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Hotspot driver mutations presented by human leukocyte antigens might be recognized by anti-tumor T cells. Based on their advantages of tumor-specificity and immunogenicity, neoantigens derived from hotspot mutations, such as PIK3CAH1047L, may serve as emerging targets for cancer immunotherapies. NetMHCpan V4.1 was utilized for predicting neoepitopes of PIK3CA hotspot mutation. Using in vitro stimulation, antigen-specific T cells targeting the HLA-A*11:01-restricted PIK3CA mutation were isolated from healthy donor-derived peripheral blood mononuclear cells. T cell receptors (TCRs) were cloned using single-cell PCR and sequencing. Their functionality was assessed through T cell activation markers, cytokine production and cytotoxic response to cancer cell lines pulsed with peptides or transduced genes of mutant PIK3CA. Immunogenic mutant antigens from PIK3CA and their corresponding CD8+ T cells were identified. These PIK3CA mutation-specific CD8+ T cells were subsequently enriched, and their TCRs were isolated. The TCR clones exhibited mutation-specific and HLA-restricted reactivity, demonstrating varying degrees of functional avidity. Identified TCR genes were transferred into CD8+ Jurkat cells and primary T cells deficient of endogenous TCRs. TCR-expressing cells demonstrated specific recognition and reactivity against the PIK3CAH1047L peptide presented by HLA-A*11:01-expressing K562 cells. Furthermore, mutation-specific TCR-T cells demonstrated an elevation in cytokine production and profound cytotoxic effects against HLA-A*11:01+ malignant cell lines harboring PIK3CAH1047L. Our data demonstrate the immunogenicity of an HLA-A*11:01-restricted PIK3CA hotspot mutation and its targeting therapeutic potential, together with promising candidates of TCR-T cell therapy.

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.

Robust allogeneic immune reactions after transplantation impede the translational pace of human embryonic stem cells (hESCs)‐based therapies. Selective genetic editing of human leucocyte antigen (HLA) molecules has been proposed to generate hESCs with immunocompatibility, which, however, has not been specifically designed for the Chinese population yet. Herein, we explored the possibility of customizing immunocompatible hESCs based on Chinese HLA typing characteristics. We generated an immunocompatible hESC line by disrupting HLA‐B, HLA‐C, and CIITA genes while retaining HLA‐A*11:01 (HLA‐A*11:01‐retained, HLA‐A11R), which covers ~21% of the Chinese population. The immunocompatibility of HLA‐A11R hESCs was verified by in vitro co‐culture and confirmed in humanized mice with established human immunity. Moreover, we precisely knocked an inducible caspase‐9 suicide cassette into HLA‐A11R hESCs (iC9‐HLA‐A11R) to promote safety. Compared with wide‐type hESCs, HLA‐A11R hESC‐derived endothelial cells elicited much weaker immune responses to human HLA‐A11+ T cells, while maintaining HLA‐I molecule‐mediated inhibitory signals to natural killer (NK) cells. Additionally, iC9‐HLA‐A11R hESCs could be induced to undergo apoptosis efficiently by AP1903. Both cell lines displayed genomic integrity and low risks of off‐target effects. In conclusion, we customized a pilot immunocompatible hESC cell line based on Chinese HLA typing characteristics with safety insurance. This approach provides a basis for establishment of a universal HLA‐AR bank of hESCs covering broad populations worldwide and may speed up the clinical application of hESC‐based therapies. Here, we first selected an human embryonic stem cell (hESC) line with homozygous human leucocyte antigen (HLA)‐A*11:01 allele, the most popular HLA‐I allele of the Chinese population, then generated a hypoimmunogenic hESC line by disrupting HLA‐B, HLA‐C, and CIITA genes while retaining HLA‐A*11:01 (named as HLA‐A11R hESCs), covering ~21% of the Chinese population. The immunocompatibility of the HLA‐A11R hESCs was verified by in vitro co‐culture assays and confirmed in humanized mice with an established human immune system. Moreover, to address safety issues, we precisely knocked a clinical trial‐grade drug‐induced caspase‐9 suicide cassette into HLA‐A11R hESCs, generating iC9‐HLA‐A11R hESCs, which were efficiently induced to apoptosis by AP1903. In summary, we customized an immunocompatible hESC cell line based on the HLA typing characteristics of the Chinese population with safety insurance, which provided the basis for the establishment of an HLA‐AR bank of hESCs covering broader populations in the world, speeding up the clinical application of a range of hESC‐based therapies.

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.

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.

Kawasaki disease (KD) is a pediatric acute multisystemic vasculitis complicated by development of coronary artery lesions. The breakthrough theory on KD etiopathogenesis points to pathogens/environmental factors triggered by northeastern wind coming from China. Natural Killer cells and T lymphocytes express the inhibitory/activating Killer Immunoglobulin-like Receptors (KIR) to elicit an immune response against pathogens by binding to human leukocyte antigens (HLA) class I epitopes. We first report on the role of KIR/HLA genetic epistasis in a sample of 100 Italian KD children. We genotyped KIR, HLA-A, HLA-B and HLA-C polymorphisms, and compared KD data with those from 270 Italian healthy donors. The HLA-A*11 ligand for KIR2DS2/2DS4/3DL2 was a KD susceptibility marker by itself (odds ratio (OR)=3.85, confidence interval (CI)=1.55–9.53, P =0.004). Although no epistasis between HLA-A*11 and KIR2DS2/S4 emerged, HLA-A*11 also engages KIR3DL2, a framework gene encoding for a pathogen sensor of CpG-oligodeoxynucleotides (CpG-ODN), and KD blood mononuclear cells are actually prone to pathogen CpG-ODN activation in the acute phase. Moreover, carriers of KIR2DS2/HLA-C1 and KIR2DL2/HLA-C1 were more frequent among KD, in keeping with data demonstrating the involvement of these HLA/KIR couples in autoimmune endothelial damage. The highest KD risk factor was observed among carriers of KIR2DL2 and two or more HLA ligands (OR=10.24, CI=1.87–56.28; P =0.007).

Background Human Papillomavirus type 18 (HPV18) is a high-risk HPV that is commonly associated with cervical cancer. HPV18 oncogenes E6 and E7 are associated with the malignant transformation of cells, thus the identification of human leukocyte antigen (HLA)-restricted E6/E7 peptide-specific CD8 + T cell epitopes and the creation of a HPV18 E6/E7 expressing cervicovaginal tumor in HLA-A2 transgenic mice will be significant for vaccine development. Methods In the below study, we characterized various human HLA class I-restricted HPV18 E6 and E7-specific CD8 + T cells mediated immune responses in HLA class I transgenic mice using DNA vaccines encoding HPV18E6 and HPV18E7. We then confirmed HLA-restricted E6/E7 specific CD8 + T cell epitopes using splenocytes from vaccinated mice stimulated with HPV18E6/E7 peptides. Furthermore, we used oncogenic DNA plasmids encoding HPV18E7E6(delD70), luciferase, cMyc, and AKT to create a spontaneous cervicovaginal carcinoma model in HLA-A2 transgenic mice. Results Therapeutic HPV18 E7 DNA vaccination did not elicit any significant CD8 + T cell response in HLA-A1, HLA-24, HLA-B7, HLA-B44 transgenic or wild type C57BL/6 mice, but it did generate a strong HLA-A2 and HLA-A11 restricted HPV18E7-specific CD8 + T cell immune response. We found that a single deletion of aspartic acid (D) at location 70 in HPV18E6 DNA abolishes the presentation of HPV18 E6 peptide (aa67-75) by murine MHC class I. We found that the DNA vaccine with this mutant HPV18 E6 generated E6-specific CD8 + T cells in HLA-A2. HLA-A11, HLA-A24 and HLA-b40 transgenic mice. Of note, HLA-A2 restricted, HPV18 E7 peptide (aa7-15)- and HPV18 E6 peptide (aa97-105)-specific epitopes are endogenously processed by HPV18 positive Hela-AAD (HLA-A * 0201/D d ) cells. Finally, we found that injection of DNA plasmids encoding HPV18E7E6(delD70), AKT, cMyc, and SB100 can result in the development of adenosquamous carcinoma in the cervicovaginal tract of HLA-A2 transgenic mice. Conclusions We characterized various human HLA class I-restricted HPV18 E6/E7 peptide specific CD8 + T cell epitopes in human HLA class I transgenic mice. We demonstrated that HPV18 positive Hela cells expressing chimeric HLA-A2 (AAD) do present both HLA-A2-restricted HPV18 E7 (aa7-15)- and HPV18 E6 (aa97-105)-specific CD8 + T cell epitopes. A mutant HPV18E6 that had a single deletion at location 70 obliterates the E6 presentation by murine MHC class I and remains oncogenic. The identification of these human MHC restricted HPV antigen specific epitopes as well as the HPV18E6/E7 expressing adenosquamous cell carcinoma model may have significant future translational potential.

Background The major histocompatibility complex (MHC) in humans includes three classical class I loci (A, B, and C), which are important biomarkers for the transplantation of organs and hematopoietic stem cells. In the MHC, polymorphism is known to be extremely high while interlocus recombination is rare. We report a rare interlocus recombination between HLA-A and HLA-H, which was analyzed using next generation sequencing and nanopore sequencing. Methods In the sample, the genotypes of HLA-A, B, C, DRB1, and DQB1 were firstly determined using the methods of sequence-specific primer, sequence-specific oligonucleotide, Sanger’s sequencing, and NGS; however, HLA-A could not be phased. Nanopore sequencing was finally utilized to distinguish the sequence of the novel allele. Results Finally, the novel HLA-A*11:335 allele was identified as an interlocus recombination involving HLA-A*11:01:01:01/126 and HLA-H*02:07/14/18 alleles; this was mainly achieved by nanopore sequencing. Conclusions The identification of the interlocus recombination indicated that nanopore sequencing can be helpful in the characterization of novel alleles with complex rearrangements. Interlocus recombination has been identified as one of the mechanisms involved in the generation of novel HLA alleles.

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 HLA‐A11 or ‐A33 allele is found in approximately 18% or 10% of the Asian population, respectively, but each of which is a minor allele worldwide, and therefore no clinical trials were previously conducted. To develop a therapeutic peptide vaccine for each of them, we investigated immunological responses of advanced cancer patients with the HLA‐A11+/A11+ (n = 18) or ‐A33+/A33+ (n = 13) allele to personalized peptide vaccine (PPV) regimens. The primary sites of HLA‐A11+/A11+ or ‐A33+/A33+ patients were the colon (n = 4 or 2), stomach (2 or 3), breast (3 or 2), lung and pancreas (2 or 2), and so on. For PPV, a maximum of four peptides were selected from nine different peptides capable of binding to HLA‐A11 and ‐A33 molecules based on the pre‐existing peptide‐specific IgG responses. There were no severe adverse events related to PPV. At the end of the first cycle, peptide‐specific CTL responses were augmented in 4/12 or 2/9 of HLA‐A11+/A11+ or ‐A33+/A33+ patients, while peptide‐specific IgG responses were augmented in 6/14 or 4/10 patients, respectively. Clinical responses consisted of four stable diseases and 14 progressive diseases in HLA‐A11+/A11+patients, versus seven and six in ‐A33+/A33+patients, respectively. Further clinical study of PPV could be recommended because of the safety and positive immunological responses. IgG boosting were observed in HLA‐A11+/A11+ patients and HLA‐A33+/A33+ patients after personalized peptide vaccine(PPV). The patients with higher rate of changes of peptide‐specific IgG titers after 2nd cycle of vaccination showed longer prognosis than those with lower rate.