carcinoma (RCC). However, the clinical success of these therapies is dampened by the fact that the majority of patients demonstrate either inherent resistance to treatment or develop secondary resistance following an initial response. One key mechanism of tumor-driven ICI resistance is the generation and recruitment of suppressive immune cell types, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), which coordinate to suppress anti-tumor T cell responses. Thus, there is a critical need for the development of novel combination strategies aimed at mitigating the presence and function of suppressive immune cells to improve the efficacy of ICIs in the clinic. Our laboratory has previously demonstrated that histone deacetylase inhibition (HDACi) is associated with a decrease in both the number and function of tumor-infiltrating Tregs and MDSCs in the RENCA tumor model. Thus, the overall goal of these studies both preclinically and clinically is to investigate the impact of the immunomodulatory effects of HDACi in combination with ICI therapy, with the overall hypothesis that HDACi will improve the efficacy of anti-PD1 therapy in RCC. Our pre-clinical data support this hypothesis by demonstrating increased tumor control and survival in RENCA tumor-bearing mice when treated with a combination of anti-PD1 and HDACi compared to single agent therapy. These increases were also associated with an increase in the activation state of anti-tumor immune cells, as demonstrated by increased surface expression of MHC Class II molecules on macrophages and a decrease in phenotypically defined monocytic MDSCs. ATACseq and RNAseq data generated from combination-treated animals also outline a gene expression signature associated with both myeloid and T cell activation. Interestingly, our clinical studies also afforded us the opportunity to investigate potential biomarkers associated with responder status, which will allow us to better predict patient outcomes and administer therapies accordingly. Accumulating evidence from two independent clinical trials run by our group suggests that high levels of suppressive CD11b+HLA-DRlo MDSCs at baseline in the peripheral blood is associated with lack of clinical response to the combination of HDACi and ICI. Overall, these data support the rationale for the combination of ICI and HDACi in the clinic, and provide a potentially useful biomarker through which we can predict the success of combination therapy. Citation Format: Sean Colligan, Li Shen, Christopher Rupert, Jonathan Bard, Roberto Pili. Developing HDAC inhibitors as immunomodulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6257.

New factors in the control of antigen presentation by MHC class I and II proteins

The process of antigen presentation by MHC molecules is one of the best studied cell biological processes. Many aspects and factors have been defined at the cell biological, biochemical and atomic level. Yet, it is still difficult to explain the exact antigens presented by MHC molecules, and it is unclear whether all factors involved in antigen presentation have been defined. We and others have performed knock-out and silencing screens to identify additional factors involved in the control of MHC class I and MHC class II expression. These have resulted in new types of biology and I will present one such an example. However, the system may not be saturated with factors. I will present two other screens for factors in control of MHC class I and MHC class II expression. These screens indicate how DNA silencers control MHC class I expression and how factors controlling the endosomal system and other systems contribute to cell surface expression of MHC class I and MHC class II molecules. We are in the process of placing these factors in the known biology of antigen presentation.

Microbial RNA, the New PAMP of Many Faces

Traditionally, pathogen-associated molecular patterns (PAMPs) were described as structural molecular motifs shared by different classes of microorganisms. However, it was later discovered that the innate immune system is also capable of distinguishing metabolically active microbes through the detection of a special class of viability-associated PAMPs (vita-PAMPs). Indeed, recognition of vita-PAMPs triggers an extra warning sign not provoked by dead bacteria. Bacterial RNA is classified as a vita-PAMP since it stops being synthesized once the microbes are eliminated. Most of the studies in the literature have focused on the pro-inflammatory capacity of bacterial RNA on macrophages, neutrophils, endothelial cells, among others. However, we, and other authors, have shown that microbial RNA also has down-modulatory properties. More specifically, bacterial RNA can reduce the surface expression of MHC class I and MHC class II on monocytes/macrophages and help evade CD8+ and CD4+ T cell-mediated immune surveillance. This phenomenon has been described for several different bacteria and parasites, suggesting that microbial RNA plays a significant immunoregulatory role in the context of many infectious processes. Thus, beyond the pro-inflammatory capacity of microbial RNA, it seems to be a crucial component in the intricate collection of immune evasion strategies. This review focuses on the different facets of the immune modulating capacity of microbial RNA.

Abstract 1661: Immune modulation by the dual-mechanism ERK inhibitor, ASTX029, in MAPK-activated tumor models

Abstract Whilst mitogen-activated protein kinase (MAPK) pathway inhibitors are approved therapies in mutant-BRAF driven cancers and have demonstrated a high response rate in the clinic, the duration of response is often short-lived. Approaches targeting the immune system have elicited durable responses and led to the approval of checkpoint inhibitors such as the anti-PD1 therapy, pembrolizumab, in indications where MAPK pathway activation is often observed, such as melanoma. Activation of the MAPK pathway has been associated with an immunosuppressive tumor microenvironment (TME). Further, preclinical studies have demonstrated that in addition to inhibiting MAPK activity in tumor cells, MAPK pathway inhibitors such as the BRAFV600E inhibitor dabrafenib or MEK inhibitor trametinib have promoted a more proinflammatory TME leading to upregulated antigen presentation on tumor cells, increased CD8+ T cell infiltration and tumor cell killing. Similar preclinical results were recently reported for a KRASG12C inhibitor, AMG 510, where treatment of in vivo models led to an increase in tumor infiltrating lymphocytes, macrophages, dendritic cells and an increase in active immune gene signatures leading to tumor immunity. We have recently described the discovery of ASTX029, which is currently undergoing clinical development in advanced solid tumors (NCT03520075). ASTX029 is a dual-mechanism ERK1/2 (ERK) inhibitor, inhibiting both the catalytic activity and phosphorylation of ERK, and shows potent inhibition of MAPK-activated tumor growth in preclinical models. Previous studies have demonstrated a difference in regulation of genes involved in response to type I interferon following treatment with dual-mechanism compared to catalytic ERK inhibitors. We investigated whether treatment with ASTX029 modulates antigen presentation and the TME in MAPK-activated tumors. Following treatment of cell lines in vitro, we observed ASTX029-dependent changes consistent with increased antigen presentation, including an increase in cell surface expression of MHC class I and the increase in gene expression of tumor-specific antigens gp100 and MART-1 following treatment of melanoma cells. We have previously demonstrated that ASTX029 has good bioavailability following oral dosing in mice. To further investigate the effect of ASTX029 on the TME, syngeneic tumors grown in immunocompetent mice dosed with ASTX029 were characterized in terms of immune cell composition of the TME and gene expression. Our results were consistent with increased immune activation, including increased interferon signaling and a change in the immune cell composition of the TME. These data demonstrate that treatment with ASTX029 leads to modulation of the TME and we hypothesize that to optimize therapeutic activity, ASTX029 could be partnered either with an immunomodulatory or tumor-directed agent. Citation Format: Chris Hindley, Andrea Biondo, Kim-Hien Dao, Matthew Davis, Lynsey Fazal, Roberta Ferraldeschi, Keisha Hearn, Vanessa Martins, Harpreet Saini, Martin Sims, Nicola Wallis, Nicola Wilsher, Harold Keer, John Lyons, Joanne Munck. Immune modulation by the dual-mechanism ERK inhibitor, ASTX029, in MAPK-activated tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1661.

Macrophage Involvement in the Response of Acute Myeloid Leukaemia to Chemotherapy

Acute myeloid leukemia (AML) is a clonally heterogeneous neoplasm with poor survival rates. Although chemotherapy often leads to clinical remission, the relapse rate of AML is very high and usually fatal. Chemotherapy is usually effective at debulking AML blasts however a small population capable of re-initiating the leukemia (LIC) survive ultimately causing relapse. Mouse models of AML suggest that LIC are protected from chemotherapy by a protective niche similar to hematopoietic stem cell (HSC) niches. As 1) bone marrow (BM) macrophages are key regulators of HSC niche function and 2) tumor-associated macrophages promote chemotherapy resistance in several models of solid tumors (e.g. breast, lung & ovarian) we investigated a potential role of BM non-malignant macrophages in promoting chemoresistance of AML blasts.Using a mouse model of AML driven by the MLL-AF9 oncogene in which mouse HSC are transduced with MSC-MLL/AF9-ires-GFP and transplanted into syngeneic recipients, we examined how AML progression alters the phenotype of non-malignant GFP-negative BM macrophages by flow cytometry. 18 days post-transplant, GFP+ leukemia burden in the BM was in the 40-60% range. In non-malignant GFPneg BM macrophages, cell surface expression of MHC class II, CD80, L-selectin and neuropilin-1 was significantly decreased suggesting that AML blasts alter the function of BM macrophages.To determine whether BM macrophages could have any role in AML response to chemotherapy, we first determine whether non-malignant BM macrophages were depleted in vivo by treatment with cytarabine (5 days, 100mg/kg) and doxorubicin (3 days, 1mg/kg). Two days after cytarabine+doxoR treatment, most CD11b+ F4/80- Ly6G+ granulocytes and CD11b+ F4/80+ CD169- monocytes were ablated from the BM with 0.1% granulocytes and 1.0 % monocytes surviving after treatment. In contrast CD11b- F4/80+ CD169+ VCAM1+ macrophages were the most resilient myeloid cells in the BM with 9.5% still present 2 days after chemotherapy.To further investigate a possible role of these chemotherapy-resilient CD169+ macrophages in promoting response of AML blasts to chemotherapy, we transplanted MLL-AF9 leukemia cells generated with wild-type HSC into CD169DTR/WT mouse recipients in which one CD169 allele is knocked-in with human diphtheria toxin receptor (DTR). This enables specific depletion of CD169+ macrophages in vivo by injecting diphtheria toxin (DT). In this model, DT treatment targets host CD169+ macrophages but not the AML blasts as they do not contain the DTR knock-in. In the absence of chemotherapy, depletion of recipient CD169+ macrophages did not alter leukemia burden in the BM, spleen or blood 48 hours post depletion and did not alter mouse survival (median survival 29-30 days in both CD169-DTR and WT recipients treated with DT or saline).However when cytarabine + doxoR were administered 48 hours after DT administration, median survival of CD169-DTR recipients was significantly increased to 39.5 days compared to wild-type recipients 35 days, p<0.001. Furthermore leukemia burden in both the blood and spleen was significantly reduced.To determine whether CD169 could by itself mediate lower response of AML to chemotherapy, we transplanted MLL-AF9 cells generated from WT HSC into CD169DTR/DTR mice which are knocked-out for both CD169 alleles. Deletion of both CD169 alleles in recipient mice did not change mouse survival in response to chemotherapy in the absence of DT. Therefore absence of CD169 is not sufficient to prolong mouse survival in response to chemotherapy.In conclusion, non-malignant CD169+ macrophages may protect MLL-AF9 leukemia blasts from the cytotoxic effects of cytarabine and doxorubicin. The mechanisms of this chemoprotection remain to be determined but do not involve a direct effect of CD169 on AML blasts. DisclosuresNo relevant conflicts of interest to declare.

Increased MHC Class I Expression on Platelets in Human and Murine Sepsis

Complications from sepsis are largely driven by maladaptive immune responses. Platelets impact innate and adaptive immune responses, but whether platelets regulate adaptive immune responses in sepsis, including CD8+ T cell responses, remains unknown. To explore a possible contribution of platelets to the altered immune response in sepsis, we used deep sequencing of ribosome-protected RNA (RPR) fragments (also called ribosome footprint profiling) to acquire a global picture of translation in platelets from healthy and septic individuals. Using this method, we identified altered translation of transcripts related to adaptive CD8+T cell responses. We further confirmed these changes using megakaryocyte cultures, and an in vivo model of murine sepsis. Finally, we employed in vitro models to examine their functional relevance.Genome-wide sequencing of ribosome-protected mRNA fragments of human platelets isolated from septic patients and healthy donors revealed more than 1,000 genes under active translation in platelets. Sepsis altered the total RNA expression and translation of more than 300 genes by at least 2 fold compared to healthy individuals. Among these, classic MHC class I genes including HLA-A, B, and C were actively translated, and increased in sepsis. For example the expression of HLA-A increased almost 3 fold (RPR of healthy 62±77 vs sepsis 166±340; N=4-5). HLA-A mRNA expression changes were confirmed by real-time qPCR. We further demonstrated consistent changes in platelet surface expression of MHC class I proteins by flow cytometry.Since platelets are anucleated and don't actively transcribe RNAs, we hypothesized that the increased mRNA of HLA-A in platelets initiated within megakaryocytes. To mimic the acute inflammation in vitro, human CD34+ derived megakaryocytes were treated with either interferon gamma at 250U/mL or LPS that was supplemented with lipid binding protein and CD14 at 100ng/mL for 16 hours. Treatments significantly increased HLA-A RNA expression (IFN-gamma 443±163% and LPS 161±41% as compared to untreated, N=5, P<0.01,) and cell surface MHC class I protein expression (MFI: IFN-gamma 220±60% and LPS 155±28% as compared to untreated, N=6, P<0.01).To examine whether sepsis directly induced MHC class I in mice, we used a murine sepsis model induced by cecal ligation and puncture (CLP). CLP induced approximately 40% mortality and a 50% decrease of platelet count. Deep sequencing of ribosome protected RNAs from control and CLP mice platelets showed a 6 fold increase in murine MHC class I expression in platelets (H2D and H2K) after CLP, (RPR counts: H-2D 36±29, 227±103; H-2K 2.5±1.3 vs 16±2.6; N=3). H2D protein increased on the platelet surface by 1.5 fold 48 h after CLP. This was associated with a significantly decreased CD8+ T cell population in the spleen.Activated platelets have been shown able to prime CD8+ T cells through MHC class I. To measure antigen cross-presentation by MHC class I, isolated platelets from CLP or sham control mice were pulsed with ovalbumin at 0.5mg/mL for 2 hours, and antigen cross-presentation was detected by an antibody specifically targeting the ovalbumin peptide SIINFEKL presented by H-2K using flow cytometry. Our studies indicated antigen cross-presentation by platelets significantly increased in the CLP group (MFI: Sham 21±2, CLP 42±24, N=3-8, P<0.05) compared to controls.Together, our studies demonstrate dynamic regulation of MHC class I on platelets. Expression of MHC class I mRNAs and active translation increase during sepsis in both human and mouse platelets. Furthermore, antigen cross-presentation by platelet MHC class I could be upregulated in sepsis and its effect on CD8+ T cell responses is under investigation. DisclosuresNo relevant conflicts of interest to declare.

Cytogenetic and Molecular Genetic Abnormalities of CIITA Gene in Patients with Primary Mediastinal (Thymic) Large B-Cell Lymphoma

Background. Primary mediastinal (thymic) large B-cell lymphoma (PMBCL) is an aggressive malignant lymphoproliférative disease which accounts for 2-3 % of all non-Hodgkin's lymphomas. In 40 % of PMBCL cases rearrangements of the MHC class II activator, i.e. CIITA gene, are observed. CIITA abnormalities lead to decreasing protein expression and surface expression of MHC class II, which results in lack of adaptive cell immunity targeted at tumor cells. Aim. To assess the rate and spectrum of cytogenetic and molecular genetic abnormalities of CIITA gene in PMBCL patients. Materials &amp; Methods. The study enrolled 37 patients with diagnosed PMBCL: 10 men and 27 women aged 21-61 years (median of 31 years). Sanger sequencing was performed in 36 patients. In 20 patients CIITA/16p13.13 FISH and in 15 patients standard cytogenetic analysis were carried out. Results. In 3 (8.3 %) out of 36 patients the sequencing method detected mutations impairing CIITA gene function, as well as microdeletion in exon 1, deletion and nucleotide substitution in a splice donor site. Multiple somatic variations in intron 1 were identified in 21 (58.3 %) patients: in 11 (52.4 %) cases there were deletions and single nucleotide variants (SNV); the other 10 (47.6 %) patients showed only SNVs. In 13 (61.9 %) out of 21 cases the abnormalities of promoter IV and/or alternative exon 1 were observed. In 5 (25 %) out of 20 patients the FISH assay identified CIITA gene translocation. Standard cytogenetic analysis detected complex karyotype in 7 (46.6 %) out of 15 patients. The comparison of data showed hypermutagenesis in 8 out of 10 patients with FISH-detected chromosome aberrations, and in 3 (37.5 %) of them complex karyotype aberrations were found as well. Conclusion. Molecular genetic methods identified different somatic variations in CIITA gene affecting its functionally important regions, which can be of special interest for further studying the biology of tumors, including PMBCL.

Calreticulin – a multifaced protein

Summary Calreticulin (CALR) is a highly conserved multi-function protein that primarily localizes within the lumen of the endoplasmic reticulum (ER). It participates in various processes in the cells, including glycoprotein chaperoning, regulation of Ca2+ homeostasis, antigen processing and presentation for adaptive immune response, cell adhesion/migration, cell proliferation, immunogenic cell death, gene expression and RNA stability. The role of CALR in the assembly, retrieval and cell surface expression of MHC class I molecules is well known. A fraction of the total cellular CALR is localized in the cytosol, following its retro-translocation from the ER. In the cell stress conditions, CALR is also expressed on the cell surface via an interaction with phosphatidylserine localized on the inner leaflet of the plasma membrane. The abovementioned mechanism is relevant for the recognition of the cells, as well as immunogenicity and phagocytic uptake of proapoptotic and apoptotic cells. Lastly, the presence of CALR exon 9 gene mutations was confirmed in patients with myeloproliferative neoplasms. Their presence results in an abnormal CALR structure due to the loss of its ER-retention sequence, CALR extra-ER localisation, the formation of a complex with thrombopoietin receptor, and oncogenic transformation of hematopoietic stem cells. It is also known that CALR exon 9 mutants are highly immunogenic and induce T cell response. Despite this fact, CALR mutant positive hematopoietic cells emerge. The last phenomenon is probably the result of the inhibition of phagocytosis of the cancer cells exposing CALR mutant protein by dendritic cells.

Plasmodium infection cure cycles induce modulation of conventional dendritic cells.

Malaria is one of the most widespread human infectious diseases worldwide and a cause of mortality. It is difficult to induce immunological memory against the malarial parasite Plasmodium. The immunity to clinical malaria disease is acquired with multiple infection and treatment cycles, along with substantial reduction in parasite burden. However, the mechanism of the acquired immunity remains largely unclear. Conventional DCs (cDCs) play a pivotal role in orchestration of immune responses. The purpose of this study is to analyze the characterization of cDCs after the infection and cure treatment cycles. Mice were infected with the lethal rodent malarial parasite Plasmodium berghei ANKA, which was followed by cure treatment with the antimalarial drug pyrimethamine. This was then followed by a challenge with live parasites. The mice that went through infection cure cycles showed significant immune response, demonstrating robust immunological memory against malaria parasites. We investigated the cytokine production capacity of splenic cDCs in both naive and infection cure mice by stimulating purified splenic cDCs with LPS (TLR4 agonist) or CpG (TLR9 agonist). The capacity of cytokine production was found to be significantly decreased in infection cure mice. The suppression of cytokine production was sustained for a long term (6 months). Moreover, the surface expression of MHC Class II molecules was significantly lower in infection cure mice than in naive mice. These results suggest that Plasmodium infection and cure treatment resulted in strong immunological memory and modulation of full functionality of cDCs.

Radiation-induced alterations in immunogenicity of a murine pancreatic ductal adenocarcinoma cell line

Pancreatic ductal adenocarcinoma (PDA) is highlighted by resistance to radiotherapy with the possible exception of hypofractionated irradiation. As single photon doses were reported to increase immunogenicity, we investigated dose-dependent irradiation effects on clonogenic survival, expression of immunologically relevant cell surface molecules and susceptibility to cytotoxic T cell (CTL) mediated killing using a murine PDA cell line. Clonogenicity decreased in a dose-responsive manner showing enhanced radioresistance at single photon doses below 5 Gy. Cell cycle analysis revealed a predominant G2/M arrest, being most pronounced 12 h after irradiation. Polyploidy increased in a dose- and time-dependent manner reaching a maximum frequency 60 h following irradiation with 10 Gy. Irradiation increased surface expression of MHC class I molecules and of immunological checkpoint molecules PDL-1 and CD73, especially at doses ≥ 5 Gy, but not of MHC class II molecules and CXCR4 receptors. Cytotoxicity assays revealed increased CTL lysis of PDA cells at doses ≥ 5 Gy. For the PDA cell line investigated, our data show for the first time that single photon doses ≥ 5 Gy effectively inhibit colony formation and induce a G2/M cell cycle arrest. Furthermore, expression levels of immunomodulatory cell surface molecules became altered possibly enhancing the susceptibility of tumour cells to CTL lysis.

An in vitro model of antibody-mediated injury to glomerular endothelial cells: Upregulation of MHC class II and adhesion molecules

Chronic active antibody-mediated rejection is a major cause of allograft failure in kidney transplantation. Microvascular inflammation and transplant glomerulopathy are defining pathologic features of chronic active antibody-mediated rejection and are associated with allograft failure. However, the mechanisms of leukocyte infiltration and glomerular endothelial cell injury remain unclear. We hypothesized MHC class II ligation on glomerular endothelial cells (GEnC) would result in upregulation of adhesion molecules and production of chemoattractants. A model of endothelial cell activation in the presence of antibodies to MHC classes I and II was used to determine the expression of adhesion molecules and chemokines. Murine GEnC were activated with IFNγ, which upregulated gene expression of β2-microglobulin (MHC class I), ICAM1, VCAM1, CCL2, CCL5, and IL-6. IFNγ stimulation of GEnC increased surface expression of MHC class I, MHC class II, ICAM1, and VCAM1. Incubation with antibodies directed at MHC class I or class II did not further enhance adhesion molecule expression. Multispectral imaging flow cytometry and confocal microscopy demonstrated MHC molecules co-localized with the adhesion molecules ICAM1 and VCAM1 on the GEnC surface. GEnC secretion of chemoattractants, CCL2 and CCL5, was increased by IFNγ stimulation. CCL2 production was further enhanced by incubation with sensitized plasma. Endothelial activation induces de novo expression of MHC class II molecules and increases surface expression of MHC class I, ICAM1 and VCAM1, which are all co-localized together. Maintaining the integrity and functionality of the glomerular endothelium is necessary to ensure survival of the allograft. IFNγ stimulation of GEnC propagates an inflammatory response with production of chemokines and co-localization of MHC and adhesion molecules on the GEnC surface, contributing to endothelial cell function as antigen presenting cells and an active player in allograft injury.

Comparative Genomic Analyses Defines Shared and Unique Features of cHL and PMBL and New Mechanisms of Sensitivity to PD-1 Blockade

Comparative Genomic Analyses Defines Shared and Unique Features of cHL and PMBL and New Mechanisms of Sensitivity to PD-1 Blockade

Metabolic Stress Triggers Immune Escape by Tumors

There is major interest in understanding the role of the tumor microenvironment in immune escape. In a recent report, Marijt etal. shed light on the signaling cascade that leads to decreased surface expression of MHC class I by tumor cells triggered by lowoxygen and glucose availability, revealing new opportunities for therapeutic intervention.

Autophagy promotes citrullination of VIM (vimentin) and its interaction with major histocompatibility complex class II in synovial fibroblasts

ABSTRACTWe aimed to investigate the involvement of macroautophagy/autophagy in autoimmunity in rheumatoid arthritis (RA) through citrullination of VIM (vimentin) and its interaction with MHC class II in synovial fibroblasts (SFs). The cell surface expression of MHC class II and B7 costimulatory molecules on SFs was analyzed by flow cytometry after treatment with IFNG/IFN-γ (interferon gamma). Intracellular citrullinated autoantigens in SFs were analyzed by immunoblotting using serum from anti-citrullinated peptide antibodies (ACPA)-positive patient as a primary antibody. SFs were incubated in serum-free medium or treated with proteasome inhibitor MG132 to induce autophagy. An autophagy inhibitor 3-methyladenin (3-MA) was used. Intracellular citrullinated VIM (cVIM) was evaluated by immunoblotting and immunocytochemistry. The interaction between MHC class II and cVIM was evaluated with co-immunoprecipitation and proximity ligation assay (PLA). We demonstrated that MHC class II, CD274/B7-H1 and PDCD1LG2/B7-DC were expressed on SFs following treatment with IFNG whereas CD276/B7-H3 was detected on SFs regardless of the presence of IFNG. ACPA-positive sera recognized a 54 kDa protein in SFs. By immunoprecipitation, the 54 kDa protein recognized by RA sera was revealed to be cVIM. Following induction of autophagy, intracellular cVIM was increased in SFs but the effect was canceled by 3-MA. The interaction between MHC class II and cVIM was demonstrated by co-immunoprecipitation. Furthermore, PLA revealed the significant increase of MHC class II-cVIM interaction following induction of autophagy. Our findings suggest that SFs may contribute to the autoimmunity in RA through citrullination of VIM and its interaction with MHC class II promoted by autophagy.Abbreviations: 3-MA: 3-methyladenine; ACPA: anti-citrullinated peptide antibodies; anti-CCP: anti-cyclic citrullinated peptide antibody; cVIM: citrullinated VIM; BECN1: beclin1; DAPI: 4ʹ,6-diamidino-2-phenylindole; FBS: fetal bovine serum; HLA: human leukocyte antigen; IFNG/IFN-γ: interferon gamma; IL6: interleukin 6; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence index; MHC: major histocompatibility complex; OA: osteoarthritis; PADI: peptidyl arginine deiminase; PepA: pepstatin A; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; RA: rheumatoid arthritis; SFs: synovial fibroblasts; siRNA: small interfering RNA; VIM: vimentin

The MHC class I-LILRB1 signalling axis as a promising target in cancer therapy.

Immune checkpoint inhibitors are among the newest, cutting-edge methods for the treatment of cancer. Currently, they primarily influence T cell adaptive immunotherapy targeting the PD-1/PD-L1 and CTLA-4/B7 signalling pathways. These inhibitors fight cancer by reactivating the patient's own adaptive immune system, with good results in many cancers. With the discovery of the "Don't Eat Me" molecule, CD47, antibody-based drugs that target the macrophage-related innate immunosuppressive signalling pathway, CD47-SIRPα, have been developed and have achieved stunning results in the laboratory and the clinic, but there remain unexplained instances of tumour immune escape. While investigating the immunological tolerance of cancer to anti-CD47 antibodies, a second "Don't Eat Me" molecule on tumour cells, beta 2 microglobulin (β2m), a component of MHC class I, was described. Some tumour cells reduce their surface expression of MHC class I to escape T cell recognition. However, other tumour cells highly express β2m complexed with the MHC class I heavy chain to send a "Don't Eat Me" signal by binding to leucocyte immunoglobulin-like receptor family B, member 1 (LILRB1) on macrophages, leading to a loss of immune surveillance. Investigating the mechanisms underlying this immunosuppressive MHC class I-LILRB1 signalling axis in tumour-associated macrophages will be useful in developing therapies to restore macrophage function and control MHC class I signalling in patient tumours. The goal is to promote adaptive immunity while suppressing the innate immune response to tumours. This work will identify new therapeutic targets for the development of pharmaceutical-based tumour immunotherapy.

Differential Roles of Dendritic Cells in Expanding CD4 T Cells in Sepsis.

Sepsis is a systemically dysregulated inflammatory syndrome, in which dendritic cells (DCs) play a critical role in coordinating aberrant immunity. The aim of this study is to shed light on the differential roles played by systemic versus mucosal DCs in regulating immune responses in sepsis. We identified a differential impact of the systemic and mucosal DCs on proliferating allogenic CD4 T cells in a mouse model of sepsis. Despite the fact that the frequency of CD4 T cells was reduced in septic mice, septic mesenteric lymph node (MLN) DCs proved superior to septic spleen (SP) DCs in expanding allogeneic CD4 T cells. Moreover, septic MLN DCs markedly augmented the surface expression of MHC class II and CD40, as well as the messaging of interleukin-1β (IL-1β). Interestingly, IL-1β-treated CD4 T cells expanded in a dose-dependent manner, suggesting that this cytokine acts as a key mediator of MLN DCs in promoting septic inflammation. Thus, mucosal and systemic DCs were found to be functionally different in the way CD4 T cells respond during sepsis. Our study provides a molecular basis for DC activity, which can be differential in nature depending on location, whereby it induces septic inflammation or immune-paralysis.

Recent Advances in Lung Cancer Immunotherapy: Input of T-Cell Epitopes Associated With Impaired Peptide Processing.

Recent advances in lung cancer treatment are emerging from new immunotherapies that target T-cell inhibitory receptors, such as programmed cell death-1 (PD-1). However, responses to anti-PD-1 antibodies as single agents are observed in fewer than 20% of non-small-cell lung cancer (NSCLC) patients, and immune mechanisms involved in the response to these therapeutic interventions remain poorly elucidated. Accumulating evidence indicates that effective anti-tumor immunity is associated with the presence of T cells directed toward cancer neoepitopes, a class of major histocompatibility complex (MHC)-bound peptides that arise from tumor-specific mutations. Nevertheless, tumors frequently use multiple pathways to escape T-cell recognition and destruction. In this regard, primary and acquired resistance to immune checkpoint blockade (ICB) therapy was associated with alterations in genes relevant to antigen presentation by MHC-class I/beta-2-microglobulin (MHC-I/β2m) complexes to CD8 T lymphocytes. Among additional known mechanisms involved in tumor resistance to CD8 T-cell immunity, alterations in transporter associated with antigen processing (TAP) play a major role by inducing a sharp decrease in surface expression of MHC-I/β2m-peptide complexes, enabling malignant cells to evade cytotoxic T lymphocyte (CTL)-mediated killing. Therefore, development of novel immunotherapies based on tumor neoantigens, that are selectively presented by cancer cells carrying defects in antigen processing and presentation, and that are capable of inducing destruction of such transformed cells, is a major challenge in translational research for application in treatment of lung cancer. In this context, we previously identified a non-mutant tumor neoepitope, ppCT16−25, derived from the preprocalcitonin (ppCT) leader sequence and processed independently of proteasomes/TAP by a mechanism involving signal peptidase (SP) and signal peptide peptidase (SPP). We also provided in vitro and in vivo proof of the concept of active immunotherapy based on ppCT-derived peptides capable of controlling growth of immune-escaped tumors expressing low levels of MHC-I molecules. Thus, non-mutant and mutant neoepitopes are promising T-cell targets for therapeutic cancer vaccines in combination with ICB. In this review, we summarize current treatments for lung cancer and discuss the promises that conserved neoantigens offer for more effective immunotherapies targeting immune-escaped tumor variants.