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.

Engineering and characterization of a novel Self Assembling Protein for Toxoplasma peptide vaccine in HLA-A*11:01, HLA-A*02:01 and HLA-B*07:02 transgenic mice.

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.

Defining the structural basis for human alloantibody binding to human leukocyte antigen allele HLA-A*11:01.

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.

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.

Human class I major histocompatibility complex alleles determine central nervous system injury versus repair.

BACKGROUND: We investigated the role of human HLA class I molecules in persistent central nervous system (CNS) injury versus repair following virus infection of the CNS. METHODS: Human class I A11+ and B27+ transgenic human beta-2 microglobulin positive (Hß2m+) mice of the H-2 b background were generated on a combined class I-deficient (mouse beta-2 microglobulin deficient, ß2m0) and class II-deficient (mouse Aß0) phenotype. Intracranial infection with Theiler's murine encephalomyelitis virus (TMEV) in susceptible SJL mice results in acute encephalitis with prominent injury in the hippocampus, striatum, and cortex. RESULTS: Following infection with TMEV, a picornavirus, the Aß0.ß2m0 mice lacking active immune responses died within 18 to 21 days post-infection. These mice showed severe encephalomyelitis due to rapid replication of the viral genome. In contrast, transgenic Hß2m mice with insertion of a single human class I MHC gene in the absence of human or mouse class II survived the acute infection. Both A11+ and B27+ mice significantly controlled virus RNA expression by 45 days and did not develop late-onset spinal cord demyelination. By 45 days post-infection (DPI), B27+ transgenic mice showed almost complete repair of the virus-induced brain injury, but A11+ mice conversely showed persistent severe hippocampal and cortical injury. CONCLUSIONS: The findings support the hypothesis that the expression of a single human class I MHC molecule, independent of persistent virus infection, influences the extent of sub frequent chronic neuronal injury or repair in the absence of a class II MHC immune response.

Pemphigus is associated with KIR3DL2 expression levels and provides evidence that KIR3DL2 may bind HLA-A3 and A11 in vivo.

Although HLA-A3 and A11 have been reported to be ligands for KIR3DL2, evidence for any in vivo relevance of this interaction is still missing. To explore the functional importance of KIR3DL2 allelic variation, we analyzed the autoimmune disease pemphigus foliaceus, previously associated (lower risk) with activating KIR genes. KIR3DL2*001 was increased in patients (odds ratio (OR) = 2.04; p = 0.007). The risk was higher for the presence of both KIR3DL2*001 and HLA-A3 or A11 (OR = 3.76, p = 0.013), providing the first evidence that HLA-A3 and A11 may interact with KIR3DL2 in vivo. The nonsynonymous single nucleotide polymorphism 1190T (rs3745902) was associated with protection (OR = 0.52, p = 0.018). This SNP results in a threonine-to-methionine substitution. Individuals who have methionine in this position exhibit a lower percentage of KIR3DL2-positive natural killer (NK) cells and also lower intensity of KIR3DL2 on expressing natural killer cells; additionally, we show that the expression of KIR3DL2 is independent of other killer cell immunoglobulin-like receptors. Pemphigus foliaceus is a very unique complex disease strongly associated with immune-related genes. It is the only autoimmune disease known to be endemic, showing a strong correlation with environmental factors. Our data demonstrate that this relatively unknown autoimmune disease may facilitate understanding of the molecular mechanisms of KIR3DL2 ligand recognition.

Activating killer cell immunoglobulin-like receptor 2DS2 binds to HLA-A*11.

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.

An immunodominant HLA-A*1101-restricted CD8+ T-cell response targeting hepatitis B surface antigen in chronic hepatitis B patients.

Hepatitis B virus (HBV) infection is a worldwide public health problem. HBV-specific CD8(+) CTLs are vital for viral clearance. Identification of immunodominant CTL epitopes from HBV-associated antigens is necessary for therapeutic vaccine development. We showed that the HLA-A*1101 allele is one of the most common alleles in both healthy individuals and chronic hepatitis B (CHB) patients in the Chongqing area, China. However, less than 10% of epitopes of HBV-associated antigens have been identified in an HLA-A*1101 context. Here, we describe an immunodominant CD8(+) T-cell response targeting a hepatitis B surface antigen determinant (HBs(295-304)) restricted by HLA-A*1101 in both healthy individuals and CHB patients. Moreover, HBs(295-304) is more immunogenic for CTL induction than a known naturally HLA-A*1101-processed epitope from hepatitis B core antigen (HBc(88-96)). Therefore, the newly identified epitope, HBs(295-304), will benefit the development of immunotherapeutic approaches for HBV infection.

Beyond HLA-A*0201: new HLA-transgenic nonobese diabetic mouse models of type 1 diabetes identify the insulin C-peptide as a rich source of CD8+ T cell epitopes.

Type 1 diabetes is an autoimmune disease characterized by T cell responses to ß cell Ags, including insulin. Investigations employing the NOD mouse model of the disease have revealed an essential role for ß cell-specific CD8(+) T cells in the pathogenic process. As CD8(+) T cells specific for ß cell Ags are also present in patients, these reactivities have the potential to serve as therapeutic targets or markers for autoimmune activity. NOD mice transgenic for human class I MHC molecules have previously been employed to identify T cell epitopes having important relevance to the human disease. However, most studies have focused exclusively on HLA-A*0201. To broaden the reach of epitope-based monitoring and therapeutic strategies, we have looked beyond this allele and developed NOD mice expressing human ß(2)-microglobulin and HLA-A*1101 or HLA-B*0702, which are representative members of the A3 and B7 HLA supertypes, respectively. We have used islet-infiltrating T cells spontaneously arising in these strains to identify ß cell peptides recognized in the context of the transgenic HLA molecules. This work has identified the insulin C-peptide as an abundant source of CD8(+) T cell epitopes. Responses to these epitopes should be of considerable utility for immune monitoring, as they cannot reflect an immune reaction to exogenously administered insulin, which lacks the C-peptide. Because the peptides bound by one supertype member were found to bind certain other members also, the epitopes identified in this study have the potential to result in therapeutic and monitoring tools applicable to large numbers of patients and at-risk individuals.