A common allele of HLA is associated with asymptomatic SARS-CoV-2 infection.

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.

Autoimmunity-associated T cell receptors recognize HLA-B*27-bound peptides.

Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))1. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8+ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public ß-chain variable region-complementary-determining region 3ß (BV9-CDR3ß) motif2-4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide-MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9-CDR3ß TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.

HLA alleles: important pieces to the COVID-19 puzzle.

Research on human leukocyte antigen (HLA) molecules in coronavirus disease 2019 (COVID-19) raised high expectations but has yielded limited results. Augusto et al.'s recent study in Nature unveils a strong association of HLA-B*15:01 with asymptomatic COVID-19, representing an important contribution to genetics in COVID-19.

The Structural Basis for Recognition of Human Leukocyte Antigen Class I Molecules by the Pan-HLA Antibody W6/32.

The central immunological role of HLA class I (HLA-I) in presenting peptide Ags to cellular components of the immune system has been the focus of intense study for >60 y. A confounding factor in the study of HLA-I has been the extreme polymorphism of these molecules. The mAb W6/32 has been a fundamental reagent bypassing the issue of polymorphism by recognizing an epitope that is conserved across diverse HLA-I allotypes. However, despite the widespread use of W6/32, the epitope of this Ab has not been definitively mapped. In this study, we present the crystal structure of the Fab fragment of W6/32 in complex with peptide-HLA-B*27:05. W6/32 bound to HLA-B*27:05 beneath the Ag-binding groove, recognizing a discontinuous epitope comprised of the α1, α2, and α3 domains of HLA-I and ß2-microglobulin. The epitope comprises a region of low polymorphism reflecting the pan-HLA-I nature of the binding. Notably, the W6/32 epitope neither overlaps the HLA-I binding sites of either T cell Ag receptors or killer cell Ig-like receptors. However, it does coincide with the binding sites for leukocyte Ig-like receptors and CD8 coreceptors. Consistent with this, the use of W6/32 to block the interaction of NK cells with HLA-I only weakly impaired inhibition mediated by KIR3DL1, but impacted HLA-LILR recognition.

Polymorphism in F pocket affects peptide selection and stability of type 1 diabetes-associated HLA-B39 allotypes.

HLA-B*39:06, HLA-B*39:01, and HLA-B*38:01 are closely related HLA allotypes differentially associated with type 1 diabetes (T1D) risk and progression. B*39:06 is highly predisposing, while B*39:01 and B*38:01 are weakly predisposing and protective allotypes, respectively. Here, we aimed to decipher molecular mechanisms underlying the differential association of these allotypes with T1D pathogenesis. We addressed peptide binding and conformational stability of HLA-B allotypes using computational and experimental approaches. Computationally, we found that B*39:06 and B*39:01 allotypes had more rigid peptide-binding grooves and were more promiscuous in binding peptides than B*38:01. Peptidomes of B*39:06 and B*39:01 contained fewer strong binders and were of lower affinity than that of B*38:01. Experimentally, we demonstrated that B*39:06 and B*39:01 had a higher capacity to bind peptides and exit to the cell surface but lower surface levels and were degraded faster than B*38:01. In summary, we propose that promiscuous B*39:06 and B*39:01 may bind suboptimal peptides and transport them the cell surface, where such unstable complexes may contribute to the pathogenesis of T1D.

Recognition of HIV-1-infected fibrocytes lacking Nef-mediated HLA-B downregulation by HIV-1-specific T cells.

Fibrocytes were reported to be host cells for HIV-1, but the immunological recognition of HIV-1-infected fibrocytes has not been studied. Here, we investigated the recognition of HIV-1-infected fibrocytes by HIV-1-specific CD8+ T cells. CD8+ T cells specific for five HIV-1 epitopes (HLA-A*24:02-restricted, HLA-B*52:01-restricted, and HLA-C*12:02-restricted epitopes) produced IFN-γ and expressed CD107a after coculture with HIV-1-infected fibrocytes. HIV-1-infected fibrocytes were effectively killed by HIV-1-specific CD8+ T cells. Although it is well known that HIV-1 Nef-mediated downregulation of HLA-A and HLA-B critically affects the T cell recognition of HIV-1-infected CD4+ T cells and HIV-1-infected macrophages, Nef downregulated HLA-A, but not HLA-B, in HIV-1-infected fibrocytes. These findings suggested that HIV-1-specific CD8+ T cells could recognize HIV-1-infected fibrocytes more strongly than HIV-1-infected CD4+ T cells or HIV-1-infected macrophages. HIV-1-infected fibrocytes were also recognized by HIV-1-specific HLA-DR-restricted T cells, indicating that HIV-1-infected fibrocytes can present HIV-1 epitopes to helper T cells. Collectively, these findings suggest that fibrocytes have an important role as antigen-presenting cells during HIV-1 infection. The present study demonstrates effective recognition of HIV-1-infected fibrocytes by HIV-1-specific T cells and suggests possible roles of fibrocytes in the induction and maintenance of HIV-1-specific T cells. IMPORTANCE: Fibrocytes were identified as unique hematopoietic cells with the features of both macrophages and fibroblasts and were demonstrated to be host cells for HIV-1. However, T cell recognition of HIV-1-infected fibrocytes has not been studied. We investigated the recognition of HIV-1-infected fibrocytes by HIV-1-specific T cells. HIV-1-infected fibrocytes were effectively recognized and killed by CD8+ T cells specific for HIV-1 epitopes presented by HLA-A, HLA-B, or HLA-C and were recognized by HIV-1-specific HLA-DR-restricted CD4+ T cells. HIV-1 Nef-mediated downregulation of HLA-A and HLA-B was found in HIV-1-infected CD4+ T cells, whereas Nef did not downregulate HLA-B in HIV-1-infected fibrocytes. These results suggest that HIV-1-specific CD8+ T cells recognize HIV-1-infected fibrocytes more strongly than HIV-1-infected CD4+ T cells. The present study suggests the importance of fibrocytes in the induction and maintenance of HIV-1-specific T cells.

Activation of Human CD8+ T Cells with Nitroso Dapsone-Modified HLA-B*13:01-Binding Peptides.

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.

Parallel T Cell Immunogenic Regions in Influenza B and A Viruses with Distinct Nuclear Export Signal Functions: The Balance between Viral Life Cycle and Immune Escape.

Influenza A viruses (IAVs) and influenza B viruses (IBVs) cause annual epidemics in human populations with seasonal circulation spikes. Peptide AM58-66GL9 located at residues 58-66 of M1 protein of IAVs has been recognized as an immunodominant T cell epitope with HLA-A*0201 restriction and broadly used as a positive reference in influenza immunity. This peptide also almost completely overlaps with a nuclear export signal (NES) 59-68 in IAV M1, which explains the limited escape mutations under the T cell immune pressure in this region. In this study, we investigated the potential immunogenicity and NES in the corresponding region of IBV. The long peptide covering this region can be recognized by specific T cells and induce robust expression of IFN-γ among HLA-B*1501 donors in vivo, but not in HLA-A*0201 donors. Among a series of truncated peptides derived from this region, we identified an immunodominant HLA-B*1501-restricted T cell epitope BM58-66AF9 (ALIGASICF) in the M1 protein of IBV. Furthermore, the structure of the HLA-B*1501/BM58-66AF9 complex shows that BM58-66AF9 performs a flat and featureless conformation that is similar to AM58-66GL9 presented by HLA-A*0201. In contrast with IAV, the sequence around residues 55-70 of IBV M1 does not contain an NES. Our comparative study on IBVs and IAVs provides new insights into the immune and evolution characteristics of IBVs and may shed light on vaccine development for influenza viruses.

Mass Spectrometric Profiling of HLA-B44 Peptidomes Provides Evidence for Tapasin-Mediated Tryptophan Editing.

The extreme polymorphisms of HLA class I proteins result in structural variations in their peptide binding sites to achieve diversity in Ag presentation. External factors could independently constrict or alter HLA class I peptide repertoires. Such effects of the assembly factor tapasin were assessed for HLA-B*44:05 (Y116) and a close variant, HLA-B*44:02 (D116), which have low and high tapasin dependence, respectively, for their cell surface expression. Analyses of the HLA-B*44:05 peptidomes in the presence and absence of tapasin reveal that peptides with C-terminal tryptophans and higher predicted affinities are preferentially selected by tapasin, coincident with reduced frequencies of peptides with other C-terminal amino acids, including leucine. Comparisons of the HLA-B*44:05 and HLA-B*44:02 peptidomes indicate the expected structure-based alterations near the peptide C termini, but also C-terminal amino acid frequency and predicted affinity changes among the unique and shared peptide groups for B*44:02 and B*44:05. Overall, these findings indicate that the presence of tapasin and the tapasin dependence of assembly alter HLA class I peptide-binding preferences at the peptide C terminus. The particular C-terminal amino acid preferences that are altered by tapasin are expected to be determined by the intrinsic peptide-binding specificities of HLA class I allotypes. Additionally, the findings suggest that tapasin deficiency and reduced tapasin dependence expand the permissive affinities of HLA class I-bound peptides, consistent with prior findings that HLA class I allotypes with low tapasin dependence have increased breadth of CD8+ T cell epitope presentation and are more protective in HIV infections.

Regulation of HLA class I expression by non-coding gene variations.

The Human Leukocyte Antigen (HLA) is a critical genetic system for different outcomes after solid organ and hematopoietic cell transplantation. Its polymorphism is usually determined by molecular technologies at the DNA level. A potential role of HLA allelic expression remains under investigation in the context of the allogenic immune response between donors and recipients. In this study, we quantified the allelic expression of all three HLA class I loci (HLA-A, B and C) by RNA sequencing and conducted an analysis of expression quantitative traits loci (eQTL) to investigate whether HLA expression regulation could be associated with non-coding gene variations. HLA-B alleles exhibited the highest expression levels followed by HLA-C and HLA-A alleles. The max fold expression variation was observed for HLA-C alleles. The expression of HLA class I loci of distinct individuals demonstrated a coordinated and paired expression of both alleles of the same locus. Expression of conserved HLA-A~B~C haplotypes differed in distinct PBMC's suggesting an individual regulated expression of both HLA class I alleles and haplotypes. Cytokines TNFα /IFNß, which induced a very similar upregulation of HLA class I RNA and cell surface expression across alleles did not modify the individually coordinated expression at the three HLA class I loci. By identifying cis eQTLs for the HLA class I genes, we show that the non-coding eQTLs explain 29%, 13%, and 31% of the respective HLA-A, B, C expression variance in unstimulated cells, and 9%, 23%, and 50% of the variance in cytokine-stimulated cells. The eQTLs have significantly higher effect sizes in stimulated cells compared to unstimulated cells for HLA-B and HLA-C genes expression. Our data also suggest that the identified eQTLs are independent from the coding variation which defines HLA alleles and thus may be influential on intra-allele expression variability although they might not represent the causal eQTLs.

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.

Development of an ARMS multiplex real-time PCR assay for the detection of HLA-B*13:01 genotype by detecting highly specific SNPs.

OBJECTIVES: HLA-B*13:01 was strongly associated with Dapsone Hypersensitivity Syndrome (DHS). This study aimed to develop and validate a rapid and economical method for HLA-B*13:01 genotyping. METHODS: Two tubes multiplex real-time PCR detection system comprising amplification refractory mutation system primers and TaqMan probes was established for HLA-B*13:01 genotyping. Sequence-based typing was applied to validate the accuracy of the assay. RESULTS: The accuracy of the assay was 100% for HLA-B*13:01 genotyping. The detection limit of the new method was 0.025 ng DNA. The positive rate of HLA-B*13:01 in the Bouyei (20%, n = 50) populations was significantly higher than that in the Uighur population (4%, n = 100), Han (4.5%, n = 200), and Tibetan (1%, n = 100) ( P  < 0.05). CONCLUSION: The proposed method is rapid and reliable for HLA-B*13:01 screening in a clinical setting.

Pharmacogenetics to prevent hypersensitivity reactions to antiepileptic drugs: is testing performed when indicated?

Extensive scientific evidence consistently demonstrates the clinical validity and utility of HLA-B*15:02 pre-screening in averting severe cutaneous adverse reactions (SCARs), namely Stevens-Johnson syndrome and toxic epidermal necrolysis, associated with carbamazepine or oxcarbazepine usage. Current practice guidelines and drug labeling actively advocate for pharmacogenetic pre-screening before initiating these antiepileptic drugs (AED), with particular emphasis on patients of Asian descent. However, there is a potential need to strengthen compliance with these recommendations. This retrospective study aimed to describe the pharmacogenetic pre-screening, documentation, and SCARs incidence for patients of Asian ancestry initiated on carbamazepine or oxcarbazepine at a large Northeastern USA healthcare system. Between 1 July 2016 and August 1, 2021, 27 patients with documented Asian heritage in the electronic health record (EHR) were included. The overall rate of HLA-B*15:02 pre-screening before carbamazepine or oxcarbazepine initiation was 4%. None who underwent pharmacogenetic pre-screening carried the associated HLA-B risk allele, and no SCARs were reported. Notably, pharmacogenetic results were not discretely entered into the EHR, and the results were only found as attached documents in the miscellaneous section of the EHR. There remains a significant opportunity for improving HLA-B*15:02 pre-screening for patients starting carbamazepine and oxcarbazepine to prevent SCARs in the USA.

Molecular insights into the HLA-B35 molecules' classification associated with HIV control.

Human leukocyte antigen (HLA) class I molecules have been shown to influence the immune response to HIV infection and acquired immunodeficiency syndrome progression. Polymorphisms within the HLA-B35 molecules divide the family into two groups, namely, Px and PY. The Px group is associated with deleterious effects and accelerated disease progression in HIV+ patients, whereas the PY group is not. The classification is based on the preferential binding of a tyrosine at the C-terminal part of the peptide in the PY group, and a nontyrosine residue in the Px group. However, there is a lack of knowledge on the molecular differences between the two groups. Here, we have investigated three HLA-B35 molecules, namely, HLA-B*35:01 (PY), HLA-B*35:03 (Px) and HLA-B*35:05 (unclassified). We selected an HIV-derived peptide, NY9, and demonstrated that it can trigger a polyfunctional CD8+ T-cell response in HLA-B*35:01+ /HIV+ patients. We determined that in the complex with the NY9 peptide, the PY molecule was more stable than the Px molecule. We solved the crystal structures of the three HLA molecules in complex with the NY9 peptide, and structural similarities with HLA-B*35:01 would classify the HLA-B*35:05 within the PY group. Interestingly, we found that HLA-B*35:05 can also bind a small molecule in its cleft, suggesting that small drugs could bind as well.

HLA-B*53:01 Is a Significant Risk Factor of Liver Injury due to Phenytoin and Other Antiepileptic Drugs in African Americans.

INTRODUCTION: To investigate human leukocyte antigen alleles associated with liver injury due to antiepileptic drugs (AEDs) in African Americans (AA). METHODS: In this study, 21 AA with AED drug-induced liver injury (DILI), 176 AA with DILI due to non-AEDs, and 5816 AA population controls were included. RESULTS: HLA-B*53:01 was significantly associated with aromatic AED-DILI (odds ratio: 4.52, 95% confidence interval: 2.42-8.44, P = 1.46 × 10 -5 ). Phenytoin DILI showed the strongest association with HLA-B*53:01 (odds ratio: 9.17; 95% confidence interval: 3.61-23.28, P = 1.1 × 10 -5 ). The HLA-B*53:01 allele was carried by 8 of 9 AA phenytoin DILI cases. DISCUSSION: HLA-B*53:01 is a significant risk factor of liver injury due to antiepileptics, particularly phenytoin, in AA.

Anti-HLA-B7/HLA-B44 strong cross immunoreactivity observed in flow cytometry HLA-B27 immunotyping.

Cross reactivities are known for human leukocyte antigen inside HLA-B7 related Cross-Reactive Group (B7CREG). Some CE-IVD flow-cytometry kits use double monoclonal antibodies (mAb) to distinguish HLA-B27 and HLA-B7 but practice reveals more complexes results. This study explores the performances of this test. Analysis of 466 consecutive cases using HLA-B27 IOTest™ kit on a Navios™ cytometer from Beckman-Coulter, partially compared to their genotypes. Expected haplotypes HLA-B27-/HLA-B7- (undoubtedly HLA-B27 negative) and HLA-B27+/HLA-B7- (undoubtedly HLA-B27+) were clearly identified according to the manufacturer's instructions. On the opposite, patients strongly labeled with anti-HLA-B7 showed three different phenotypes regarding anti-HLA-B27 labeling: (1) most of the cases were poorly labeled in accordance with cross reactivity inside B7CREG (HLA-B27-/HLA-B7+ haplotype); (2) rare cases had strong B7 and B27 labeling corresponding to HLA-B27+/HLA-B7+ haplotype; (3) even less cases had strong labeling by anti-HLA-B7 but non for anti-HLA-B27, all expressing HLA-B44 and no B7CREG molecules. Surprisingly, more cases were not labeled with anti-HLA-B7 antibody but partially labeled with anti-HLA-B27 suggesting another cross reactivity out of B7CREG. mAb HLA typing suggests new, cross reactivities of anti-HLA-B27 antibody to more molecules out of B7CREG and of anti-HLA-B7 antibody but not anti-HLA-B27 to HLA-B44 molecule also out of B7CREG. HLA-B27 could surely be excluded in most samples labeled with HLA-B27, below a "grey zone" on intermediate intensity. More comparison is needed in future studies.

TCR clonotypes modulate the protective effect of HLA class I molecules in HIV-1 infection.

The human leukocyte antigens HLA-B27 and HLA-B57 are associated with protection against progression of disease that results from infection with human immunodeficiency virus type 1 (HIV-1), yet most people with alleles encoding HLA-B27 and HLA-B57 are unable to control HIV-1. Here we found that HLA-B27-restricted CD8(+) T cells in people able to control infection with HIV-1 (controllers) and those who progress to disease after infection with HIV-1 (progressors) differed in their ability to inhibit viral replication through targeting of the immunodominant epitope of group-associated antigen (Gag) of HIV-1. This was associated with distinct T cell antigen receptor (TCR) clonotypes, characterized by superior control of HIV-1 replication in vitro, greater cross-reactivity to epitope variants and enhanced loading and delivery of perforin. We also observed clonotype-specific differences in antiviral efficacy for an immunodominant HLA-B57-restricted response in controllers and progressors. Thus, the efficacy of such so-called 'protective alleles' is modulated by specific TCR clonotypes selected during natural infection, which provides a functional explanation for divergent HIV-1 outcomes.

HLA informs risk predictions after haploidentical stem cell transplantation with posttransplantation cyclophosphamide.

Hematopoietic cell transplantation from HLA-haploidentical related donors is increasingly used to treat hematologic cancers; however, characteristics of the optimal haploidentical donor have not been established. We studied the role of donor HLA mismatching in graft-versus-host disease (GVHD), disease recurrence, and survival after haploidentical donor transplantation with posttransplantation cyclophosphamide (PTCy) for 1434 acute leukemia or myelodysplastic syndrome patients reported to the Center for International Blood and Marrow Transplant Research. The impact of mismatching in the graft-versus-host vector for HLA-A, -B, -C, -DRB1, and -DQB1 alleles, the HLA-B leader, and HLA-DPB1 T-cell epitope (TCE) were studied using multivariable regression methods. Outcome was associated with HLA (mis)matches at individual loci rather than the total number of HLA mismatches. HLA-DRB1 mismatches were associated with lower risk of disease recurrence. HLA-DRB1 mismatching with HLA-DQB1 matching correlated with improved disease-free survival. HLA-B leader matching and HLA-DPB1 TCE-nonpermissive mismatching were each associated with improved overall survival. HLA-C matching lowered chronic GVHD risk, and the level of HLA-C expression correlated with transplant-related mortality. Matching status at the HLA-B leader and HLA-DRB1, -DQB1, and -DPB1 predicted disease-free survival, as did patient and donor cytomegalovirus serostatus, patient age, and comorbidity index. A web-based tool was developed to facilitate selection of the best haploidentical-related donor by calculating disease-free survival based on these characteristics. In conclusion, HLA factors influence the success of haploidentical transplantation with PTCy. HLA-DRB1 and -DPB1 mismatching and HLA-C, -B leader, and -DQB1 matching are favorable. Consideration of HLA factors may help to optimize the selection of haploidentical related donors.

Incongruity between T cell receptor recognition of breast cancer hotspot mutations ESR1 Y537S and D538G following exogenous peptide loading versus endogenous antigen processing.

T cell receptor engineered T cell (TCR T) therapies have shown recent efficacy against certain types of solid metastatic cancers. However, to extend TCR T therapies to treat more patients across additional cancer types, new TCRs recognizing cancer-specific antigen targets are needed. Driver mutations in AKT1, ESR1, PIK3CA, and TP53 are common in patients with metastatic breast cancer (MBC) and if immunogenic could serve as ideal tumor-specific targets for TCR T therapy to treat this disease. Through IFN-γ ELISpot screening of in vitro expanded neopeptide-stimulated T cell lines from healthy donors and MBC patients, we identified reactivity towards 11 of 13 of the mutations. To identify neopeptide-specific TCRs, we then performed single-cell RNA sequencing of one of the T cell lines following neopeptide stimulation. Here, we identified an ESR1 Y537S specific T cell clone, clonotype 16, and an ESR1 Y537S/D538G dual-specific T cell clone, clonotype 21, which were HLA-B*40:02 and HLA-C*01:02 restricted, respectively. TCR Ts expressing these TCRs recognized and killed target cells pulsed with ESR1 neopeptides with minimal activity against ESR1 WT peptide. However, these TCRs failed to recognize target cells expressing endogenous mutant ESR1. To investigate the basis of this lack of recognition we performed immunopeptidomics analysis of a mutant-overexpressing lymphoblastoid cell line and found that the ESR1 Y537S neopeptide was not endogenously processed, despite binding to HLA-B*40:02 when exogenously pulsed onto the target cell. These results indicate that stimulation of T cells that likely derive from the naïve repertoire with pulsed minimal peptides may lead to the expansion of clones that recognize non-processed peptides, and highlights the importance of using methods that selectively expand T cells with specificity for antigens that are efficiently processed and presented.