Increased Major Histocompatibility Complex Class Research Articles

Targeted demethylation and activation of NLRC5 augment cancer immunogenicity through MHC class I

Impaired expression of MHC (major histocompatibility complex) class I in cancers constitutes a major mechanism of immune evasion. It has been well documented that the low level of MHC class I is associated with poor prognosis and resistance to checkpoint blockade therapies. However, there is lmited approaches to specifically induce MHC class I to date. Here, we show an approach for robust and specific induction of MHC class I by targeting an MHC class I transactivator (CITA)/NLRC5, using a CRISPR/Cas9-based gene-specific system, designated TRED-I (Targeted reactivation and demethylation for MHC-I). The TRED-I system specifically recruits a demethylating enzyme and transcriptional activators on the NLRC5 promoter, driving increased MHC class I antigen presentation and accelerated CD8+ T cell activation. Introduction of the TRED-I system in an animal cancer model exhibited tumor-suppressive effects accompanied with increased infiltration and activation of CD8+ T cells. Moreover, this approach boosted the efficacy of checkpoint blockade therapy using anti-PD1 (programmed cell death protein) antibody. Therefore, targeting NLRC5 by this strategy provides an attractive therapeutic approach for cancer.

Abstract C083: HDAC6 inhibition increases proteasome activity and modulates the myeloma immunopeptidome to promote cytotoxic T-cell activity

Abstract The proteasome functions as an essential component of immune surveillance by generating peptides from intracellular antigens that are then presented to cytotoxic T-cells. The vast majority of defined antitumor T-cell responses involves the proteasomal processing of intracellular proteins and their presentation in the context of major histocompatibility complex (MHC) class I molecules to antigenic peptide-specific CD8+ T-cells. Such antigenic peptides generated by the proteasome are generally 8-10 amino acids in length and mirror the linear sequence of the parental protein. However, immune evasion is a hallmark of cancer cells that disrupt the antigen processing and presentation machinery. Tumors develop strategies to evade host immunity and resist current immunotherapies, while others display a low tumor mutation burden limiting the presence of tumor antigenicity. Here, we postulated that pharmacologic enhancement of proteasome catalytic activity would increase MHC class I antigen presentation, modulate the myeloma immunopeptidome, and promote CD8+ T-cells anti-myeloma activity. A cell-based, high-throughput screen revealed that the histone deacetylase 6 (HDAC6) inhibitors tubastatin-A, ACY-738 and ACY-1215 increased proteasome activity in multiple myeloma (MM) cells. HDAC6 inhibitors increased pan-MHC class-I expression in MM cells as well as a panel of cancer cell lines. Treatment of MM patient tumor cells with HDAC6 inhibitors increased the cytotoxic effect of autologous patient-derived T-cells. Mechanism-based studies demonstrated that pharmacologic blockade or genetic ablation of the HDAC6 ubiquitin-binding (BUZ) domain disrupted HDAC6 association with HR23B, a substrate-shuttle factor that delivers ubiquitinylated cargo to proteasomes. HDAC6 inhibitors or HDAC6-BUZ domain deletion reduced the association of HR23B with HDAC6 and promoted binding of HR23B to proteasomes and a consequent increase in proteasome activity. The results validate the HDAC6-BUZ domain as an attractive drug discovery target and demonstrate that proteasome activators enhance antitumor immunity. Proteasome activators represent a paradigm-shifting approach to overcome mechanisms of immune escape in cancer cells. FDA-approved drugs that increase proteasome activity and boost antigen presentation and can be repositioned as cancer immunotherapeutics to overcome the existing bottlenecks in drug development. Taken together, our results highlight the breadth and magnitude of the proteasome in governing fundamental cellular processes and the immense potential of therapeutics that exploit proteasomes to treat human disease. Citation Format: Priyanka S. Rana, James J. Ignatz-Hoover, James J. Driscoll. HDAC6 inhibition increases proteasome activity and modulates the myeloma immunopeptidome to promote cytotoxic T-cell activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C083.

An Immunoproteasome Activator That Increases MHC Class I Antigen Presentation to Enhance Anti-Tumor Immunity

Evasion of immune destruction is a hallmark of cancer that thwarts the efficacy of immunotherapy and reduces patient benefit. The development of immunotherapies has sparked a revolution in oncology by releasing the power of the human immune system, but the identity of novel, actionable antigens exclusively presented on cancer cells remains elusive. Proteasomes are multi-subunit proteolytic complexes that selectively degrade intracellular proteins. Proteasomes play a critical in controlling myriad cellular processes including metabolism, cell cycle progression, signal transduction, protein quality control and differentiation. Importantly, proteasomes are also the source of most antigenic peptides presented to the immune system on major histocompatibility complex (MHC)-class I molecules. In this process, intracellular and viral proteins are degraded in the cytosol to 8 and 9-amino acid peptides, which are then transported into the endoplasmic reticulum, where they associate with MHC-class I molecules and are presented to the cell surface. Immune cells contain a highly specialized, cytokine-inducible proteasome variant, known as the immunoproteasome, in which the three constitutive catalytic subunits are replaced by homologous catalytic subunits. Immunoproteasomes are specially adapted for a role in MHC class I antigen processing and presentation to CD8 + T-cells. Here, we performed a high-through screen (HTS) to detect novel molecular entities that activated immuno-proteasomal catalytic activity. We identified a curated panel of molecules which not only enhanced hydrolysis of the cell-permeable substrate LLVY-R110 but also enhanced the presentation of MHC class I antigens. E.G7-Ova is a mouse lymphoma cell line derived by electroporation of EL4 cells (C57BL/6, H-2b, T lymphoma) with chicken ovalbumin ( OVA) cDNA. Proteasomes degrade OVA to generate the peptide SIINFEKL that is presented by murine MHC-class I H-2Kb molecules. E.G7-Ova cells were pretreated with compound A and co-cultured with the T-cell hybridoma B3Z that had been genetically-engineered to express a SIINFEKL-restricted t-cell receptor (TCR). Pretreatment of E.G7-Ova cells with compound A significantly increased tumor cell lysis by B3Z cells. The treatment of multiple myeloma (MM) cells with compound A also dramatically enhanced the expression of pan-MHC class I antigens. Treatment of MM cells with compound A increased the intracellular level of the proteasome activator PA28α/β, which is expressed by PSME1/2. Compound A treatment also enhanced hydrolysis of the fluorogenic peptide substrate Ac-ANW-AMC, which is selectively cleaved by the immunoproteasome β5-associated catalytic activity. Treatment of MM cells with compound A followed by mass spectrometry (MS) revealed that immunoproteasome activation modulated the relative presentation of individual antigenic peptides bound to MHC class I molecules. Pre-treatment of MM cells with compound A also significantly enhanced the anti-myeloma activity of human cytotoxic T-cells. Taken together, our results demonstrate that immunoproteasome activation has the potential to overcome immune escape and promote host immunity. In addition, our model systems now allows us to identify actionable tumor antigens on patient tumor cells as an indispensable tool for the development of cancer immunotherapies, e.g., T cell receptor (TCR)-transduced T-cells and patient-specific mRNA and peptide vaccines. Finally, immunoproteasome activators have the potential to contribute not only to cytotoxic T-cell mediated tumor lysis but also the treatment of intracellular infections and autoimmune and neurodegenerative proteinopathies characterized by the pathologic accumulation of toxic, proteinaceous aggregates.

A rationally designed cancer vaccine based on NIR-II fluorescence image-guided light-triggered remote control of antigen cross-presentation and autophagy.

Cancer vaccines represent a promising immunotherapeutic treatment modality. The promotion of cross-presentation of extracellular tumor-associated antigens on the major histocompatibility complex (MHC) class I molecules and dendritic cell maturation at the appropriate time and place is crucial for cancer vaccines to prime cytolytic T cell response with reduced side effects. Current vaccination strategies, however, are not able to achieve the spatiotemporal control of antigen cross-presentation. Here, we report a liposomal vaccine loading the second near-infrared window (NIR-II, 1000-1700nm) fluorophore BPBBT with an efficient photothermal conversion effect that offers an NIR-light-triggered endolysosomal escape under the imaging guidance. The NIR-II image-guided vaccination strategy specifically controls the cytosolic delivery of antigens for cross-presentation in the draining lymph nodes (DLNs). Moreover, the photothermally induced endolysosomal rupture initiates autophagy. We also find that the adjuvant simvastatin acts as an autophagy activator through inhibiting the PI3K/AKT/mTOR pathway. The light-induced autophagy in the DLNs together with simvastatin treatment cooperatively increase MHC class II expression by activating autophagy machinery for dendritic cell maturation. This study presents a paradigm of NIR-II image-guided light-triggered vaccination. The approach for remote control of antigen cross-presentation and autophagy represents a new strategy for vaccine development.

The Toll-like Receptor 7-Mediated Ro52 Antigen-Presenting Pathway in the Salivary Gland Epithelial Cells of Sjögren's Syndrome.

To investigate whether stimulation with toll-like receptor (TLR) 7 leads to pathways that proceed to tripartite motif-containing protein 21 (TRIM21) or Ro52/SS-A antigen presentation through major histocompatibility complex (MHC) class I in salivary gland epithelial cells (SGECs) from Sjögren's syndrome (SS) patients. Cultured SGECs from SS patients were stimulated with TLR7 agonist, loxoribine, and interferon-β. Cell lysates immunoprecipitated by anti-MHC class I antibody were analyzed by Western blotting. The immunofluorescence of salivary gland tissue from SS and non-SS subjects and cultured TLR7-stimulated SGECs was examined. Significantly increased MHC class I expression was observed in SS patients' ducts versus non-SS ducts; no significant difference was detected for ubiquitin. Upregulated MHC class I in the cell membrane and cytoplasm and augmented Ro52 expression were observed in SGECs stimulated with TLR7. The formation of peptide-loading complex (PLC), including tapasin, calreticulin, transporter associated with antigen processing 1, and endoplasmic reticulum-resident protein 57 in labial salivary glands (LSGs) from SS patients, was dominantly observed and colocalized with MHC class I, which was confirmed in TLR7-stimulated SGEC samples. These findings suggest that the TLR7 stimulation of SS patients' SGECs advances the process toward the antigen presentation of TRIM21/Ro52-SS-A via MHC class I.

Abstract B041: AR suppresses MHC Class I expression and T-cell response in prostate cancer

Abstract Immunotherapy is a treatment option that has had limited success with prostate cancer patients. The major histocompatibility complex (MHC) Class I plays a pivotal role in the adaptive immune response by presenting neo-antigens on the surface of cancer cells to CD8+ T-cells. Prostate cancer cells have markedly lower expression of MHC Class I genes compared to immune-responsive cancers. Loss of MHC Class I is also associated with more aggressive disease and immune evasion in prostate cancer. However, the molecular mechanisms that control MHC Class I downregulation in prostate cancer are still unknown. We hypothesize that increasing MHC Class I expression in prostate cancer cells will increase antigen presentation and improve the efficacy of immunotherapy agents. To investigate the mechanism of MHC Class I regulation in prostate cancer cells, we conducted a whole-genome CRISPRi flow cytometry screen. In this screen, C42B cells containing a non-catalytic Cas9 (dCas9) were infected with a lentivirus that contained a sgRNA library of 100,000 guides targeting 20,000 genes. Infected cells were selected and stained with a pan-Class I MHC antibody, sorted, and the highest and lowest 25-30% of MHC Class I expressing cells were collected for analysis. As expected, knockdown of MHC Class I genes such as B2M, HLA-A, and TAP1 decreased MHC Class I expression. Surprisingly, knockdown of AR and AR co-factors GRHL2 and FOXA1 resulted in a dramatic increase of MHC Class I surface expression. Additional AR inhibition experiments using enzalutamide, an AR degrader, or charcoal-stripped serum also showed increased MHC Class I expression over time. These in vitro results were substantiated with RNA expression analyses of patient biopsy samples taken before or after neoadjuvant enzalutamide treatment, which showed significantly increased MHC Class I expression post-treatment. Results from a small library CRISPR screen, showed that elimination of androgen response elements upstream of MHC Class I genes increased MHC expression, providing a mechanism by which AR regulates MHC Class I. Importantly, AR knockdown was shown to increase antigen-specific T-cell response in co-culture and transgenic mouse models. These observations were further validated in a mCRPC phase II clinical trial. Bulk RNA expression analyses comparing responders and non-responders to anti-PD1 therapy in enzalutamide treated patients, showed decreased expression of AR and increased expression of MHC Class I in patients who responded to immunotherapy. Overall, our data show that AR suppresses MHC Class I expression in prostate cancer, and that androgen-targeted therapies can increase antigen presentation and improve T-cell response. By understanding how AR regulates MHC Class I expression, we can identify new combination treatments utilizing AR signaling inhibition and immunotherapy that lead to a improved anti-cancer response. Citation Format: Lisa Chesner, Julie Graff, Fanny Polesso, Alexis Smith, Arian Lundberg, Martin Sjoestroem, Zheng Xia, Simon Linder, Andries Bergman, Alan Ashworth, David Quigley, Wilbert Zwart, Luke Gilbert, Amy Moran, Felix Feng. AR suppresses MHC Class I expression and T-cell response in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B041.

MHC Class I Enables MSCs to Evade NK-Cell-Mediated Cytotoxicity and Exert Immunosuppressive Activity.

Allogeneic mesenchymal stem/stromal cells (MSCs) are frequently used in clinical trials due to their low expression of major histocompatibility complex (MHC) class I and lack of MHC class II. However, the levels of MHC classes I and II in MSCs are increased by inflammatory stimuli, raising concerns over potential adverse effects associated with allogeneic cell therapy. Also, it is unclear how the host immune response to MHC-mismatched MSCs affects the therapeutic efficacy of the cells. Herein, using strategies to manipulate MHC genes in human bone marrow-derived MSCs via the CRISPR-Cas9 system, plasmids, or siRNAs, we found that inhibition of MHC class I-not MHC class II-in MSCs lowered the survival rate of MSCs and their immunosuppressive potency in mice with experimental autoimmune uveoretinitis, specifically by increasing MSC vulnerability to natural killer (NK)-cell-mediated cytotoxicity. A subsequent survey of MSC batches derived from 6 human donors confirmed a significant correlation between MSC survival rate and susceptibility to NK cells with the potency of MSCs to increase MHC class I level upon stimulation. Our overall results demonstrate that MHC class I enables MSCs to evade NK-cell-mediated cytotoxicity and exert immunosuppressive activity.

Abstract 1817: Epidermal growth factor receptor alters major histocompatibility complex class I expression on pancreatic cancer cells

Abstract Pancreatic cancer is one of the deadliest neoplasms, with a poor 5-year survival rate of just 10%. The ability of pancreatic cancer cells to evade the immune system hinders an anti-tumor response and contributes to the poor survival rate. In some cases, down regulation of major histocompatibility complex (MHC) class I cell-surface expression aids in immune evasion by preventing endogenous tumor antigens from being presented to cytotoxic T cells. The goal of this study is to evaluate the role epidermal growth factor receptor (EGFR) plays in the regulation of MHC class I expression by pancreatic cancer cells. Pancreatic cancer cell lines were treated with epidermal growth factor (EGF) or transforming growth factor alpha (TGF-alpha) to activate EGFR or with the small molecule drug erlotinib to inhibit EGFR. Immunoprecipitation, western blot analysis, and flow cytometry were used to analyze cells' MHC class I expression post treatment. Human pancreatic cancer cell lines (BxPC-3, T3M4, S2-013, Panc-1) were found to decrease MHC class I expression after EGFR activation by EGF. However, activation of EGFR via TGF-alpha led to differential effects on MHC class I expression in a cell line-dependent manner. EGFR inhibition by erlotinib led to increased MHC class I expression by greater than 2.5-fold on both human and mouse pancreatic cancer cells. Mechanistic studies were done to evaluate the role of src homology 2 phosphatase (SHP2) in EGFR's regulation of MHC class I in pancreatic cancer cells, since in head and neck cancer cells SHP2 can be phosphorylated by activated EGFR and subsequently dephosphorylate STAT1 and reduce MHC class I expression. Following SHP2 knockdown by siRNA transfection, a slight increase in MHC class I expression was observed on pancreatic cancer cells, suggesting SHP2 has a minor down-regulatory role in MHC class I expression in these cells. EGFR was found to co-immunoprecipitate with MHC class I, indicating that EGFR might directly affect MHC class I expression by facilitating its endocytosis. However, after EGFR inhibition, no change in the MHC class I cell surface turnover rate was observed. Other potential mechanisms for EGFR regulation of MHC class I expression are currently being evaluated. Our discoveries defining a role for activated EGFR in reducing MHC class I expression and revealing that EGFR inhibitors can boost MHC class I levels on cancer cells could facilitate the adoption of new, more effective therapeutic tactics that combine the use of EGFR inhibitors with immunotherapies to augment patients' immune systems for effective targeting and killing of cancer cells. Citation Format: Shelby M. Knoche, Joyce C. Solheim. Epidermal growth factor receptor alters major histocompatibility complex class I expression on pancreatic cancer cells [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 1817.

IL-10 Restores MHC Class I Expression and Interferes With Immunity in Papillary Thyroid Cancer With Hashimoto Thyroiditis.

The incidence of papillary thyroid cancer (PTC) with concomitant Hashimoto thyroiditis (HT) is increasing. Interleukin (IL)-10 is a cytokine previously reported to be elevated in this condition. Evidence from multiple human malignancies showed IL-10 participated in tumor immunity and exhibited therapeutic potential. The aim of this study is to investigate whether IL-10 interferes with tumor immunity in PTC with concomitant HT. Expression of IL-10 and major histocompatibility complex (MHC) class Ⅰ were compared with PTC tissues with or without concomitant HT. PTC cell lines K1 and TPC-1 were stimulated with IL-10 and analyzed for MHC class Ⅰ expression afterward. T-cell activation, production of IL-2 and interferon (IFN)-γ and programmed death-1 (PD-1) expression were assessed by coculture of donor peripheral blood lymphocytes (PBLs) with IL-10-pretreated PTC cells. Programmed death-ligand 1 (PD-L1) expression was measured in PTC tissues and IL-10-pretreated cells of K1 and TPC-1. Increased levels of IL-10 and MHC class Ⅰ were observed in PTC with concomitant HT. IL-10 stimulation increased MHC class Ⅰ expression of PTC cells in vitro. Coculture of PBLs with IL-10-pretreated PTC cells enhanced T-cell activation (% cluster of differentiation [CD]25+ of CD3+T cells) and increased IL-2 production along with decreased IFN-γ secretion and PD-1 expression. Reduced PD-L1 expression was seen in PTC + HT tissue samples and IL-10-stimulated PTC cell lines. Elevated IL-10 expression in PTC with concomitant HT restores MHC class Ⅰ expression and interferes with tumor immunity. The potential mechanism of IL-10 in tumor immunity needs further investigation.

IL-17A increases MHC class I expression and promotes T cell activation in papillary thyroid cancer patients with coexistent Hashimoto\u2019s thyroiditis

BackgroundThe incidence of coexisting papillary thyroid cancer (PTC) and Hashimoto’s thyroiditis (HT) is increasing. The impact of HT on PTC prognosis and its possible mechanism remains controversial. Interleukin-17A (IL-17A) has been reported to participate in the pathogenesis of multiple autoimmune diseases and cancers. The aim of this study is to investigate the role of IL-17A in PTC with coexistent HT and evaluate the changes in tumor antigenicity.MethodsExpression of IL-17A and major histocompatibility complex (MHC) class I molecules were compared on PTC tissue samples with or without HT. PTC cell lines K1 and TPC-1 were stimulated with IL-17A and analyzed for MHC class I expression afterwards. Cluster of differentiation (CD) 8+T cell activation, production of Interleukin-2 (IL-2) and Interferon-gamma (IFN-γ) as well as the programmed death-1 (PD-1) expression on lymphocytes were assessed by coculture of donor peripheral blood lymphocytes (PBLs) with IL-17A pretreated PTC cells.ResultsElevated IL-17A and MHC class I expression were observed in PTC tissue samples with coexistent HT. Stimulation of PTC cells with IL-17A effectively increased MHC class I expression in vitro. Coculture of PBLs with IL-17A pretreated PTC cells resulted in enhanced T cell activation (%CD25+ of CD3+T cells) and increased IL-2 production along with decreased IFN-γ secretion and PD-1 expression of the lymphocytes.ConclusionsPapillary thyroid cancer with coexisting Hashimoto’s thyroiditis presents elevated MHC class I expression, which may be the result of IL-17A secretion. T cell activation is enhanced in vitro by IL-17A and may provide future utility in PTC immunotherapy.

Autoimmunity in Parkinson's Disease: The Role of α-Synuclein-Specific T Cells.

Evidence from a variety of studies implicates a role for the adaptive immune system in Parkinson's disease (PD). Similar to multiple sclerosis (MS) patients who display a high number of T cells in the brain attacking oligodendrocytes, PD patients show higher numbers of T cells in the ventral midbrain than healthy, age-matched controls. Mouse models of the disease also show the presence of T cells in the brain. The role of these infiltrating T cells in the propagation of disease is controversial; however, recent studies indicate that they may be autoreactive in nature, recognizing disease-altered self-proteins as foreign antigens. T cells of PD patients can generate an autoimmune response to α-synuclein, a protein that is aggregated in PD. α-Synuclein and other proteins are post-translationally modified in an environment in which protein processing is altered, possibly leading to the generation of neo-epitopes, or self-peptides that have not been identified by the host immune system as non-foreign. Infiltrating T cells may also be responding to such modified proteins. Genome-wide association studies (GWAS) have shown associations of PD with haplotypes of major histocompatibility complex (MHC) class II genes, and a polymorphism in a non-coding region that may increase MHC class II in PD patients. We speculate that the inflammation observed in PD may play both pathogenic and protective roles. Future studies on the adaptive immune system in neurodegenerative disorders may elucidate steps in disease pathogenesis and assist with the development of both biomarkers and treatments.

IRG1 increases MHC class I level in macrophages through STAT-TAP1 axis depending on NADPH oxidase mediated reactive oxygen species

The major histocompatibility complex (MHC) is the connection between innate immunity and acquired immune system. Recently, many studies reported that the immunoresponsive gene 1 (IRG1) play an important role on innate immunity including reactive oxygen species (ROS), antiviral effect and expression of inflammatory factors. However, the function of IRG1 in antigen presenting remains unclear. In this study, we found that overexpressed-IRG1 promoted MHC I level instead of MHC II in macrophages membrane. Besides, IRG1 increased expression of some transporter proteins associated with antigen processing involving TAP1, PSMB9 depending on ROS. By detecting the activation of glucose-6-phosphate dehydrogenase (G6PD), we confirmed that IRG1 could increase ROS level by promoting pentose phosphate pathway (PPP). DPI, an inhibitor of NADPH oxidase (NOX), also significant attenuated TAP1 and MHC I level in IRG1-overexpressed macrophages. Finally, results showed that phosphorylation of STAT1/3 involved in IRG1-mediated TAP1 and MHC I expression. In conclusion, IRG1 increased MHC class I level in macrophages through STAT1/3-TAP1 axis depending on PPP and NOX mediated ROS.

Recipient Dendritic Cells Are Regulated By MiR-146a during Acute GvHD

Acute Graft-versus-host disease (GvHD) is a major immunological complication following allogeneic hematopoietic cell transplantation (allo-HCT) occurring in 30-40% of patients. Although immunosuppression with corticosteroids serves as standard first-line therapy in acute GvHD, sustained responses are achieved in less than 50% of patients. Therefore, the identification of risk factors and a better understanding of the molecular mechanisms underlying the disease are indispensable to develop novel therapeutic strategies. MicroRNAs control various key biological processes, including innate and adaptive immune responses. In particular, microRNA-146a (miR-146a) is emerging as a central regulator of innate immune cell signaling.Here we found that a G/C single nucleotide polymorphism (SNP) within the pre-miR-146a gene, which reduces the amount of miR-146a produced from the C allele, was strongly associated with the risk to develop severe acute GvHD. Patients with the rs2910164 CC genotype had a significantly higher risk to develop acute GvHD grade III-IV than all other allo-HCT recipients (P =0.002) as well as an overall increased histopathological GvHD severity. In order to functionally dissect the role of miR-146a in recipient cells during GvHD, we used gene targeted (miR-146a-/-) mice and a major histocompatibility complex (MHC)-mismatched experimental model for acute GvHD. MiR-146a deficiency of the recipient mice resulted in increased GvHD severity, elevated serum levels of pro-inflammatory cytokines and accelerated mortality compared to wild type (WT) recipients after allo-HCT. Chimeric mice with a lack of miR-146a specifically in the hematopoietic system showed an even more distinct disease exacerbation, whereas miR-146a deficiency only in non-hematopoietic recipient cells did not affect GvHD severity. Moreover, co-transplantation of recipient type miR-146a-/-dendritic cells (DCs) elicited a more aggressive course of GvHD, while overexpression of miR-146a in DCs using a specific miR-146a mimic ameliorated disease. Phenotypically, miR-146a-/- DCs displayed a more activated phenotype with increased MHC class II, CD80 and CD86 surface expression levels after endotoxin stimulation when compared to WT DCs. Global gene expression analysis revealed upregulation of the JAK-STAT signaling pathway in miR-146a-/- DCs, which we confirmed at the protein and phosphoprotein level using Western blot and flow cytometry. Importantly, inhibition of JAK-STAT signaling in DCs using the JAK1/2 inhibitor ruxolitinib prevented miR-146a-/- DC-induced GvHD exacerbation.In conclusion, our findings indicate that miR-146a acts as a central regulator of recipient type DCs during GvHD, by dampening cytokine receptor signaling via the JAK-STAT pathway. We detected the SNP rs2910164 as a marker to identify patients at high risk for GvHD before allo-HCT, facilitating the opportunity to apply pre-emptive interventions. DisclosuresNo relevant conflicts of interest to declare.

Mechanisms of action underlying the immunotherapeutic activity of Allovectin in advanced melanoma

Allovectin (velimogene aliplasmid) is a cancer immunotherapeutic currently completing a pivotal phase 3 study for metastatic melanoma. Consisting of a bicistronic plasmid encoding both major histocompatibility complex (MHC) class I heavy and light chains (HLA-B7 and β2-microglobulin, respectively) formulated with a cationic lipid-based system, it is designed for direct intratumoral administration. Following injection into a single lesion, the product is intended to induce anti-tumor immune responses against both treated and distal lesions. Both the plasmid and lipid components of Allovectin contribute to the biological activity of the drug product, and its therapeutic activity is hypothesized to derive from multiple mechanisms of actions (MOAs). These include the induction of both cytotoxic T-cell and innate immune responses directed against allogeneic as well as tumor-derived targets, consequences of both an increased MHC class I expression on tumor cells and the induction of a localized immune/inflammatory response. In this paper, we review Allovectin's proposed MOAs, placing their contributions in the context of anti-tumor immunity and highlighting both preclinical and clinical supporting data.

Leishmania amazonensis impairs DC function by inhibiting CD40 expression via A2B adenosine receptor activation

Dendritic cells (DCs) play an essential role in the modulation of immune responses and several studies have evaluated the interactions between Leishmania parasites and DCs. While extracellular ATP exhibits proinflammatory properties, adenosine is an important anti-inflammatory mediator. Here we investigated the effects of Leishmania infection on DC responses and the participation of purinergic signalling in this process. Bone marrow-derived dendritic cells (BMDCs) from C57BL/6J mice infected with Leishmania amazonensis, Leishmania braziliensis or Leishmania major metacyclic promastigotes showed decreased major histocompatibility complex (MHC) class II and CD86 expression and increased ectonucleotidase expression as compared with uninfected cells. In addition, L. amazonensis-infected DCs, which had lower CD40 expression, exhibited a decreased ability to induce T-cell proliferation. The presence of MRS1754, a highly selective A(2B) adenosine receptor antagonist at the time of infection increased MHC class II, CD86 and CD40 expression in L. amazonensis-infected DCs and restored the ability of the infected DCs to induce T-cell proliferation. Similar results were obtained through the inhibition of extracellular ATP hydrolysis using suramin. In conclusion, we propose that A(2B) receptor activation may be used by L. amazonensis to inhibit DC function and evade the immune response.

Interspecific hybridization increases MHC class II diversity in two sister species of newts

Our understanding of the evolutionary mechanisms generating variation within the highly polymorphic major histocompatibility complex (MHC) genes remains incomplete. Assessing MHC variation across multiple populations, of recent and ancient divergence, may facilitate understanding of geographical and temporal aspects of variation. Here, we applied 454 sequencing to perform a large-scale, comprehensive analysis of MHC class II in the closely related, hybridizing newts, Lissotriton vulgaris (Lv) and Lissotriton montandoni (Lm). Our study revealed an extensive (299 alleles) geographically structured polymorphism. Populations at the southern margin of the Lv distribution, inhabited by old and distinct lineages (southern Lv), exhibited moderate MHC variation and strong population structure, indicating little gene flow or extensive local adaptation. Lissotriton vulgaris in central Europe and the northern Balkans (northern Lv) and almost all Lm populations had a high MHC variation. A much higher proportion of MHC alleles was shared between Lm and northern Lv than between Lm and southern Lv. Strikingly, the average pairwise F(ST) between northern Lv and Lm was significantly lower than between northern and southern Lv for MHC, but not for microsatellites. Thus, high MHC variation in Lm and northern Lv may result from gene flow between species. We hypothesize that the interspecific exchange of MHC genes may be facilitated by frequency-dependent selection. A marginally significant correlation between the MHC and microsatellite allelic richness indicates that demographic factors may have contributed to the present-day pattern of MHC variation, but unequivocal signatures of adaptive evolution in MHC class II sequences emphasize the role of selection on a longer timescale.

MHC class II upregulation and colocalization with Fas in experimental models of immune-mediated bone marrow failure

To test the hypothesis that γ-interferon (IFN-γ) promotes major histocompatibility complex (MHC) class II expression on bone marrow (BM) cell targets that facilitate T-cell-mediated BM destruction in immune-mediated BM failure. Allogeneic lymph node (LN) cells were infused into MHC- or minor histocompatibility antigen-mismatched hosts to induce BM failure. MHC class II and Fas expression and cell apoptosis were analyzed by flow cytometry. MHC class II-Fas colocalization was detected by ImageStream Imaging Flow Cytometry and other cell-to-cell associations were visualized by confocal microscopy. T-cell-mediated BM cell apoptosis and effects of IFN-γ on MHC class II-Fas colocalization on normal BM cells were studied using cell culture invitro followed by conventional and imaging flow cytometry. BM failure animals had significantly upregulated MHC class II expression on CD4(-)CD8(-)CD11b(-)CD45R(-) residual BM cells and significantly increased MHC class II-Fas colocalization on BM CD150(+) and CD34(+) hematopoietic cells. MHC class II(+)Fas(+) BM cells were closely associated with CD4(+) T cells in the BM of affected animals, and they were significantly more responsive to T-cell-mediated cell apoptosis relative to MHC class II(-)Fas(-) BM cells. Infusion of IFN-γ-deficient LN cells into minor histocompatibility antigen-mismatched recipients resulted in no MHC class II-Fas upregulation and no clinically overt BM failure. Treatment with recombinant IFN-γ significantly increased both MHC class II-Fas coexpression and colocalization on normal BM cells. Elevation of the inflammatory cytokine IFN-γ-stimulated MHC class II expression and MHC class II-Fas colocalization, which may facilitate T-cell-mediated cell destruction.

Basophils as TH2‐inducing APCs: the dog can sing but is it a diva?

Basophils as T_H2‐inducing APCs: the dog can sing but is it a diva?

Cytotoxic T-lymphocyte–mediated killing of human pancreatic islet cells in vitro

Cytotoxic T lymphocytes (CTL) are believed to play an essential role in beta-cell destruction leading to development of type 1 diabetes and allogeneic islet graft failure. We aimed to identify the mechanisms used by CTL to kill human beta cells. CTL clones that recognize epitopes from influenza virus and Epstein-Barr virus restricted by human leukocyte antigen (HLA)-A0201 and -B0801, respectively, were used to investigate the susceptibility of human beta cells to CTL. In a short-term (5-hour) assay, CTL killed human islet cells of the appropriate major histocompatibility complex (MHC) class I type that had been pulsed with viral peptides. Killing was increased by pretreating islets with interferon gamma that increases MHC class I on target cells. Killing was abolished by incubation of CTL with the perforin inhibitor concanamycin A. The Fas pathway did not contribute to killing because blocking with neutralizing anti-Fas ligand antibody did not significantly reduce beta-cell killing. In conclusion, we report a novel way of investigating the interaction between CTL and human islets. Human islets were rapidly killed in vitro by MHC class I-restricted CTL predominantly by the granule exocytosis pathway.

Carbon black nanoparticles promote the maturation and function of mouse bone marrow-derived dendritic cells

Particulate matter including carbon black (CB) nanoparticles can enhance antigen-related inflammation and immunoglobulin production in vivo. Dendritic cells (DC) as antigen-presenting cells (APC) are the most capable inducers of immune responses. The present study was designed to determine whether CB nanoparticles affect the maturation/activation and function of DC in vitro. DC were differentiated from bone marrow (BM) cells of BALB/c mice by culture with granulocyte macrophage colony stimulating factor (GM-CSF). At day 8 of culture, BM-derived DC (BMDC) were exposed to CB nanoparticles with a diameter of 14 nm or 56 nm for 24 h. The expression of major histocompatibility complex (MHC) class II, DEC205, CD80, and CD86 (maturation/activation markers of BMDC) was measured by flow cytometry. BMDC function was evaluated by an allogeneic mixed lymphocyte reaction (MLR) assay. CB nanoparticles significantly increased the expression of DEC205 and CD86 in BMDC and tended to increase MHC class II and CD80 expression; however, a size-dependent effect was not observed. On the other hand, BMDC-mediated MLR was significantly enhanced by the CB nanoparticles and the enhancement was greater by 14 nm CB nanoparticles than by 56 nm CB nanoparticles. Taken together, CB nanoparticles can promote the maturation/activation and function of BMDC, which could be related to their effects on allergic diseases and/or responses. In addition, BMDC-mediated MLR might be useful assay for in vitro screening for adjuvant activity of environmental toxicants.