A subset of MHC-associated self-peptides presented by the recipient's cells and immunologically foreign to the donor can induce an allogeneic immune response after hematopoietic stem cell transplantation (HSCT). These immunogenic peptides originate from the genomic polymorphisms and are known as minor histocompatibility antigens (MiHA). MiHA mismatches trigger the post-transplant immune response, which could manifest in both the deleterious "graft-vs.-host" disease and the beneficial "graft-vs.-leukemia" effect. Importantly, some MiHAs are considered to be promising targets for posttransplant T-cell immunotherapy of hematopoietic malignancies. This creates a demand for a robust and fast approach to genotyping MiHA-encoding polymorphisms. We report a multiplex real-time PCR method for the genotyping of 20 polymorphisms that are encoding HLA-A*02:01-restricted MiHAs. This method uses allele-specific primers and gene-specific hydrolysis probes. In 1 h it allows for the detection of MiHA mismatches in a donor-recipient pair without the need for electrophoresis, sequencing, or other time-consuming techniques. We validated the method with Sanger and NGS sequencing and demonstrated good performance over a wide range of DNA concentrations. We propose our protocol as a fast and accurate method of identifying mismatched MiHAs. The information on the MiHA mismatches is useful for studying the allogeneic immune response following HSCT and for selecting the targets for post-transplant T-cell therapy.

Patients undergoing allogeneic stem cell transplantation as treatment for hematological diseases face the risk of Graft-versus-Host Disease as well as relapse. Graft-versus-Host Disease and the favorable Graft-versus-Leukemia effect are mediated by donor T cells recognizing polymorphic peptides, which are presented on the cell surface by HLA molecules and result from single nucleotide polymorphism alleles that are disparate between patient and donor. Identification of polymorphic HLA-binding peptides, designated minor histocompatibility antigens, has been a laborious procedure, and the number and scope for broad clinical use of these antigens therefore remain limited. Here, we present an optimized whole genome association approach for discovery of HLA class I minor histocompatibility antigens. T cell clones isolated from patients who responded to donor lymphocyte infusions after HLA-matched allogeneic stem cell transplantation were tested against a panel of 191 EBV-transformed B cells, which have been sequenced by the 1000 Genomes Project and selected for expression of seven common HLA class I alleles (HLA-A∗01:01, A∗02:01, A∗03:01, B∗07:02, B∗08:01, C∗07:01, and C∗07:02). By including all polymorphisms with minor allele frequencies above 0.01, we demonstrated that the new approach allows direct discovery of minor histocompatibility antigens as exemplified by seven new antigens in eight different HLA class I alleles including one antigen in HLA-A∗24:02 and HLA-A∗23:01, for which the method has not been originally designed. Our new whole genome association strategy is expected to rapidly augment the repertoire of HLA class I-restricted minor histocompatibility antigens that will become available for donor selection and clinical use to predict, follow or manipulate Graft-versus-Leukemia effect and Graft-versus-Host Disease after allogeneic stem cell transplantation.

HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced a 9-42 fold increase of all six HLA-A,-B,-C gene transcripts. Interestingly, prior to stimulation, gene transcripts for all but two alleles were present in similar amounts suggesting that post-transcriptional mechanisms regulate the constitutive expression of HLA-A,-B, and -C. Locus-restricted expression of HLA-A, -B and -C challenges our current understanding of the function of these molecules as regulators of CD8(+) T-cell and NK-cell function and should lead to further inquiries into their expression on other cell types.

Certain phosphorylated peptides are differentially presented by major histocompatibility complex (MHC) molecules on cancer cells characterized by aberrant phosphorylation. Phosphopeptides presented in complex with the human leukocyte antigen HLA-A*02:01 provide a stability advantage over their non-phosphorylated counterparts. This stability is thought to contribute to enhanced immunogenicity. Whether tumor-associated phosphopeptides presented by other common alleles exhibit immunogenicity and structural characteristics similar to those presented by A*02:01 is unclear. Therefore, we determined the identity, structural features, and immunogenicity of phosphopeptides presented by the prevalent alleles HLA-A*03:01, HLA-A*11:01, HLA-C*07:01, and HLA-C*07:02.

T-cell immunotherapies are often thwarted by the limited presentation of tumor-specific antigens abetted by the downregulation of human leukocyte antigen (HLA). We showed that drugs inhibiting ALK and RET produced dose-related increases in cell-surface HLA in tumor cells bearing these mutated kinases in vitro and in vivo, as well as elevated transcript and protein expression of HLA and other antigen-processing machinery. Subsequent analysis of HLA-presented peptides after ALK and RET inhibitor treatment identified large changes in the immunopeptidome with the appearance of hundreds of new antigens, including T-cell epitopes associated with impaired peptide processing (TEIPP) peptides. ALK inhibition additionally decreased PD-L1 levels by 75%. Therefore, these oncogenes may enhance cancer formation by allowing tumors to evade the immune system by downregulating HLA expression. Altogether, RET and ALK inhibitors could enhance T-cell-based immunotherapies by upregulating HLA, decreasing checkpoint blockade ligands, and revealing new, immunogenic, cancer-associated antigens.

Certain phosphorylated peptides are differentially presented by MHC molecules on cancer cells characterized by aberrant phosphorylation. Phosphopeptides presented in complex with the human leukocyte antigen HLA-A*02:01 provide a stability advantage over their nonphosphorylated counterparts. This stability is thought to contribute to enhanced immunogenicity. Whether tumor-associated phosphopeptides presented by other common alleles exhibit immunogenicity and structural characteristics similar to those presented by A*02:01 is unclear. Therefore, we determined the identity, structural features, and immunogenicity of phosphopeptides presented by the prevalent alleles HLA-A*03:01, -A*11:01, -C*07:01, and - C*07:02.

Trypanosoma cruzi antigens TSA-1 and Tc24 have shown promise as vaccine candidates in animal studies. We evaluated here the recall immune response these antigens induce in Chagasic patients, as a first step to test their immunogenicity in humans. We evaluated the in vitro cellular immune response after stimulation with recombinant TSA-1 (rTSA-1) or recombinant Tc24 (rTc24) in mononuclear cells of asymptomatic Chagasic chronic patients (n = 20) compared to healthy volunteers (n = 19) from Yucatan, Mexico. Proliferation assays, intracellular cytokine staining, cytometric bead arrays, and memory T cell immunophenotyping were performed by flow cytometry. Peripheral blood mononuclear cells (PBMC) from Chagasic patients showed significant proliferation after stimulation with rTc24 and presented a phenotype of T effector memory cells (CD45RA-CCR7-). These cells also produced IFN-γ and, to a lesser extent IL10, after stimulation with rTSA-1 and rTc24 proteins. Overall, both antigens recalled a broad immune response in some Chagasic patients, confirming that their immune system had been primed against these antigens during natural infection. Analysis of HLA-A and HLA-B allele diversity by PCR-sequencing indicated that HLA-A03 and HLA-B07 were the most frequent supertypes in this Mexican population. Also, there was a significant difference in the frequency of HLA-A01 and HLA-A02 supertypes between Chagasic patients and controls, while the other alleles were evenly distributed. Some aspects of the immune response, such as antigen-induced IFN-γ production by CD4+ and CD8+ T cells and CD8+ proliferation, showed significant association with specific HLA-A supertypes, depending on the antigen considered. In conclusion, our results confirm the ability of both TSA-1 and Tc24 recombinant proteins to recall an immune response induced by the native antigens during natural infection in at least some patients. Our data support the further development of these antigens as therapeutic vaccine against Chagas disease.

Hepatitis B chronic carriage remains as a major public health problem. Protein and DNA vaccines are now widely used in therapeutic vaccine candidates. Although, the hepatitis B surface antigen (HBsAg) based vaccines have been largely studied, candidates comprising both HBsAg and core (HBcAg) either protein- or DNA-based approaches deserve further immunological characterization. In the present study, a repeated dose administration schedule for protein or DNA immunogens was conducted in order to characterize the resulting immune response in a humanized and HBsAg-tolerized setting. A novel transgenic (Tg) mice that express the HBsAg, human MHC class I (HLA-A*0201) and MHC class II (HLA-DRB1*01) molecules and devoid of endogenous murine class I and II molecules was used as a model of HBV chronic carrier. Mice were immunized by subcutaneous (protein) or intramuscular (DNA) routes and the humoral and cellular responses were evaluated. Protein or DNA immunization induced humoral immune responses against both HBsAg and HBcAg. The systematic analysis of epitopes that activate CD4+ and CD8+ T lymphocytes confirmed the accuracy of the model. Cellular immune responses were detected differing in their nature. CD8 T-cell responses were induced mostly after DNA immunization while CD4 T-cell responses were predominant in protein based immunizations. In addition, the intensity of HLA-A2-restricted CD8+ T cell responses was reduced in Tg mice expressing HBsAg when compared to control Tg mice. In conclusion, our results indicate that cellular immune responses necessary for the development of protective immunity can be achieved by DNA or protein immunization. However, important differences in their nature arise when immunogens are administered several times. How to cite this article: Mancini-Bourgine M, Guillen G, Michel ML, Aguilar JC. Impact of the Immunogen Nature on the Immune Response against the Major HBV Antigens in an HBsAg and HLA-humanized Transgenic Mouse Model. Euroasian J Hepato-Gastroenterol 2014;4(1):36-44.

Host CD8 T cell response to viral infections involves recognition of 8-10-mer peptides presented by MHC-I molecules. However, proteasomes generate predominantly 2-7-mer peptides, but the role of these peptides is largely unknown. Here, we show that single short peptides of <8-mer from Latent Membrane Protein 2 (LMP2) of Epstein Barr Virus (EBV) can bind HLA-A*11:01 and stimulate CD8+ cells. Surprisingly, two peptide fragments between 4-7-mer derived from LMP2(340-349) were able to complement each other, forming combination epitopes that can stimulate specific CD8+ T cell responses. Moreover, peptides from self-antigens can complement non-self peptides within the HLA binding cleft, forming neoepitopes. Solved structures of a tetra-complex comprising two peptides, HLA and β2-microglobulin revealed the free terminals of the two peptides to adopt an upward conformation directed towards the T cell receptor. Our results demonstrate a previously unknown mix-and-match combination of dual peptide occupancy in HLA that can generate vast combinatorial complexity.

The detection of HLA antibodies is important in clinical practice, such as platelet transfusion refractoriness and transfusion-related lung injury. However, difficulties are associated with the preparation of panel cells for conventional HLA detection systems using intact cells, such as the immunocomplex capture fluorescence analysis (ICFA). Based on an ICFA analysis, HEK293 cells stably transfected with the HLA-A locus were used instead of peripheral blood mononuclear cells (PBMC). The reactivity, sensitivity, and stability of transfectants were examined. All 20 antisera to HLA-A identified by LABScreen® Single Antigen class I (LS-SA1) were reactive to our modified-ICFA (m-ICFA) and showed the same specificities as those in LS-SA1, indicating the cell surface expression and correct antigenicity of the HLA-A locus in transfectants. The expression of HLA class I antigens was similar between transfectants frozen for 6 years and those prior to freezing. In the reaction of the anti-A24 or anti-A33 antibody vs each transfectant, the index of m-ICFA was higher than that of WAKFlow® ICFA. Our m-ICFA also showed that false negative reactions sometimes observed in capture assays may be avoided. By using HLA-A transfectants as ICFA targets, we herein developed m-ICFA. Our m-ICFA may avoid false negative reactions of capture assay like enzyme-linked immunosorbent assay and can also be carried out in almost any laboratory without cell culture facilities.

HLA molecules are cell-surface glycoproteins that present peptides, derived from intracellular protein antigens, for surveillance by T lymphocytes. Secreted HLA (sHLA) technology is a powerful approach for studying these peptides, since it facilitates large-scale production of HLA-bound peptides. We compared secreted and membrane-bound forms of HLA A2 in terms of intracellular trafficking and their bound peptide repertoire (termed the immunopeptidome). We demonstrate that sHLA and membrane bound HLA (mHLA) negotiate intracellular compartments with similar maturation kinetics. Moreover, mass spectrometry revealed a substantial overlap in the immunopeptidome was observed when HLA A2-bound peptides were purified from various sources of sHLA and mHLA. By combining machine based algorithms with manual validation, we identified 1266 non-redundant peptides. Analysis of these peptides revealed a number bearing post-translational modifications, although some of these may arise spontaneously others represent modifications performed within the cell that survive antigen processing. Peptides bearing some of these modifications have not previously been described for HLA ligands, therefore, this compendium of 1266 non-redundant peptide sequences adds greatly to the existing database of HLA A2 ligands. Peptides from all sources displayed comparable HLA A2 consensus binding motifs, peptide lengths, predicted HLA A2 binding affinities and putative source antigens. We conclude that sHLA is a valid and useful technique for studying the immunopeptidome.

The classical class I human leukocyte antigens (HLA-A, -B, and -C) present allele-specific self- or pathogenic peptides originated by intracellular processing to CD8(+) immune effector cells. Even a single mismatch in the heavy chain (hc) of an HLA class I molecule can impact on the peptide binding profile. Since HLA class I molecules are highly polymorphic and most of their polymorphisms affect the peptide binding region (PBR), it becomes obvious that systematic HLA matching is crucial in determining the outcome of transplantation. The opposite holds true for the nonclassical HLA class I molecule HLA-E. HLA-E polymorphism is restricted to two functional versions and is thought to present a limited set of highly conserved peptides derived from class I leader sequences. However, HLA-E appears to be a ligand for the innate and adaptive immune system, where the immunological response to peptide-HLA-E complexes is dictated through the sequence of the bound peptide. Structural investigations clearly demonstrate how subtle amino acid differences impact the strength and response of the cognate CD94/NKG2 or T cell receptor.

The host's immune system develops in equilibrium with both cellular self-antigens and non-self-antigens derived from microorganisms which enter the body during lifetime. In addition, during the years, a tumor may arise presenting to the immune system an additional pool of non-self-antigens, namely tumor antigens (tumor-associated antigens, TAAs; tumor-specific antigens, TSAs).

HLA-E shares several peptide sequences with HLA-class Ia molecules. Therefore, anti-HLA-E antibodies that recognize the shared sequences may bind to HLA-class Ia alleles. This hypothesis was validated with a murine anti-HLA-E monoclonal antibody (mAb) MEM-E/02, which reacted with microbeads coated with several HLA-B and HLA-C antigens. In this report, the hypothesis was reexamined with another mAb 3D12, considered to be specific for HLA-E. The antibody binding is evaluated by measuring mean fluorescence index [MFI] with Luminex Multiplex Flow-Cytometric technology. The peptide-inhibition experiments are carried out with synthetic shared peptides, most prevalent to HLA-E and HLA-Ia alleles. The results showed that mAb 3D12 simulated MEM-E/02 in recognizing several HLA-B and HLA-C antigens. Both 3D12 and MEM-E/02 did not bind to HLA-A, HLA-F and HLA-G molecules. As observed with MEM-E/02, binding of 3D12 to HLA-E is inhibited by the peptides sequences (115)QFAYDGKDY(123) and (137)DTAAQI(142). Decrease in binding of mAb 3D12 to HLA class Ia, after heat treatment of antigen coated microbeads, supports the contention that the epitope may be located at the outside of the "thermodynamically stable" α-helix conformations of HLA-E. Several sequence and structure-based web-tools were employed to validate the discontinuous epitopes recognized by the mAbs. The scores obtained by these web-tools distinguished the shared peptide sequences that inhibited the mAb binding to HLA-E. Furthermore, ElliPro web tool points out that both mAbs recognize the conformational discontinuous epitopes (the shared inhibitory peptide sequences) in the secondary structure of the HLA-E molecule. The study favors the contention that the domain of the shared inhibitory peptide sequences may be the most immunogenic site of HLA-E molecule. It also postulates and clarifies that amino acid substitution on or near the binding domains may account for the lack of cross reactivity of 3D12 and MEM-E/02 with HLA-A, HLA-F and HLA-G molecules.

Genes of the Major Histocompatibility Complex (MHC) have recently been shown to have neuronal functions in the thalamus and hippocampus. Common genetic variants in the Human Leukocyte Antigens (HLA) region, human homologue of the MHC locus, are associated with small effects on susceptibility to schizophrenia, while volumetric changes of the thalamus and hippocampus have also been linked to schizophrenia. We therefore investigated whether common variants of the HLA would affect volumetric variation of the thalamus and hippocampus. We analysed thalamus and hippocampus volumes, as measured using structural magnetic resonance imaging, in 1.265 healthy participants. These participants had also been genotyped using genome-wide single nucleotide polymorphism (SNP) arrays. We imputed genotypes for single nucleotide polymorphisms at high density across the HLA locus, as well as HLA allotypes and HLA amino acids, by use of a reference population dataset that was specifically targeted to the HLA region. We detected a significant association of the SNP rs17194174 with thalamus volume (nominal P=0.0000017, corrected P=0.0039), as well as additional SNPs within the same region of linkage disequilibrium. This effect was largely lateralized to the left thalamus and is localized within a genomic region previously associated with schizophrenia. The associated SNPs are also clustered within a potential regulatory element, and a region of linkage disequilibrium that spans genes expressed in the thalamus, including HLA-A. Our data indicate that genetic variation within the HLA region influences the volume and asymmetry of the human thalamus. The molecular mechanisms underlying this association may relate to HLA influences on susceptibility to schizophrenia.

Cancer-Testis antigens (CTA) are named after the tissues where they are mainly expressed: in germinal and in cancer cells, a process that mimics many gametogenesis features. Mapping accurately the CTA gene expression signature in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) is a prerequisite for downstream immune target-discovery projects. In this study, we take advantage of the use of azacitidine to treat high-risk MDS and CMML to draw the CTAs landscape, before and after treatment, using an ad hoc targeted RNA sequencing (RNA-seq) design for this group of low transcript genes. In 19 patients, 196 CTAs were detected at baseline. Azacitidine did not change the number of CTAs expressed, but it significantly increased or decreased expression in nine and five CTAs, respectively. TFDP3 and DDX53, emerged as the main candidates for immunotherapeutic targeting, as they showed three main features: i) a significant derepression on day +28 of cycle one in those patients who achieved complete remission with hypomethylating treatment (FC = 6, p = .008; FC = 2.1, p = .008, respectively), ii) similar dynamics at the protein level to what was observed at the RNA layer, and iii) to elicit significant specific cytotoxic immune responses detected by TFDP3 and DDX53 HLA-A*0201 tetramers. Our study addresses the unmet landscape of CTAs expression in MDS and CMML and revealed a previously unrecognized TFDP3 and DDX53 reactivation, detectable in plasma and able to elicit a specific immune response after one cycle of azacitidine.

Hantavirus infections cause severe emerging diseases in humans and are associated with high mortality rates; therefore, they have become a global public health concern. Our previous study showed that the CD8(+) T-cell epitope aa129-aa137 (FVVPILLKA, FA9) of the Hantaan virus (HTNV) nucleoprotein (NP), restricted by human leukocyte antigen (HLA)-A*02, induced specific CD8(+) T-cell responses that controlled HTNV infection in humans. However, the in vivo immunogenicity of peptide FA9 and the effect of FA9-specific CD8(+) T-cell immunity remain unclear. Here, based on a detailed structural analysis of the peptide FA9/HLA-A*0201 complex and functional investigations using HLA-A2.1/K(b) transgenic (Tg) mice, we found that the overall structure of the peptide FA9/HLA-A*0201 complex displayed a typical MHC class I fold with Val2 and Ala9 as primary anchor residues and Val3 and Leu7 as secondary anchor residues that allow peptide FA9 to bind tightly with an HLA-A*0201 molecule. Residues in the middle portion of peptide FA9 extruding out of the binding groove may be the sites that allow for recognition by T-cell receptors. Immunization with peptide FA9 in HLA-A2.1/K(b) Tg mice induced FA9-specific cytotoxic T-cell responses characterized by the induction of high expression levels of interferon-γ, tumor necrosis factor-α, granzyme B, and CD107a. In an HTNV challenge trial, significant reductions in the levels of both the antigens and the HTNV RNA loads were observed in the liver, spleen, and kidneys of Tg mice pre-vaccinated with peptide FA9. Thus, our findings highlight the ability of HTNV epitope-specific CD8(+) T-cell immunity to control HTNV and support the possibility that the HTNV-NP FA9 peptide, naturally processed in vivo in an HLA-A*02-restriction manner, may be a good candidate for the development HTNV peptide vaccines.

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor related antigens is a promising approach for malignant tumor immunotherapy. TC2N, a recently identified tumor associated antigen from human glioblastoma, is regarded as a promising target of tumor-specific immunotherapy. As one of the most widely used histocompatibility molecules in Chinese is HLA-A*0201, we were able to identify the TC2N peptides that are provided by this molecular type. A panel of antigenic peptides produced from TC2N were predicted by using a computer tool. The binding affinities of three peptides with the highest predicted score to the HLA-A*0201 molecule were evaluated after synthesis. In vitro and in vivo stimulation of the main T-cell response against the predicted peptides. The results demonstrated that TC2N (152-160) was able to release IFN-γ and lyse U251 cells in vitro as well as in vivo by eliciting peptide-specific CTLs. Our results indicated that peptide TC2N (152-160) (RLYGSVCDL) was a novel HLA-A2.1-restricted CTL epitope capable of inducing TC2N specific CTLs in vitro. As TC2N might qualify as a viable target for immunotherapeutic approaches for patients with GBM, we speculated that the newly identified epitope RLYGSVCDL would be of potential use in peptide-based, cancer-specific immunotherapy against GBM.

Generalized vitiligo is an autoimmune disease characterized by melanocyte loss, which results in patchy depigmentation of skin and hair. Recent studies suggested the key role of CD8+T lymphocytes for mediating immune response in vitiligo through melanocyte differentiation antigens, including tyrosinase, gp100 and MelanA/Mart-1. However, the specific epitopes of these auto-antigens are still unknown. In our study, we predicted the possible HLA-A*0201-restricted nonapeptides overlaying the full-length amino acid sequences of these three known antigens and investigated the lymphocytes reactivity to these nonapeptides by Elispot assay. In addition, we evaluated the abilities of these nonapeptides to activate CD8+T cells. We screened out 5 possible epitopes originated from tyrosinase and gp100, numbered P28, P41, P112, P118 and P119. Among these 5 epitopes, notably, P28 and P119 played the dominant role in activating CTLs, with a significant increase in proliferation rate and Interferon-γ (IFN-γ) production of CD8+T cells. Nevertheless, antigen-specific T cell reactivity was not detected in MelanA/Mart-1 peptides. Our studies identified two novel epitopes originated from proteins of gp100 and tyrosinase, which may have implications for the development of immunotherapies for vitiligo.

The identification of universal tumor-specific antigens shared between multiple patients and/or multiple tumors is of great importance to overcome the practical limitations of personalized cancer immunotherapy. Recent studies support the involvement of DEPDC1 in many aspects of cancer traits, such as cell proliferation, resistance to induction of apoptosis and cell invasion, suggesting that it may play key roles in the oncogenic process. In this study, we report that DEPDC1 expression is upregulated in most types of human tumors, and closely linked to a poorer prognosis; therefore, it might be regarded as a novel universal oncoantigen potentially suitable for targeting many different cancers. In this regard, we report the identification of a HLA-A*0201 allele-restricted immunogenic DEPDC1-derived epitope, which is able to induce cytotoxic T lymphocytes (CTL) exerting a strong and specific functional response in vitro toward not only peptide-loaded cells but also triple negative breast cancer (TNBC) cells endogenously expressing the DEPDC1 protein. Such CTL are also therapeutically active against human TNBC xenografts in vivo upon adoptive transfer in immunodeficient mice. Overall, these data provide evidence that this DEPDC1-derived antigenic epitope can be exploited as a new tool for developing immunotherapeutic strategies for HLA-A*0201 patients with TNBC, and potentially many other cancers.