Major Histocompatibility Complex Class II Genes Research Articles

Andrographolide exhibits antinociceptive effects in neuropathic rats via inhibiting class Ⅱ MHC associated response and regulating synaptic plasticity

BackgroundNeuropathic pain (NP) due to nerve injury, disrupts neural plasticity by triggering the release of inflammatory mediators. Alongside the hypothesis that neuro-inflammation contributes to this disruption, Andrographolide (Andro), a traditional bioactive compound derived from Andrographis paniculata, has garnered attention for its potent anti-inflammatory properties. However, whether Andro could ameliorate NP by regulating neuroinflammation remains unknown. PurposeThis study aimed to investigate whether and how Andro regulates neuroinflammation and alleviates NP. MethodsThe analgesic effects of Andro on NP were evaluated using both the spinal nerve ligation (SNL) and formalin rat models. A combination of network pharmacology, RNA sequencing, and experimental validation was employed to elucidate the underlying mechanism behind Andro's analgesic effects. Additionally, various techniques such as functional ultrasound, immunohistochemistry, quantitative real-time polymerase chain reaction (qPCR), patch clamp, and electron microscopy were employed to investigate the specific neural cell types, neural functions, and changes in neural plasticity influenced by Andro. ResultsNetwork pharmacology analysis unveiled the crucial roles played by shared targets of Andro and pain in regulating pain-related inflammation, including microglia activation, neuroinflammation, immune modulation, and synaptic transmission. Furthermore, we confirmed Andro's superior efficacy in pain relief compared to the traditional analgesic drug, Gabapentin. In these models, Andro was observed to modulate the haemodynamic response triggered by SNL. Transcriptome analysis and molecular docking studies indicated the involvement of major histocompatibility complex class II (MHCII) genes (Db1, Da, and Bb). Electron microscopy revealed improvements in synaptic ultrastructure, and electrophysiological investigations showed a selective reduction in glutamatergic transmission in neuropathic rats after following Andro treatment. The integration of systems pharmacology analysis and biological validation collectively demonstrated that the mechanism of pain relief involves immune modulation, enhancement of synaptic plasticity, and precise regulation of excitatory neurotransmission. ConclusionIn conclusion, this study has demonstrated that Andro, by targeting MHCII genes, may serve as a promising therapeutic candidate for neuropathic pain.

Abstract 4049: The beta-carboline Harmine sensitives microsatellite stable colorectal cancer mouse model to anti-PD-1 through upregulating MHC-I dependent antigen presentation machinery and improving the infiltration of CD8 T cells into the tumor microenvironment

Abstract Colorectal cancer (CRC) is the most common gastrointestinal cancer and the third leading cause of cancer-related death in both sexes. Mismatch repair deficient/microsatellite instability-high (MSI) colorectal cancer patients benefit from immune checkpoint blockades (ICBs)-based immunotherapy; however, this benefit has not been translated into microsatellite stable (MSS) colorectal cancer. It has been proposed that the loss of major histocompatibility complex class I (MHC-I) in tumor cells limits the use of ICB in colorectal cancer. Harmine displays a number of biological and pharmacological properties affecting the immune response and tumor phenotype. Here, we assessed the impact of combining harmine, also referred to as ACB1801 molecule, on improving the responsiveness of ICB in the mouse MSS CRC model. Our results showed that combining ACB1801 significantly improves the therapeutic benefit of anti-PD-1 in the CT26 CRC mouse model by decreasing tumor growth and improving the survival of tumor-bearing mice. The improvement of anti-PD-1-based therapy by combining ACB-1801 was associated with a modification of the immune landscape of tumors as evidenced by an increase in the CD8+ T cells and a decrease in the Treg infiltrations into the tumor microenvironment. By profiling the cytokine/chemokine network, we showed that ACB1801 induced the expression and the release of the proinflammatory chemokine CXCL10 by CRC tumor cells in vitro, which could be responsible for driving CD8+ T cells to the tumor microenvironment. Mechanistically, we showed that ACB1801 increased the expression of MHC-I genes, including TAP1 and TAPASIN, and improved the antigen loading on MHC-I in CRC cells in vitro. Overall, our study highlights the value of combining ACB1801 and anti-PD-1 as a therapeutic opportunity to convert MSS colorectal cancer into an “immune hot” cancer, which may define the future treatment paradigm of colorectal cancer for which there is a great unmet need. Citation Format: Ruize Gao, Kris Van Moer, Coralie Pulido, Anaïs Oudin, Diane Murera, Teresa L. Ramos, Margaux Poussard, Andreas Schläpfer, Annette Ives, Christian Auclair, Bassam Janji. The beta-carboline Harmine sensitives microsatellite stable colorectal cancer mouse model to anti-PD-1 through upregulating MHC-I dependent antigen presentation machinery and improving the infiltration of CD8 T cells into the tumor microenvironment [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 4049.

Abstract 6554: Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with a very poor prognosis and limited therapeutic options. Repression of the MHC-I and reduced tumor infiltration of cytotoxic T-lymphocytes is often associated with immunotherapy resistance in SCLC. Lurbinectedin is FDA-approved as a second-line treatment for SCLC and we previously showed lurbinectedin as an effective therapeutic strategy in SCLC preclinical models. There are ongoing clinical trials combining immune checkpoint blockade with lurbinectedin in ES-SCLC. However, there is no insight into the effect of lurbinectedin on the immune microenvironment in SCLC. In this study, we evaluated the effect of lurbinectedin on the immune microenvironment and the anti-tumor effect with or without PD-L1 blockade. Results: To determine the effect of lurbinectedin in SCLC models, we treated immunocompetent flank RPP (Rb1, Trp53, and p130) and RPM (Rb1, Trp53, and MYCT58A) tumor-bearing mice with lurbinectedin (0.2 mg/kg, 1/7days) and/or anti-PD-L1 (300µg, 1/7days) for 3 weeks. Mice treated with anti-PD-L1 alone showed no anti-tumor response, and single-agent lurbinectedin caused a delay in tumor growth. However, 6 of the 10 mice treated with the combination of lurbinectedin and anti-PD-L1, had a complete response (100% reduction) and the other 4 had 90% tumor regression. Tumors were resected and analyzed by multicolor flow cytometry for changes in tumor-infiltrating lymphocytes (TILs). Furthermore, analysis of immune cells infiltrating into the tumors post- treatment showed significant induction of cytotoxic T-cells and a reduction of exhausted and regulatory T cells in the combination treatment arm. Similarly, pro-inflammatory M1 type macrophages and dendritic cells were increased, while immunosuppressive M2 type macrophages and MDSC cells were decreased. These effects are consistent with data showing that lurbinectedin treatment leads to an activation of the cGAS/STING pathway, type I/II interferons (IFNα/β), and pro-inflammatory chemokines (CCL5, CXCL10) in tumors. Interestingly, lurbinectedin treatment led to significant upregulation of mRNA and surface expression of MHC class-I genes (HLA-A/B/C) in vitro and in vivo. Finally, bulk RNA sequencing as well as RT-PCR of pre- and post-lurbinectedin treatment demonstrated an increase in DAMPs associated with immunogenic cell death. Conclusion: We provide the first mechanistic insight into the lurbinectedin-induced multimodal immune modulation in SCLC leading to a dramatic anti-tumor activity accompanied by the establishment of a strong anti-tumor immune microenvironment. Since lurbinectedin is already approved as a second-line agent in SCLC, our preclinical data provide a strong rationale for combining this regimen with inhibitors of the PD-L1 pathway and also highlight the immune subsets that the combination targets. Citation Format: Subhamoy Chakraborty, Yazhini Mahendravarman, Utsav Sen, Avisek Banerjee, Charles Coleman, Deniz Demircioglu, Dan Hasson, Triparna Sen. Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung cancer [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 6554.

Only rare classical MHC-I alleles are highly expressed in the European house sparrow.

The exceptional polymorphism observed within genes of the major histocompatibility complex (MHC), a core component of the vertebrate immune system, has long fascinated biologists. The highly polymorphic classical MHC class-I (MHC-I) genes are maintained by pathogen-mediated balancing selection (PMBS), as shown by many sites subject to positive selection, while the more monomorphic non-classical MHC-I genes show signatures of purifying selection. In line with PMBS, at any point in time, rare classical MHC alleles are more likely than common classical MHC alleles to confer a selective advantage in host-pathogen interactions. Combining genomic and expression data from the blood of wild house sparrows Passer domesticus, we found that only rare classical MHC-I alleles were highly expressed, while common classical MHC-I alleles were lowly expressed or not expressed. Moreover, highly expressed rare classical MHC-I alleles had more positively selected sites, indicating exposure to stronger PMBS, compared with lowly expressed classical alleles. As predicted, the level of expression was unrelated to allele frequency in the monomorphic non-classical MHC-I alleles. Going beyond previous studies, we offer a fine-scale view of selection on classical MHC-I genes in a wild population by revealing differences in the strength of PMBS according to allele frequency and expression level.

Abstract B034: Characterization of MHC-I-mediated immune evasion in triple-negative breast cancer using human transcriptomic datasets and immunocompetent mouse models

Abstract In Triple-Negative Breast Cancer (TNBC) patients, adaptive immune system activity in the tumor microenvironment is associated with improved prognosis. However, tumors can evolve to evade immune detection, likely leading to worse patient outcomes. Multi-omic data of paired primary and metastatic breast tumors demonstrate that TNBC metastases frequently lose expression of genes involved in major histocompatibility complex class I (MHC-I) antigen presentation, a key pathway for adaptive immune activation (PMID: 36585450). Therefore, understanding and addressing MHC-I-mediated immune evasion may have profound clinical benefits for TNBC patients. We hypothesize that breast tumor MHC-I loss results in a unique immune-evasive phenotype, which we are characterizing using human clinical datasets and immunocompetent mouse models of breast cancer. First, using large transcriptomic datasets (i.e., SCAN-B and TCGA), we are characterizing molecular and clinical features of MHC-I-low human breast tumors using expression of the MHC-I gene human leukocyte antigen A (HLA-A) as a continuous variable. We found HLA-A expression varies significantly between PAM50 molecular and clinical subtypes of breast cancer and has differing impacts on patient survival based on clinical subtype. In TNBCs, low HLA-A expression is associated with worse outcomes, while in HR+/HER2- tumors, low HLA-A predicts improved outcomes. We also identified genes highly correlated with HLA-A regardless of molecular subtype. This list was functionally rich, related to antigen processing machinery, adaptive immune-mediated cytotoxicity, and T cell exhaustion features. To complement human genomic data, we are empirically testing the role of MHC-I in syngeneic mouse mammary tumor models by individually knocking out murine MHC-I genes–H2-K, H2-D, and B2m–through CRISPR/Cas9-mediated genome editing. We have generated and validated knockouts (KOs) of H2-K and B2m, as well as empty vector control clones from the “immune-hot” and immune checkpoint inhibitor (ICI) responsive mammary tumor cell lines KPB25L-UV and T11-APOBEC. RNA sequencing of KPB25L-UV H2-K and B2m KO versus wildtype cell lines in vitro identified lists of differentially expressed genes, many of which are related to proteolytic and peptide antigen processing. Next, these CRISPR-modified cell lines were injected into the mammary fat pad of syngeneic immunocompetent mice, and the resulting tumors were harvested for RNA sequencing. Differentially expressed genes in KPB25L-UV H2-K KO versus wildtype tumors were related to increased MHC Class II antigen presentation and dendritic cell activity. Preliminary data suggests that mice bearing KPB25L-UV H2-K KO tumors have shortened survival compared to H2-K wildtype tumors. Experiments are ongoing to further characterize the growth dynamics, metastatic potential, single-cell transcriptomic profiles, and ICI sensitivities of these KO tumors in vivo. Our long-term goal is to identify therapeutic means to overcome this common immune-evasive phenotype to improve TNBC patient outcomes. Citation Format: Constandina E O'Connell, Brooke M Felsheim, Aranzazu Fernandez-Martinez, Kevin R Mott, Charles M Perou. Characterization of MHC-I-mediated immune evasion in triple-negative breast cancer using human transcriptomic datasets and immunocompetent mouse models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B034.

An Ancestral Major Histocompatibility Complex Organization in Cartilaginous Fish: Reconstructing MHC Origin and Evolution.

Cartilaginous fish (sharks, rays, and chimeras) comprise the oldest living jawed vertebrates with a mammalian-like adaptive immune system based on immunoglobulins (Ig), T-cell receptors (TCRs), and the major histocompatibility complex (MHC). Here, we show that the cartilaginous fish "adaptive MHC" is highly regimented and compact, containing (i) a classical MHC class Ia (MHC-Ia) region containing antigen processing (antigen peptide transporters and immunoproteasome) and presenting (MHC-Ia) genes, (ii) an MHC class II (MHC-II) region (with alpha and beta genes) with linkage to beta-2-microglobulin (β2m) and bromodomain-containing 2, (iii) nonclassical MHC class Ib (MHC-Ib) regions with 450 million-year-old lineages, and (iv) a complement C4 associated with the MHC-Ia region. No MHC-Ib genes were found outside of the elasmobranch MHC. Our data suggest that both MHC-I and MHC-II genes arose after the second round of whole-genome duplication (2R) on a human chromosome (huchr) 6 precursor. Further analysis of MHC paralogous regions across early branching taxa from all jawed vertebrate lineages revealed that Ig/TCR genes likely arose on a precursor of the huchr9/12/14 MHC paralog. The β2m gene is linked to the Ig/TCR genes in some vertebrates suggesting that it was present at 1R, perhaps as the donor of C1 domain to the primordial MHC gene. In sum, extant cartilaginous fish exhibit a conserved and prototypical MHC genomic organization with features found in various vertebrates, reflecting the ancestral arrangement for the jawed vertebrates.

Pegylated Long-Acting Interferon Gamma Treatment Enhances MHC Class II Expression and the Graft-Versus-Leukemia Effect in Preclinical Models of AML Relapse

Relapse after allogeneic hematopoietic stem cell transplantation (HCT)represents the most common type of treatment failure and is thought to result in part from AML cell escape from immune pressure exerted by donor immune cells (the “Graft-versus-leukemia” effect, GvL). We and others have previously reported downregulation of MHC class II (MHC-II) expression in up to half of AML cases relapsing after transplantation, but it is unclear whether a) loss of MHC-II contributes mechanistically to loss of GvL, and b) whether reversal of MHC-II downregulation can restore GvL. We tested the role of MHC-II in mediating GvL in a mouse model of minor-antigen mismatch HCT where C57BL/6 mice (B6) bearing syngeneic AML cells (H-2b) were co-transplanted with or without CD3+ T cells from C3.SW-H2b/SnJ mice (H-2b). Since murine AML cells do not generally express MHC-II at baseline, we transduced MLL-AF9-driven B6 AML cells with a fluorescently-tagged retrovirus expressing CIITA the main transcriptional regulator of MHC-II genes. MHC-II-expressing AML cells were selectively deleted when co-transplanted with donor T cells, in contrast to animals that received no T cells (Figure 1, right) or mice that received AML cells transduced with empty vector (Figure 1, left). To extend these findings to human cells, we used both MHC-II blocking antibodies and CRISPR-mediated disruption of CIITA in human AML cell lines and primary samples. Loss or blocking of MHC-II led to diminished the ability of AML cells to stimulate MHC-mismatched donor T cells in a mixed lymphocyte reaction. Loss of MHC-II expression in AML cells can be reversed in vitro by Interferon gamma (IFNG). We tested the ability of IFNG to enhance the GvL by culturing AML cell lines and primary patient samples with or without IFNG for 48 hours. IFNG-treatment enhanced the ability of AML cells from some-but not all-samples to stimulate third-party T cells. Interestingly, enhanced stimulation was observed in both CD4 and CD8 cells, suggesting that IFNG may also potentiate GvL through MHC-II-independent pathways. Since the use of IFNG in vivo may be limited by its short half-life, we tested a novel long-acting pegylated human interferon gamma (peg-IFNG, Bolder BioTechnology) in a xenograft model of AML. Immunodeficient mice were engrafted with MHC-II-low human AML cells. After engraftment, mice (n=3 each group) received six doses of IFNG, peg-IFNG, or vehicle (15mcg given s.c. M-W-F). After treatment, mice were sacrificed and MHC-II expression was quantified on human AML cells isolated from bone marrow of treated mice. IFNG treatment increased MHC-II expression modestly compared to vehicle (2.7-fold, p=0.04), while peg-IFNG much more robustly stimulated MHC-II stimulation on human AML cells in this xenograft model (12-fold, p=0.02). Finally, we tested whether peg-IFNG can enhance AML cell clearance in a xenograft model. Luciferin-labelled THP1 cells (low MHC-II expression) were injected on day 0 into immundeficient mice (n=8-9 each group). On day 7, mice started treatment with peg-IFNG or vehicle (15mcg given s.c. M-W-F for two weeks). On day 21, baseline tumor burden was measured using bioluminescence, and healthy third-party donor T cells were injected. Mice were followed with weekly CBC and bioluminescence measurments, and moribund mice were sacrificed and banked. Mice treated with peg-IFNG plus T cells had decreased tumor burden during the course of the experiment compared to untreated controls as well as mice treated with T cells plus vehicle only (Figure 2, p<0.01 by ANOVA for both comparisons). In addition, mice treated with peg-IFNG had significantly improved survival compared to control groups (median survival 79 days compared to 63 days and 67 days, p<0.05 by Log-Rank test). Together, these findings suggest that long-acting IFNG analogues may effectively enhance GvL in AML patients after HCT.

Characterization of Multiple Myeloma Subpopulations Associated with Treatment Resistance By Single-Cell RNA Sequencing

Multiple myeloma (MM) has extensive genomic and transcriptomic heterogeneity with identical variable-diversity-joining (VDJ) region recombination of immunoglobulin loci (called repertoire). Even though new treatment approaches have been developed, most cases of MM remain incurable. Recent evidence suggests that epigenetic cell plasticity and changes in cell state underlie the therapy resistance, but the mechanisms remain largely unknown. We attempted to understand the comprehensive architecture of the entire tumor cell populations using repertoire clonality as a fingerprint. First, we subdivided bone marrow samples of primary MM at different stages (n=8) into 11 fractions and performed bulk RNA sequencing (bulk RNA-seq). Analysis of the repertoire revealed that tumor cells were present at very low frequencies in some minor fractions other than the main fraction (Lineage marker - (Lin -)/CD19 -/CD38 +/CD138 +) as the disease stage progressed. Importantly, MM cells were consistently present in CD138 negative fraction (Lin -/CD19 -/CD38 +/CD138 -) regardless of disease stage. Flow cytometry (FCM) analysis confirmed that CD138negative MM cells were present at a rate of less than 10% across the disease stages. Compared with CD138 positive cells, they were slowly cycling and showed unique morphology with large cytoplasm (n=59). To achieve a selective and comprehensive evaluation of MM population, we performed single-cell RNA sequencing (scRNA-seq) coupled with VDJ targeted sequencing (scVDJ-seq) on Lin -/CD19 -/CD38 +(without CD138 gating) samples from 11 MM patients, including those at diagnosis and in remission with minimal residual disease (MRD). This method allowed us to discriminate MM cells (20,084 cells) clearly from others by clonal repertoire sequence even in the post-treatment samples with extremely small populations of tumor cells. Because mRNA levels of CD138 did not correspond to the differential intensity in FCM, we defined gene signatures of CD138 positive and negative cells respectively by bulk RNA-seq (n=6). As a result of integration of these gene signatures using Gene Set Enrichment Analysis (GSEA), the heterogeneity on the CD138 axis was also unraveled in scRNA-seq data. Interestingly, gene ontology (GO) analysis of bulk RNA-seq data revealed that several terms related to H3K4 methylation were positively enriched in CD138 negative fraction, which included KMT2A, KMT2B, KMT2C and KMT2D. Western blotting confirmed significant reductions in H3K4me1 and H3K4me3 levels in CD138 negative cells. Furthermore, primary MM cell culture indicated the plasticity between CD138 positive and negative cells, which was also suggested by trajectory/velocity analysis of scRNA-seq data. Collectively, it was suggested that epigenetic changes, including those in histone modifications, contribute to the heterogeneity along the CD138 axis. We next compared the size of each cluster before and after treatment in scRNA-seq data to identify the MM populations associated with treatment resistance. Remarkably, we identified unique clusters which tended to remain at high proportions against treatment in the CD138 negative population. They were characterized by low expression of MHC class-I genes and TNFRSF17/BCMA, as well as 198 significantly upregulated genes including MCL1, MALAT1 and NEAT1, which have been previously implicated in treatment resistance of MM. To systematically interrogate their contribution in MM cell survival, we performed CRISPR/Cas9 knockout screening of selected 100 genes, which were differentially up-regulated in the resistant clusters and associated with poor prognosis in a MMRF CoMMpass Study database. GO analysis of indispensable genes reproducibly identified among three MM cell lines revealed enrichment of several pathways related to mRNA splicing. These results suggested that the RNA splicing pathway is a promising therapeutic target to eliminate resistant populations. Indeed, we confirmed that inhibition of a splicing factor by CRISPR/Cas9-mediated knockout and pharmacological protein degradation suppresses MM cell proliferation effectively. Taken together, we achieved a complete view of MM diversity by using repertoire clonality and confirmed heterogeneity along the CD138 axis, which may be characterized by epigenetic plasticity. Our results highlight a novel subpopulation as a candidate therapeutic target for cure.

Abdominal surgery induces long-lasting changes in expression and binding of CTCF with impact on Major Histocompatibility Complex II transcription in circulating human monocytes.

Postoperative immunosuppression has been recognized as an important driver of surgery-related morbidity and mortality. It is characterized by lymphocyte depression and impaired monocyte capability to present foreign antigens to T-cells via Major Histocompatibility Complex, Class II (MHC-II) molecules. In patients with postoperative abdominal sepsis, we previously detected a persisting differential binding of the CCCTC-Binding Factor (CTCF), a superordinate regulator of transcription, inside the MHC-II region with specific impact on human leucocyte antigen (HLA) gene expression. In this prospective exploratory study, we investigated to which extent major surgery affects the MHC-II region of circulating CD14+-monocytes. In non-immunocompromised patients undergoing elective major abdominal surgery, a postoperative loss of monocyte HLA-DR surface receptor density was accompanied by a decline in the transcription levels of the classical MHC-II genes HLA-DRA, HLA-DRB1, HLA-DPA1 and HLA-DPB1. The surgical event decreased the expression of the transcriptional MHC-II regulators CIITA and CTCF and led to a lower CTCF enrichment at an intergenic sequence within the HLA-DR subregion. During the observation period, we found a slow and only incomplete restoration of monocyte HLA-DR surface receptor density as well as a partial recovery of CIITA, HLA-DRA and HLA-DRB1 expression. In contrast, transcription of HLA-DPA1, HLA-DPB1, CTCF and binding of CTCF within the MHC-II remained altered. In circulating monocytes, major surgery does not globally affect MHC-II transcription but rather induces specific changes in the expression of selected HLA genes, followed by differential recovery patterns and accompanied by a prolonged reduction of CTCF expression and binding within the MHC-II region. Our results hint toward a long-lasting impact of a major surgical intervention on monocyte functionality, possibly mediated by epigenetic changes that endure the life span of the individual cell.

FBXO11 constitutes a major negative regulator of MHC class II through ubiquitin-dependent proteasomal degradation of CIITA

Major histocompatibility complex (MHC) class I and II molecules play critical roles in the activation and regulation of adaptive immunity through antigen presentation to CD8+ and CD4+ T cells, respectively. Strict regulation of MHC expression is critical for proper immune responses. CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, is a master regulator of MHC class II (MHC-II) gene transcription. Although it has been known that CIITA activity is regulated at the transcriptional and protein levels, the mechanism to determine CIITA protein level has not been elucidated. Here, we show that FBXO11 is a bona fide E3 ligase of CIITA and regulates CIITA protein level through ubiquitination-mediated degradation. A nonbiased proteomic approach for CIITA-binding protein identified FBXO11, a member of the Skp1-Cullin-1-F-boxE3 ligase complex, as a binding partner of CIITA but not MHC class I transactivator, NLRC5. The cycloheximide chase assay showed that the half-life of CIITA is mainly regulated by FBXO11 via the ubiquitin-proteasome system. The expression of FBXO11 led to the reduced MHC-II at the promoter activity level, transcriptional level, and surface expression level through downregulation of CIITA. Moreover, human and mouse FBXO11-deficient cells display increased levels of MHC-II and related genes. In normal and cancer tissues, FBXO11 expression level is negatively correlated with MHC-II. Interestingly, the expression of FBXO11, along with CIITA, is associated with prognosis of cancer patients. Therefore, FBXO11 is a critical regulator to determine the level of MHC-II, and its expression may serve as a biomarker for cancer.

MHC architecture in amphibians - ancestral reconstruction, gene rearrangements and duplication patterns.

The hypervariable major histocompatibility complex (MHC) is a crucial component of vertebrate adaptive immunity, but large-scale studies on MHC macroevolution in non-model vertebrates have long been constrained by methodological limitations. Here, we used rapidly accumulating genomic data to reconstruct macroevolution of the MHC region in amphibians. We retrieved contigs containing the MHC region from genome assemblies of 32 amphibian species and examined major structural rearrangements, duplication patterns and gene structure across the amphibian phylogeny. Based on the few available caecilian and urodele genomes we showed that the structure of ancestral MHC region in amphibians was probably relatively simple and compact, with a close physical linkage between MHC-I and MHC-II regions. This ancestral MHC architecture was generally conserved in anurans, although the evolution of class I subregion proceeded towards more extensive duplication and rapid expansion of gene copy number, providing evidence for dynamic evolutionary trajectories. Although in anurans we recorded tandems of duplicated MHC-I genes outside the core subregion, our phylogenetic analyses of MHC-I sequences provided little support for an expansion of nonclassical MHC-Ib genes across amphibian families. Finally, we found that intronic regions of amphibian classical MHC genes were much longer when compared to other tetrapod lineages (birds and mammals), which could partly be driven by the expansion of genome size. Our study reveals novel evolutionary patterns of the MHC region in amphibians and provides a comprehensive framework for further studies on the MHC macroevolution across vertebrates.

Conserved and cell type-specific transcriptional responses to IFN-γ in the ventral midbrain

Dysregulated inflammation within the central nervous system (CNS) contributes to neuropathology in infectious, autoimmune, and neurodegenerative disease. With the exception of microglia, major histocompatibility complex (MHC) proteins are virtually undetectable in the mature, healthy central nervous system (CNS). Neurons have generally been considered incapable of antigen presentation, and although interferon gamma (IFN-γ) can elicit neuronal MHC class I (MHC-I) expression and antigen presentation in vitro, it has been unclear whether similar responses occur in vivo. Here we directly injected IFN-γ into the ventral midbrain of mature mice and analyzed gene expression profiles of specific CNS cell types. We found that IFN-γ upregulated MHC-I and associated mRNAs in ventral midbrain microglia, astrocytes, oligodendrocytes, and GABAergic, glutamatergic, and dopaminergic neurons. The core set of IFN-γ-induced genes and their response kinetics were similar in neurons and glia, but with a lower amplitude of expression in neurons. A diverse repertoire of genes was upregulated in glia, particularly microglia, which were the only cells to undergo cellular proliferation and express MHC classII (MHC-II) and associated genes. To determine if neurons respond directly via cell-autonomous IFN-γ receptor (IFNGR) signaling, we produced mutant mice with a deletion of the IFN-γ-binding domain of IFNGR1 in dopaminergic neurons, which resulted in a complete loss of dopaminergic neuronal responses to IFN-γ. Our results demonstrate that IFN-γ induces neuronal IFNGR signaling and upregulation of MHC-I and related genes in vivo, although the expression level is low compared to oligodendrocytes, astrocytes, and microglia.

NLRC5-CIITA Fusion Protein as an Effective Inducer of MHC-I Expression and Antitumor Immunity.

Aggressive tumors evade cytotoxic T lymphocytes by suppressing MHC class-I (MHC-I) expression that also compromises tumor responsiveness to immunotherapy. MHC-I defects strongly correlate to defective expression of NLRC5, the transcriptional activator of MHC-I and antigen processing genes. In poorly immunogenic B16 melanoma cells, restoring NLRC5 expression induces MHC-I and elicits antitumor immunity, raising the possibility of using NLRC5 for tumor immunotherapy. As the clinical application of NLRC5 is constrained by its large size, we examined whether a smaller NLRC5-CIITA fusion protein, dubbed NLRC5-superactivator (NLRC5-SA) as it retains the ability to induce MHC-I, could be used for tumor growth control. We show that stable NLRC5-SA expression in mouse and human cancer cells upregulates MHC-I expression. B16 melanoma and EL4 lymphoma tumors expressing NLRC5-SA are controlled as efficiently as those expressing full-length NLRC5 (NLRC5-FL). Comparison of MHC-I-associated peptides (MAPs) eluted from EL4 cells expressing NLRC5-FL or NLRC5-SA and analyzed by mass spectrometry revealed that both NLRC5 constructs expanded the MAP repertoire, which showed considerable overlap but also included a substantial proportion of distinct peptides. Thus, we propose that NLRC5-SA, with its ability to increase tumor immunogenicity and promote tumor growth control, could overcome the limitations of NLRC5-FL for translational immunotherapy applications.

CD74: a prospective marker for reactive microglia?

CD74: a prospective marker for reactive microglia?

Molecular Characterization of MHC Class I Alpha 1 and 2 Domains in Asian Seabass (Lates calcarifer).

The Asian seabass is of importance both as a farmed and wild animal. With the emergence of infectious diseases, there is a need to understand and characterize the immune system. In humans, the highly polymorphic MHC class I (MHC-I) molecules play an important role in antigen presentation for the adaptive immune system. In the present study, we characterized a single MHC-I gene in Asian seabass (Lates calcarifer) by amplifying and sequencing the MHC-I alpha 1 and alpha 2 domains, followed by multi-sequence alignment analyses. The results indicated that the Asian seabass MHC-I α1 and α2 domain sequences showed an overall similarity within Asian seabass and retained the majority of the conserved binding residues of human leukocyte antigen-A2 (HLA-A2). Phylogenetic tree analysis revealed that the sequences belonged to the U lineage. Mapping the conserved binding residue positions on human HLA-A2 and grass carp crystal structure showed a high degree of similarity. In conclusion, the availability of MHC-I α1 and α2 sequences enhances the quality of MHC class I genetic information in Asian seabass, providing new tools to analyze fish immune responses to pathogen infections, and will be applicable in the study of the phylogeny and the evolution of antigen-specific receptors.

Role of the major histocompatibility complex class II protein presentation pathway in bone immunity imbalance in postmenopausal osteoporosis.

Bone immunity regulates osteoclast differentiation and bone resorption and is a potential target for the treatment of postmenopausal osteoporosis (PMOP). The molecular network between bone metabolism and the immune system is complex. However, the molecular mechanism underlying the involvement of the major histocompatibility complex class II (MHC-II) molecule protein presentation pathway in PMOP remains to be elucidated. The MHC-II molecule is a core molecule of the protein presentation pathway. It is combined with the processed short peptide and presented to T lymphocytes, thereby activating them to become effector T cells. T-cell-derived inflammatory factors promote bone remodeling in PMOP. Moreover, the MHC-II molecule is highly expressed in osteoclast precursors. MHC-II transactivator (CIITA) is the main regulator of MHC-II gene expression and the switch for protein presentation. CIITA is also a major regulator of osteoclast differentiation and bone homeostasis. Therefore, we hypothesized that the MHC-II promotes osteoclast differentiation, providing a novel pathogenic mechanism and a potential target for the treatment of PMOP.

Deciphering the immunopeptidome in vivo reveals new tumour antigens.

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules1-5. Current approaches to profiling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specific patterns of antigen presentation in vivo6. To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53fl/fl mouse model (KP/KbStrep)7. This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8+ T cell responses in naive mice and tumour-bearing mice. Many peptides specific to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance8. Beyond cancer, the KbStrep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity.

Abstract 1370: Targeting LSD1 rescues MHC-I antigen presentation in small cell lung cancer

Abstract Purpose: Small cell lung cancer (SCLC) is a highly aggressive cancer with early primary resistance and modest clinical benefit to immune checkpoint blockade (ICB). Mutation or transcriptional repression of the major histocompatibility complex class I (MHC-I) is a key mechanism driving resistance to T cell-based therapies. Lysine Demethylase 1 (LSD1) regulates gene expression through modulating mono- and di- methylated lysine 4 and 9 of histone H3. LSD1 is a therapeutic target of interests in SCLC due to its oncogenic requirement for tumor growth, and there has been growing evidence pointing to LSD1 as a repressor of tumor-intrinsic immunogenicity. Here investigated the role of LSD1 as a transcriptional regulator of antigen presentation in SCLC. Method: To perturb LSD1 function, we employed the pharmacological inhibitor ORY-1001 and shRNA-mediated knockdown. We then assessed changes in MHC-I expression on SCLC cell lines by flow cytometry and western blot. To analyze transcriptional changes following LSD1 inhibition, we performed RNA-seq on SCLC cells pre- and post-treatment with ORY-1001. To observe for antigen-specific T cell killing, we engineered SCLC cells to express endogenous antigens and co-cultured these cells with primary cognate CD8+ T cells. Finally, we treated genetically engineered mouse model (GEMM)-derived tumors on immunocompetent syngeneic mice with ORY-1001 with or without anti-PD-L1 blockade to assess for anti-tumor effects. Results: We discovered a significant and strong negative correlation between expression of LSD1 and genes encoding the MHC-I antigen presentation pathway. Inhibition of LSD1 induces expression of MHC-I genes and restores expression of genes encoding the antigen presentation machinery. Perturbation of LSD1 further activates interferon signaling and interferon-inducible expression of NLRC5, a well-characterized transcriptional activator of MHC-I genes. We further showed that targeting LSD1 sensitizes SCLC cells to MHC-I-restricted T cell-dependent cytolysis and enhances ICB efficacy in refractory SCLC tumors. Conclusion: Our data define a role for LSD1 as a potent negative regulator of MHC-I-mediated antigen presentation and provide rationale for the use of LSD1 inhibitors to augment response to immunotherapy in SCLC. Citation Format: Minh N. Nguyen, Hirokazu Taniguchi, Andrew Chow, Yingqian A. Zhan, Triparna Sen, Charles M. Rudin. Targeting LSD1 rescues MHC-I antigen presentation in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1370.

Sequence Diversity and Differences at the Highly Duplicated MHC-I Gene Reflect Viral Susceptibility in Sympatric Pinniped Species.

Differences in disease susceptibility among species can result from rapid host-pathogen coevolution and differences in host species ecology that affect the strength and direction of natural selection. Among 2 sympatric pinniped species that differ in sociality and putative disease exposure, we investigate observed differences in susceptibility through an analysis of a highly variable, duplicated gene family involved in the vertebrate immune response. Using high-throughput amplicon sequencing, we characterize diversity at the 2 exons that encode the peptide binding region of the major histocompatibility complex class I (MHC-I) gene in harbor (N = 60) and gray (N = 90) seal populations from the Northwest Atlantic. Across species, we identified 106 full-length exon 2 and 103 exon 3 sequence variants and a minimum of 11 duplicated MHC-I loci. The sequence variants clustered in 15 supertypes defined by the physiochemical properties of the peptide binding region, including a putatively novel Northwest Atlantic MHC-I diversity sublineage. Trans-species polymorphisms, dN/dS ratios, and evidence of gene conversion among supertypes are consistent with balancing selection acting on this gene. High functional redundancy suggests particularly strong selection among gray seals at the novel Northwest Atlantic MHC-I diversity sublineage. At exon 2, harbor seals had a significantly greater number of variants per individual than gray seals, but fewer supertypes. Supertype richness and private supertypes are hypothesized to contribute to observed differences in disease resistance between species, as consistently, across the North Atlantic and many disease outbreaks, gray seals appear to be more resistant to respiratory viruses than harbor seals.

Histone methyltransferase WHSC1 loss dampens MHC-I antigen presentation pathway to impair IFN-γ-stimulated antitumor immunity.

IFN-γ–stimulated MHC class I (MHC-I) antigen presentation underlies the core of antitumor immunity. However, sustained IFN-γ signaling also enhances the programmed death ligand 1 (PD-L1) checkpoint pathway to dampen antitumor immunity. It remains unclear how these opposing effects of IFN-γ are regulated. Here, we report that loss of the histone dimethyltransferase WHSC1 impaired the antitumor effect of IFN-γ signaling by transcriptional downregulation of the MHC-I machinery without affecting PD-L1 expression in colorectal cancer (CRC) cells. Whsc1 loss promoted tumorigenesis via a non-cell-autonomous mechanism in an Apcmin/+ mouse model, CRC organoids, and xenografts. Mechanistically, we found that the IFN-γ/STAT1 signaling axis stimulated WHSC1 expression and, in turn, that WHSC1 directly interacted with NLRC5 to promote MHC-I gene expression, but not that of PD-L1. Concordantly, silencing Whsc1 diminished MHC-I levels, impaired antitumor immunity, and blunted the effect of immune checkpoint blockade. Patient cohort analysis revealed that WHSC1 expression positively correlated with enhanced MHC-I expression, tumor-infiltrating T cells, and favorable disease outcomes. Together, our findings establish a tumor-suppressive function of WHSC1 that relays IFN-γ signaling to promote antigen presentation on CRC cells and provide a rationale for boosting WHSC1 activity in immunotherapy.