The MHC Class II Antigen-Processing and Presentation Pathway Is Dysregulated in Type 1 Diabetes.

Peptide loading of MHC class II (MHCII) molecules is facilitated by HLA-DM (DM), which catalyzes CLIP release, stabilizes empty MHCII, and edits the MHCII-bound peptide repertoire. HLA-DO (DO) binds to DM and modulates its activity, resulting in an altered set of peptides presented at the cell surface. MHCII-peptide presentation in individuals with type 1 diabetes (T1D) is abnormal, leading to a breakdown in tolerance; however, no direct measurement of the MHCII pathway activity in T1D patients has been performed. In this study, we measured MHCII Ag-processing pathway activity in humans by determining MHCII, MHCII-CLIP, DM, and DO levels by flow cytometry for peripheral blood B cells, dendritic cells, and monocytes from 99 T1D patients and 97 controls. Results showed that MHCII levels were similar for all three APC subsets. In contrast, MHCII-CLIP levels, independent of sex, age at blood draw, disease duration, and diagnosis age, were significantly increased for all three APCs, with B cells showing the largest increase (3.4-fold). DM and DO levels, which usually directly correlate with MHCII-CLIP levels, were unexpectedly identical in T1D patients and controls. Gene expression profiling on PBMC RNA showed that DMB mRNA was significantly elevated in T1D patients with residual C-peptide. This resulted in higher levels of DM protein in B cells and dendritic cells. DO levels were also increased, suggesting that the MHCII pathway maybe differentially regulated in individuals with residual C-peptide. Collectively, these studies show a dysregulation of the MHCII Ag-processing pathway in patients with T1D.

MHC-II dynamics are maintained in HLA-DR allotypes to ensure catalyzed peptide exchange.

Presentation of antigenic peptides by major histocompatibility complex class II (MHC-II) proteins determines T helper cell reactivity. The MHC-II genetic locus displays a large degree of allelic polymorphism influencing the peptide repertoire presented by the resulting MHC-II protein allotypes. During antigen processing, the human leukocyte antigen (HLA) molecule HLA-DM (DM) encounters these distinct allotypes and catalyzes exchange of the placeholder peptide CLIP by exploiting dynamic features of MHC-II. Here, we investigate 12 highly abundant CLIP-bound HLA-DRB1 allotypes and correlate dynamics to catalysis by DM. Despite large differences in thermodynamic stability, peptide exchange rates fall into a target range that maintains DM responsiveness. A DM-susceptible conformation is conserved in MHC-II molecules, and allosteric coupling between polymorphic sites affects dynamic states that influence DM catalysis. As exemplified for rheumatoid arthritis, we postulate that intrinsic dynamic features of peptide-MHC-II complexes contribute to the association of individual MHC-II allotypes with autoimmune disease.

HLA-DMB alleles and haplotypes in Ecuador (Cuenca) Amerindians: Importance for HLA and disease studies.

HLA-DMB allele frequencies and HLA-DBM-DRB1-DQB1 extended haplotypes were studied for the first time in Amerindians (Cuenca city area, Ecuador). It was found that most common extended haplotypes gathered the most frequent HLA-DRB1 Amerindian alleles. HLA-DMB polymorphism studies may be important to uncover HLA and diseases pathogenesis and also in an extended HLA haplotype frameshift. HLA-DM molecule has a crucial role together with CLIP protein in HLA class II peptide presentation. HLA extended haplotypes including complement and non classical genes alleles are proposed to HLA and disease studies.

HLA-II-Associated HIV-1 Adaptation Decreases CD4+ T-Cell Responses in HIV-1 Vaccine Recipients.

Epitopes with evidence of HLA-II-associated adaptation induce poorly immunogenic CD4+ T-cell responses in HIV-positive (HIV+) individuals. Many such escaped CD4+ T-cell epitopes are encoded by HIV-1 vaccines being evaluated in clinical trials. Here, we assessed whether this viral adaptation adversely impacts CD4+ T-cell responses following HIV-1 vaccination, thereby representing escaped epitopes. When evaluated in separate peptide pools, vaccine-encoded adapted epitopes (AE) induced CD4+ T-cell responses less frequently than nonadapted epitopes (NAE). We also demonstrated that in a polyvalent vaccine, where both forms of the same epitope were encoded, AE were less immunogenic. NAE-specific CD4+ T cells had increased, albeit low, levels of interferon gamma (IFN-γ) cytokine production. Single-cell transcriptomic analyses showed that NAE-specific CD4+ T cells expressed interferon-related genes, while AE-specific CD4+ T cells resembled a Th2 phenotype. Importantly, the magnitude of NAE-specific CD4+ T-cell responses, but not that of AE-specific responses, was found to positively correlate with Env-specific antibodies in a vaccine efficacy trial. Together, these findings show that HLA-II-associated viral adaptation reduces CD4+ T-cell responses in HIV-1 vaccine recipients and suggest that vaccines encoding a significant number of AE may not provide optimal B-cell help for HIV-specific antibody production. IMPORTANCE Despite decades of research, an effective HIV-1 vaccine remains elusive. Vaccine strategies leading to the generation of broadly neutralizing antibodies are likely needed to provide the best opportunity of generating a protective immune response against HIV-1. Numerous studies have demonstrated that T-cell help is necessary for effective antibody generation. However, immunogen sequences from recent HIV-1 vaccine efficacy trials include CD4+ T-cell epitopes that have evidence of immune escape. Our study shows that these epitopes, termed adapted epitopes, elicit lower frequencies of CD4+ T-cell responses in recipients from multiple HIV-1 vaccine trials. Additionally, the counterparts to these epitopes, termed nonadapted epitopes, elicited CD4+ T-cell responses that correlated with Env-specific antibodies in one efficacy trial. These results suggest that vaccine-encoded adapted epitopes dampen CD4+ T-cell responses, potentially impacting both HIV-specific antibody production and efficacious vaccine efforts.

Distribution of HLA-A, -B, -C, -DRB1, -DQB1, -DPB1 allele frequencies in patients with COVID-19 bilateral pneumonia in Russians, living in the Chelyabinsk region (Russia).

In this population-based case-control study conducted in the Chelyabinsk region of Russia, we examined the distribution of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1, in a group of 100 patients with confirmed COVID-19 bilateral pneumonia. Typing was performed by NGS and statistical calculations were carried out with the Arlequin program. HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles were compared between patients with COVID-19 and 99 healthy controls. We identified that COVID-19 susceptibility is associated with alleles and genotypes rs9277534A (disequilibrium with HLA-DPB1*02:01, -02:02, -04:01, -04:02, -17:01 alleles) with low expression of protein products HLA-DPB1 (pc < 0.028) and homozygosity at HLA-C*04 (p = 0.024, pc = 0.312). Allele HLA-A*01:01 was decreased in a group of patients with severe forms of bilateral pneumonia, and therefore it may be considered as a protective factor for the development of severe symptoms of COVID-19 (p = 0.009, pc = 0.225). Our studies provide further evidence for the functional association between HLA genes and COVID-19.

Multiple-Allele MHC Class II Epitope Engineering by a Molecular Dynamics-Based Evolution Protocol.

Epitopes that bind simultaneously to all human alleles of Major Histocompatibility Complex class II (MHC II) are considered one of the key factors for the development of improved vaccines and cancer immunotherapies. To engineer MHC II multiple-allele binders, we developed a protocol called PanMHC-PARCE, based on the unsupervised optimization of the epitope sequence by single-point mutations, parallel explicit-solvent molecular dynamics simulations and scoring of the MHC II-epitope complexes. The key idea is accepting mutations that not only improve the affinity but also reduce the affinity gap between the alleles. We applied this methodology to enhance a Plasmodium vivax epitope for multiple-allele binding. In vitro rate-binding assays showed that four engineered peptides were able to bind with improved affinity toward multiple human MHC II alleles. Moreover, we demonstrated that mice immunized with the peptides exhibited interferon-gamma cellular immune response. Overall, the method enables the engineering of peptides with improved binding properties that can be used for the generation of new immunotherapies.

Marginal zone B cells acquire dendritic cell functions by trogocytosis.

Marginal zone (MZ) B cells produce broad-spectrum antibodies that protect against infection early in life. In some instances, antibody production requires MZ B cells to display pathogen antigens bound to major histocompatibility complex class II (MHC II) molecules to T cells. We describe the trogocytic acquisition of these molecules from conventional dendritic cells (cDCs). Complement component 3 (C3) binds to murine and human MHC II on cDCs. MZ B cells recognize C3 with complement receptor 2 (CR2) and trogocytose the MHC II-C3 complexes, which become exposed on their cell surface. The ubiquitin ligase MARCH1 limits the number of MHC II-C3 complexes displayed on cDCs to prevent their elimination through excessive trogocytosis. Capture of C3 by MHC II thus enables the transfer of cDC-like properties to MZ B cells.

Tuning DO:DM Ratios Modulates MHC Class II Immunopeptidomes.

Major histocompatibility complex class II (MHC-II) antigen presentation underlies a wide range of immune responses in health and disease. However, how MHC-II antigen presentation is regulated by the peptide-loading catalyst HLA-DM (DM), its associated modulator, HLA-DO (DO), is incompletely understood. This is due largely to technical limitations: model antigen-presenting cell (APC) systems that express these MHC-II peptidome regulators at physiologically variable levels have not been described. Likewise, computational prediction tools that account for DO and DM activities are not presently available. To address these gaps, we created a panel of single MHC-II allele, HLA-DR4-expressing APC lines that cover a wide range of DO:DM ratio states. Using a combined immunopeptidomic and proteomic discovery strategy, we measured the effects DO:DM ratios have on peptide presentation by surveying over 10,000 unique DR4-presented peptides. The resulting data provide insight into peptide characteristics that influence their presentation with increasing DO:DM ratios. These include DM sensitivity, peptide abundance, binding affinity and motif, peptide length, and choice of binding register along the source protein. These findings have implications for designing improved HLA-II prediction algorithms and research strategies for dissecting the variety of functions that different APCs serve in the body.

HLA-DR, -DP, -DQ expression status of parathyroid tissue as a potential parathyroid donor selection criteria and review of literature.

BACKGROUND: Basic and clinical studies about parathyroid allotransplantation have to be utilized with more definitive criteria for longer graft survival. Several reports demonstrated different isolation and cultivation methods for parathyroid cells to minimize their immunogenicity. In this study, we aim to compare and evaluate the clinical characteristics and the status of HLA class II expression changes in parathyroid tissue. METHODS: A total of 22 parathyroid hyperplasia tissue donors was included in this study. Clinical characteristics were evaluated and compared with the HLA-DR, -DP, -DQ mRNA, and protein expression levels which were determined by qRT-PCR and Western blot. RESULTS: We have compared the clinical characteristics (age, dialysis duration, frequency, recurrency of hyperparathyroidism and, calcimimetic usage) and HLA class II expression. HLA class II mRNA and protein levels showed varied expression patterns between tissues. Only, the HLA-DP has high mRNA expression levels without affecting the protein level when compared with the ages of the tissue donors. In addition, the HLA-DR, HLA-DP, and HLA-DQα1 protein expression levels showed a permanent and varied expression rate between tissues. CONCLUSION: Expression of HLA class II molecules in parathyroid cells appears to constitute a decisive factor. Despite the lack of clinical outcomes, present data proposes new insight with a detailed understanding of parathyroid immunogenicity. In the future, randomized controlled clinical trials are needed for the accurate assessment of the effect of the varied HLA class II expression profiles in parathyroid tissue.

Epstein-Barr Virus and the Origin of Myalgic Encephalomyelitis or Chronic Fatigue Syndrome.

Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) affects approximately 1% of the general population. It is a chronic, disabling, multi-system disease for which there is no effective treatment. This is probably related to the limited knowledge about its origin. Here, we summarized the current knowledge about the pathogenesis of ME/CFS and revisit the immunopathobiology of Epstein-Barr virus (EBV) infection. Given the similarities between EBV-associated autoimmune diseases and cancer in terms of poor T cell surveillance of cells with EBV latency, expanded EBV-infected cells in peripheral blood and increased antibodies against EBV, we hypothesize that there could be a common etiology generated by cells with EBV latency that escape immune surveillance. Albeit inconclusive, multiple studies in patients with ME/CFS have suggested an altered cellular immunity and augmented Th2 response that could result from mechanisms of evasion to some pathogens such as EBV, which has been identified as a risk factor in a subset of ME/CFS patients. Namely, cells with latency may evade the immune system in individuals with genetic predisposition to develop ME/CFS and in consequence, there could be poor CD4 T cell immunity to mitogens and other specific antigens, as it has been described in some individuals. Ultimately, we hypothesize that within ME/CFS there is a subgroup of patients with DRB1 and DQB1 alleles that could confer greater susceptibility to EBV, where immune evasion mechanisms generated by cells with latency induce immunodeficiency. Accordingly, we propose new endeavors to investigate if anti-EBV therapies could be effective in selected ME/CFS patients.

The similarity of class II HLA genotypes defines patterns of autoreactivity in idiopathic bone marrow failure disorders.

Idiopathic aplastic anemia (IAA) is a rare autoimmune bone marrow failure (BMF) disorder initiated by a human leukocyte antigen (HLA)-restricted T-cell response to unknown antigens. As in other autoimmune disorders, the predilection for certain HLA profiles seems to represent an etiologic factor; however, the structure-function patterns involved in the self-presentation in this disease remain unclear. Herein, we analyzed the molecular landscape of HLA complexes of a cohort of 300 IAA patients and almost 3000 healthy and disease controls by deeply dissecting their genotypic configurations, functional divergence, self-antigen binding capabilities, and T-cell receptor (TCR) repertoire specificities. Specifically, analysis of the evolutionary divergence of HLA genotypes (HED) showed that IAA patients carried class II HLA molecules whose antigen-binding sites were characterized by a high level of structural homology, only partially explained by specific risk allele profiles. This pattern implies reduced HLA binding capabilities, confirmed by binding analysis of hematopoietic stem cell (HSC)-derived self-peptides. IAA phenotype was associated with the enrichment in a few amino acids at specific positions within the peptide-binding groove of DRB1 molecules, affecting the interface HLA-antigen-TCR ß and potentially constituting the basis of T-cell dysfunction and autoreactivity. When analyzing associations with clinical outcomes, low HED was associated with risk of malignant progression and worse survival, underlying reduced tumor surveillance in clearing potential neoantigens derived from mechanisms of clonal hematopoiesis. Our data shed light on the immunogenetic risk associated with IAA etiology and clonal evolution and on general pathophysiological mechanisms potentially involved in other autoimmune disorders.

Mouse Homologue of Human HLA-DO Does Not Preempt Autoimmunity but Controls Murine Gammaherpesvirus MHV68.

H2-O (human HLA-DO) is a relatively conserved nonclassical MHC class II (MHCII)-like molecule. H2-O interaction with human HLA-DM edits the repertoire of peptides presented to TCRs by MHCII. It was long hypothesized that human HLA-DM inhibition by H2-O provides protection from autoimmunity by preventing binding of the high-affinity self-peptides to MHCII. The available evidence supporting this hypothesis, however, was inconclusive. A possibility still remained that the effect of H2-O deficiency on autoimmunity could be better revealed by using H2-O-deficient mice that were already genetically predisposed to autoimmunity. In this study, we generated and used autoimmunity-prone mouse models for systemic lupus erythematosus and organ-specific autoimmunity (type 1 diabetes and multiple sclerosis) to definitively test whether H2-O prevents autoimmune pathology. Whereas our data failed to support any significance of H2-O in protection from autoimmunity, we found that it was critical for controlling a γ-herpesvirus, MHV68. Thus, we propose that H2-O editing of the MHCII peptide repertoire may have evolved as a safeguard against specific highly prevalent viral pathogens.

The landscape of DNA methylation in asthma: a data mining and validation.

Human asthma is caused by interactions between a range of genetic and environmental factors. However, the specific pathogenesis of asthma remains controversial. This study explored the contribution of DNA methylation to asthma using computer learning methods. Relevant datasets and information related to patients with asthma were collected from the Gene Expression Omnibus (GEO) database. A multivariate linear regression model was established. Differentially expressed genes and DNA methylation sites were identified. The results showed that the expression of 169 genes was significantly different between the two groups. Through differential analysis of methylation and differential analysis of gene expression, 44 differentially expressed genes that may be affected by DNA methylation modification were identified. The results of the multiple linear regression model showed that DNA methylation could explain 9.81% of the variation in gene expression. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that the differentially expressed genes, HLA-DMB, IL4, HLA-DPB1, and CD40LG, were related to the occurrence of asthma, and HLA-DMB expression was significantly reduced in allergic asthma. There was a positive correlation between cg04933135 and HLA-DMB expression, and cg04933135 was a differential site for DNA methylation. Using blood samples from asthma patients, we confirmed that HLA-DMB expression is down-regulated, which may be affected by abnormal DNA methylation. DNA methylation plays an important role in the development of asthma, and HLA-DMB which modified by abnormal DNA methylation can be regarded as a new biomarker of asthma.

Comprehensive Analysis of RNA Expression Profile Identifies Hub miRNA-circRNA Interaction Networks in the Hypoxic Ischemic Encephalopathy.

Hypoxic ischemic encephalopathy (HIE) is classified as a sort of serious nervous system syndrome that occurs in the early life period. Noncoding RNAs had been confirmed to have crucial roles in human diseases. So far, there were few systematical and comprehensive studies towards the expression profile of RNAs in the brain after hypoxia ischemia. In this study, 31 differentially expressed microRNAs (miRNAs) with upregulation were identified. In addition, 5512 differentially expressed mRNAs, long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs) were identified in HIE groups. Bioinformatics analysis showed these circRNAs and mRNAs were significantly enriched in regulation of leukocyte activation, response to virus, and neutrophil degranulation. Pathway and its related gene network analysis indicated that HLA - DPA1, HLA - DQA2, HLA - DQB1, and HLA - DRB4 have a more crucial role in HIE. Finally, miRNA-circRNA-mRNA interaction network analysis was also performed to identify hub miRNAs and circRNAs. We found that miR-592 potentially targeting 5 circRNAs, thus affecting 15 mRNA expressions in HIR. hsa_circ_0068397 and hsa_circ_0045698 were identified as hub circRNAs in HIE. Collectively, using RNA-seq, bioinformatics analysis, and circRNA/miRNA interaction prediction, we systematically investigated the differentially expressed RNAs in HIE, which could give a new hint of understanding the pathogenesis of HIE.

HLA Does Not Impact on Short-Medium-Term Antibody Response to Preventive Anti-SARS-Cov-2 Vaccine.

The response to anti-SARS-Cov-2 preventive vaccine shows high interpersonal variability at short and medium term. One of the explanations might be the individual HLA allelic variants. Indeed, B cell response is stimulated and sustained by CD4+ T helper cells activated by antigens presented by HLA-class II alleles on antigen-presenting cells (APCs). The impact of the number of antigens binding to HLA class-II alleles on the antibody response to the COVID vaccine has been assessed in a cohort of 56 healthcare workers who received the full schedule of the Pfizer-BioNTech BNT162b2 vaccine. Such vaccine is based on the entire spike protein of the SARS-CoV-2. Ab titers have been evaluated 2 weeks after the first dose as well as 2 weeks and 4 months after the boosting dose. HLA-DRB1 and DBQ1 for each of the vaccinees have been assessed, and strong binders have been predicted. The analysis showed no significant correlation between the short-medium-term Ab titers and the number of strong binders (SB) for each individual. These results indicate that levels of Ab response to the spike glycoprotein is not dependent on HLA class II allele, suggesting an equivalent efficacy at global level of the currently used vaccines. Furthermore, the pattern of persistence in Ab titer does not correlate with specific alleles or with the number of SBs.

HLA Class II Polymorphisms Modulate Gut Microbiota and Experimental Autoimmune Encephalomyelitis Phenotype.

Multiple sclerosis (MS) is an autoimmune disease of the CNS in which the interaction between genetic and environmental factors plays an important role in disease pathogenesis. Although environmental factors account for 70% of disease risk, the exact environmental factors associated with MS are unknown. Recently, gut microbiota has emerged as a potential missing environmental factor linked with the pathobiology of MS. Yet, how genetic factors, such as HLA class II gene(s), interact with gut microbiota and influence MS is unclear. In the current study, we investigated whether HLA class II genes that regulate experimental autoimmune encephalomyelitis (EAE) and MS susceptibility also influence gut microbiota. Previously, we have shown that HLA-DR3 transgenic mice lacking endogenous mouse class II genes (AE-KO) were susceptible to myelin proteolipid protein (91-110)-induced EAE, an animal model of MS, whereas AE-KO.HLA-DQ8 transgenic mice were resistant. Surprisingly, HLA-DR3.DQ8 double transgenic mice showed higher disease prevalence and severity compared with HLA-DR3 mice. Gut microbiota analysis showed that HLA-DR3, HLA-DQ8, and HLA-DR3.DQ8 double transgenic mice microbiota are compositionally different from AE-KO mice. Within HLA class II transgenic mice, the microbiota of HLA-DQ8 mice were more similar to HLA-DR3.DQ8 than HLA-DR3. As the presence of DQ8 on an HLA-DR3 background increases disease severity, our data suggests that HLA-DQ8-specific microbiota may contribute to disease severity in HLA-DR3.DQ8 mice. Altogether, our study provides evidence that the HLA-DR and -DQ genes linked to specific gut microbiota contribute to EAE susceptibility or resistance in a transgenic animal model of MS.

In vitro immunogenicity prediction: bridging between innate and adaptive immunity.

Development of antidrug antibodies (ADAs) is an undesirable potential outcome of administration of biotherapeutics and involves the innate and adaptive immune systems. ADAs can have detrimental clinical consequences: they can reduce biotherapeutic efficacy or produce adverse events. Because animal models are considered poor predictors of immunogenicity in humans, in vitro assays with human innate and adaptive immune cells are commonly used alternatives that can reveal cell-mediated unwanted immune responses. Multiple methods have been developed to assess the immune cell response following exposure to biotherapeutics and estimate the potential immunogenicity of biotherapeutics. This review highlights the role of innate and adaptive immune cells as the drivers of immunogenicity and summarizes the use of these cells in assays to predict clinical ADA.

Analysis of B Cell Receptor-Mediated Antigen Extraction by B Lymphocytes from Plasma Membrane Sheets Using Confocal Microscopy.

High-resolution confocal imaging has provided new insights in the process of receptor-mediated endocytosis in variety of cell types. We describe here the protocol for investigating B cell receptor (BCR)-mediated internalization of membrane bound antigens using confocal microscopy. We describe the method to prepare plasma membrane sheets (PMS) in a small area, bind fluorescently tagged antigens to the PMS and activate B cells on the PMS. We also describe the method for analyzing antigen internalization using confocal microscopy and computational image analysis. This protocol is useful for the study of antigen internalization by B cells and can be applied for studying receptor-mediated endocytosis in other cells as well. The setup we describe here is especially useful for studying rare cell types when the number of cells available is limiting.