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We previously identified papillomavirus binding factor (PBF) as an osteosarcoma antigen recognized by an autologous cytotoxic T lymphocyte clone. Vaccination with PBF-derived peptide presented by HLA-A24 (PBF peptide) elicited strong immune responses. In the present study, we generated T cell receptor-engineered T cells (TCR-T cells) directed against the PBF peptide (PBF TCR-T cells). PBF TCR was successfully transduced into T cells and detected using HLA-A*24:02/PBF peptide tetramer. PBF TCR-T cells generated from a healthy donor were highly expanded and recognized T2-A24 cells pulsed with PBF peptide, HLA-A24 + 293T cells transfected with PBF cDNA, and sarcoma cell lines. To establish an adoptive cell therapy model, we modified the PBF TCR by replacing both α and β constant regions with those of mice (hybrid PBF TCR). Hybrid PBF TCR-T cells also showed reactivity against T2-A24 cells pulsed with PBF peptide and to HLA-A24 + 293T cells transfected with various lengths of PBF cDNA including the PBF peptide sequence. Subsequently, we generated target cell lines highly expressing PBF (MFH03-PBF [short] epitope [+]) containing PBF peptide with in vivo tumorigenicity. Hybrid PBF TCR-T cells exhibited antitumor effects compared with mock T cells in NSG mice xenografted with MFH03-PBF (short) epitope (+) cells. CD45 + T cells significantly infiltrated xenografted tumors only in the hybrid PBF TCR T cell group and most of these cells were CD8-positive. CD8 + T cells also showed Ki-67 expression and surrounded the CD8-negative tumor cells expressing Ki-67. These findings suggest that PBF TCR-T cell therapy might be a candidate immunotherapy for sarcoma highly expressing PBF., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

MHC class I molecules regulate brain development and plasticity in mice and HLA class I molecules are associated with brain disorders in humans. We investigated the relationship between plasma-derived soluble human HLA class I molecules (sHLA class I), HLA class I serotypes and dementia. A cohort of HLA class I serotyped elderly subjects with no dementia/pre-dementia (NpD, n = 28), or with dementia (D, n = 28) was studied. Multivariate analysis was used to examine the influence of dementia and HLA class I serotype on sHLA class I levels, and to compare sHLA class I within four groups according to the presence or absence of HLA-A23/A24 and dementia. HLA-A23/A24 and dementia, but not age, significantly influenced the level of sHLA class I. Importantly, the concurrent presence of HLA-A23/A24 and dementia was associated with higher levels of sHLA class I (p < 0.001). This study has shown that the simultaneous presence of HLA-A23/HLA-A24 and dementia is associated with high levels of serum sHLA class I molecules. Thus, sHLA class I could be considered a biomarker of neurodegeneration in certain HLA class I carriers., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder. The pathogenesis of IBS has not yet been fully elucidated, and the relationship between human leukocyte antigen (HLA) class I molecules and IBS is not clear. The present case-control study investigated the correlation between HLA-A and HLA-B genes and IBS. Peripheral blood samples were collected from 102 IBS patients and 108 healthy volunteers at Nanning First People's Hospital. DNA was extracted using a routine procedure, and HLA-A and HLA-B gene polymorphisms were identified by polymerase chain reaction with sequence-specific primers to determine the genotype and distribution frequency of HLA-A and HLA-B in IBS patients and healthy controls. Susceptibility and protective genes for IBS were identified using univariate and multivariate analyses. The frequency of HLA-A11 gene expression in the IBS group was significantly higher than that in the healthy control group, while the frequencies of HLA-A24, 26, and 33 gene expression were significantly higher in the healthy control group than in the IBS group (all P < .05). The frequencies of HLA-B56 and 75 (15) gene expression in the IBS group were significantly higher than those in the healthy control group, while the frequencies of HLA-B46 and 48 gene expression were significantly higher in the healthy control group than in the IBS group (all P < .05). Genes that may be related to the prevalence of IBS were included in the multivariate logistic regression, and the results suggested that the HLA-B75 (15) gene is a susceptibility gene for IBS (P = .031, odds ratio [OR] = 2.625, 95% confidence interval [CI]: 1.093-6.302), while the HLA-A24 (P = .003, OR = 0.308, 95% CI: 0.142-0.666), A26 (P = .009, OR = 0.162, 95% CI: 0.042-0.629), A33 (P = .012, OR = 0.173, 95% CI: 0.044-0.679), and B48 (P = .008, OR = 0.051, 95% CI: 0.006-0.459) genes are protective genes for IBS., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

Although human hepatocyte-transplanted immunodeficient mice support infection with hepatitis viruses, these mice fail to develop viral hepatitis due to the lack of an adaptive immune system. In this study, we generated new immunodeficiency cDNA-urokinase-type plasminogen activator (uPA)/SCID/Rag2 -/- /Jak3 -/- mice and established a mouse model with both a humanized liver and immune system. Transplantation of human hepatocytes with human leukocyte antigen (HLA)-A24 resulted in establishment of a highly replaced liver in cDNA-uPA/SCID/Rag2 -/- /Jak3 -/- mice. These mice were successfully infected with hepatitis B virus (HBV) and hepatitis C virus (HCV) for a prolonged period and facilitate analysis of the effect of anti-HCV drugs. Administration of peripheral blood mononuclear cells (PBMCs) obtained from an HLA-A24 donor resulted in establishment of 22.6%-81.3% human CD45-positive mononuclear cell chimerism in liver-infiltrating cells without causing graft-versus-host disease in cDNA-uPA/SCID/Rag2 -/- /Jak3 -/- mice without human hepatocyte transplantation. When mice were transplanted with human hepatocytes and then administered HLA-A24-positive human PBMCs, an alloimmune response between transplanted human hepatocytes and PBMCs occurred, with production of transplanted hepatocyte-specific anti-HLA antibody. In conclusion, we succeeded in establishing a humanized liver/immune system characterized by an allo-reaction between transplanted human immune cells and human liver using a novel cDNA-uPA/SCID/Rag2 -/- /Jak3 -/- mouse. This mouse model can be used to generate a chronic hepatitis mouse model with a human immune system with application not only to hepatitis virus virology but also to investigation of the pathology of post-transplantation liver rejection., (© 2023 John Wiley&Sons Ltd.)

Cross-recognized public TCRs against HIV epitopes have been proposed to be important for the control of AIDS disease progression and HIV variants. The overlapping Nef138-8 and Nef138-10 peptides from the HIV Nef protein are HLA-A24-restricted immunodominant T cell epitopes, and an HIV mutant strain with a Y139F substitution in Nef protein can result in immune escape and is widespread in Japan. Here, we identified a pair of public TCRs specific to the HLA-A24-restricted Nef-138-8 epitope using PBMCs from White and Japanese patients, respectively, namely TD08 and H25-11. The gene use of the variable domain for TD08 and H25-11 is TRAV8-3, TRAJ10 for the α-chain and TRBV7-9, TRBD1*01, TRBJ2-5 for the β-chain. Both TCRs can recognize wild-type and Y2F-mutated Nef138-8 epitopes. We further determined three complex structures, including TD08/HLA-A24-Nef138-8, H25-11/HLA-A24-Nef138-8, and TD08/HLA-A24-Nef138-8 (2F). Then, we revealed the molecular basis of the public TCR binding to the peptide HLA, which mostly relies on the interaction between the TCR and HLA and can tolerate the mutation in the Nef138-8 peptide. These findings promote the molecular understanding of T cell immunity against HIV epitopes and provide an important basis for the engineering of TCRs to develop T cell-based immunotherapy against HIV infection., (Copyright © 2022 by The American Association of Immunologists, Inc.)

The immunogenicity of a T cell Ag is correlated with the ability of its antigenic epitope to bind HLA and be stably presented to T cells. This presents a challenge for the development of effective cancer immunotherapies, as many self-derived tumor-associated epitopes elicit weak T cell responses, in part due to weak binding affinity to HLA. Traditional methods to increase peptide-HLA binding affinity involve modifying the peptide to reflect HLA allele binding preferences. Using a different approach, we sought to analyze whether the immunogenicity of wild-type peptides could be altered through modification of the HLA binding pocket. After analyzing HLA class I peptide binding pocket alignments, we identified an alanine 81 to leucine (A81L) modification within the F binding pocket of HLA-A*24:02 that was found to heighten the ability of artificial APCs to retain and present HLA-A*24:02-restricted peptides, resulting in increased T cell responses while retaining Ag specificity. This modification led to increased peptide exchange efficiencies for enhanced detection of low-avidity T cells and, when expressed on artificial APCs, resulted in greater expansion of Ag-specific T cells from melanoma-derived tumor-infiltrating lymphocytes. Our study provides an example of how modifications to the HLA binding pocket can enhance wild-type cognate peptide presentation to heighten T cell activation., (Copyright © 2022 by The American Association of Immunologists, Inc.)

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., (© 2022. The Author(s).)

T-cell receptor (TCR)-like Abs that specifically recognize antigenic peptides presented on MHC molecules have been developed for next-generation cancer immunotherapy. Recently, we reported a rapid and efficient method to generate TCR-like Abs using a rabbit system. We humanized previously generated rabbit-derived TCR-like Abs reacting Epstein-Barr virus peptide (BRLF1p, TYPVLEEMF) in the context of HLA-A24 molecules, produced chimeric antigen receptor (CAR)-T cells, and evaluated their antitumor effects using in vitro and in vivo tumor models. Humanization of the rabbit-derived TCR-like Abs using the complementarity-determining region grafting technology maintained their specificity and affinity. We prepared a second-generation CAR using single-chain variable fragment of the humanized TCR-like Abs and then transduced them into human T cells. The CAR-T cells specifically recognized BRLF1p/MHC molecules and lysed the target cells in an antigen-specific manner in vitro. They also demonstrated antitumor activity in a mouse xenograft model. We report the generation of CAR-T cells using humanized rabbit-derived TCR-like Abs. Together with our established and efficient generation procedure for TCR-like Abs using rabbits, our platform for the clinical application of humanized rabbit-derived TCR-like Abs to CAR-T cells will help improve next-generation cancer immunotherapy., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: HLA-B*58:01 is a well-known risk factor for allopurinol-induced severe cutaneous adverse reactions (SCARs). However, only a minority of HLA-B*58:01 carriers suffer SCARs after taking allopurinol. The aim of this study was to investigate subsidiary genetic markers that could identify those at further increased risk of developing allopurinol-induced drug reaction with eosinophilia and systemic symptoms (DRESS) in subjects with HLA-B*58:01., Methods: Subjects with B*58:01 were enrolled (21 allopurinol-induced DRESS and 52 allopurinol-tolerant control). HLA-A, -B, -C and -DRB1 alleles were compared. Comparison of risk between HLAs and allopurinol-induced SCAR in separate populations was performed to support the results. Kruskal-Wallis test, Pearson's chi-square test, Fisher's exact test and binary logistic regression were used to analyze the risk of SCAR development., Results: Frequencies of A*24:02 (71.4 vs. 17.3%, p < 0.001, odds ratio [OR] = 12.0; 95% confidence interval [CI], 3.6-39.2) were significantly higher in B*58:01 (+) DRESS than B*58:01 (+) tolerant controls. In addition, DRB1*13:02 further increased the risk of DRESS. The phenotype frequency of A*24:02/DRB1*13:02 was significantly higher in the B*58:01 (+) DRESS group than in the B*58:01 (+) tolerant controls (52.4% vs. 5.8%, p < 0.001, OR, 66.0; 95% CI, 6.1-716.2). In 2782 allopurinol user cohort, the overall prevalence of DRESS was 0.22%, which increased to 1.62% and 2.86% in the presence of B*58:01 and B*58:01/A*24:02, respectively., Conclusion: The additional secondary screening with A*24:02 and DRB1*13:02 alleles may identify those at further increased risk of allopurinol-induced DRESS in B*58:01 carriers., Competing Interests: The authors declare that they have no competing interests., (© 2022 The Authors. Clinical and Translational Allergy published by John Wiley and Sons Ltd on behalf of European Academy of Allergy and Clinical Immunology.)

HLA-A*24:02:138 differs from HLA-A*24:02:01:01 by one nucleotide substitution at position 549 C>T., (© 2022 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

Background: HLA-B27 and -B57 were found in people with low levels of HIV-1 DNA, suggesting that HLA class I molecules may influence the size of HIV-1 reservoir. Aim of the study was to explore the association between HLA class I molecules and HIV-1 DNA in people with chronic HIV-1 infection., Methods: Post-hoc analysis of the APACHE trial, on adults with chronic HIV-1 infection, prolonged suppressive antiretroviral therapy and good immunological profile. HIV-1 DNA was quantified in peripheral blood mononuclear cells (PBMCs); HLA-A, -B and -C were tested on genomic DNA. Crude odds ratios (OR) with their respective 95% Wald confidence intervals (95% CIs) were estimated by univariable logistic regression for HLAs with a p-value <0.10., Results: We found 78 and 18 patients with HIV-1 DNA ≥100 copies/106PBMCs and with HIV-1 DNA <100 copies/106PBMCs, respectively. HLA-A24 was present in 21 (29.6%) participants among subjects with HIV-1 DNA ≥100 copies/106PBMCs and 1 (5.9%) among those with HIV-1 DNA <100 copies/106PBMCs (OR = 5.67, 95%CI = 0.79-46.03; p = 0.105); HLA-B39 was present in 1 (1.4%) with HIV-1 DNA ≥100 copies/106PBMCs and in 3 (17.6%) with HIV-1 DNA <100 copies/106PBMCs (OR = 13.71, 95%CI = 1.33-141.77; p = 0.028) and HLA-B55 in 3 (4.2%) and 3 (17.6%), respectively (OR = 4.43, 95%CI = 0.81-24.29; p = 0.087). All the three patients with HLA-B39 and HIV-1 DNA <100 copies/106PBMCs did not have HLA-A24., Conclusions: In patients with HIV-1 infection who maintained a good virological and immunological profile, HLA-B39 and -B55 may be associated with lower levels of HIV-1 DNA., Competing Interests: The authors have declared that no competing interests exist.

γδT cell receptors (γδTCRs) recognize a broad range of malignantly transformed cells in mainly a major histocompatibility complex (MHC)-independent manner, making them valuable additions to the engineered immune effector cell therapy that currently focuses primarily on αβTCRs and chimeric antigen receptors (CARs). As an exception to the rule, we have previously identified a γδTCR, which exerts antitumor reactivity against HLA-A*24:02-expressing malignant cells, however without the need for defined HLA-restricted peptides, and without exhibiting any sign of off-target toxicity in humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mouse models. This particular tumor-HLA-A*24:02-specific Vγ5Vδ1TCR required CD8αα co-receptor for its tumor reactive capacity when introduced into αβT cells engineered to express a defined γδTCR (TEG), referred to as TEG011; thus, it was only active in CD8 + TEG011. We subsequently explored the concept of additional redirection of CD4 + T cells through co-expression of the human CD8α gene into CD4 + and CD8 + TEG011 cells, later referred as TEG011&#95;CD8α. Adoptive transfer of TEG011&#95;CD8α cells in humanized HLA-A*24:02 transgenic NSG (NSG-A24:02) mice injected with tumor HLA-A*24:02 + cells showed superior tumor control in comparison to TEG011, and to mock control groups. The total percentage of mice with persisting TEG011&#95;CD8α cells, as well as the total number of TEG011&#95;CD8α cells per mice, was significantly improved over time, mainly due to a dominance of CD4 + CD8 + double-positive TEG011&#95;CD8α, which resulted in higher total counts of functional T cells in spleen and bone marrow. We observed that tumor clearance in the bone marrow of TEG011&#95;CD8α-treated mice associated with better human T cell infiltration, which was not observed in the TEG011-treated group. Overall, introduction of transgenic human CD8α receptor on TEG011 improves antitumor reactivity against HLA-A*24:02 + tumor cells and further enhances in vivo tumor control., Competing Interests: DB, ZS, and JK are inventors on different patents with γδTCR sequences, recognition mechanisms, and isolation strategies. JK is cofounder and shareholder of Gadeta (www.gadeta.nl). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Johanna, Hernández-López, Heijhuurs, Scheper, Bongiovanni, de Bruin, Beringer, Oostvogels, Straetemans, Sebestyen and Kuball.)

In-depth understanding of human T-cell-mediated immunity in coronavirus disease 2019 (COVID-19) is needed if we are to optimize vaccine strategies and immunotherapies. Identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T-cell epitopes and generation of peptide-human leukocyte antigen (peptide-HLA) tetramers facilitate direct ex vivo analyses of SARS-CoV-2-specific T cells and their T-cell receptor (TCR) repertoires. We utilized a combination of peptide prediction and in vitro peptide stimulation to validate novel SARS-CoV-2 epitopes restricted by HLA-A*24:02, one of the most prominent HLA class I alleles, especially in Indigenous and Asian populations. Of the peptides screened, three spike-derived peptides generated CD8 + IFNγ + responses above background, S 1208-1216 (QYIKWPWYI), S 448-456 (NYNYLYRLF) and S 193-201 (VFKNIDGYF), with S 1208 generating immunodominant CD8 + IFNγ + responses. Using peptide-HLA-I tetramers, we performed direct ex vivo tetramer enrichment for HLA-A*24:02-restricted CD8 + T cells in COVID-19 patients and prepandemic controls. The precursor frequencies for HLA-A*24:02-restricted epitopes were within the range previously observed for other SARS-CoV-2 epitopes for both COVID-19 patients and prepandemic individuals. Naïve A24/SARS-CoV-2-specific CD8 + T cells increased nearly 7.5-fold above the average precursor frequency during COVID-19, gaining effector and memory phenotypes. Ex vivo single-cell analyses of TCRαβ repertoires found that the A24/S 448 + CD8 + T-cell TCRαβ repertoire was driven by a common TCRβ chain motif, whereas the A24/S 1208 + CD8 + TCRαβ repertoire was diverse across COVID-19 patients. Our study provides an in depth characterization and important insights into SARS-CoV-2-specific CD8 + T-cell responses associated with a prominent HLA-A*24:02 allomorph. This contributes to our knowledge on adaptive immune responses during primary COVID-19 and could be exploited in vaccine or immunotherapeutic approaches., (© 2021 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Aim: This study is looking for a common pathogenicity between SARS-CoV-2 and Plasmodium species, in individuals with certain HLA serotypes., Methods: 1. Tblastx searches of SARS-CoV-2 are performed by limiting searches to five Plasmodium species that infect humans. 2. Aligned sequences in the respective organisms' proteomes are searched with blastp. 3. Binding predictions of the identified SARS-CoV-2 peptide to HLA supertype representatives are performed. 4. Blastp searches of predicted epitopes that bind strongly to the identified HLA allele are performed by limiting searches to H. sapiens and Plasmodium species, separately. 5. Peptides with minimum 60% identity to the predicted epitopes are found in results. 6. Peptides among those, which bind strongly to the same HLA allele, are predicted. 7. Step-4 is repeated by limiting searches to H. sapiens, followed by the remaining steps until step-7, for peptides sourced by Plasmodium species after step-6., Results: SARS-CoV-2 peptide with single letter amino acid code CFLGYFCTCYFGLFC has the highest identity to P. vivax. Its YFCTCYFGLF part is predicted to bind strongly to HLA-A*24:02. Peptides in the human proteome both homologous to YFCTCYFGLF and with a strong binding affinity to HLA-A*24:02 are YYCARRFGLF, YYCHCPFGVF, and YYCQQYFFLF. Such peptides in the Plasmodium species' proteomes are FFYTFYFELF, YFVACLFILF, and YFPTITFHLF. The first one belonging to P. falciparum has a homologous peptide (YFYLFSLELF) in the human proteome, which also has a strong binding affinity to the same HLA allele., Conclusion: Immune responses to the identified-peptides with similar sequences and strong binding affinities to HLA-A*24:02 can be related to autoimmune response risk in individuals with HLA-A*24:02 serotypes, upon getting infected with SARS-CoV-2 or P. falciparum., (Copyright © 2021. Published by Elsevier B.V.)

Compared with HLA-A*24:02:01:01, the alleles HLA-A*24:02:129 and HLA-A*24:02:135 each show one nucleotide substitution, respectively., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

One nucleotide replacement in codon 233 of HLA-A*24:02:01:01 results in a novel allele, HLA-A*24:02:31., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective To explore the structure and function of β2 microglobulin-linker 2-human leukocyte antigen A24 (β2m-linker 2-HLA A24) by bioinformatics, and to obtain the purified protein by eukaryotic expression. Methods The physicochemical properties of β2m-linker 2-HLA A24 was predicted by ProtParam. The predicting software of phosphorylation site (NetPhos) and the predicting software of the secondary structure (SOPMA) were used to analyze the phosphorylation site and the secondary structure of the protein, respectively. And the predicting tool of modeling the proteins' structure (SWISS-MODEL) was used to model its tertiary structure. Then the recombinant plasmid of pcDNA3.4/β2m-linker 2-HLA A24-His tag was constructed and transfected into Expi293F cells. The protein of interest was purified through Ni-affinity chromatography. And the purity and properties of the protein were identified by SDS-PAGE, Western blot analysis and indirect ELISA. Results The β2m-linker 2-HLA A24 protein contained 44 phosphorylated sites. The protein was predicted to be a hydrophilic protein, of which the secondary structure mainly consists of irregular curl, and the modeling of the tertiary structure was successful. The recombinant plasmids of pcDNA3.4/β2m-linker 2-HLA A24-His tag was successfully constructed and expressed in the Expi293F cells. Conclusion The β2m-linker 2-HLA A24 protein has been successfully constructed. And bioinformatics results show that the protein has the function of binding to antigen peptides and activating T cells. It has established a foundation for activating antigen-specific CD8 + T cell stimulated by this protein and peptide in the subsequent experiments.

Objectives: To identify antigenic peptides of cancer stem-like cells (CSCs) antigen, DNAJB8, and establish a mouse CSCs-targeting immunotherapy model., Materials and Methods: To induce DNAJB8-specific immune reaction, we stimulated human CD8 + lymphocytes with antigen-presenting cells pulsed with a cocktail of three candidate HLA-A*24:02 restricted peptides and assessed peptide specific human cytotoxic T lymphocytes (CTLs) induction. One of the antigenic peptides showed identical amino acid sequence as corresponding mouse DNAJB8. We evaluated CTL induction with the peptide immunization in mouse model., Results: We confirmed peptide-specific interferon-γ secretions and cytotoxic activities of induced human CTLs. In vivo immunization with the peptide to mice, peptide-specific CTL response could be observed in mouse CD8 + T cells. Furthermore, immunization with the peptide showed significant anti-tumor effects compared with negative controls., Conclusion: DNAJB8-derived peptide is a novel candidate for CSCs-targeting immunotherapy, and mouse models can be used to evaluate CSCs-targeting immunotherapy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cell division associated 1 (CDCA1) was screened as an oncogene that is overexpressed on several cancers, including prostate cancer. A highly immunogenic HLA-A*2402-restricted epitope peptide corresponding to part of the CDCA1 protein was also identified. A phase I clinical trial was conducted for patients with castration resistant prostate cancer (CRPC) using a CDCA1 peptide vaccination. Twelve patients having HLA-A*2402 with CRPC after failure of docetaxel chemotherapy were enrolled. They received subcutaneous administration of the CDCA1 peptide as an emulsion with Montanide ISA51VG once a week in a dose-escalation manner (doses of 1.0 or 3.0 mg/body, six patients received each dose). The primary endpoint was safety, and the secondary endpoints were the immunological and clinical responses. Vaccination with CDCA1 peptide was well tolerated without any serious adverse events. Peptide-specific cytotoxic T lymphocyte (CTL) responses using ELISPOT assay and dextramer assay were observed in three patients receiving the 1.0 mg dose and five patients receiving the 3.0 mg dose. The median overall survival time was 11.0 months and specific CTL reacting to CDCA1 peptide were recognized in long-surviving patients. CDCA1-derived peptide vaccine treatment was tolerable and might effectively induce peptide-specific CTLs for CRPC patients. This novel peptide vaccine therapy for CRPC appears promising. (ClinicalTrials.gov number, NCT01225471)., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Survivin, a member of the inhibitor of apoptosis protein (IAP) family containing a single baculovirus IAP repeat domain, is highly expressed in cancerous tissues but not in normal counterparts. Our group identified an HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), that is recognized by CD8 + CTLs and functions as an immunogenic molecule in patients with cancers of various histological origins such as colon, breast, lung, oral, and urogenital malignancies. Subsequent clinical trials with this epitope peptide alone resulted in clinical and immunological responses. However, these were not strong enough for routine clinical use as a therapeutic cancer vaccine, and our previous study of colon cancer patients indicated that treatment with a vaccination protocol of survivin-2B80-88 plus incomplete Freund's adjuvant (IFA) and α-interferon (IFNα) conferred overt clinical improvement and enhanced the immunological responses of patients. In the current study, we further investigated whether this vaccination protocol could efficiently provide not only improved immune responses but also better clinical outcomes for advanced pancreatic cancers. Tetramer and enzyme-linked immunosorbent spot analysis data indicated that more than 50% of the patients had positive clinical and immunological responses. In contrast, assessment of treatment with IFNα only to another group of cancer patients resulted in no obvious increase in the frequency of survivin-2B80-88 peptide-specific CTLs. Taken together, our data clearly indicate that a vaccination protocol of survivin-2B80-88 plus IFA and IFNα is very effective and useful in immunotherapy for this type of poor-prognosis neoplasm. This trial was registered with the UMIN Clinical Trials Registry, no. UMIN000000905., (© 2012 Japanese Cancer Association.)

The human CD8 functions as a co-receptor for specific T cell recognition, and only one complex structure of human CD8αα binding to HLA-A*0201 has been solved, revealing the molecular basis of CD8 interacting with its ligand pHLA. Here, we present the complex structures of human CD8αα bound to HLA-A*2402, which demonstrate two opposite α3 domain CD loop shifts (either pull or push) in the HLA heavy chain upon CD8 engagement. Taking the previously reported mouse CD8-pMHC complex structures into account, from the structural view, all of the data indicate the plasticity of CD8 binding to pMHC/HLA, which facilitates its co-receptor function for T cells. The plasticity of CD8 binding appears not to affect the specificity of TCR recognition, as no peptide conformation change extends to the pMHC interface for TCR contacting., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In a structural aberration analysis of patients with arthritis mutilans, a 50 kb deletion near the HLA-A locus with HLA-A*24:02 allele was detected. It was previously reported that HLA-A*24:02 haplotype harbored a large-scale deletion telomeric of the HLA-A gene in healthy individuals. In order to confirm that the deletion are the same in patients with arthritis mutilans and in healthy individuals, and to identify the break point of this deletion, the boundary sequences across the deletion in A*24:02 was amplified by polymerase chain reaction (PCR) as a 3.7 kb genomic fragment and subjected to nucleotide sequence determination. A comparison of these genomic sequences with those of the non-A*24:02 haplotype revealed that the deleted genomic region spanning 50 kb was flanked by 3.7 kb repetitive element-rich segments homologous to each other on both sides in non-A*24. The nucleotide sequences of the PCR products were identical in patients with arthritis mutilans and in healthy individuals, revealing that the deletion linked to A*24:02 is irrelevant to the onset of arthritis mutilans. The deletion was detected in all other A*24 alleles so far examined but not in other HLA-A alleles, except A*23:01. This finding, along with the phylogenic tree of HLA-A alleles and the presence of the 3.7 kb highly homologous segments at the boundary of the deleted genomic region in A*03 and A*32, may suggest that this HLA-A*24:02-linked deletion was generated by homologous recombination within two 3.7 kb homologous segments situated 50 kb apart in the ancestral A*24 haplotype after divergence from the A*03 and A*32 haplotypes.

Background: The object of this study was to investigate the clinical predictive capability of peripheral myeloid dendritic cells (DCs) in Wilms' tumor 1 (WT1) vaccine therapy for patients with gynaecological cancer., Patients and Methods: Six patients with WT1/human leukocyte antigen (HLA)-A*2402-positive gynaecological cancer were included in this study. The patients received intradermal injections of a modified 9-mer WT1 peptide every week for 12 weeks. Peripheral blood samples were obtained at 0, 4, 8 and 12 weeks after the initial vaccination. Circulating DCs were detected by flow cytometry., Results: The frequencies of CD14(+)CD16(+)CD33(+)CD85(+) myeloid DCs were significantly higher in the therapeutically effective group than in therapeutically inert group (p<0.05)., Conclusion: These results suggested that myeloid DCs, which should be associated with inducing cytotoxic T-cells, provided additional prognostic information in the use of cancer peptide vaccine.

Cytotoxic γδ T-cells recognize antigens directly without the need for antigen processing and presentation. Recently, it was reported that they can also present antigens and proliferate in vitro. In this study, we examined whether γδ T-cells isolated from patients with advanced cancer can be used for immunotherapy. Twenty-two inoperable patients with multiple cancer metastases were enrolled in the study. There was no significant difference in the ratio of γδ T-cells within the peripheral blood mononuclear cell population isolated from healthy volunteers and cancer patients. γδ T-Cells isolated from cancer patients were expanded 2- to 5-fold using zoledronic acid or 2-methyl-3butenyl-1-pyrophosphate and IL-2. Autologous CD8(+) T-cells co-cultured with expanded CEA peptide-pulsed γδ T-cells from cancer patients with HLA-A24 killed more CEA-positive HLA-A24-matched gastric cancer cells and secreted higher levels of interferon-γ. These results suggest that γδ T-cells from cancer patients may be ideal candidates for adoptive immunotherapy.

Unlabelled: We report on Wilms tumor (WT1) peptide immunotherapy in a patient with intractable ovarian cancer patient over an extended period., Case Report: Immunotherapy using WT1 peptide has been undergoing clinical trials for gynecological cancer. We used WT1 peptide vaccination to treat a 53-year-old woman suffering from ovarian cancer with peritoneal dissemination. After 2 months, her pleural and cardiac effusion had disappeared, and the sum of the longest diameter of the target lesion (in the pelvic mass) was reduced. There was a weak positive correlation between CA125 and mononuclear phagocyte/lymphocyte ratio (Spearman's ϱ=0.275, p=0.015). Intradermally administered WT1 peptide vaccination in a case of intractable ovarian cancer stabilized the disease over the course of one year. However, the immunotherapeutic mechanism of WT1 peptide and immunological escape mechanism for carcinoma cells remain to be elucidated.

Background: Takayasu arteritis and ulcerative colitis are immune-mediated inflammatory diseases; genetic factors are assumed to play an important role in the pathogenesis of these 2 diseases. However, the coexistence of these 2 diseases has rarely been reported., Case Report: In this report, we present a rare case of a 29-year-old man with a 4 years history of ulcerative colitis who developed Takayasu arteritis. He was found to carry the following human leukocyte antigens (HLA): A11, A24, B52, B62, DR4, and DR9., Conclusions: We present a case report and review of the pertinent literature on serological analysis of HLA haplotype of the patients who exhibit both these diseases. In patients with both Takayasu arteritis and ulcerative colitis, high frequency of HLA-A24, B52, and DR 2 is observed. The pathological relevance of HLA-A24, B52, and DR2 to concomitant Takayasu arteritis and ulcerative colitis requires further investigation.

We previously identified a human leukocyte antigen (HLA)-A24-restricted antigenic peptide, survivin-2B80-88, recognized by CD8+ cytotoxic T lymphocytes (CTL). Subsequently, we attempted clinical trials with this epitope peptide alone for some malignancies, resulting in clinical and immunological responses, although their potential was not strong enough for routine clinical use as a cancer vaccine. In the current study, to assess whether immunogenicity of the survivin-2B80-88 peptide could be enhanced with other vaccination protocols, we performed clinical trials in advanced colon cancer patients with two vaccination protocols: (i) survivin-2B80-88 plus incomplete Freund's adjuvant (IFA); and (ii) survivin-2B80-88 plus IFA and a type-I interferon (IFN), IFNα. Our data clearly indicated that, although the effect of survivin-2B80-88 plus IFA was not significantly different from that with survivin-2B80-88 alone, treatment with the vaccination protocol of survivin-2B80-88 plus IFA and IFNα resulted in clinical improvement and enhanced immunological responses of patients. Tetramer analysis of survivin-2B80-88 peptide-specific CTL demonstrated that such CTL were increased at least twofold after vaccination with this protocol in four of eight patients. In these patients, enzyme-linked immunosorbent spot (ELISPOT) results were also enhanced. Subsequent study of single-cell clone separation by cell sorting of peptide-specific CTL showed that each CTL clone was indeed not only peptide-specific but also cytotoxic against human cancer cells in the context of the expression of both HLA-A24 and survivin molecules. Taken together, these results indicate that vaccination of colon cancer patients with survivin-2B80-88 plus IFA and IFNα can be considered to be a very potent immunotherapeutic regimen, and that this protocol might work for other cancers., (© 2011 Japanese Cancer Association.)

Aim: To study the antigen specific anti-tumor effect of cytotoxic T lymphocytes(CTLs), which was induced by human telomerase reverse transcriptase (hTERT)-related multiple epitope peptides impulsed myeloid dendritic cells(mDCs), against human leukocyte antigen (HLA)-A24(+) tumor cells., Methods: Four branches of multiple antigen peptides (MAPs) of hTERT epitopes and three separate peptides were solid-phase artificially synthesized, phlebotomize peripheral blood from HLA-A24(+) healthy volunteers, sorted the blood through MACS MicroBeads and cultured mDCs, Nylon fiber column purified T lymphocytes, mDCs impulsed with each type of peptides were co-cultured with T lymphocytes to induce CTLs specifically killing effect, and the resultant CTLs were used as effector cells, SMMC-7721 with hTERT and HLA-A24 positive and SKOV3 which are hTERT-positive but HLA-A24-negative tumor cells were used as target cells. The level of human IL-12, TNF-α in the culture supernatant was determined by ELISA. Flow cytometry assay was used to assess the killing ability of CTLs against tumor cells., Results: MAPs of hTERT epitopes including I540 (ILAKFLHWL), V461 (VYGFVRACL), L766 (LTDLQPYMRQFVAHL) and three separate peptides could impulse mDCs and then induce CTLs to specifically kill SMMC-7721, CTLs induced by MAPs had stronger cytotoxic effect compared with three separate peptides mixed(P < 0.05)., Conclusion: mDCs-impulsed with hTERT-associated MAPs can induce production and proliferation of allogenic CTLs, which show antigen specific anti-tumor effect against HLA-A24(+) tumor cells. This result has significantly meaning in tumor immunotherapy.

ERas is a recently identified oncogene involved in the tumorgenic growth of embryonic stem cells. We examined the significance of ERas expression in scirrhous gastric carcinoma, and the possibility of ERas as a tumor-associated antigen of gastric cancer for developing a cancer vaccine. ERas expression was determined in scirrhous gastric carcinoma specimens by immunohistochemical staining. To assess the possibility of the ERas protein as an anticancer vaccine target, we examined whether ERas for HLA-A-restricted epitope peptides were capable of eliciting cytotoxic T lymphocyte activity. Immunohistochemical analysis identified ERas protein in the nucleus and cytoplasm of cancer cells, yet ERas was not expressed in normal gastric epithelium. By western blotting, lysates of the scirrhous gastric cancer cell lines, OCUM-8, OCUM-2MD3 and OCUM-2M were shown to contain a 25-kDa band of ERas protein. ERas mRNA was detected in these cell lines by RT-PCR. To investigate cytotoxicity, we successfully established cytotoxic T lymphocyte clones stimulated by HLA-A*2402-restricted ERas peptides (FALDDPSSL). These peptides have specific cytotoxicity against corresponding HLA-A*2402-positive target cells pulsed with the candidate peptide. We found that the cytotoxic T lymphocyte clones demonstrated cytotoxic activity against OCUM-8 cells that endogenously express ERas. Our results suggest that ERas is a novel tumor-associated antigen with the potential application to be a vaccine against scirrhous gastric cancer., (© 2011 Japanese Cancer Association.)

Background: To evaluate the safety, tolerability, immune response, and antitumor activity of a combination of personalized peptide vaccination (PPV) and estramustine phosphate (EMP) in patients with castration-resistant prostate cancer (CRPC)., Methods: In a phase I dose-escalation study, four peptides showing the highest levels of peptide-specific immunoglobulin G (IgG) to 14 vaccine candidates (ITK-1) were subcutaneously injected every week in three different dose settings (1, 3, and 5 mg per peptide) for 6 weeks with a low dose of EMP, and the patients were followed by maximum 2 years extension study either weekly or bi-weekly six times PPV as one course with a low dose of EMP., Results: Fifteen patients were enrolled in the phase I study. No serious treatment-related adverse events were observed. The most common adverse events were grade 2 skin reactions at the injection sites. The maximum acceptable dose of ITK-1 was 8.643 mg. There were no treatment-related systemic adverse events of grade 3 or more, and maximum tolerated dose could not be determined. Cytotoxic T lymphocyte responses measured by interferon-γ release assay were boosted in 10 of 15 (67%) patients, and IgG responses were boosted in 7 of 15 (47%) patients. Twelve patients proceeded to the extension study, and the median survival time was 23.8 months during a median follow-up of 23.8 months., Conclusions: PPV treatment for HLA-A24 positive patients with CRPC could be recommended for further stages of clinical trials because of its safety and the higher frequency of boosting immune responses., (Copyright © 2010 Wiley-Liss, Inc.)

Carcinoembryonic antigen (CEA) is a very common tumor marker because many types of solid cancer usually produce a variety of CEA and a highly sensitive measuring kit has been developed. However, immunological responses associated with CEA have not been fully characterized, and specifically a weak immunogenicity of CEA protein as a tumor antigen is reported in human leukocyte antigen (HLA)-A24-restricted CEA peptide-based cancer immunotherapy. These observations demonstrated that immunogenic and potent HLA-A24-restricted CTL epitope peptides derived from CEA protein are seemingly difficult to predict using a conventional bioinformatics approach based on primary amino acid sequence. In the present study, we developed an in silico docking simulation assay system of binding affinity between HLA-A24 protein and A24-restricted peptides using two software packages, AutoDock and MODELLER, and a crystal structure of HLA-A24 protein obtained from the Protein Data Bank. We compared the current assay system with HLA-peptide binding predictions of the bioinformatics and molecular analysis section (BIMAS) in terms of the prediction capability using MHC stabilization and peptide-stimulated CTL induction assays for CEA and other HLA-A24 peptides. The MHC stabilization score was inversely correlated with the affinity calculated in the docking simulation alone (r = -0.589, P = 0.015), not with BIMAS score or the IFN-γ production index. On the other hand, BIMAS was not significantly correlated with any other parameters. These results suggested that our in silico assay system has potential advantages in efficiency of epitope prediction over BIMAS and ease of use for bioinformaticians., (© 2011 Japanese Cancer Association.)

The present study attempted to identify a useful tumor-associated antigen (TAA) for lung cancer immunotherapy and potential immunogenic peptides derived from the TAA. We focused on cell division cycle 45-like (CDC45L), which has a critical role in the initiation and elongation steps of DNA replication, as a novel candidate TAA for immunotherapy based on a genome-wide cDNA microarray analysis of lung cancer. The CDC45L was overexpressed in the majority of lung cancer tissues, but not in the adjacent non-cancerous tissues or in many normal adult tissues. We examined the in vitro and in vivo anti-tumor effects of cytotoxic T-lymphocytes (CTL) specific to CDC45L-derived peptides induced from HLA-A24 (A*24:02)-positive donors. We identified three CDC45L-derived peptides that could reproducibly induce CDC45L-specific and HLA-A24-restricted CTL from both healthy donors and lung cancer patients. The CTL could effectively lyse lung cancer cells that endogenously expressed both CDC45L and HLA-A24. In addition, we found that CDC45L (556) KFLDALISL(564) was eminent in that it induced not only HLA-A24 but also HLA-A2 (A*02:01)-restricted antigen specific CTL. Furthermore, the adoptive transfer of the CDC45L-specific CTL inhibited the growth of human cancer cells engrafted into immunocompromised mice. These results suggest that these three CDC45L-derived peptides are highly immunogenic epitopes and CDC45L is a novel TAA that might be a useful target for lung cancer immunotherapy., (© 2011 Japanese Cancer Association.)

We encountered a case that exhibited a discrepancy in human leukocyte antigen-A (HLA-A) type determined by sequence-based typing (SBT) and sequence-specific primer (SSP) molecular typing. The child of this case was identified as A* 02:01 homozygote and A* 02, A* 24, respectively. The HLA-A type of his father was A* 02:01, 26:01, but low-resolution SSP also showed unexpected amplification with A* 24 primers as with the child. Serologic typing of the child and the father was A2/blank and A2/A26, respectively. Sequencing analysis of the A* 24 variant in the child and the father showed a complete deletion of all introns of the A* 24:02 allele. Though rare, this type of processed pseudogene variant can be one of the causes of discrepancies between high- and low-resolution HLA typing., (© 2011 John Wiley & Sons A/S.)

Objective: This study clarified characteristics of interferon-associated type 1 diabetes., Research Design and Methods: The study compared 12 patients with interferon-associated type 1 diabetes with 128 patients with type 1A diabetes with respect to clinical characteristics, and with 10 patients without diabetes despite interferon therapy and 136 normal controls with respect to HLA allele distributions., Results: Patients with interferon-associated type 1 diabetes retained higher levels of fasting serum C peptide as well as GAD65 antibodies than those with type 1A diabetes until 2 to 4 years after onset. HLA-A*2402 was increased among patients with interferon-associated type 1 diabetes compared with those without diabetes, despite interferon therapy (odds ratio [OR] 4.00 [95% CI 1.09-17.26]). The haplotype of DRB1*1302-DQA1*0102-DQB1*0604 was increased in these two groups combined compared with normal controls (OR 5.64 [95% CI 2.67-11.81])., Conclusions: Interferon-associated type 1 diabetes is characterized clinically by high titers of GAD65 antibodies and preserved β-cell function, and genetically by addition of HLA-A*2402to DRB1*1302-DQA1*0102-DQB1*0604.

Cancer immunotherapy is a potential therapeutic strategy, in addition to surgical treatment, radiotherapy, and chemotherapy. Cancer-specific immunotherapy, such as the MAGE peptide vaccine, has been utilized clinically. How-ever, there are inherent limits to the effectiveness of vaccinotherapy using a single antigen because of the expression frequency of cancer-specific antigens on tumor cells. Thus, identification of a new cancer-specific antigen is needed. In this study, we examined the possibility of using cancer-specific immunotherapy based upon mitotic centromere-associated kinesin (MCAK) which was previously identified as a novel cancer/testis antigen. To evaluate the feasibility of developing cancer immunotherapy using MCAK peptides, we studied HLA-A*0201 and *2402 as targets for CTLs in the context of HLA class I molecules. By using a peptide with a sequence of AINPELLQL (amino acid positions 63-71 in MCAK, HLA-A*0201) and FFEIYNGKL (amino acid positions 401-409 in MCAK, HLA-A*2402), CTL responses could be induced from unseparated PBMCs by stimulation of freshly isolated, peptide-pulsed PBMCs as antigen-presenting cells (APCs) and also by using interleukin-7 and keyhole limpet hemocyanin in primary culture. The induced CTLs could lyse HLA-A-*0201/*2402 colon and gastric cancer cells expressing MCAK, as well as the peptide-pulsed target cells, in an HLA class l, and CD8 restricted manner. The identification of the MCAK/HLA-A*0201 and *2402 peptides suggests the possibility of designing peptide-based immunotherapeutic approaches that might prove effective in treating patients with MCAK-positive cancer.

In our previous study, we demonstrated that a peptide derived from the novel centrosome residing protein Cep55/c10orf3 can be targeted by the cytotoxic T lymphocytes (CTLs) in peripheral blood mononuclear cells (PBMCs) of breast carcinoma patients. In this report, we evaluated the feasibility of cancer immunotherapy using Cep55/c10orf3 peptide for colorectal carcinoma (CRC). To evaluate the expression of Cep55/c10orf3 in CRC tissues, we performed immunohistochemical staining of using anti-Cep55/c10orf3 monoclonal antibody. Sixty-three percent cases showed weak positive for Cep55/c10orf3 in total 70 CRC cases. The Cep55/c10orf3 expression intention was collated with high histological grade of CRC. Thus, we hypothesized that Cep55/c10orf3 can also be the target of CTLs in CRC cases. We generated CTLs from PBMCs of human leukocyte antigen (HLA)-A24-positive colorectal carcinoma patients using HLA-A24-restricted Cep55/c10orf3 peptides. Two of 6 colorectal cancer patients were reactive for the Cep55/c10orf3&#95;193(10) peptide, which was the only immunogenic peptide in breast carcinoma patients. CTL clone specific for Cep55/c10orf3&#95;193(10) recognized and lysed HLA-A24 (+) and Cep55/c10orf3 (+) colorectal carcinoma cell lines. In addition, 1 of 6 colorectal carcinoma patients was reactive for the Cep55/c10orf3&#95;402(11) and Cep55/c10orf3&#95;283(12) peptides, but not for Cep55/c10orf3&#95;193(10) with the ELISPOT assay. These observations suggest that the antigenic peptide repertoire presented by HLA-A24 in colorectal carcinoma might be different from that in breast carcinoma. Thus, these peptide vaccination peptide mixture of Cep55/c10orf3&#95;193(10), Cep55/c10orf3&#95;402(11) and Cep55/c10orf3&#95;283(12) might be more effective than a single peptide in the treatment of colorectal carcinoma patients., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Purpose: Personalized selection of suitable peptides for patients could offer a novel approach to developing cancer vaccines that boost anticancer immunity. We present the results of a phase I trial of 14 kinds of vaccine candidates (ITK-1) in patients with recurrent or progressive glioblastoma multiforme (GBM)., Patients and Methods: From January 2006 to January 2008, 12 patients from eight Japanese hospitals who were positive for human leukocyte antigen-A24, including 10 patients refractory to temozolomide (TMZ), were enrolled. The dose escalation trial included three dose groups (1, 3, and 5 mg) to determine the safety and tolerability of ITK-1 peptides. Immunologic response was monitored by measuring levels of cytotoxic T-lymphocyte precursors and peptide-specific immunoglobulin G. In another ITK-1 phase I trial for advanced prostate cancer, the vaccination schedule was skipped or discontinued in all three patients receiving the highest dose (5 mg/peptide) because of injection site reactions. This trial was therefore ended without enrollment for the highest dose, and data were analyzed by intention to treat., Results: No serious adverse drug reactions were encountered, and treatment was well tolerated. The vaccine induced dose-dependent immune boosting. The recommended dose of ITK-1 peptides is 3 mg/peptide., Conclusion: Personalized vaccination with ITK-1 peptides could be recommended in further stages of clinical trials. The safety and increased frequency of immune boosting offers potential clinical benefits in cases of recurrent or progressive GBM, even in TMZ-refractory settings.

Human leukocyte antigen (HLA) typing is essential to carry out HLA-class I restricted antigenic peptide-based cancer immunotherapy. To establish a one-step polymerase chain reaction-sequence-specific primer (PCR-SSP) method, we designed two novel HLA-A24-specific primer sets and determined the optimal conditions for specific amplification. Then, we performed HLA-A24 typing of two healthy donors' and 17 cancer patients' peripheral blood with serological typing and PCR-SSP typing. Eleven of the 19 cases were determined HLA-A24-positive by the PCR-SSP method precisely; however, five cases showed false positive with serological analysis. Thus, for HLA-A24 typing in the Japanese population, the PCR-SSP method is faster and more accurate than serological typing.

An unusual haplotype without a detectable human leukocyte antigen (HLA)-A allele by serologic or molecular typing methods segregates in a Caucasian family. Microsatellite analysis and fluorescence in situ hybridization implicated that the deletion encompasses a narrow region. To identify the deleted region, five different fragments in close proximity to HLA-A, known to be highly polymorphic, were amplified and sequenced. The presence of heterozygous sequences in all five fragments of the individuals carrying the haplotype with the HLA-A deletion, indicates that the fragments are not involved in the deletion. Therefore, the 5' primer from the fragment closest to the centromeric side of HLA-A was combined with the 3' primer closest to the telomeric side encompassing an 11-kb region. Sequencing revealed that a deletion of 4089 bp was present, located upstream of HLA-A, including exons and introns 1-3 of the HLA gene. Sequence information of the 3' part of HLA-A, downstream the deletion, identified that the deleted allele originates from an A*24 allele. Although different repeat sequences are present in the region both inside and outside the deletion, no evidence points to a retrotransposon mechanism. The detected partial deletion of HLA-A turns this functional gene into a pseudogene., (Copyright © 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Adult T-cell leukemia (ATL) is a lymphoproliferative malignancy associated with human T-cell lymphotropic virus type 1 (HTLV-1) infection. Recently, it has been shown that allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for ATL, and that HTLV-1 Tax-specific CD8(+) cytotoxic T cells (CTL) contribute to the graft-versus-ATL effect. In the present study, we, for the first time, analyzed the T-cell receptor (TCR) repertoire of isolated Tax(301-309) (SFHSLHLLF)-specific CTLs in HLA-A*2402(+) ATL patients before and after allo-HSCT by single-cell reverse transcription-PCR. The Tax(301-309)-specific CTLs in bone marrow and peripheral blood showed highly restricted oligoclonal diversity. In addition, a unique conserved amino acid motif of "P-D/P-R" in TCR-beta complementarity-determining region 3 in either BV7- or BV18-expressing CTLs was observed not only in all of the samples from ATL patients, but also in samples from the same patient before and after HSCT. Furthermore, the P-D/P-R motif-bearing CTL clones established from peripheral blood samples after HSCT exhibited strong killing activity against the HTLV-1-infected T cells of the patient. CTL clones were not established in vitro from samples prior to allo-HSCT. In addition, CTL clones with a strong killing activity were enriched in vivo after HSCT in the patient. Hence, Tax(301-309)-specific CTLs in ATL patients might have a preference for TCR construction and induce strong immune responses against the HTLV-1-infected T cells of patients, which contribute to the graft-versus-ATL effects after allo-HSCT. However, further analyses with a larger number of patients and more frequent sampling after allo-HSCT is required to confirm these findings.

HLA-A*240220 has one nucleotide change from HLA-A*24020101 at position 201 in exon 2 from G to C.

Wilms tumor gene (WT1) can be overexpressed in childhood cancers. We evaluated the efficacy of WT1 vaccination for five children with solid cancer or acute leukemia. WT1 vaccine was administered to HLA-A*2402-positive patients with WT1-overexpressing residual tumor despite prior conventional treatment. One patient showed complete response and one patient showed stable disease according to the Response Evaluation Criteria in Solid Tumors; the remaining three showed progressive disease. Treatment-related adverse effects were limited to local injection site erythema. These results suggest that WT1 vaccination has therapeutic potential, but any beneficial effect may be insufficient in the presence of gross residual disease., ((c) 2010 Wiley-Liss, Inc.)

Unlabelled: Neonatal alloimmune thrombocytopenia (NAIT) occurs as a result of maternal alloimmunization against paternally inherited antigens on foetal platelets. Platelets express platelet specific antigens (HPA) along with human leucocyte antigens (HLA) class I. Although anti-HLA class I antibodies are often detectable in pregnant women, their role in NAIT is considered controversial. We report a case of NAIT where the most sensitive serological analysis and molecular methods could not detect platelet specific antibodies. Only HLA incompatibility and presence of anti-HLA-A24 antibodies in both the mother's and the newborn's serum were proven., Conclusion: This case supports the idea that some anti-HLA class I antibodies could cause NAIT.

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is regularly expressed in all proliferating virus-infected cells and is therefore an interesting target for immunotherapy. Alleles of the human leucocyte antigen (HLA) -A2 family are dominantly expressed in Caucasians so we sought to identify EBNA1-specific cytotoxic T-lymphocyte (CTL) responses restricted through this allele. We report on the characterization of the LQTHIFAEV (LQT) epitope. LQT-specific memory CTL responses were reactivated in three of 14 healthy EBV seropositive donors (21%) whereas responses to HLA-A2-restricted epitopes, two derived from LMP2 and one from EBNA3A, were detected in 93%, 71% and 42% of the donors, respectively. The LQT-specific CTL clones did not lyse EBV-carrying lymphoblastoid cell lines and Burkitt's lymphoma cell lines nor EBNA1-transfected Burkitt's lymphoma cells but specifically released interferon-gamma upon stimulation with HLA-matched EBNA1-expressing cells and this response was enhanced by deletion of the Gly-Ala repeat domain that inhibits proteasomal degradation. The poor presentation of the endogenously expressed LQT epitope was not affected by inhibition of peptidases that trim antigenic peptides in the cytosol but full presentation was achieved in cells expressing a trojan antigen construct that releases the epitope directly into the endoplasmic reticulum. Hence, inefficient proteasomal processing appears to be mainly responsible for the poor presentation of this epitope.

Malignant gliomas are the most aggressive human primary brain tumors and are currently incurable. Immunotherapies have the potential to target glioma and glioma stem cells (GSCs) that are resistant to conventional therapies. We previously identified SOX6 as a human glioma antigen and demonstrated that vaccination with SOX6 DNA induced cytotoxic T lymphocytes (CTLs) specific for glioma, thereby exerting therapeutic antitumor responses in glioma-bearing mice. In this study, we attempted to identify SOX6-derived peptides as specific targets for effective and safe T-cell-mediated immunotherapy targeting SOX6-positive glioma and GSCs. In vitro stimulation with human leukocyte antigen (HLA)-A*2402 (A24)-restricted peptides, RFENLGPQL (SOX6(504)) and PYYEEQARL (SOX6(628)) or the HLA-A*0201 (A2)-restricted peptide, ALFGDQDTV (SOX6(447)) was capable of inducing SOX6 peptide-specific CTLs in peripheral blood mononuclear cells derived from healthy donors and glioma patients. These CTLs were able to lyse a majority of glioma cell lines and a GSC line derived from human glioblastoma in an HLA Class I-restricted and an antigen-dependent manner. Furthermore, peptide vaccines of SOX6(628), which was conserved in the murine SOX6 protein and expected to bind to major histocompatibility complex (MHC) H-2(d), induced CTLs specific for SOX6(628) in H-2(d) mice. Normal autologous cells from mice, in which SOX6-specific immune responses were generated, were not destroyed. These results suggest that these SOX6 peptides are potnetially immunogenic in HLA-A24 or -A2 positive glioma patients and should be considered as a promising strategy for safe and effective T-cell-based immunotherapy of patients with gliomas.

Cytotoxic T lymphocytes (CTLs) play an essential role in immunological responses for tumor rejection. In the past decade, many tumor-associated antigens (TAAs) have been identified predominantly in melanomas. Several clinical trials based on such antigenic peptides with or without adjuvants brought about partially favorable results, suggesting that identification of more immunogenic TAAs is needed. We show here the successful establishment of human leukocyte antigen (HLA)-A24-restricted CTL (TcLHK2 line1) from a pleural effusion of lung cancer patient, using B7.1 (CD80) transduced autologous lung cancer cells as an antigen-presenting cell (APC). TcLHK2 line1 recognized autologous lung adenocarcinoma cell line LHK2 in an HLA-A24-restricted fashion. Moreover, this CTL line also recognized allogeneic HLA-A24-positive lung adenocarcinoma cell line, gastric carcinoma cell line and melanoma cell line. These data raise the possibility that co-stimulatory molecule B7.1 (CD80) plays important role to overcome the immunological tolerance. Furthermore, TcLHK2 line1 is a useful tool for the identification of widely expressed shared antigens restricted by HLA-A24. Further analysis of this CTL and autologous cancer cell line will bring about novel TAAs., (Copyright 2009 Elsevier Inc. All rights reserved.)

Tuberculosis is caused by an intracellular pathogen Mycobacterium tuberculosis (Mtb) and poses a persistent threat to global health. MHC class I-restricted CD8 cytotoxic T lymphocytes (CTL) are essential for protective immunity to Tuberculosis. Information for CTL epitopes derived from Mtb is desirable for vaccine design and assessment of T cell responses. However, the knowledge about CTL epitopes of Mtb, particularly those non-A2 HLA alleles restricted is rare. In this study, beta-2-microglobulin (beta 2m, beta(2)m) linked HLA-A24 single chain was expressed on RMA-S cell line defective in the endogenous antigen processing and applied for screening of peptides which could stabilize the HLA-A24 complex on the cell surface. From a group of peptides predicted as binders by a computer algorithm, five peptides were shown to bind to HLA-A24 protein on the cell surface. As comparison we have also identified a dozen Mtb proteins derived peptides that bind to HLA-A2 specifically. The cell line and HLA binders present here would be useful for further identification of CD8 restricted Mtb epitopes.

Alpha-methylacyl-CoA racemase (AMACR) is an enzyme playing an important role in the beta-oxidation of branched-chain fatty acids and fatty acid derivatives. High expression levels of AMACR have been described in various cancers, including prostate cancer, colorectal cancer and kidney cancer. Because of its cancer-specific and frequent expression, AMACR could be an attractive target for cytotoxic T-lymphocyte (CTL)-based immunotherapy for cancer. In the present study, we examined the induction of AMACR-specific CTLs from prostate cancer patients' peripheral blood mononuclear cells (PBMCs) and determined HLA-A24-restricted CTL epitopes.RT-PCR and immunohistochemical analysis revealed that AMACR was strongly expressed in prostate cancer cell lines and tissues as compared with benign or normal prostate tissues. Four AMACR-derived peptides carrying the HLA-A24-binding motif were synthesized from the amino acid sequence of this protein and analyzed to determine their binding affinities to HLA-A24. By stimulating patient's PBMCs with the peptides, specific CTLs were successfully induced in 6 of 11 patients. The peptide-specific CTLs exerted significant cytotoxic activity against AMACR-expressing prostate cancer cells in the context of HLA-A24. Our study demonstrates that AMACR could become a target antigen for prostate cancer immunotherapy, and that the AMACR-derived peptides might be good peptide vaccine candidates for HLA-A24-positive AMACR-expressing cancer patients.

To evaluate the usefulness of monocyte-derived dendritic cells transfected with tumor antigen mRNA for dendritic cell-based antitumor immunotherapy, we attempted to generate antigen-specific cytotoxic T cells by priming lymphocytes with monocyte-derived dendritic cells transfected with in vitro-transcribed tumor antigen mRNA. Mature monocyte-derived dendritic cells were generated from microbeads-separated CD14(+) cells by culturing with GM-CSF/IL-4 for 7 days and with TNF-alpha, IL-1alpha, IL-6, and PGE(2) for the last one day. Monocyte-derived dendritic cells, lymphocytes, and target cells, which were positive for HLA-A24, were used in the present study. Although lymphocytes prestimulated with untransfected monocyte-derived dendritic cells did not possess the cytotoxic ability against the target cells in a (51)Cr-release cytotoxicity assay, lymphocytes primed with tumor antigen RNA-transfected monocyte-derived dendritic cells were cytotoxic against the tumor antigen-expressing cells but not against the target cells without the expression of the antigen. The cytotoxic ability of the lymphocytes was blocked by the addition of antibodies against MHC class I but not by antibodies against MHC class II. These findings revealed that monocyte-derived dendritic cells transfected with WT1 or SART1 mRNA are able to induce tumor antigen-specific cytotoxic T cells and applicable for antitumor dendritic cell-based cellular immunotherapy.