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Our understanding of the conformational and electrostatic determinants that underlie targeting of human leukocyte antigens (HLA) by anti-HLA alloantibodies is principally based upon in silico modelling. Here we provide a biochemical/biophysical and functional characterization of a human monoclonal alloantibody specific for a common HLA type, HLA-A*11:01. We present a 2.4 Å resolution map of the binding interface of this antibody on HLA-A*11:01 and compare the structural determinants with those utilized by T-cell receptor (TCR), killer-cell immunoglobulin-like receptor (KIR) and CD8 on the same molecule. These data provide a mechanistic insight into the paratope−epitope relationship between an alloantibody and its target HLA molecule in a biological context where other immune receptors are concomitantly engaged. This has important implications for our interpretation of serologic binding patterns of anti-HLA antibodies in sensitized individuals and thus, for the biology of human alloresponses. Anti-human leukocyte antigen (HLA) antibodies are important mediators of alloresponses, but structural insights on antibody:HLA interaction are still lacking. Here the authors provide a 2.4 Å structure of antibody:HLA complex, and also analyse HLA features important for other HLA-interacting molecules, to enhance our understanding of alloimmunity.

Inhibitory killer cell Ig-like receptors (KIRs) are known to recognize HLA ligands mainly of the HLA-C and Bw4 groups, but the ligands for KIRs are poorly understood. We report here the identification of the cognate ligand for the activating KIR 2DS2 as HLA-A*11:01. The crystal structure of the KIR2DS2–HLA-A*11:01 complex was solved at 2.5-Å resolution and revealed residue-binding characteristics distinct from those of inhibitory KIRs with HLA-C and the critical role of residues Tyr45 and Asp72 in shaping binding specificity to HLA-A*11:01. Using KIR2DS2 tetramers, binding to surface HLA-A*11:01 on live cells was demonstrated and, furthermore, that binding can be altered by residue changes at p8 of the peptide, indicating the influence of peptide sequence on KIR–HLA association. In addition, heteronuclear single quantum coherence NMR was used to map the involvement of critical residues in HLA binding at the interface of KIR and HLA, and validates the data observed in the crystal structure. Our data provide structural evidence of the recognition of A*11:01 by the activating KIR2DS2 and extend our understanding of the KIR–HLA binding spectrum.

Osteosarcoma is the most common malignancy of bone that affects young people. Neoadjuvant chemotherapy and surgery have significantly improved the prognosis. However, the prognosis of non‐responders to chemotherapy is still poor. To develop peptide‐based immunotherapy for osteosarcoma, we previously identified CTL epitopes derived from papillomavirus binding factor (PBF) in the context of human leukocyte antigen (HLA)‐A2, HLA‐A24 and HLA‐B55. In the present study, we identified two novel CTL epitopes, QVT (QVTVWLLEQK) and LSA (LSALPPPLHK), in the context of HLA‐A11 using a sequence of screenings based on the predicted affinity of peptides, in vitro folding ability of peptide/HLA‐A11 complex, reactivity of peptide/HLA‐A11 tetramer and interferon (IFN)‐γ production of T cells that was induced by mixed lymphocyte peptide culture under a limiting dilution condition. CTL clones directed to QVT and LSA peptides showed specific cytotoxicity against HLA‐A11+PBF+ osteosarcoma (HOS‐A11) cells. In contrast, another epitope, ASV (ASVLSRRLGK), could highly induce cognate tetramer‐positive CTL. This might be because the ASV peptide mimics the peptide ASV (R6Q) (ASVLSQRLGK) derived from bacterial polypeptides, ROK family proteins. However, ASV‐induced CTL did not show cytokine production against the cognate peptide. In conclusion, the CTL epitopes QVT and LSA peptides might be useful for the development of immunotherapy targeting PBF for patients with osteosarcoma. We identified two novel CTL epitopes derived from osteosarcoma antigen PBF in the context of HLA‐A11 using a sequence of screenings based on the predicted affinity of peptides, in vitro folding ability of peptide/HLA‐A11 complex, reactivity of MHC‐tetramer and IFN‐γ production of T cells that were induced by mixed lymphocyte peptide culture under limiting dilution conditions.

Over the last decades, organ transplantation has made rapid progress as a curative therapy for organ failure. However, the adaptive immune system—alloreactive T cells and antibodies targeting human leukocyte antigens (HLA)—is the leading cause of graft rejection. The presence of anti-donor HLA antibodies is considered a risk factor that disqualifies a particular donor-recipient pair. However, alloantibodies are found in some long-term graft survivors, suggesting a protective blocking function of some alloantibodies. Therefore, whether alloantibodies can have a positive as well as a negative effect in transplantation remains unclear. Here, HLA-A*11:01-specific monoclonal antibodies were generated from a human non-immune antibody library, and the effect of these antibodies was investigated on activation of A*11:01- specific T cells. We identified an A*11:01-specific monoclonal antibody with the capacity to block TCR recognition, TCR recruitment to the immune synapse, and T cell activation. The antibody reduced translocation of the transcription factor NFAT1 and phosphorylation of the MAP kinase ERK, which are both required for T cell effector function and TCR signal transduction. Cross-linking mass spectrometry was used to identify the epitope, demonstrating that this alloantibody can inhibit TCR from binding to the HLA molecule. These findings indicate that some HLA-specific alloantibodies can reduce T cell responses to the allograft. This has significant implications for interpretation of the existence of donor-specific antibodies, since some of them can protect the graft. Moreover, such antibodies may have therapeutic potential as specific treatments targeting mismatched donor HLA molecules. Graphical abstract

Epstein-Barr Virus (EBV) is closely linked to nasopharyngeal carcinoma (NPC), notably prevalent in southern China. Although type II latency of EBV plays a crucial role in the development of NPC, some lytic genes and intermittent reactivation are also critical for viral propagation and tumor progression. Since T cell-mediated immunity is effective in targeted killing of EBV-positive cells, it is important to identify EBV-derived peptides presented by highly prevalent human leukocyte antigen class I (HLA-I) molecules throughout the EBV life cycle. Here, we constructed an EBV-positive NPC cell model to evaluate the presentation of EBV lytic phase peptides on streptavidin-tagged specific HLA-I molecules. Utilizing a mass spectrometry (LC-MS/MS)-based immunopeptidomic approach, we characterized eleven novel EBV peptides as well as two previously identified peptides. Furthermore, we determined these peptides were immunogenic and could stimulate PBMCs from EBV VCA/NA-IgA positive donors in an NPC endemic southern Chinese population. Overall, this work demonstrates that highly prevalent HLA-I-specific EBV peptides can be captured and functionally presented to elicit immune responses in an in vitro model, which provides insight into the epitopes presented during EBV lytic cycle and reactivation. It expands the range of viral targets for potential NPC early diagnosis and treatment.

Fighting smart diseases requires smart vaccines. Novel ways to present protective immunogenic peptide epitopes to human immune systems are needed. Herein, we focus on Self Assembling Protein Nanoparticles (SAPNs) as scaffolds/platforms for vaccine delivery that produce strong immune responses against Toxoplasma gondii in HLA supermotif, transgenic mice. Herein, we present a useful platform to present peptides that elicit CD4 + , CD8 + T and B cell immune responses in a core architecture, formed by flagellin, administered in combination with TLR4 ligand-emulsion (GLA-SE) adjuvant. We demonstrate protection of HLA-A*11:01, HLA-A*02:01, and HLA-B*07:02 mice against toxoplasmosis by (i) this novel chimeric polypeptide, containing epitopes that elicit CD8 + T cells , CD4 + T helper cells, and IgG2b antibodies, and (ii) adjuvant activation of innate immune TLR4 and TLR5 pathways. HLA-A*11:01, HLA-A*02:01, and HLA-B*07:02q11 transgenic mouse splenocytes with peptides demonstrated predicted genetic restrictions. This creates a new paradigm-shifting vaccine approach to prevent toxoplasmosis, extendable to other diseases.

The major histocompatibility complex (MHC) loci, the most polymorphic regions within the human genome, encode protein complexes responsible for antigen presentation and CD4+ and CD8+ cell activation. In prostate cancer (PCa), the second most diagnosed cancer in the male population, MHC loci undergo significant changes in their expression patterns, which affect the ability of the immune system to attack and eliminate malignant cells. The purpose of this study was to explore the genetic diversity of human leukocyte antigen (HLA)-A and HLA-B in patients with PCa and healthy controls (HCs) by performing HLA genotyping using NGS technology. The analysis highlighted statistically significant differences (p < 0.05) in the prevalence of three alleles (A*11:01, A*24:02, and B*18:01). Among the HCs analyzed, 14.89% had A*11:01, 20.21% had A*24:02, and 30.61% had B*18:01; while 5.21% of patients with PCa presented A*11:01, 9.38% presented A*24:02, 18.08% presented B*18:01. Odds ratio (OR) calculations underlined a negative association between the three alleles and the risk of PCa (OR < 1). The results presented in this study suggest a protective role of A*11:01, A*24:02, and B*18:01 in PCa.

Dengue virus presents a growing threat to public health in the developing world. Four major serotypes of dengue virus have been characterized, and epidemiological evidence shows that dengue hemorrhagic fever (DHF), the more serious manifestation of the disease, occurs more frequently upon reinfection with a second serotype. We have studied dengue virus–specific T-cell responses in Thai children. During acute infection, few dengue-responsive CD8 + T cells were recovered; most of those present showed an activated phenotype and were undergoing programmed cell death. Many dengue-specific T cells were of low affinity for the infecting virus and showed higher affinity for other, probably previously encountered strains. Profound T-cell activation and death may contribute to the systemic disturbances leading to DHF, and original antigenic sin in the T-cell responses may suppress or delay viral elimination, leading to higher viral loads and increased immunopathology.

The majority of peptides presented by MHC class I result from proteasomal protein turnover. The specialized immunoproteasome, which is induced during inflammation, plays a major role in antigenic peptide generation. However, other cellular proteases can, either alone or together with the proteasome, contribute peptides to MHC class I loading non-canonically. We used an immunopeptidomics workflow combined with prediction software for proteasomal cleavage probabilities to analyze how inflammatory conditions affect the proteasomal processing of immune epitopes presented by A549 cells. The treatment of A549 cells with IFNγ enhanced the proteasomal cleavage probability of MHC class I ligands for both the constitutive proteasome and the immunoproteasome. Furthermore, IFNγ alters the contribution of the different HLA allotypes to the immunopeptidome. When we calculated the HLA allotype-specific proteasomal cleavage probabilities for MHC class I ligands, the peptides presented by HLA-A*30:01 showed characteristics hinting at a reduced C-terminal proteasomal cleavage probability independently of the type of proteasome. This was confirmed by HLA-A*30:01 ligands from the immune epitope database, which also showed this effect. Furthermore, two additional HLA allotypes, namely, HLA-A*03:01 and HLA-A*11:01, presented peptides with a markedly reduced C-terminal proteasomal cleavage probability. The peptides eluted from all three HLA allotypes shared a peptide binding motif with a C-terminal lysine residue, suggesting that this lysine residue impairs proteasome-dependent HLA ligand production and might, in turn, favor peptide generation by other cellular proteases.