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Studies have demonstrated that at least 20% of individuals infected with SARS-CoV-2 remain asymptomatic1-4. Although most global efforts have focused on severe illness in COVID-19, examining asymptomatic infection provides a unique opportunity to consider early immunological features that promote rapid viral clearance. Here, postulating that variation in the human leukocyte antigen (HLA) loci may underly processes mediating asymptomatic infection, we enrolled 29,947 individuals, for whom high-resolution HLA genotyping data were available, in a smartphone-based study designed to track COVID-19 symptoms and outcomes. Our discovery cohort (n = 1,428) comprised unvaccinated individuals who reported a positive test result for SARS-CoV-2. We tested for association of five HLA loci with disease course and identified a strong association between HLA-B*15:01 and asymptomatic infection, observed in two independent cohorts. Suggesting that this genetic association is due to pre-existing T cell immunity, we show that T cells from pre-pandemic samples from individuals carrying HLA-B*15:01 were reactive to the immunodominant SARS-CoV-2 S-derived peptide NQKLIANQF. The majority of the reactive T cells displayed a memory phenotype, were highly polyfunctional and were cross-reactive to a peptide derived from seasonal coronaviruses. The crystal structure of HLA-B*15:01-peptide complexes demonstrates that the peptides NQKLIANQF and NQKLIANAF (from OC43-CoV and HKU1-CoV) share a similar ability to be stabilized and presented by HLA-B*15:01. Finally, we show that the structural similarity of the peptides underpins T cell cross-reactivity of high-affinity public T cell receptors, providing the molecular basis for HLA-B*15:01-mediated pre-existing immunity.

PurposeUltrahigh resolution (UHR) HLA matching is reported to result in better outcomes following unrelated donor hematopoietic cell transplantation, improving survival and reducing post-transplant complications. However, most studies included relatively small numbers of patients. Here we report the findings from a large, multicenter validation study.MethodsUHR HLA typing was available on 5,140 conventionally 10 out of 10 HLA-matched patients with malignant disease transplanted between 2008 and 2017.ResultsAfter UHR HLA typing, 82% of pairs remained 10 out of 10 UHR-matched; 12.3% of patients were 12 out of 12 UHR HLA-matched. Compared with 12 out of 12 UHR-matched patients, probabilities of grade 2-4 acute graft-versus-host disease (aGVHD) were significantly increased with UHR mismatches (overall P = .0019) and in those patients who were HLA-DPB1 T-cell epitope permissively mismatched or nonpermissively mismatched (overall P = .0011). In the T-cell-depleted subset, the degree of UHR HLA mismatch was only associated with increased transplant-related mortality (TRM) (overall P = .0068). In the T-cell-replete subset, UHR HLA matching was associated with a lower probability of aGVHD (overall P = .0020); 12 out of 12 UHR matching was associated with reduced TRM risk when compared with HLA-DPB1 T-cell epitope permissively mismatched patients, whereas nonpermissive mismatching resulted in a greater risk (overall P = .0003).ConclusionThis study did not confirm that UHR 12 out of 12 HLA matching increases the probability of overall survival but does demonstrate that aGVHD risk, and in certain settings TRM, is lowest in UHR HLA-matched pairs and thus warrants consideration when multiple 10 out of 10 HLA-matched donors of equivalent age are available.

The goals of the KIR component of the 17th International HLA and Immunogenetics Workshop (IHIW) were to encourage and educate researchers to begin analyzing KIR at allelic resolution, and to survey the nature and extent of KIR allelic diversity across human populations. To represent worldwide diversity, we analyzed 1269 individuals from ten populations, focusing on the most polymorphic KIR genes, which express receptors having three immunoglobulin (Ig)-like domains (KIR3DL1/S1, KIR3DL2 and KIR3DL3). We identified 13 novel alleles of KIR3DL1/S1, 13 of KIR3DL2 and 18 of KIR3DL3. Previously identified alleles, corresponding to 33 alleles of KIR3DL1/S1, 38 of KIR3DL2, and 43 of KIR3DL3, represented over 90% of the observed allele frequencies for these genes. In total we observed 37 KIR3DL1/S1 allotypes, 40 for KIR3DL2 and 44 for KIR3DL3. As KIR allotype diversity can affect NK cell function, this demonstrates potential for high functional diversity worldwide. Allelic variation further diversifies KIR haplotypes. We determined KIR3DL3 ∼ KIR3DL1/S1 ∼ KIR3DL2 haplotypes from five of the studied populations, and observed multiple population-specific haplotypes in each. This included 234 distinct haplotypes in European Americans, 191 in Ugandans, 35 in Papuans, 95 in Egyptians and 86 in Spanish populations. For another 35 populations, encompassing 642,105 individuals we focused on KIR3DL2 and identified another 375 novel alleles, with approximately half of them observed in more than one individual. The KIR allelic level data gathered from this project represents the most comprehensive summary of global KIR allelic diversity to date, and continued analysis will improve understanding of KIR allelic polymorphism in global populations. Further, the wealth of new data gathered in the course of this workshop component highlights the value of collaborative, community-based efforts in immunogenetics research, exemplified by the IHIW.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a curative option for blood cancers, but the coupled effects of graft-versus-tumor and graft-versus-host disease (GVHD) limit its broader application. Outcomes improve with matching at HLAs, but other factors are required to explain residual risk of GVHD. In an effort to identify genetic associations outside the major histocompatibility complex, we conducted a genome-wide clinical outcomes study on 205 acute myeloid leukemia patients and their fully HLA-A-, HLA-B-, HLA-C-, HLA-DRB1-, and HLA-DQB1-matched (10/10) unrelated donors. HLA-DPB1 T-cell epitope permissibility mismatches were observed in less than half (45%) of acute GVHD cases, motivating a broader search for genetic factors affecting clinical outcomes. A novel bioinformatics workflow adapted from neoantigen discovery found no associations between acute GVHD and known, HLA-restricted minor histocompatibility antigens (MiHAs). These results were confirmed with microarray data from an additional 988 samples. On the other hand, Y-chromosome-encoded single-nucleotide polymorphisms in 4 genes (PCDH11Y, USP9Y, UTY, and NLGN4Y) did associate with acute GVHD in male patients with female donors. Males in this category with acute GVHD had more Y-encoded variant peptides per patient with higher predicted HLA-binding affinity than males without GVHD who matched X-paralogous alleles in their female donors. Methods and results described here have an immediate impact for allo-HCT, warranting further development and larger genomic studies where MiHAs are clinically relevant, including cancer immunotherapy, solid organ transplant, and pregnancy.

The HLA gene complex on human chromosome 6 is one of the most polymorphic regions in the human genome and contributes in large part to the diversity of the immune system. Accurate typing of HLA genes with short-read sequencing data has historically been difficult due to the sequence similarity between the polymorphic alleles. Here, we introduce an algorithm, xHLA, that iteratively refines the mapping results at the amino acid level to achieve 99-100% four-digit typing accuracy for both class I and II HLA genes, taking only [Formula: see text]3 min to process a 30× whole-genome BAM file on a desktop computer.

Purpose Disease relapse remains a major challenge to successful outcomes in patients who undergo allogeneic hematopoietic cell transplantation (HCT). Donor natural killer (NK) cell alloreactivity in HCT can control leukemic relapse, but capturing alloreactivity in HLA-matched HCT has been elusive. HLA expression on leukemia cells-upregulated in the post-HCT environment-signals for NK cell inhibition via inhibitory killer immunoglobulin-like (KIR) receptors and interrupts their antitumor activity. We hypothesized that varied strengths of inhibition among subtypes of the ubiquitous KIR3DL1 and its cognate ligand, HLA-B, would titrate NK reactivity against acute myelogenous leukemia (AML). Patients and Methods By using an algorithm that was based on polymorphism-driven expression levels and specificities, we predicted and tested inhibitory and cytotoxic NK potential on the basis of KIR3DL1/HLA-B subtype combinations in vitro and evaluated their impact in 1,328 patients with AML who underwent HCT from 9/10 or 10/10 HLA-matched unrelated donors. Results Segregated by KIR3DL1 subtype, NK cells demonstrated reproducible patterns of strong, weak, or noninhibition by target cells with defined HLA-B subtypes, which translated into discrete cytotoxic hierarchies against AML. In patients, KIR3DL1 and HLA-B subtype combinations that were predictive of weak inhibition or noninhibition were associated with significantly lower relapse (hazard ratio [HR], 0.72; P = .004) and overall mortality (HR, 0.84; P = .030) compared with strong inhibition combinations. The greatest effects were evident in the high-risk group of patients with all KIR ligands (relapse: HR, 0.54; P < .001; and mortality: HR, 0.74; P < .008). Beneficial effects of weak and noninhibiting KIR3DL1 and HLA-B subtype combinations were separate from and additive to the benefit of donor activating KIR2DS1. Conclusion Consideration of KIR3DL1-mediated inhibition in donor selection for HLA-matched HCT may achieve superior graft versus leukemia effects, lower risk for relapse, and an increase in survival among patients with AML.

The immune responses of natural killer cells are regulated, in part, by killer cell immunoglobulin-like receptors (KIR). The 16 closely-related genes in the KIR gene system have been diversified by gene duplication and unequal crossing over, thereby generating haplotypes with variation in gene copy number. Allelic variation also contributes to diversity within the complex. In this study, we estimated allele-level haplotype frequencies and pairwise linkage disequilibrium statistics for 14 KIR loci. The typing utilized multiple methodologies by four laboratories to provide at least 2x coverage for each allele. The computational methods generated maximum-likelihood estimates of allele-level haplotypes. Our results indicate the most extensive allele diversity was observed for the KIR framework genes and for the genes localized to the telomeric region of the KIR A haplotype. Particular alleles of the stimulatory loci appear to be nearly fixed on specific, common haplotypes while many of the less frequent alleles of the inhibitory loci appeared on multiple haplotypes, some with common haplotype structures. Haplotype structures cA01 and/or tA01 predominate in this cohort, as has been observed in most populations worldwide. Linkage disequilibrium is high within the centromeric and telomeric haplotype regions but not between them and is particularly strong between centromeric gene pairs KIR2DL5∼KIR2DS3S5 and KIR2DS3S5∼KIR2DL1, and telomeric KIR3DL1∼KIR2DS4. Although 93% of the individuals have unique pairs of full-length allelic haplotypes, large genomic blocks sharing specific sets of alleles are seen in the most frequent haplotypes. These high-resolution, high-quality haplotypes extend our basic knowledge of the KIR gene system and may be used to support clinical studies beyond single gene analysis.

The HLA gene complex on human chromosome 6 is one of the most polymorphic regions in the human genome and contributes in large part to the diversity of the immune system. Accurate typing of HLA genes with short-read sequencing data has historically been difficult due to the sequence similarity between the polymorphic alleles. Here, we introduce an algorithm, xHLA, that iteratively refines the mapping results at the amino acid level to achieve 99-100% four-digit typing accuracy for both class I and II HLA genes, taking only ~3 min to process a 30x whole-genome BAM file on a desktop computer. [ABSTRACT FROM AUTHOR]

Aim The knowledge of complete haplotype structure and underlying genetic variation is often key in understanding gene function, phenotype, and medically relevant traits such as susceptibility to disease and individual response to drugs. However, genome wide SNP arrays and shotgun sequencing methods are limited in their ability to identify phased genetic variations at high resolution over extended localized regions. Methods To address this, we improved methods for targeted DNA isolation using fosmid technology and single-molecule, long-sequence-read generation that combine for complete, haplotype-resolved resequencing across extended genomic subregions. As specific applications, we targeted subregions of the human major histocompatibility complex (MHC) and the killer Ig-like receptor (KIR) locus, two of the most polymorphic genomic regions in humans. Results For the MHC region, an 800 kb segment of the MHC class II region including the highly polymorphic DR, DQ, and DP genes was targeted. Complete phased sequences were obtained encompassing the DR4 common extended haplotype – a haplotype associated with autoimmune disorders - from four class II heterozygous individuals. For the KIR locus, which spans some 200 kb including tandem repeats of highly homologous polymorphic loci, we targeted several KIR haplotype heterozygous individuals. Conclusions Complete phased sequences for all haplotypes were obtained from all individuals with a high level of data accuracy. The results demonstrate the approach is amenable to cost-effective scale-up to generate scores to hundreds of phased chromosomal sequences of extended lengths that encompass targeted genomic regions associated with disease. [ABSTRACT FROM AUTHOR]

Aim: ARS identical alleles are considered high resolution matched for unrelated donor stem cell transplant; however, it is unknown if mismatches outside of the ARS region impact transplant outcome. We investigated ARS identical allele pairs, HLA-A∗02:01/09, HLA-B∗44:02/27, HLA-C∗07:01/06 and C∗07:01/18, to determine if these alleles exist on the same haplotype in the Be the Match Registry®. The goal was to identify cases for in vitro cellular assays of alloreactivity to ARS identical alleles. Methods: Over nine million donors on the Be the Match Registry were queried. Haplotypes were selected where both ARS alleles have been reported previously. These haplotypes were paired with common haplotypes within each race. High resolution sequence based typing was completed on 40 Caucasian donors for A∗02:01/09, 58 Caucasian donors for B∗44:02/27, 87 Asian Pacific Islander donors for C∗07:01/06, and 64 African American donors for C∗07:01/18. Results: A∗02:01/09 was evaluated in two haplotype pairs: 98% carried A∗02:01, 2% were neither A∗02:01 nor A∗02:09 and none carried A∗02:09. B∗44:02/27 was evaluated in two haplotype pairs: 69% were B∗44:02, 22% were B∗44:27 and 9% were neither B∗44:02 nor B∗44:27; however, B∗44:02 and B∗44:27 alleles were never found on the same haplotype. Within two haplotype pairs tested for C∗07:01/06, 98% of donors carried the C∗07:06 allele, 2% carried neither C allele of interest, while none carried C∗07:01. For the C∗07:01/18, eleven haplotype pairs were investigated, 53% of donors carried C∗07:18 and none carried C∗07:01. The remaining 47% of donors carried other C alleles. Conclusions: The results obtained from this study for A∗02:01/09, B∗44:02/27, C∗07:01/06 and C∗07:01/18 suggest that the chance of encountering an isolated mismatch between these ARS identical alleles on the haplotypes investigated in an unrelated donor transplant setting would be quite low. Work will continue to identify isolated ARS mismatches for evaluation of alloreactivity in in vitro cellular assays. [ABSTRACT FROM AUTHOR]

Aim: For hematopoietic stem cell transplantation (HSCT) HLA identical sibling donors or HLA matched unrelated donors are the most ideal donors. As 10/10 (HLA-A, -B, -C,-DRB1, -DQB1) matched donors, are not available for all patients, the identification of permissive/non-immunogenic mismatches is important to inmprove the outcome of HLA mismatched transplants. Alleles that share the amino acid sequence of the antigen recognition site (ARS)(exon 2 and 3 for HLA class I or exon 2 for HLA-DRB1) or differences outside the peptide binding groove or α-helices (as the HLA-C*03:03 vs C*03:04 mismatch) are good candidates for permissive mismatches as these differences are not likely to induce T cell alloreactivity. Methods: An inventory of the functional impact of such mismatches was performed by in vitro cellular assays including the Cytotoxic T Lymphocyte precursor test (CTLp) for HLA-class I allele differences and the MLC, IFN-γ elispot and Primed Lymphocyte Test (PLT) for HLA-DRB1 differences. Results: CTLp assays were performed for: HLA-A*23:17 vs 23:01 (differ at pos 283, exon 5), HLA-C*07:06 vs *07:01 (pos 307 & 324 exon 5 & 6), HLA-C*04:30 vs 04:01 (pos 327, exon 7), HLA-C*03:62 vs 03:03:01 (pos 197, exon 4), HLA-C*03:03 vs 03:04 (pos 91, exon 3) and HLA-B*35:02 vs 35:04 (pos 109, exon 3). The only positive CTLp test was for HLA-C*07:06 vs *07:01. DRB1*14:01 vs DRB1*14:54 differ at pos 112 and a 2nd mismatch of DRB3*02:01 vs DRB3*02:02, respectively, yielded complicated results. Five of 6 combinations tested did not respond in the MLC and 3 of 6 in the Elispot against the combined DRB1/DRB3 mismatch and only in one direction; DRB1*14:01/DRB3*02:01 against DRB1*14:54/DRB3*02:02. PLT results suggested that the DRB1* mismatch was responsible for the response. Conclusions: In conclusion, mismatches involving positions outside the ARS are not very immunogenic. However, some mismatches can lead to T cell reactivity in vitro. The impact of these mismatches on clinical outcome of HSCT remains to be established. [Copyright &y& Elsevier]

Aim: Recent data show that DP matching plays an important role in hematopoietic cell transplantation. Our previous work demonstrated that in European Americans haplotypic associations for the DP heterodimer are determined by near complete linkage disequilibrium between amino acid motifs on DPA1 (pos 31) and DPB1 (pos 85–87). We aimed to generate motif and allele-level haplotype frequencies (HF) for four race groups to extend these insights to minorities. Methods: We calculated DPA1∼DPB1 HF at allele and motif level from 16,802 Be the Match Registry® members typed by DNA methods, resolving phase, allelic and motif ambiguity using expectation maximization. Motif frequencies were calculated for position 31 on DPA1 and positions 85–87 on DPB1, which play a critical role in the P1 pocket of the peptide binding region. Normalized linkage disequilibrium (LD) was calculated on each haplotype (D′) and globally (Wn) for each population. Results: The top three allele-level haplotypes in all populations account for over 66% of the HF, with DPA1∗01:03 DPB1∗04:01 most common in the Caucasian (CAU), Asian Pacific Islander (API) and Hispanic (HIS) populations and DPA1∗02:02 DPB1∗01:01 in the African American (AFA) population. Wn for DPA1∼DPB1 allele associations for CAU, HIS, API, and AFA populations was .55, .45, .49, and .52 respectively. At the motif level there were only four possible haplotypes. M GPM and Q EAV were the two most common in every population. M GPM was the most common in CAU, HIS and API with frequencies 69%, 68% and 56% respectively, with D′= 0.97 for CAU (complete LD). The most common haplotype for AFA was Q EAV, at 48% and D′=.99. Conclusions: Low global LD was observed at the allele level for all populations. However, at the motif level haplotypes were in near complete LD suggesting that for the DPA1 DPB1 heterodimer the unit of selection for all populations is the combined amino acid motif rather than the allele, as previously observed in European Americans. [ABSTRACT FROM AUTHOR]