HLA-DM and HLA-DO interplay for the peptide editing of HLA class II in healthy tissues and leukemia.

HLA class II antigen presentation is modulated by the activity of the peptide editor HLA-DM and its antagonist HLA-DO, with their interplay controlling the peptide repertoires presented by normal and malignant cells. The role of these molecules in allogeneic hematopoietic cell transplantation (alloHCT) is poorly investigated. Balanced expression of HLA-DM and HLA-DO can influence the presentation of leukemia-associated antigens and peptides targeted by alloreactive T cells, therefore affecting both anti-leukemia immunity and the potential onset of Graft versus Host Disease. We leveraged on a large collection of bulk and single cell RNA sequencing data, available at different repositories, to comprehensively review the level and distribution of HLA-DM and HLA-DO in different cell types and tissues of the human body. The resulting expression atlas will help future investigations aiming to dissect the dual role of HLA class II peptide editing in alloHCT, and their potential impact on its clinical outcome.

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.

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.

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.

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.

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.

High-resolution HLA allele and haplotype frequencies of the Saudi Arabian population based on 45,457 individuals and corresponding stem cell donor matching probabilities.

We estimated HLA allele and haplotype frequencies of the Saudi Arabian population from a sample of 45,457 registered stem cell donors. The most frequent HLA alleles were A*02:01g (18.5%), C*06:02g (16.1%), B*51:01g (14.1%), DRB1*07:01g (16.2%), DQB1*02:01g (30.5%), and DPB1*04:01g (33.6%). The most frequent 5-locus haplotypes were A*02:05g~C*06:02g~B*50:01g~DRB1*07:01g~DQB1*02:01g (1.73%), A*02:01g~C*06:02g~B*50:01g~DRB1*07:01g~DQB1*02:01g (1.66%), and A*26:01g~C*07:02g~B*08:01g~DRB1*03:01g~DQB1*02:01g (1.38%). Furthermore, we used the calculated haplotype frequencies to estimate stem cell donor matching probabilities for Saudi Arabian donor and patient populations under various matching requirements. These results are relevant for strategic donor registry planning in the Kingdom of Saudi Arabia.

Comprehensive Analysis of CD4+ T Cell Response Cross-Reactive to SARS-CoV-2 Antigens at the Single Allele Level of HLA Class II.

Common human coronaviruses have been circulating undiagnosed worldwide. These common human coronaviruses share partial sequence homology with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); therefore, T cells specific to human coronaviruses are also cross-reactive with SARS-CoV-2 antigens. Herein, we defined CD4+ T cell responses that were cross-reactive with SARS-CoV-2 antigens in blood collected in 2016-2018 from healthy donors at the single allele level using artificial antigen-presenting cells (aAPC) expressing a single HLA class II allotype. We assessed the allotype-restricted responses in the 42 individuals using the aAPCs matched 22 HLA-DR alleles, 19 HLA-DQ alleles, and 13 HLA-DP alleles. The response restricted by the HLA-DR locus showed the highest magnitude, and that by HLA-DP locus was higher than that by HLA-DQ locus. Since two alleles of HLA-DR, -DQ, and -DP loci are expressed co-dominantly in an individual, six different HLA class II allotypes can be used to the cross-reactive T cell response. Of the 16 individuals who showed a dominant T cell response, five, one, and ten showed a dominant response by a single allotype of HLA-DR, -DQ, and -DP, respectively. The single allotype-restricted T cells responded to only one antigen in the five individuals and all the spike, membrane, and nucleocapsid proteins in the six individuals. In individuals heterozygous for the HLA-DPA and HLA-DPB loci, four combinations of HLA-DP can be expressed, but only one combination showed a dominant response. These findings demonstrate that cross-reactive T cells to SARS-CoV-2 respond with single-allotype dominance.

Permissive HLA-DPB1 mismatches in HCT depend on immunopeptidome divergence and editing by HLA-DM.

In hematopoietic cell transplantation (HCT), permissive HLA-DPB1 mismatches between patients and their unrelated donors are associated with improved outcomes compared with nonpermissive mismatches, but the underlying mechanism is incompletely understood. Here, we used mass spectrometry, T-cell receptor-ß (TCRß) deep sequencing, and cellular in vitro models of alloreactivity to interrogate the HLA-DP immunopeptidome and its role in alloreactive T-cell responses. We find that permissive HLA-DPB1 mismatches display significantly higher peptide repertoire overlaps compared with their nonpermissive counterparts, resulting in lower frequency and diversity of alloreactive TCRß clonotypes in healthy individuals and transplanted patients. Permissiveness can be reversed by the absence of the peptide editor HLA-DM or the presence of its antagonist, HLA-DO, through significant broadening of the peptide repertoire. Our data establish the degree of immunopeptidome divergence between donor and recipient as the mechanistic basis for the clinically relevant permissive HLA-DPB1 mismatches in HCT and show that permissiveness is dependent on HLA-DM-mediated peptide editing. Its key role for harnessing T-cell alloreactivity to HLA-DP highlights HLA-DM as a potential novel target for cellular and immunotherapy of leukemia.

HLA class II eplet mismatch load improves prediction of dnDSA development after living donor kidney transplantation.

HLA donor-specific antibodies developed de novo after transplant remain a major cause of chronic allograft dysfunction. Our study main purpose was to determine whether HLA MM, assessed traditionally and by HLA total and AbVer eplet mismatch load (EptMM and EpvMM) assessed with HLAMatchMaker, had impact on dnDSA development after living donor kidney transplantation (LDKT). We retrospectively analysed a cohort of 96 LDKT between 2008 and 2017 performed in Hospital Santo António. Seven patients developed dnDSA-II and EpvMM and EptMM were greater in dnDSA-II group compared to the no dnDSA-II (18.0 ± 8.7 versus 9.9 ± 7.9, p = .041 and 41.3 ± 18.9 versus 23.1 ± 16.7, p = .018), which is not observed for AgMM (2.29 versus 1.56; p = .09). In a multivariate analysis, we found that preformed DSA (HR = 7.983; p = .023), living unrelated donors (HR = 8.052; p = .024) and retransplantation (HR = 14.393; p = .009) were predictors for dnDSA-II (AUC = 0.801; 0.622-0.981). HLA-II EpvMM (HR = 1.105; p = .028; AUC = 0.856) showed to be a superior predictor of dnDSA-II, when compared to AgMM (HR = 1.740; p = .113; AUC = 0.783), when adjusted for these clinical variables. Graft survival was significantly lower within dnDSA-II patient group (36% versus 88%, p < .001). HLA molecular mismatch analysis is extremely important to minimize risk for HLA-II dnDSA development improving outcome and increasing chance of retransplant lowering allosensitization.

Autoantibody-negative insulin-dependent diabetes mellitus after SARS-CoV-2 infection: a case report.

Here we report a case where the manifestations of insulin-dependent diabetes occurred following SARS-CoV-2 infection in a young individual in the absence of autoantibodies typical for type 1 diabetes mellitus. Specifically, a 19-year-old white male presented at our emergency department with diabetic ketoacidosis, C-peptide level of 0.62 µg l-1, blood glucose concentration of 30.6 mmol l-1 (552 mg dl-1) and haemoglobin A1c of 16.8%. The patient´s case history revealed probable COVID-19 infection 5-7 weeks before admission, based on a positive test for antibodies against SARS-CoV-2 proteins as determined by enzyme-linked immunosorbent assay. Interestingly, the patient carried a human leukocyte antigen genotype (HLA DR1-DR3-DQ2) considered to provide only a slightly elevated risk of developing autoimmune type 1 diabetes mellitus. However, as noted, no serum autoantibodies were observed against islet cells, glutamic acid decarboxylase, tyrosine phosphatase, insulin and zinc-transporter 8. Although our report cannot fully establish causality between COVID-19 and the development of diabetes in this patient, considering that SARS-CoV-2 entry receptors, including angiotensin-converting enzyme 2, are expressed on pancreatic ß-cells and, given the circumstances of this case, we suggest that SARS-CoV-2 infection, or COVID-19, might negatively affect pancreatic function, perhaps through direct cytolytic effects of the virus on ß-cells.

HLA-DM catalytically enhances peptide dissociation by sensing peptide-MHC class II interactions throughout the peptide-binding cleft.

Human leukocyte antigen-DM (HLA-DM) is an integral component of the major histocompatibility complex class II (MHCII) antigen-processing and -presentation pathway. HLA-DM shapes the immune system by differentially catalyzing peptide exchange on MHCII molecules, thereby editing the peptide-MHCII (pMHCII) repertoire by imposing a bias on the foreign and self-derived peptide cargos that are presented on the cell surface for immune surveillance and tolerance induction by CD4+ T cells. To better understand DM selectivity, here we developed a real-time fluorescence anisotropy assay to delineate the pMHCII intrinsic stability, DM-binding affinity, and catalytic turnover, independent kinetic parameters of HLA-DM enzymatic activity. We analyzed prominent pMHCII contacts by differentiating the kinetic parameters in pMHCII homologs, observing that peptide interactions throughout the MHCII-binding cleft influence both the rate of peptide dissociation from the DM-pMHCII catalytic complex and the binding affinity of HLA-DM for a pMHCII. We show that the intrinsic stability of a pMHCII linearly correlates with DM catalytic turnover, but is nonlinearly correlated with its binding affinity. Surprisingly, interactions at the peptides N terminus up to and including MHCII position one (P1) anchor affected the catalytic turnover, suggesting that the active DM-pMHCII catalytic complex operates on pMHCII complexes with full peptide occupancy. Furthermore, interactions at the peptide C terminus modulated DM-binding affinity, suggesting distal communication between peptide interactions with the MHCII and the DM-pMHCII binding interface. Our results imply an intimate linkage between the DM-pMHCII interface and peptide-MHCII interactions throughout the peptide-binding cleft.

Distinct editing functions of natural HLA-DM allotypes impact antigen presentation and CD4+ T cell activation.

Classical human leukocyte antigen (HLA) molecules of the major histocompatibility class II (MHCII) complex present peptides for the development, surveillance and activation of CD4+ T cells. The nonclassical MHCII-like protein HLA-DM (DM) catalyzes the exchange and loading of peptides onto MHCII molecules, thereby shaping MHCII immunopeptidomes. Natural variations of DM in both chains of the protein (DMA and DMB) have been hypothesized to impact peptide presentation, but no evidence for altered function has been reported. Here we define the presence of DM allotypes in human populations covered by the 1000 Genomes Project and probe their activity. The functional properties of several allotypes are investigated and show strong enhancement of peptide-induced T cell activation for a particular combination of DMA and DMB. Biochemical evidence suggests a broader pH activity profile for the new variant relative to that of the most commonly expressed DM allotype. Immunopeptidome analysis indicates that the compartmental activity of the new DM heterodimer extends beyond the late endosome and suggests that the natural variation of DM has profound effects on adaptive immunity when antigens bypass the canonical processing pathway.

Association of HLA-A, -B, DRB, and DQB Alleles with Persistent HPV-16 Infection in Women from Tamil Nadu, India.

Women with persistent human papillomavirus (HPV) infections have a high risk of developing cervical cancer (CaCx). HPV-16 alone accounts for more than 60% of CaCx worldwide. Most of the HPV infections are transient and only a subset of women develop persistent HPV-16 infection. Many studies have shown associations of different human leukocyte antigen (HLA) alleles with HPV-mediated CaCx, but there are only a few studies globally that relate to persistent HPV-16 infection. Furthermore, such studies from India are sparse. Hence, we investigated the association of HLA-A, B, DRB, and DQB alleles with persistent HPV-16 infection and HPV-16-positive CaCx in south India (Tamil Nadu). HPV-16 persistent infection was observed in 7% of normal women. A total of 50 women with HPV-16-positive CaCx, 21 women with HPV-16 persistent infection, and 74 HPV-16-negative normal women were recruited for this study. Low-resolution typing of HLA-A, B, DRB, and DQB alleles was performed. HLA-B*44 and DRB1*07 showed a significant association with persistent HPV-16 infection (odds ratio, p-value = 26.3, 0.03 and 4.7, 0.01, respectively). HLA-B*27 and DRB1*12 were significantly associated with both HPV-16+ CaCx and persistent HPV-16 infection (23.8, 0.03; 52.9, 0.01; 9.8, 0.0009; and 13.8, 0.009; respectively). HLA-B*15 showed a negative association with HPV-16-positive CaCx (0.1, 0.01), whereas DRB1*04 exhibited protection to both HPV-16-positive CaCx and persistent HPV-16 infection (0.3, 0.0001 and 0.1, 0.0002, respectively). Thus, we show HLA allelic association with HPV-16 infection in Tamil Nadu. Larger studies on high-resolution HLA typing coupled with HPV-16 genome diversity will offer further insights into host/pathogen genome coevolution.

Synergy between B cell receptor/antigen uptake and MHCII peptide editing relies on HLA-DO tuning.

B cell receptors and surface-displayed peptide/MHCII complexes constitute two key components of the B-cell machinery to sense signals and communicate with other cell types during antigen-triggered activation. However, critical pathways synergizing antigen-BCR interaction and antigenic peptide-MHCII presentation remain elusive. Here, we report the discovery of factors involved in establishing such synergy. We applied a single-cell measure coupled with super-resolution microscopy to investigate the integrated function of two lysosomal regulators for peptide loading, HLA-DM and HLA-DO. In model cell lines and human tonsillar B cells, we found that tunable DM/DO stoichiometry governs DMfree activity for exchange of placeholder CLIP peptides with high affinity MHCII ligands. Compared to their naïve counterparts, memory B cells with less DMfree concentrate a higher proportion of CLIP/MHCII in lysosomal compartments. Upon activation mediated by high affinity BCR, DO tuning is synchronized with antigen internalization and rapidly potentiates DMfree activity to optimize antigen presentation for T-cell recruitment.

Acetaldehyde suppresses the display of HBV-MHC class I complexes on HBV-expressing hepatocytes.

Hepatitis B virus (HBV) infection and alcoholism are major public health problems worldwide, contributing to the development of end-stage liver disease. Alcohol intake affects HBV infection pathogenesis and treatment outcomes. HBV-specific cytotoxic T lymphocytes (CTLs) play an important role in HBV clearance. Many previous studies have focused on alcohol-induced impairments of the immune response. However, it is not clear whether alcohol alters the presentation of HBV peptide-major histocompatibility complex (MHC) class I complexes on infected hepatocytes resulting in escape of its recognition by CTLs. Hence, the focus of this study was to investigate the mechanisms by which ethanol metabolism affects the presentation of CTL epitope on HBV-infected hepatocytes. As demonstrated here, although continuous cell exposure to acetaldehyde-generating system (AGS) increased HBV load in HepG2.2.15 cells, it decreased the expression of HBV core peptide 18-27-human leukocyte antigen-A2complex (CTL epitope) on the cell surface. Moreover, we observed AGS-induced suppression of chymotrypsin- and trypsin-like proteasome activities necessary for peptide processing by proteasome as well as a decline in IFNγ-stimulated immunoproteasome (IPR) function and expression of PA28 activator and immunoproteasome subunits LMP7 and LMP2. Furthermore, IFNγ-induced activation of peptide-loading complex (PLC) components, such as transporter associated with antigen processing (TAP1) and tapasin, were suppressed by AGS. The attenuation of IPR and PLC activation was attributed to AGS-triggered impairment of IFNγ signaling in HepG2.2.15 cells. Collectively, all these downstream events reduced the display of HBV peptide-MHC class I complexes on the hepatocyte surface, which may suppress CTL activation and the recognition of CTL epitopes on HBV-expressing hepatocytes by immune cells, thereby leading to persistence of liver inflammation.NEW & NOTEWORTHY Our study shows that in HBV-expressing HepG2.2.15 cells, acetaldehyde alters HBV peptide processing by suppressing chymotrypsin- and trypsin-like proteasome activities and decreases IFNγ-stimulated immunoproteasome function and expression of PA28 activator and immunoproteasome subunits. It also suppresses IFNγ-induced activation of peptide-loading complex (PLC) components due to impairment of IFNγ signaling via the JAK-STAT1 pathway. These acetaldehyde-induced dysfunctions reduced the display of HBV peptide-MHC class I complexes on the hepatocyte surface, thereby leading to persistence of HBV infection.

What to do with HLA-DO/H-2O two decades later?

The main objective of antigen processing is to orchestrate the selection of immunodominant epitopes for recognition by CD4 T cells. To achieve this, MHC class II molecules have evolved with a flexible peptide-binding groove in need of a bound peptide. Newly synthesized MHC-II molecules bind a class II invariant chain (Ii) upon synthesis and are shuttled to a specialized compartment, where they encounter exogenous antigens. Ii serves multiple functions, one of which is to maintain the shape of the MHC-II groove so that it can readily bind exogenous antigens upon dissociation of the Ii peptide in MHC- II compartment. MIIC contains processing enzymes, one or both accessory molecules, HLA-DM/H2-M (DM) and HLA-DO/H2-O (DO), and optimal denaturing conditions. In a process known as "editing," DM facilitates the dissociation of the invariant chain peptide, CLIP, for exchange with exogenous antigens. Despite the availability of mechanistic insights into DM functions, understanding how DO contributes to epitope selection has proven to be more challenging. The current dogma assumes that DO inhibits DM, whereas an opposing model suggests that DO fine-tunes the epitope selection process. Understanding which of these, or potentially other models of DO function is important, as DO variants have been linked to autoimmunity, cancer, and the generation of broadly neutralizing antibodies to viruses. This review therefore attempts to evaluate experimental evidence in support of these hypotheses, with an emphasis on the less discussed model, and to explore intriguing questions about the importance of DO in biology.