Major Histocompatibility Complex Regulation Research Articles

Abstract 860: Pan-tissue master regulator inference reveals mechanisms of MHC alterations in cancers

Abstract Introduction: Major histocompatibility complex (MHC) genes are an integral part of the adaptive immune system. The T-cells can detect and kill cancer cells through the recognition of mutant peptides on the cell surface via the MHC. As a result, many cancers evade the immune system by altering their MHC regulation which can also lead to resistance to immune checkpoint blockade (ICB). Therefore, understanding MHC regulation in both baseline (normal state) and cancer is critical for uncovering mechanisms behind MHC alterations. Here, we have undertaken a pan-tissue evaluation to comprehensively decode MHC transcriptional regulation in healthy tissues and various cancers using a large compendium of single-cell RNA-seq (scRNA-seq) datasets. Methods: To study MHC class I (MHC-I) and MHC class II (MHC-II) transcriptional regulation at baseline we leveraged a large-scale scRNA-seq dataset encompassing 18 tissues and 124 epithelial cell types. Upon re-clustering and annotation, we investigated MHC expression across cell types and tissues and quantified its heterogeneity using the Gini coefficient. Next, we sought to identify transcription factors (TF) predictive of MHC-I and MHC-II expression, for that, we employed a master regulator inference that utilizes uni- and multi- regression models. Further, to investigate MHC and nominated TFs dysregulation in cancer, we curated a compendium of scRNA-seq datasets comprising 43 datasets, 20 cancer types, across 538 patients. Results: In normal epithelial cell types, we observed a large range (~8 fold) in MHC-I expression. We also detected MHC-II expression in unexpected cell types such as club cells in the prostate. We found that lung epithelial cells showed elevated MHC-II expression. Immunohistochemistry was performed to validate these findings. Using master regulator inference, we nominated 104 and 107 TFs which predict MHC-I and MHC-II expression, respectively. The nominated TFs showed significant enrichment (p < 0.001) for known TFs, alongside putative novel transcriptional regulators such as YBX1 (MHC-I p < 2e-16, MHC-II p = 8e-07) and XBP1 (MHC-I p < 2e-16, MHC-II p = 7e-04). Using our single-cell compendium of 20 cancer types we also observed a pan-cancer repression in MHC genes (p < 0.001). Next, we utilized an ICB cohort to identify specific TFs that could predict therapeutic benefits. Interestingly, XBP1 (p < 1e-16) and SP100 (p < 2.2e-08) were strong predictors of patients' survival. We also observed a large heterogeneity in MHC-II expression among colon cancer patients. Upon investigation, we found that patients with high MHC-II expression were mismatch repair deficient, this implicates MHC-II upregulation as an immune evasion mechanism. Conclusion: In conclusion, we have comprehensively evaluated MHC expression in 124 epithelial cell types and across 20 cancers identifying TFs predictive of MHC-I and MHC-II expression and their effects on patient outcomes. Citation Format: Mahnoor N. Gondal, Rahul Mannan, Yi Bao, Jing Hu, Marcin Cieslik, Arul M. Chinnaiyan. Pan-tissue master regulator inference reveals mechanisms of MHC alterations in cancers [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 860.

Interleukin-22 suppresses major histocompatibility complex II in mucosal epithelial cells.

Major histocompatibility complex (MHC) II is dynamically expressed on mucosal epithelial cells and is induced in response to inflammation and parasitic infections, upon exposure to microbiota, and is increased in chronic inflammatory diseases. However, the regulation of epithelial cell-specific MHC II during homeostasis is yet to be explored. We discovered a novel role for IL-22 in suppressing epithelial cell MHC II partially via the regulation of endoplasmic reticulum (ER) stress, using animals lacking the interleukin-22-receptor (IL-22RA1), primary human and murine intestinal and respiratory organoids, and murine models of respiratory virus infection or with intestinal epithelial cell defects. IL-22 directly downregulated interferon-γ-induced MHC II on primary epithelial cells by modulating the expression of MHC II antigen A α (H2-Aα) and Class II transactivator (Ciita), a master regulator of MHC II gene expression. IL-22RA1-knockouts have significantly higher MHC II expression on mucosal epithelial cells. Thus, while IL-22-based therapeutics improve pathology in chronic disease, their use may increase susceptibility to viral infections.

Endothelial mechanical stretch regulates the immunological synapse interface of renal endothelial cells in a sex-dependent manner.

Increased mechanical endothelial cell stretch contributes to the development of numerous cardiovascular and renal pathologies. Recent studies have shone a light on the importance of sex-dependent inflammation in the pathogenesis of renal disease states. The endothelium plays an intimate and critical role in the orchestration of immune cell activation through upregulation of adhesion molecules and secretion of cytokines and chemokines. While endothelial cells are not recognized as professional antigen-presenting cells, in response to cytokine stimulation, endothelial cells can express both major histocompatibility complex (MHC) I and MHC II. MHCs are essential to forming a part of the immunological synapse interface during antigen presentation to adaptive immune cells. Whether MHC I and II are increased under increased mechanical stretch is unknown. Due to hypertension being multifactorial, we hypothesized that increased mechanical endothelial stretch promotes the regulation of MHCs and key costimulatory proteins on mouse renal endothelial cells (MRECs) in a stretch-dependent manner. MRECs derived from both sexes underwent 5%, 10%, or 15% uniaxial cyclical stretch, and immunological synapse interface proteins were determined by immunofluorescence microscopy, immunoblot analysis, and RNA sequencing. We found that increased endothelial mechanical stretch conditions promoted downregulation of MHC I in male MRECs but upregulation in female MRECs. Moreover, MHC II was upregulated by mechanical stretch in both male and female MRECs, whereas CD86 and CD70 were regulated in a sex-dependent manner. By bulk RNA sequencing, we found that increased mechanical endothelial cell stretch promoted differential gene expression of key antigen processing and presentation genes in female MRECs, demonstrating that females have upregulation of key antigen presentation pathways. Taken together, our data demonstrate that mechanical endothelial stretch regulates endothelial activation and immunological synapse interface formation in renal endothelial cells in a sex-dependent manner.NEW & NOTEWORTHY Endothelial cells contribute to the development of renal inflammation and have the unique ability to express antigen presentation proteins. Whether increased endothelial mechanical stretch regulates immunological synapse interface proteins remains unknown. We found that antigen presentation proteins and costimulatory proteins on renal endothelial cells are modulated by mechanical stretch in a sex-dependent manner. Our data provide novel insights into the sex-dependent ability of renal endothelial cells to present antigens in response to endothelial mechanical stimuli.

Blockade of endothelin-1 receptor B regulates molecules of the major histocompatibility complex in sickle cell disease.

Major Histocompatibility Complex (MHC) molecules have been proposed to play a role in Sickle Cell Disease (SCD) pathophysiology. Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion, and excess levels of endothelin-1 (ET-1) contributing to vaso-occlusive crises. ET-1 activates endothelial cells, induces oxidative stress and inflammation, and alters erythrocyte volume homeostasis. However, the role of ET-1 on MHC regulation in SCD is unclear. We first studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βS-Antilles and Berkeley (BERK) mice. We observed significant increases in H2-Aa mRNA levels in spleens, lungs, and kidneys from transgenic sickle mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). Mice treated for 14 days with ET-1 receptor antagonists significantly reduced H2-Aa mRNA levels. We characterized the effect of ET-1 on MHC class II expression in the human endothelial cell line EA.hy926. We observed dose-dependent increases in the expression of MHC class II (HLA-DRA) and MHC transcription factor (CIITA) that were significantly blocked by treatment with BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the HLA-DRA promoter, an event blocked by BQ788 treatment. These results implicate ET-1 as a novel regulator of MHC class II molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD.

Abstract 015: Hypertensive Stretch On The Regulation Of The Immunological Synapse And Secretomes Of Male And Female Renal Endothelial Cells

Immune cells and inflammation contribute to the development of hypertension and renal injury. While endothelial cells are recognized as professional antigen presenting cells, they can express both Major Histocompatibility Complex (MHC) I and II and possess antigen-presenting functions. Endothelial cell activation and inflammation are critical in the development of hypertension. However, the mechanisms by which inflammatory hypertensive stimuli regulate renal endothelial immunological synapse proteins remains unknown. We hypothesize that inflammatory hypertensive mechanical stretch promotes the regulation of MHC I and II and costimulatory molecules on mouse renal endothelial cells (MRECs) in a sex-dependent manner. MRECs derived from both sexes underwent normotensive (5%; 1 Hz), hypertensive (10%, 1 Hz), and supra-hypertensive (15% 1 Hz) uniaxial cyclical stretch for 48 hours. We then assessed CD80, CD86, MHC II, MHC I, CD70, and CD40 by immunoblot analysis. A proteome multiplex assay was used to analyze male and female MREC secretomes during 5%, 10%, and 15% stretch. Hypertensive 10% stretch significantly increased only CD40 expression in males, while it upregulated CD80 and CD70 expression in females (p<0.05). Under 15% stretch, males significantly upregulated MHC I, CD70, and CD40 compared to I-Ab, CD80, CD86, and CD70 in females MRECs (p<0.05). We investigated sex differences and found that females significantly increased CD70 in both 10% and 15% stretch compared to males MRECs (p<0.05). We compared sex differences from our secretome proteomics in male and female MRECs at 10% and 15% stretch. We found that pathways associated with wound healing, Th17 activation, IL-6, and NF-κB signaling were upregulated in male MRECs undergoing 10% stretch. Interestingly, Th17 activation, natural killer cell signaling, and IL-23 signaling were downregulated in female MRECs undergoing 10% stretch. Under 15% stretch, IL-13 signaling, HMBG1 signaling, B cell signaling, and IL-13 signaling were downregulated in MRECs from both sexes. Our data provides novel insights into how inflammatory hypertensive stretch regulates immunological synapse proteins and secretomes in renal endothelial cells by stretch and sex-dependent manner.

Tight Regulation of Major Histocompatibility Complex I for the Spatial and Temporal Expression in the Hippocampal Neurons.

The expression and function of immune molecules, such as major histocompatibility complex (MHC), within the developing and adult brain have been discovered over the past few years. Studies utilizing classical class I MHC knockout animals suggest that these molecules, in fact, play essential roles in the establishment, function, and modification of synapses in the CNS. Altered neuronal expression of class I MHC, as has been reported in pathological conditions, leads to aberrations in neuronal development and repair. In the hippocampus, cellular and molecular mechanisms that regulate synaptic plasticity have heretofore been extensively studied. It is for this reason that multiple studies directed at better understanding the expression, regulation, and function of class I MHC within the hippocampus have been undertaken. Since several previous reviews have addressed the roles of class I MHC in the formation and function of hippocampal connections, the present review will focus on describing the spatial and temporal expression of class I MHC in developing, healthy adult, and aging hippocampus. Herein, we also review current literatures exploring mechanisms that regulate class I MHC expression in murine hippocampus. With this review, we aim to facilitate a deeper mechanistic understanding into the complex tight regulation of MHC I expression in hippocampus, which are needed as we explore the potential for targeting MHC I for therapeutic intervention in normal aging and in neurodegenerative diseases in the future.

Haplotype-Specific Expression Analysis of MHC Class II Genes in Healthy Individuals and Rheumatoid Arthritis Patients.

HLA-DRB1 alleles have been associated with several autoimmune diseases. For anti-citrullinated protein antibody positive rheumatoid arthritis (RA), HLA-DRB1 shared epitope (SE) alleles are the major genetic risk factors. In order to study the genetic regulation of major histocompatibility complex (MHC) Class II gene expression in immune cells, we investigated transcriptomic profiles of a variety of immune cells from healthy individuals carrying different HLA-DRB1 alleles. Sequencing libraries from peripheral blood mononuclear cells, CD4+ T cells, CD8+ T cells, and CD14+ monocytes of 32 genetically pre-selected healthy female individuals were generated, sequenced and reads were aligned to the standard reference. For the MHC region, reads were mapped to available MHC reference haplotypes and AltHapAlignR was used to estimate gene expression. Using this method, HLA-DRB and HLA-DQ were found to be differentially expressed in different immune cells of healthy individuals as well as in whole blood samples of RA patients carrying HLA-DRB1 SE-positive versus SE-negative alleles. In contrast, no genes outside the MHC region were differentially expressed between individuals carrying HLA-DRB1 SE-positive and SE-negative alleles, thus HLA-DRB1 SE alleles have a strong cis effect on gene expression. Altogether, our findings suggest that immune effects associated with different allelic forms of HLA-DR and HLA-DQ may be associated not only with differences in the structure of these proteins, but also with differences in their expression levels.

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.

Abstract LB236: Utilizing endometrial tumor organoids to model cancer immunomodulation

Abstract Endometrial cancer is the most common gynecologic malignancy in the United States. Most women present with early stage disease and have a favorable prognosis. However, the treatment options for recurrent endometrial cancer is limited. Immunotherapy has become increasingly popular in the treatment of recurrent endometrial cancer. A hallmark of malignant cells is immune-evasion at both the genetic level and in the tumor microenvironment. We hypothesized that an endometrial cancer organoid model could be utilized to study the tumor immune microenvironment. Major Histocompatibility Complex (MHC) is a protein receptor recognized with its antigen by immune cells to screen either non-self or tumorous cells. It has a crucial role in eliminating non-self cells, such as cancer cells. We first assessed the genes involved in the regulation of MHC I and II in cultured normal and tumor organoids. RNA-Sequencing of fifty-six patients from our living organoid biobank were analyzed and we found an immunologic cluster of high-grade endometrial cancers that display significant downregulation in both MHC I and II. This was confirmed with real time-PCR which showed that high-grade cancers organoids had downregulated MHC I and II compare to organoids derived from low-grade cancer tissues and benign tissues. We next sought to demonstrate that both normal and endometrial cancer organoids retain the ability to present external antigen by using DQ-Ovalbumin. We demonstrated that endometrial organoids retain the ability to present antigen, but cancer organoids display reduced efficiency in the antigen processing. Next, we stimulated endometrial cancer organoids with interferon-gamma (IFNg), a well known cytokine for enhancing immunological response, to increase the expression of MHC I and II in cancer organoids. IFNg led to a 2-6 fold increase in MHC expression in low-grade cancer organoids, but a 5-40 fold increase in expression in high-grade cancer organoids utilizing real time-PCR. Finally, we implement the organoid-immune cell co-culture system with patient-derived organoids and CD8+ cytotoxic T-Cells in the allogeneic and syngeneic settings. Endometrial cancer organoids and matched patient-derived cytotoxic T-Cells were co-cultured for 48 hours and imaged for 16 hours. Both migratory effect and tumor killing effect were compared. As expected, in the allogeneic setting CD8+ T-Cells efficiently colocalized and killed cancer cells in the vehicle and IFNg stimulated groups. In the syngeneic setting, CD8+ T-Cells did not colocalize or kill cancer cells in the vehicle, whereas they significantly colocalized and killed cancer cells when stimulated with IFNg. Our data demonstrate that high-grade endometrial cancer cells downregulate MHC I and II, suggesting that they can evade the host immune system. We also showed that this effect is patient-specific and can be overcome by stimulating with interferon-gamma. Furthermore, we demonstrated that increasing MHC I and II among cancer organoids enhances the target cell recognition and apoptosis induction by CD8+ T-cells. Collectively, our finding offers an approach to study the tumor microenvironment that can be individualized for each patient through the use of patient-derived cancer organoids. This technology can be further developed to study targeted therapies and provides a much needed pre-clinical model for the study of endometrial cancer. Citation Format: Aaron Nizam, Charlie Chung, Gary L. Goldberg, Semir Beyaz. Utilizing endometrial tumor organoids to model cancer immunomodulation [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 LB236.

NLRC5 promotes transcription of BTN3A1-3 genes and Vγ9Vδ2 Tcell-mediated killing.

SummaryBTN3A molecules—BTN3A1 in particular—emerged as important mediators of Vγ9Vδ2 T cell activation by phosphoantigens. These metabolites can originate from infections, e.g. with Mycobacterium tuberculosis, or by alterations in cellular metabolism. Despite the growing interest in the BTN3A genes and their high expression in immune cells and various cancers, little is known about their transcriptional regulation. Here we show that these genes are induced by NLRC5, a regulator of MHC class I gene transcription, through an atypical regulatory motif found in their promoters. Accordingly, a robust correlation between NLRC5 and BTN3A gene expression was found in healthy, in M. tuberculosis-infected donors' blood cells, and in primary tumors. Moreover, forcing NLRC5 expression promoted Vγ9Vδ2 T-cell-mediated killing of tumor cells in a BTN3A-dependent manner. Altogether, these findings indicate that NLRC5 regulates the expression of BTN3A genes and hence open opportunities to modulate antimicrobial and anticancer immunity.

Major histocompatibility complexes are up-regulated in glomerular endothelial cells via activation of c-Jun N-terminal kinase in 5/6 nephrectomy mice.

Background and PurposeThis study aims to explore the mechanism underlying the up‐regulation of major histocompatibility complex (MHC) proteins in glomerular endothelial cells in 5/6 nephrectomy mice.Experimental ApproachC57/BL6 mice were randomly allocated to sham‐operated (2K) and 5/6 nephrectomy (5/6Nx) groups. Mouse splenic lymphocytes, from either syngeneic or allogeneic background, were injected into 5/6Nx mice after total body irradiation. Human glomerular endothelial cells (HGECs) were cultured for experiments in vitro. Western blots, PCR, immunohistochemical and fluorescent staining were used, along with assays of tissue cytokines, lymphocyte migration and renal function.Key ResultsFour weeks after nephrectomy, expression of both mRNA and protein of MHC II, CD80, and CD86 were increased in 5/6Nx glomerular endothelial cells. After total body irradiation, 5/6Nx mice injected with lymphocytes from Balb/c mice, but not those from C57/BL6 mice, exhibited increased creatinine levels, indicating that allograft lymphocyte transfer impaired renal function. In HGECs, the protein levels of MHC and MHC Class II transactivator (CIITA) were increased by stimulation with TNF‐α or IFN‐γ, which promoted human lymphocytes movement. These increases were reduced by JNK inhibitors. In the 5/6Nx mice, JNK inhibition down‐regulated MHC II protein in glomerular endothelial cells, suggesting that JNK signalling participates in the regulation of MHC II protein.Conclusion and ImplicationsChronic inflammation in mice subjected to nephrectomy induces the up‐regulation of MHC molecules in glomerular endothelial cells. This up‐regulation is reduced by inhibition of JNK signalling.

Targeting MHC Regulation Using Polycyclic Polyprenylated Acylphloroglucinols Isolated from Garcinia bancana

Modulation of major histocompatibility complex (MHC) expression using drugs has been proposed to control immunity. Phytochemical investigations on Garcinia species have allowed the isolation of bioactive compounds such as polycyclic polyprenylated acylphloroglucinols (PPAPs). PPAPs such as guttiferone J (1), display anti-inflammatory and immunoregulatory activities while garcinol (4) is a histone acetyltransferases (HAT) p300 inhibitor. This study reports on the isolation, identification and biological characterization of two other PPAPs, i.e., xanthochymol (2) and guttiferone F (3) from Garcinia bancana, sharing structural analogy with guttiferone J (1) and garcinol (4). We show that PPAPs 1–4 efficiently downregulated the expression of several MHC molecules (HLA-class I, -class II, MICA/B and HLA-E) at the surface of human primary endothelial cells upon inflammation. Mechanistically, PPAPs 1–4 reduce MHC proteins by decreasing the expression and phosphorylation of the transcription factor STAT1 involved in MHC upregulation mediated by IFN-γ. Loss of STAT1 activity results from inhibition of HAT CBP/p300 activity reflected by a hypoacetylation state. The binding interactions to p300 were confirmed through molecular docking. Loss of STAT1 impairs the expression of CIITA and GATA2 but also TAP1 and Tapasin required for peptide loading and transport of MHC. Overall, we identified new PPAPs issued from Garcinia bancana with potential immunoregulatory properties.

Targeting the coding sequence: opposing roles in regulating classical and non-classical MHC class I molecules by miR-16 and miR-744.

BackgroundTo control gene expression, microRNAs (miRNAs) are of key importance and their deregulation is associated with the development and progression of various cancer types. In this context, a discordant messenger RNA/protein expression pointing to extensive post-transcriptional regulation of major histocompatibility complex (MHC) class I molecules was already shown. However, only a very limited number of miRNAs targeting these molecules have yet been identified. Despite an increasing evidence of coding sequence (CDS)-located miRNA binding sites, there exists so far, no detailed study of the interaction of miRNAs with the CDS of MHC class I molecules.MethodsUsing an MS2-tethering approach in combination with small RNA sequencing, a number of putative miRNAs binding to the CDS of human leukocyte antigen (HLA)-G were identified. These candidate miRNAs were extensively screened for their effects in the HLA-G-positive JEG3 cell line. Due to the high sequence similarity between HLA-G and classical MHC class I molecules, the impact of HLA-G candidate miRNAs on HLA class I surface expression was also analyzed. The Cancer Genome Atlas data were used to correlate candidate miRNAs and HLA class I gene expression.ResultsTransfection of candidate miRNAs revealed that miR-744 significantly downregulates HLA-G protein levels. In contrast, overexpression of the candidate miRNAs miR-15, miR-16, and miR-424 sharing the same seed sequence resulted in an unexpected upregulation of HLA-G. Comparable results were obtained for classical MHC class I members after transfection of miRNA mimics into HEK293T cells. Analyses of The Cancer Genome Atlas data sets for miRNA and MHC class I expression further validated the results.ConclusionsOur data expand the knowledge about MHC class I regulation and showed for the first time an miRNA-dependent control of MHC class I antigens mediated by the CDS. CDS-located miRNA binding sites could improve the general use of miRNA-based therapeutic approaches as these sites are highly independent of structural variations (e.g. mutations) in the gene body. Surprisingly, miR-16 family members promoted MHC class I expression potentially in a gene activation-like mechanism.

Interleukin-dependent modulation of the expression of MHC class I and MHC class II genes in chicken HD11 cells

Interleukins (ILs) regulate cell surface antigens known as activation markers, which have distinct functional roles. However, the regulation of major histocompatibility complex (MHC) class I, MHC class II, and related genes by cytokines in chickens is not well understood. In the present study, we evaluated the influence of certain recently discovered chicken interleukins—i.e., IL-11, IL-12B, IL-17A, IL-17B, IL-26, and IL-34—on the expression and regulation of genes related to MHC class I, MHC class II, and the associated proteins in an HD11 chicken macrophage cell line. We used quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunocytochemical, and flow cytometric analyses to assess dose- and time-dependent expression in the HD11 cell line and found that the ILs induced MHC class I, MHC class II, and associated protein. As NF-κB is actively involved in cell activation and is constitutively activated in many immune cells, we also determined whether NF-κB regulates MHC class I, MHC class II, and related gene expression in the HD11 cell line. The NF-κB inhibitor sulfasalazine (Sz) dose-dependently inhibited MHC class I and MHC class II in the HD11 cell line. Sz also downregulated the expression of MHC class I, MHC class II, and the associated proteins in the IL-induced HD11 cell line. The expression of MHC class I, MHC class II, and associated genes was accompanied by the Sz-sensitive degradation of the p65 (RelA) and p50 subunits of NF-κB and IκBα. Our results indicate that the different effects of each IL on the expression of genes related to MHC class I, MHC class II, and the associated proteins are involved with the regulation of the dose and duration of antigenic peptide presentation and, thus, also influence Th1, Th2, and Th17 production.

Kidney hyperfusion upregulates major histocompatibility complex expression in glomerular endothelial cells through c‐Jun N‐terminal kinase

ObjectivesThe endothelium is the first line of defense while contacting with the immune system. The present study was investigated roles of antigen representing in glomerular endothelial cells.MethodsC57/BL6 mice were randomly grouped into sham‐operated (2K) and 5/6 nephrectomy groups (5/6Nx). After whole body irradiation, mice were received splenic lymphocytes from either C57/BL6 or Balb/c mice. Human glomerular endothelial cells were cultured in vitro. Blood and renal cortex, and glomeruli were collected for measurement of kidney function, Western blotting, fluorescence‐activated cell sorting and enzyme‐linked immunosorbent assays.ResultsFour weeks after nephrectomy 5/6Nx mice had a normal renal function, but a significantly higher level of renal blood flow, compared with 2K groups, indicating that mice in 5/6Nx have hyper‐perfusion in the kidney. Both mRNA and protein expressions of major histocompatibility complex (MHC) II, CD80, CD86, and interferon γ were upregulated in glomerular endothelial cells of the 5/6Nx mice. Mice injected with lymphocytes from Balb/c mice, but not C57/BL6, had significantly higher levels of serum creatinine, confirming that kidney hyper‐perfusion initiate the immune reaction and the allograft lymphocytes transplant accelerates renal dysfunction. In cultured glomerular endothelial cells, interferon γ significantly upregulated protein expression of MHC II and MHC class I chain‐related genes A (MICA), but not MHC I. Interferon γ also increased transcription factor CIITA protein expression, as well as phosphorylation JNK. JNK inhibitor, SP600125 or JNK peptide, reduced protein expressions of CIITA, MHC II and MICA in cultured glomerular endothelial cells. In the 5/6Nx mice SP600125 or JNK peptide, reduced MHC II expression, but not CD80 or CD86 in the 5/6Nx mice, suggesting that JNK signalling plays a role in the regulation of MHC II protein.ConclusionIn the present study, nephrectomy induces hemodynamic changes in the kidney, leading to increases in major histocompatibility complex protein expression in glomerular endothelial cells. Inhibition of JNK signalling reduces MHC expression in glomerular endothelial cells in vitro and in vivo.Support or Funding InformationThis work was supported by the National Nature Science Foundation of China (No 81573418) and ZSYXRC‐014This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evolving Insights for MHC Class II Antigen Processing and Presentation in Health and Disease

Recent findings regarding major histocompatibility complex (MHC) class II polymorphisms, sources of peptides, and regulation of expression on human pathologies are described with regards to impacts on human health. Studies of MHC class II polymorphisms associated with disease indicate that beyond the selection of loaded peptide, sequence variations affect the ability of certain MHC class II to associate with chaperones that assist with peptide editing or to assume conformations needed for function. That exogenous and endogenous sources of protein are both sampled by MHC II is revealing an increasing level of overlap between classical MHC classes I and II pathways of peptide acquisition. Regulation of MHC II expression, controlled by CIITA, is also emerging as a previously underappreciated determinant of immune homeostasis. The concept of MHC class II antigen processing and presentation to CD4 T cells has developed over the past 40 years. As research on MHC class II continues, we are beginning to discern relationships between allelic polymorphisms, peptide selection, and regulation of expression on immune health and disease.

Emerging Major Histocompatibility Complex Class I-Related Functions of NLRC5.

Recent evidence demonstrates a key role for the nucleotide-binding oligomerization domain-like receptor (NLR) family member NLRC5 (NLR family, CARD domain containing protein 5) in the transcriptional regulation of major histocompatibility complex (MHC) class I and related genes. Detailed information on NLRC5 target genes in various cell types and conditions is emerging. Thanks to its analogy to CIITA (class II major MHC transactivator), a NLR family member known for over 20 years to be the master regulator of MHC class II gene transcription, also the molecular mechanisms underlying NLRC5 function are being rapidly unraveled. MHC class I molecules are crucial in regulating innate and adaptive cytotoxic responses. Whereas CD8+ T cells detect antigens presented on MHC class I molecules by infected or transformed cells, natural killer (NK) lymphocytes eliminate target cells with downregulated MHC class I expression. Data uncovering the relevance of NLRC5 in homeostasis and activity of these two lymphocyte subsets have been recently reported. Given the importance of CD8+ T and NK cells in controlling infection and cancer, it is not surprising that NLRC5 is also starting to emerge as a central player in these diseases. This chapter summarizes and discusses novel insights into the molecular mechanisms underlying NLRC5 activity and its relevance to pathological conditions. A thorough understanding of both aspects is essential to evaluate the clinical significance and therapeutic potential of NLRC5.

Endothelin-1 Regulates Molecules of the Major Histocompatibility Complex: Role in Sickle Cell Disease

Molecules of the Major Histocompatibility Complex (MHC), and in particular specific human leukocyte antigen (HLA) alleles, have been proposed in the pathophysiology of immune and vascular alterations leading to vasoocclusive crises (VOC) and stroke in Sickle Cell Disease (SCD). Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion and excess levels of the potent mitogen, endothelin-1 (ET-1). ET-1 activates endothelial cells, induces oxidative stress and inflammation in the vascular wall and regulates erythrocyte homeostasis. However, the role of ET-1 on MHC regulation in SCD is not clear. We first characterized the effect of ET-1 on MHC expression in the human endothelial cell line, EA.hy926. We observed dose-dependent increases in the expression of MHC class I (HLA-A2 4.8 ± 2.1 folds p<0.01 n=4), MHC class II (HLA-DR 4.4 ± 1.7 folds p<0.01 n=4) and MHC transcription factor (CIITA 3.5 ± 1.8 folds p<0.05 n=4) in EA.hy926 cells. ET-1-stimulated expression of HLA-A2, HLA-DR and CIITA were significantly blocked by pre-incubation of cells with 10 µM BQ788, a selective blocker of ET-1 type B receptors (p<0.01, n=4). Chromatin immunoprecipitation (ChIP) studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the promoter region within MHC molecules (HLA-A2 62% ± 5%, HLA-DRB 33% ± 18%, p<0.01, n=4); an event that was likewise blocked by BQ788. We then studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βSAntilles and Berkeley (BERK) mice, respectively. We observed significant increases in MHC molecule, H2-Aa mRNA levels (n=7; p<0.01) in spleens from sickle transgenic mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). We then treated BERK, βSAntilles and HbA mice for 14 days with ET-1 receptor antagonists and observed significant reductions in H2-Aa mRNA levels in spleen tissue from sickle transgenic mice but not in HbA mice (n=7; p<0.05). These results implicate ET-1 as a novel regulator of MHC molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD. DisclosuresNo relevant conflicts of interest to declare.

Regulation of protein synthesis and autophagy in activated dendritic cells: implications for antigen processing and presentation.

Antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules originate from the degradation of both self and non-self proteins. T cells can therefore recognize at the surface of surveyed cells, the self-peptidome produced by the cell itself (mostly inducing tolerance) or immunogenic peptides derived from exogenous origins. The initiation of adaptive immune responses by dendritic cells (DCs), through the antigenic priming of naïve T cells, is associated to microbial pattern recognition receptors engagement. Activation of DCs by microbial product or inflammatory cytokines initiates multiple processes that maximize DC capacity to present exogenous antigens and stimulate T cells by affecting major metabolic and membrane traffic pathways. These include the modulation of protein synthesis, the regulation of MHC and co-stimulatory molecules transport, as well as the regulation of autophagy, that, all together promote exogenous antigen presentation while limiting the display of self-antigens by MHC molecules.

Genomic mapping of the MHC transactivator CIITA using an integrated ChIP-seq and genetical genomics approach.

BackgroundThe master transactivator CIITA is essential to the regulation of Major Histocompatibility Complex (MHC) class II genes and an effective immune response. CIITA is known to modulate a small number of non-MHC genes involved in antigen presentation such as CD74 and B2M but its broader genome-wide function and relationship with underlying genetic diversity has not been resolved.ResultsWe report the first genome-wide ChIP-seq map for CIITA and complement this by mapping inter-individual variation in CIITA expression as a quantitative trait. We analyse CIITA recruitment for pathophysiologically relevant primary human B cells and monocytes, resting and treated with interferon-gamma, in the context of the epigenomic regulatory landscape and DNA-binding proteins associated with the CIITA enhanceosome including RFX, CREB1/ATF1 and NFY. We confirm recruitment to proximal promoter sequences in MHC class II genes and more distally involving the canonical CIITA enhanceosome. Overall, we map 843 CIITA binding intervals involving 442 genes and find 95% of intervals are located outside the MHC and 60% not associated with RFX5 binding. Binding intervals are enriched for genes involved in immune function and infectious disease with novel loci including major histone gene clusters. We resolve differentially expressed genes associated in trans with a CIITA intronic sequence variant, integrate with CIITA recruitment and show how this is mediated by allele-specific recruitment of NF-kB.ConclusionsOur results indicate a broader role for CIITA beyond the MHC involving immune-related genes. We provide new insights into allele-specific regulation of CIITA informative for understanding gene function and disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0494-z) contains supplementary material, which is available to authorized users.