HLA Genes: A Hallmark of Functional Genetic Variation and Complex Evolution.

The major histocompatibility complex (MHC) with its highly polymorphic HLA genes represents one of the most intensely studied genomic regions in the genome. MHC proteins play a key role in antigen-specific immunity and are associated with a wide range of complex diseases. Despite decades of research and many advances in the field, the characterization and interpretation of its genetic and genomic variability remain challenging. Here an overview is provided of the MHC, the nature of its exceptional variability, and the complex evolutionary processes assumed to drive this variability. Highlighted are also recent advances in the field that promise to improve our understanding of the variability in the MHC and in antigen-specific immunity more generally.

MAIT cell-MR1 reactivity is highly conserved across multiple divergent species.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by major histocompatibility complex class I related protein 1 (MR1), via an αß T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved major histocompatibility complex-I-like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology, thereby allowing the use of selected species-mismatched MR1-antigen (MR1-Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1-Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1-Ag tetramers and utilized these alongside previously developed human, mouse, and pig-tailed macaque MR1-Ag tetramers to characterize cross-species tetramer reactivities. MR1-Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1-Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1-Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1-Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1-Ag tetramers. Collectively, our results validate the use and define the limitations of species-mismatched MR1-Ag tetramers in comparative immunology studies.

Quantitative Measurement of Plasma Membrane Protein Internalisation and Recycling in Heterogenous Cellular Samples by Flow Cytometry.

Plasma membrane proteins mediate important aspects of physiology, including nutrient acquisition, cell-cell interactions, and monitoring homeostasis. The trafficking of these proteins, involving internalisation from and/or recycling back to the cell surface, is often critical to their functions. These processes can vary among different proteins and cell types and states and are still being elucidated. Current strategies to measure surface protein internalisation and recycling are typically microscopy or biochemical assays; these are accurate but generally limited to analysing a homogenous cell population and are often low throughput. Here, we present flow cytometry-based methods involving probe-conjugated antibodies that enable quantification of internalisation or recycling rates at the single-cell level in complex samples. To measure internalisation, we detail an assay where the protein of interest is labelled with a specific antibody conjugated to a fluorescent oligonucleotide-labelled probe. To measure recycling, a specific antibody conjugated to a cleavable biotin group is employed. These probes permit the differentiation of molecules that have been internalised or recycled from those that have not. When combined with cell-specific marker panels, these methods allow the quantitative study of plasma membrane protein trafficking dynamics in a heterogenous cell mixture at the single-cell level. Key features • These assays allow sensitive quantification of internalised or recycled surface molecules using oligonucleotide or cleavable biotin-conjugated probes, respectively, and detected by flow cytometry. • They can be adapted to any membrane protein that transits via the cell surface and for which a specific purified antibody is available. • The dynamics of a cell surface protein can be measured in heterogenous cell populations simultaneously, including various cellular activation states. • The internalisation assay builds upon the method developed by Liu et al. [1,2] and extends its application to heterogenous human peripheral blood mononuclear cells. • These assays have been extensively used on suspension cells but have not been tested on adherent cells.

Regulatory SVA retrotransposons and classical HLA genotyped-transcripts associated with Parkinson's disease.

Introduction: Parkinson's disease (PD) is a neurodegenerative and polygenic disorder characterised by the progressive loss of neural dopamine and onset of movement disorders. We previously described eight SINE-VNTR-Alu (SVA) retrotransposon-insertion-polymorphisms (RIPs) located and expressed within the Human Leucocyte Antigen (HLA) genomic region of chromosome 6 that modulate the differential co-expression of 71 different genes including the HLA classical class I and class II genes in a Parkinson's Progression Markers Initiative (PPMI) cohort. Aims and methods: In the present study, we (1) reanalysed the PPMI genomic and transcriptomic sequencing data obtained from whole blood of 1521 individuals (867 cases and 654 controls) to infer the genotypes of the transcripts expressed by eight classical HLA class I and class II genes as well as DRA and the DRB3/4/5 haplotypes, and (2) examined the statistical differences between three different PD subgroups (cases) and healthy controls (HC) for the HLA and SVA transcribed genotypes and inferred haplotypes. Results: Significant differences for 57 expressed HLA alleles (21 HLA class I and 36 HLA class II alleles) up to the three-field resolution and four of eight expressed SVA were detected at p<0.05 by the Fisher's exact test within one or other of three different PD subgroups (750 individuals with PD, 57 prodromes, 60 individuals who had scans without evidence of dopamine deficits [SWEDD]), when compared against a group of 654 HCs within the PPMI cohort and when not corrected by the Bonferroni test for multiple comparisons. Fourteen of 20 significant alleles were unique to the PD-HC comparison, whereas 31 of the 57 alleles overlapped between two or more different subgroup comparisons. Only the expressed HLA-DRA*01:01:01 and -DQA1*03:01:01 protective alleles (PD v HC), the -DQA1*03:03:01 risk (HC v Prodrome) or protective allele (PD v Prodrome), the -DRA*01:01:02 and -DRB4*01:03:02 risk alleles (SWEDD v HC), and the NR_SVA_381 present genotype (PD v HC) at a 5% homozygous insertion frequency near HLA-DPA1, were significant (Pc<0.1) after Bonferroni corrections. The homologous NR_SVA_381 insertion significantly decreased the transcription levels of HLA-DPA1 and HLA-DPB1 in the PPMI cohort and its presence as a homozygous genotype is a risk factor (Pc=0.012) for PD. The most frequent NR_SVA_381 insertion haplotype in the PPMI cohort was NR_SVA_381/DPA1*02/DPB1*01 (3.7%). Although HLA C*07/B*07/DRB5*01/DRB1*15/DQB1*06 was the most frequent HLA 5-loci phased-haplotype (n, 76) in the PPMI cohort, the NR_SVA_381 insertion was present in only six of them (8%). Conclusions: These data suggest that expressed SVA and HLA gene alleles in circulating white blood cells are coordinated differentially in the regulation of immune responses and the long-term onset and progression of PD, the mechanisms of which have yet to be elucidated.

The balance between nuclear import and export of NLRC5 regulates MHC class I transactivation.

Major histocompatibility complex (MHC) class I molecules play an essential role in regulating the adaptive immune system by presenting antigens to CD8 T cells. CITA (MHC class I transactivator), also known as NLRC5 (NLR family, CARD domain-containing 5), regulates the expression of MHC class I and essential components involved in the MHC class I antigen presentation pathway. While the critical role of the nuclear distribution of NLRC5 in its transactivation activity has been known, the regulatory mechanism to determine the nuclear localization of NLRC5 remains poorly understood. In this study, a comprehensive analysis of all domains in NLRC5 revealed that the regulatory mechanisms for nuclear import and export of NLRC5 coexist and counterbalance each other. Moreover, GCN5 (general control non-repressed 5 protein), a member of HATs (histone acetyltransferases), was found to be a key player to retain NLRC5 in the nucleus, thereby contributing to the expression of MHC class I. Therefore, the balance between import and export of NLRC5 has emerged as an additional regulatory mechanism for MHC class I transactivation, which would be a potential therapeutic target for the treatment of cancer and virus-infected diseases.

Investigating the genetic makeup of the major histocompatibility complex (MHC) in the United Arab Emirates population through next-generation sequencing.

The Human leukocyte antigen (HLA) molecules are central to immune response and have associations with the phenotypes of various diseases and induced drug toxicity. Further, the role of HLA molecules in presenting antigens significantly affects the transplantation outcome. The objective of this study was to examine the extent of the diversity of HLA alleles in the population of the United Arab Emirates (UAE) using Next-Generation Sequencing methodologies and encompassing a larger cohort of individuals. A cohort of 570 unrelated healthy citizens of the UAE volunteered to provide samples for Whole Genome Sequencing and Whole Exome Sequencing. The definition of the HLA alleles was achieved through the application of the bioinformatics tools, HLA-LA and xHLA. Subsequently, the findings from this study were compared with other local and international datasets. A broad range of HLA alleles in the UAE population, of which some were previously unreported, was identified. A comparison with other populations confirmed the current population's unique intertwined genetic heritage while highlighting similarities with populations from the Middle East region. Some disease-associated HLA alleles were detected at a frequency of > 5%, such as HLA-B*51:01, HLA-DRB1*03:01, HLA-DRB1*15:01, and HLA-DQB1*02:01. The increase in allele homozygosity, especially for HLA class I genes, was identified in samples with a higher level of genome-wide homozygosity. This highlights a possible effect of consanguinity on the HLA homozygosity. The HLA allele distribution in the UAE population showcases a unique profile, underscoring the need for tailored databases for traditional activities such as unrelated transplant matching and for newer initiatives in precision medicine based on specific populations. This research is part of a concerted effort to improve the knowledge base, particularly in the fields of transplant medicine and investigating disease associations as well as in understanding human migration patterns within the Arabian Peninsula and surrounding regions.

Major histocompatibility complex genes exhibit a potential immunological role in mixed Eimeria-infected broiler cecum analyzed using RNA sequencing.

OBJECTIVE: This study was conducted to investigate the differential expression of the major histocompatibility complex (MHC) gene region in Eimeria-infected broiler. METHODS: We profiled gene expression of Eimeria-infected and uninfected ceca of broilers sampled at 4, 7, and 21 days post-infection (dpi) using RNA sequencing. Differentially expressed genes (DEGs) between two sample groups were identified at each time point. DEGs located on chicken chromosome 16 were used for further analysis. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis was conducted for the functional annotation of DEGs. RESULTS: Fourteen significant (false discovery rate <0.1) DEGs were identified at 4 and 7 dpi and categorized into three groups: MHC-Y class I genes, MHC-B region genes, and non-MHC genes. In Eimeria-infected broilers, MHC-Y class I genes were upregulated at 4 dpi but downregulated at 7 dpi. This result implies that MHC-Y class I genes initially activated an immune response, which was then suppressed by Eimeria. Of the MHC-B region genes, the DMB1 gene was upregulated, and TAP-related genes significantly implemented antigen processing for MHC class I at 4 dpi, which was supported by KEGG pathway analysis. CONCLUSION: This study is the first to investigate MHC gene responses to coccidia infection in chickens using RNA sequencing. MHC-B and MHC-Y genes showed their immune responses in reaction to Eimeria infection. These findings are valuable for understanding chicken MHC gene function.

Adaptation of HLA testing to characterize the cynomolgus macaque MHC polymorphisms and alloantibody signatures.

Nonhuman primates are the closest animal models to humans with respect to genetics and physiology. Consequently, a critical component of immunogenetics research relies on drawing inferences from the cynomolgus macaque to inform human trials. Despite the conserved organization of the Major Histocompatibility Complex (MHC) between cynomolgus macaques and humans, MHC genotyping of cynomolgus macaques is challenging due to high rates of copy number variants, duplications, and rearrangements, particularly at the MHC class I loci. Furthermore, the limited availability of commercial reagents specific to cynomolgus macaques that can be used to characterize anti-MHC class I and class II antibody (Ab) specificities in cynomolgus macaques presents a major bottleneck in translational research. Here we successfully characterized cynomolgus macaque Mafa class I and class II serologic specificities in 86 animals originating from various geographical regions using the complement dependent cytotoxicity (CDC) assay with human HLA class I and class II monoclonal antibody (mAb) typing trays. Further, we successfully induced and characterized anti-Mafa class I and class II alloantibody specificity using HLA single antigen bead assays. We also subsequently tracked the alloAb burden in the animals during treatment with anti-B lymphocyte stimulator (BLyS) treatment. Altogether, these methods can be easily used in translational research to serotype MHC class I and class II specificity in macaques, characterize their alloAb specificity, and evaluate the efficacy of novel therapeutic modalities in depleting circulating alloAbs in these animals.

Intracellular Major Histocompatibility Complex Class II and C-X-C Motif Chemokine Ligand 10-Expressing Neutrophils Indicate the State of Anti-Tumor Activity Induced by Bacillus Calmette-Guérin.

(1) Background: Inflammatory responses induce the formation of both anti-tumor and pro-tumor neutrophils known as myeloid-derived suppressor cells (MDSCs). Intermittent intravesical infusion of Bacillus Calmette-Guérin (BCG) is an established cancer immunotherapy for non-muscle-invasive bladder cancer (NMIBC). However, the types of neutrophils induced via the inflammatory response to both tumor-bearing and BCG remain unclear. (2) Methods: We therefore analyzed neutrophil dynamics in the peripheral blood and urine of patients with NMIBC who received BCG therapy. Further, we analyzed the effects of BCG in a mouse intraperitoneal tumor model. (3) Results: BCG therapy induced the formation of CXCL10 and MHC class II-positive neutrophils in the urine of patients with NMIBC but did not induce MDSC formation. CXCL10- and MHC class II-expressing neutrophils were detected in peritoneal exudate cells formed after BCG administration. Partial neutrophil depletion using an anti-Ly6G antibody suppressed the upregulation of CXCL10 and MHC class II in neutrophils and reversed the anti-tumor activity of BCG in mouse models. (4) Conclusions: These results indicated that intracellular MHC class II- and CXCL10-expressing neutrophils indicate the state of anti-tumor activity induced via BCG. The status of neutrophils in mixed inflammation of immunosuppressive and anti-tumor responses may therefore be useful for evaluating immunological systemic conditions.

tidytcells: standardizer for TR/MH nomenclature.

T cell receptors (TR) underpin the diversity and specificity of T cell activity. As such, TR repertoire data is valuable both as an adaptive immune biomarker, and as a way to identify candidate therapeutic TR. Analysis of TR repertoires relies heavily on computational analysis, and therefore it is of vital importance that the data is standardized and computer-readable. However in practice, the usage of different abbreviations and non-standard nomenclature in different datasets makes this data pre-processing non-trivial. tidytcells is a lightweight, platform-independent Python package that provides easy-to-use standardization tools specifically designed for TR nomenclature. The software is open-sourced under the MIT license and is available to install from the Python Package Index (PyPI). At the time of publishing, tidytcells is on version 2.0.0.

C-Terminal Region of Caveolin-3 Contains a Stretch of Amino Acid Residues Capable of Diminishing Symptoms of Experimental Autoimmune Encephalomyelitis but Not Rheumatoid Arthritis Modeled in Rats.

A short synthetic peptide from the C-terminal part of the caveolin-3 structure was tested for experimental autoimmune encephalomyelitis (EAE) treatment in rats. The structure-function similarity established between the novel synthetic peptide of pCav3 and the well-known immunomodulator immunocortin determined pCav3's ability to reduce EAE symptoms in Dark Agouti (DA) rats injected with pCav3 (500 µg/kg). pCav3 was found to interfere with the proliferation of lymphocytes extracted from the LNs of DA rats primed with homogenate injection, with IC50 = 0.42 µM (2.35 mcg/mL). pCav3 affected EAE in a very similar manner as immunocortin. The high degree of homology between the amino acid sequences of pCav3 and immunocortin corresponded well with the therapeutic activities of both peptides, as demonstrated on EAE. The latter peptide, possessing a homologous structure to pCav3, was also tested on EAE to explore whether there were structural restrictions between these peptides implied by the MHC-involved cell machinery. Consequently, immunocortin was further examined with a different autoimmune disease model, collagen-induced arthritis (CIA), established in Sprague-Dawley rats. CIA was established using an intentionally different genetic platform than EAE. Based on the results, it was concluded that the effectiveness of pCav3 and immunocortin peptides in EAE rat model was almost identical, but differed in the rat model of rheumatoid arthritis; thus, efficacy may be sensitive to the MHC type of animals used to establish the autoimmune disease model.

A Chicken Tapasin ortholog can chaperone empty HLA-B∗37:01 molecules independent of other peptide-loading components.

Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC), and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange in vitro, limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in a stable form, independent of co-chaperones. chTapasin can bind the human HLA-B∗37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved ß2m epitope on HLA-B∗37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B∗37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.

Assessing micro-macroparasite selective pressures and anthropogenic disturbance as drivers of immune gene diversity in a Neotropical wild cat.

Anthropogenic environmental change is reducing available habitat for wild species, providing novel selection pressures such as infectious diseases and causing species to interact in new ways. The potential for emerging infectious diseases and zoonoses at the interface between humans, domestic animals, and wild species is a key global concern. In vertebrates, diversity at the major histocompatibility complex MHC is critical to disease resilience, and its study in wild populations provides insights into eco-evolutionary dynamics that human activities alter. In natural populations, variation at MHC loci is partly maintained by balancing selection, driven by pathogenic selective pressures. We hypothesize that MHC genetic diversity differs between guigna populations inhabiting human-dominated landscapes (higher pathogen pressures) versus more natural habitats (lower pathogen pressures). We predict that MHC diversity in guignas would be highest in human-dominated landscapes compared with continuous forest habitats. We also expected to find higher MHC diversity in guignas infected with micro and macro parasites (higher parasite load) versus non infected guignas. We characterized for the first time the genetic diversity at three MHC class I and II exons in 128 wild guignas (Leopardus guigna) across their distribution range in Chile (32-46° S) and Argentina, representing landscapes with varying levels of human disturbance. We integrated MHC sequence diversity with multiple measures of anthropogenic disturbance and both micro and macro parasite infection data. We also assessed signatures of positive selection acting on MHC genes. We found significantly higher MHC class I diversity in guignas inhabiting landscapes where houses were present, and with lower percentage of vegetation cover, and also in animals with more severe cardiorespiratory helminth infection (richness and intensity) and micro-macroparasite co-infection. This comprehensive, landscape-level assessment further enhances our knowledge on the evolutionary dynamics and adaptive potential of vertebrates in the face of emerging infectious disease threats and increasing anthropogenic impacts.

High-Resolution Genotyping of Expressed Equine MHC Reveals a Highly Complex MHC Structure.

The Major Histocompatibility Complex (MHC) genes play a key role in a number of biological processes, most notably in immunological responses. The MHCI and MHCII genes incorporate a complex set of highly polymorphic and polygenic series of genes, which, due to the technical limitations of previously available technologies, have only been partially characterized in non-model but economically important species such as the horse. The advent of high-throughput sequencing platforms has provided new opportunities to develop methods to generate high-resolution sequencing data on a large scale and apply them to the analysis of complex gene sets such as the MHC. In this study, we developed and applied a MiSeq-based approach for the combined analysis of the expressed MHCI and MHCII repertoires in cohorts of Thoroughbred, Icelandic, and Norwegian Fjord Horses. The approach enabled us to generate comprehensive MHCI/II data for all of the individuals (n = 168) included in the study, identifying 152 and 117 novel MHCI and MHCII sequences, respectively. There was limited overlap in MHCI and MHCII haplotypes between the Thoroughbred and the Icelandic/Norwegian Fjord horses, showcasing the variation in MHC repertoire between genetically divergent breeds, and it can be inferred that there is much more MHC diversity in the global horse population. This study provided novel insights into the structure of the expressed equine MHC repertoire and highlighted unique features of the MHC in horses.

Liquid Biopsy, a Potential New Detection Method in Heart Allograft Rejection.

Allografts rejection remains the most important reason causing allograft dysfunction in heart transplantation recipients. Currently, the golden standard for detecting graft rejection is endomyocardial biopsy (EMB). As a new noninvasive technique, liquid biopsy emerges along with the great developments of droplet-based digital PCR and the various optimizations of next-generation sequencing technologies, which is also cheaper than EMB. This review introduces several types of liquid biopsy and its application in heart transplantation.

Discovery of novel MHC-B haplotypes in Chantecler chickens.

The major histocompatibility complex (MHC) is a highly polymorphic cluster of genes which contribute to immune response. Located on chromosome 16, the chicken MHC has great influence over disease resistance and susceptibility. Through the use of a high-density SNP panel which encompasses the MHC-B region, haplotypes can be easily identified. This study aims to use an MHC-B SNP panel to evaluate the MHC-B variability in the Chantecler breed. This breed is native to Quebec, Canada, and is a dual-purpose breed known for its strong resistance to extreme cold temperatures. The Chantecler breed faced a near extinction event in the 1970s, which most likely resulted in a genetic bottleneck and loss of diversity. Despite this, SNP haplotype diversity was observed among 4 Chantecler populations. A total of 8 haplotypes were observed. Of these haplotypes, 6 were previously defined in other breeds, and the other 2 were unique to the Chantecler. Within the populations, the number of haplotypes ranged from 4 to 7, with 3 haplotypes, including the novel BSNP-Chant01, being present in all the groups. This study shows existence of reasonable diversity in the MHC-B region of the Chantecler breed and our results further contribute to understanding the variability of this region in chickens.

Complimentary electrostatics dominate T-cell receptor binding to a psoriasis-associated peptide antigen presented by human leukocyte antigen C∗06:02.

Psoriasis is a chronic skin disease characterized by hyperproliferative epidermal lesions infiltrated by autoreactive T cells. Individuals expressing the human leukocyte antigen (HLA) C∗06:02 allele are at highest risk for developing psoriasis. An autoreactive T cell clone (termed Vα3S1/Vß13S1) isolated from psoriatic plaques is selective for HLA-C∗06:02, presenting a peptide derived from the melanocyte-specific autoantigen ADAMTSL5 (VRSRRCLRL). Here we determine the crystal structure of this psoriatic TCR-HLA-C∗06:02 ADAMTSL5 complex with a stabilized peptide. Docking of the TCR involves an extensive complementary charge network formed between negatively charged TCR residues interleaving with exposed arginine residues from the self-peptide and the HLA-C∗06:02 α1 helix. We probed these interactions through mutagenesis and activation assays. The charged interface spans the polymorphic region of the C1/C2 HLA group. Notably the peptide-binding groove of HLA-C∗06:02 appears exquisitely suited for presenting highly charged Arg-rich epitopes recognized by this acidic psoriatic TCR. Overall, we provide a structural basis for understanding the engagement of melanocyte antigen-presenting cells by a TCR implicated in psoriasis while simultaneously expanding our knowledge of how TCRs engage HLA-C.

Infant gut microbiota and environment associate with juvenile idiopathic arthritis many years prior to disease onset, especially in genetically vulnerable children.

BACKGROUND: The etiology of juvenile idiopathic arthritis (JIA) is poorly understood. This study investigated genetic and environmental factors and infant gut microbiota in a prospective birth cohort to assess disease risk. METHODS: Data was collected from the All Babies in Southeast Sweden (ABIS) population-based cohort (n = 17,055), 111 of whom later acquired JIA (ABISJIA). Stool samples were collected at one year of age for 10.4%. To determine disease association, 16S rRNA gene sequences were analyzed, with and without confound adjustment. Genetic and environmental risks were assessed. FINDINGS: ABISJIA had higher abundance of Acidaminococcales, Prevotella 9, and Veillonella parvula and lower abundance of Coprococcus, Subdoligranulum, Phascolarctobacterium, Dialister spp., Bifidobacterium breve, Fusicatenibacter saccharivorans, Roseburia intestinalis, and Akkermansia muciniphila (q's < 0.05). Parabacteroides distasonis greatly increased the odds of later contracting JIA (OR = 6.7; 1.81-24.84, p = 0.0045). Shorter breastfeeding duration and increased antibiotic exposure compounded risk in a dose-dependent manner, especially in those with genetic predisposition. INTERPRETATION: Microbial dysregulation in infancy may trigger or accelerate JIA development. Environmental risk factors have a stronger impact on genetically predisposed children. This study is the first to implicate microbial dysregulation in JIA at such an early age, with many bacterial taxa associated with risk factors. These findings provide opportunities for intervention or early screening and offer new insights into JIA pathogenesis. FUNDING: Barndiabetesfonden; Swedish Council for Working Life and Social Research; Swedish Research Council; Östgöta Brandstodsbolag; Medical Research Council of Southeast Sweden; JDRF-Wallenberg Foundation; Linköping.

High-affinity CD8 variants enhance the sensitivity of pMHCI antigen recognition via low-affinity TCRs.

CD8+ T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity, and the CD8 coreceptor, which stabilizes the TCR/pMHCI complex. Earlier work has shown that the sensitivity of antigen recognition can be regulated in vitro by altering the strength of the pMHCI/CD8 interaction. Here, we characterized two CD8 variants with moderately enhanced affinities for pMHCI, aiming to boost antigen sensitivity without inducing non-specific activation. Expression of these CD8 variants in model systems preferentially enhanced pMHCI antigen recognition in the context of low-affinity TCRs. A similar effect was observed using primary CD4+ T cells transduced with cancer-targeting TCRs. The introduction of high-affinity CD8 variants also enhanced the functional sensitivity of primary CD8+ T cells expressing cancer-targeting TCRs, but comparable results were obtained using exogenous wild-type CD8. Specificity was retained in every case, with no evidence of reactivity in the absence of cognate antigen. Collectively, these findings highlight a generically applicable mechanism to enhance the sensitivity of low-affinity pMHCI antigen recognition, which could augment the therapeutic efficacy of clinically relevant TCRs.

Differential pairing of transmembrane domain GxxxG dimerization motifs defines two HLA-DR MHC class II conformers.

MHC class II molecules function to present exogenous antigen-derived peptides to CD4 T cells to both drive T cell activation and to provide signals back into the class II antigen-presenting cell. Previous work established the presence of multiple GxxxG dimerization motifs within the transmembrane domains of MHC class II α and ß chains across a wide range of species and revealed a role for differential GxxxG motif pairing in the formation of two discrete mouse class II conformers with distinct functional properties (i.e., M1-and M2-paired I-Ak class II). Biochemical and mutagenesis studies detailed herein extend this model to human class II by identifying an anti-HLA-DR mAb (Tü36) that selectively binds M1-paired HLA-DR molecules. Analysis of the HLA-DR allele reactivity of the Tü36 mAb helped define other HLA-DR residues involved in mAb binding. In silico modeling of both TM domain interactions and whole protein structure is consistent with the outcome of biochemical/mutagenesis studies and provides insight into the possible structural differences between the two HLA-DR conformers. Cholesterol depletion studies indicate a role for cholesterol-rich membrane domains in the formation/maintenance of Tü36 mAb reactive DR molecules. Finally, phylogenetic analysis of the amino acid sequences of Tü36-reactive HLA-DR ß chains reveals a unique pattern of both Tü36 mAb reactivity and key amino acid polymorphisms. In total, these studies bring the paradigm M1/M2-paired MHC class II molecules to the human HLA-DR molecule and suggest that the functional differences between these conformers defined in mouse class II extend to the human immune system.