Identification of an HLA-A*11:01-restricted neoepitope of mutant PIK3CA and its specific T cell receptors for cancer immunotherapy targeting hotspot driver mutations.

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.

Identification of HLA-A*11:01 and A*02:01-Restricted EBV Peptides Using HLA Peptidomics.

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.

Genome editing HLA alleles for a pilot immunocompatible hESC line in a Chinese hESC bank for cell therapies.

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.

HLA-H*02:07 Is a Membrane-Bound Ligand of Denisovan Origin That Protects against Lysis by Activated Immune Effectors.

The biological relevance of genes initially categorized as "pseudogenes" is slowly emerging, notably in innate immunity. In the HLA region on chromosome 6, HLA-H is one such pseudogene; yet, it is transcribed, and its variation is associated with immune properties. Furthermore, two HLA-H alleles, H*02:07 and H*02:14, putatively encode a complete, membrane-bound HLA protein. Here we thus hypothesized that HLA-H contributes to immune homeostasis similarly to tolerogenic molecules HLA-G, -E, and -F. We tested if HLA-H*02:07 encodes a membrane-bound protein that can inhibit the cytotoxicity of effector cells. We used an HLA-null human erythroblast cell line transduced with HLA-H*02:07 cDNA to demonstrate that HLA-H*02:07 encodes a membrane-bound protein. Additionally, using a cytotoxicity assay, our results support that K562 HLA-H*02:07 inhibits human effector IL-2-activated PBMCs and human IL-2-independent NK92-MI cell line activity. Finally, through in silico genotyping of the Denisovan genome and haplotypic association with Denisovan-derived HLA-A*11, we also show that H*02:07 is of archaic origin. Hence, admixture with archaic humans brought a functional HLA-H allele into modern European and Asian populations.

HLA-A*11:01:46, a novel HLA-A*11 variant, detected in a Chinese individual.

HLA-A*11:01:46 has one synonymous nucleotide change from HLA-A*11:01:01:01 at nucleotide 807 (codon 245 Alanine).

HLA-B*13:64, a novel allele, identified in a Chinese individual.

HLA-B*13:64 has one amino acid change from HLA-B*13:02:01:01 where 94 Threonine is changed to Isoleucine.

Identification of novel human leukocyte antigen-A*11:01-restricted cytotoxic T-lymphocyte epitopes derived from osteosarcoma antigen papillomavirus binding factor.

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.

Identification of novel HLA-A11-restricted T-cell epitopes in the Ebola virus nucleoprotein.

The Ebola virus (EBOV) is a very contagious virus that is highly fatal in humans and animals. The largest epidemic was in West Africa in 2014, in which over 11,000 people died. However, to date, there are no licensed vaccines against it. Studies show that CD4+ and CD8+ T-cell responses, especially cytotoxic T-lymphocyte (CTL) responses, play key roles in protecting individuals from EBOV infection. Since HLA-restricted epitope vaccines are likely to be effective and safe immunization strategies for infectious diseases, the present study screened for CTL epitopes in the EBOV-nucleoprotein that are restricted by HLA-A11 (a common allele in Chinese people). Predictive computer analysis of the amino-acid sequence of EBOV-nucleoprotein identified ten putative HLA-A11-restricted epitopes. ELISPOT assay of immunized HLA-A11/DR1 transgenic mice showed that five (GR-9, VR-9, EK-9, PK-9, and RK-9) induced effective CTL responses. Additional epitope analyses will aid the design of epitope vaccines against EBOV.

The novel allele, HLA-A*11:264, was identified in a Chinese individual by sequence-based typing.

The novel allele, A*11:264, was identified in a Chinese individual by sequence-based typing.

A novel HLA class I allele: HLA-A*11:01:49.

The HLA-A*11:01:49 allele differs from A*11:01:01:01 by one synonymous nucleotide substitution at position 837.

Identification of HLA-B*58:01:21, a novel allele in a Korean individual.

B*58:01:21 has a single nucleotide change, c.264A>G (ACA→ACG at codon 88) compared with B*58:01:01:01.

α3-Deletion Isoform of HLA-A11 Modulates Cytotoxicity of NK Cells: Correlations with HIV-1 Infection of Cells.

Alternative splicing occurs frequently in many genes, especially those involved in immunity. Unfortunately, the functions of many alternatively spliced molecules from immunologically relevant genes remain unknown. Classical HLA-I molecules are expressed on almost all nucleated cells and play a pivotal role in both innate and adaptive immunity. Although splice variants of HLA-I genes have been reported, the details of their functions have not been reported. In the current study, we determined the characteristics, expression, and function of a novel splice variant of HLA-A11 named HLA-A11svE4 HLA-A11svE4 is located on the cell surface without ß2-microglobulin (ß2m). Additionally, HLA-A11svE4 forms homodimers as well as heterodimers with HLA-A open conformers, instead of combining with ß2m. Moreover, HLA-A11svE4 inhibits the activation of NK cells to protect target cells. Compared with ß2m and HLA-A11, the heterodimer of HLA-A11svE4 and HLA-A11 protected target cells from lysis by NK cells more effectively. Furthermore, HLA-AsvE4 expression was upregulated by HIV-1 in vivo and by HSV, CMV, and hepatitis B virus in vitro. In addition, our findings indicated that HLA-A11svE4 molecules were functional in activating CD8+ T cells through Ag presentation. Taken together, these results suggested that HLA-A11svE4 can homodimerize and form a novel heterodimeric complex with HLA-A11 open conformers. Furthermore, the data are consistent with HLA-A11svE4 playing a role in the immune escape of HIV-1.

Identification of the novel HLA-A*11:141 allele in a Chinese bone marrow donor.

HLA-A*11:141 has 1 nucleotide change from HLA-A*11:01:01 at position 470G>C.

HLA-A*11:256Q, a novel HLA-A*11 variant, detected in a Taiwanese individual.

Nucleotide deletions from residues 409 to 417 of HLA-A*11:02:01 results in a novel allele, HLA-A*11:256Q.

Two new HLA class I alleles described in a Spanish individual, HLA-A*11:01:01:04 and HLA-B*35:330.

Two new HLA class I alleles, HLA-A*11:01:01:04 and HLA-B*35:330, were characterized in a Spanish patient.

Detection of a novel HLA-A*11 variant, A*11:255, in a Taiwanese individual.

One nucleotide substitution at residue 532 of HLA-A*11:02:01 results in a novel allele, HLA-A*11:255.

HLA-A*11:134, a novel HLA-A*11 variant, identified by sequence-based typing in a Taiwanese individual.

A one nucleotide replacement in codon 31 of HLA-A*11:01:01 results in a novel allele, HLA-A*11:134.

Two new HLA class I alleles - A*11:241 and C*08:132.

HLA-A*11:241 differs from A*11:01:01:01 (215G > A, exon 2, R48Q); HLA-C*08:132 from C*08:02:01:01 (587 T > A exon 3, L172Q).

Recognition of a novel HLA-A*11 variant, HLA-A*11:196, in a Taiwanese unrelated bone marrow stem cell donor.

One nucleotide substitution at residue 484 of HLA-A*11:01:01:01 results in a novel allele, HLA-A*11:196.

HLA-A*11:235Q, a novel HLA-A*11 variant, detected in a Taiwanese individual.

Nucleotide deletions from residue 120 to 125 of HLA-A*11:01:01:01 result in a novel allele, HLA-A*11:235Q.