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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.

Direct-acting antivirals (DAAs) have recently revolutionized the eradication of chronic hepatitis C virus (HCV) infection. However, the effects of DAAs on the development of hepatocellular carcinoma (HCC) remain unknown. Therefore, the present study aimed to investigate immune responses to HCC influenced by DAAs in HCV-infected patients and elucidate the underlying mechanisms. We compared immune responses to 19 different HCC-related tumor-associated antigen (TAA)-derived peptides and host immune cell profiles before and 24 weeks after a treatment with DAAs in 47 HLA-A24-positive patients. The relationships between the different immune responses and phenotypic changes in immune cells were also examined. The treatment with DAAs induced four types of immune responses to TAAs and markedly altered host immune cell profiles. Prominently, reductions in the frequencies of PD-1+CD4+ and PD-1+CD8+ T cells by DAAs were associated with enhanced immune responses to TAAs. The HCV F protein was identified as contributing to the increased frequency of PD-1+ T cells, which may be decreased after eradication by DAAs. DAAs altered the immune responses of patients to HCC by decreasing the frequency of PD-1-expressing CD4+ and CD8+ T cells.

Background Invariant chain (CD74, Ii) is a multifunctional protein expressed in antigen presenting cells. It assists the ER exit of various cargos and serves as a receptor for the macrophage migration inhibitory factor. The newly translated Ii chains trimerize, a structural feature that is not readily understood in the context of its MHCII chaperoning function. Two segments of Ii, the luminal C-terminal region (TRIM) and the transmembrane domain (TM), have been shown to participate in the trimerization process but their relative importance and impact on the assembly with MHCII molecules remains debated. Here, we addressed the requirement of these domains in the trimerization of human Ii as well as in the oligomerization with MHCII molecules. We used site-directed mutagenesis to generate series of Ii and DR mutants. These were transiently transfected in HEK293T cells to test their cell surface expression and analyse their interactions by co-immunoprecipitations. Results Our results showed that the TRIM domain is not essential for Ii trimerization nor for intracellular trafficking with MHCII molecules. We also gathered evidence that in the absence of TM, TRIM allows the formation of multi-subunit complexes with HLA-DR. Similarly, in the absence of TRIM, Ii can assemble into high-order structures with MHCII molecules. Conclusions Altogether, our data show that trimerization of Ii through either TM or TRIM sustains nonameric complex formation with MHCII molecules.

HLA-A*24:02:138 differs from HLA-A*24:02:01:01 by one nucleotide substitution at position 549 CT.

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+ IFNI³+ responses above background, S1208-1216 (QYIKWPWYI), S448-456 (NYNYLYRLF) and S193-201 (VFKNIDGYF), with S1208 generating immunodominant CD8+ IFNI³+ 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. Naive 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 TCRαs repertoires found that the A24/S448 + CD8+ T-cell TCRαs repertoire was driven by a common TCRs chain motif, whereas the A24/S1208 + CD8+ TCRαs 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.

The promising immunotherapy effects of a multiple antigenic peptide on glioblastoma (GBM) in a previous study encourage the use of adjuvants to enhance its therapeutic efficacy. Among adjuvants, pan HLA-DR-binding epitope (PADRE) and anti-programmed cell death protein 1 (anti-PD1) have potentially been tested for cancer immunotherapy. Therefore, here we evaluated the ability of PADRE and anti-PD1 to enhance the function of the branched multipeptide against GBM. The potential utility of tumor-associated antigens (ErbB-2 and WT-1) targeting GBM with HLA-A24 was confirmed and a branched multipeptide was constructed from these antigens. The effects of the branched multipeptide and PADRE on immunophenotyping and polarized Th cytokine production in dendritic cells were clarified. The expression of PD1 on T cells and PDL1 on GBM cells was also investigated. The interferon-γ enzyme-linked immunospot and lactate dehydrogenase release assays were performed to determine the function of GBM peptide antigen-specific cytotoxic T cells against GBM cells. Overall, this study showed that both ErbB-2 and WT-1 are potential candidates for branched multipeptide construction. The branched multipeptide and PADRE enhanced the expression of major histocompatibility complex and co-stimulatory molecules and the production of polarized Th1 cytokines in dendritic cells. The increase in the number of interferon-γ+ effector T cells was consistent with the increase in the percentage specific lysis of GBM target cells by GBM peptide antigen-specific cytotoxic T cells in the presence of the branched multipeptide, PADRE, and anti-PD1. Our study suggests the combination of branched multipeptide and adjuvants such as PADRE and anti-PD1 can potentially enhance the effects of immunotherapy for GBM treatment.

Rhesus monkeys have evolved MHC-encoded class I allomorphs such as Mamu-B∗098 that are capable of binding N-myristoylated short lipopeptides rather than conventional long peptides; however, it remains unknown whether such antigen-binding molecules exist in other species, including humans. We herein demonstrate that human leukocyte antigen (HLA)-A∗24:02 and HLA-C∗14:02 proteins, which are known to bind conventional long peptides, also have the potential to bind N-myristoylated short lipopeptides. These HLA class I molecules shared a serine at position 9 (Ser9) with Mamu-B∗098, in contrast to most MHC class I molecules that harbor a larger amino acid residue, such as tyrosine, at this position. High resolution X-ray crystallographic analyses of lipopeptide-bound HLA-A∗24:02 and HLA-C∗14:02 complexes indicated that Ser9 was at the bottom of the B pocket with its small hydroxymethyl side chain directed away from the B-pocket cavity, thereby contributing to the formation of a deep hydrophobic cavity suitable for accommodating the long-chain fatty acid moiety of lipopeptide ligands. Upon peptide binding, however, we found the hydrogen-bond network involving Ser9 was reorganized, and the remodeled B pocket was able to capture the second amino acid residue (P2) of peptide ligands. Apart from the B pocket, virtually no marked alterations were observed for the A and F pockets upon peptide and lipopeptide binding. Thus, we concluded that the structural flexibility of the large B pocket of HLA-A∗2402 and HLA-C∗1402 primarily accounted for their previously unrecognized capacity to bind such chemically distinct ligands as conventional peptides and N-myristoylated lipopeptides.

Background Preeclampsia is an idiopathic disease during pregnancy. This study explores the correlation between HLA-A polymorphism and the onset of preeclampsia. Methods The Illumina HiSeq2500 sequencing platform was used to genotyping HLA-A allele in venous blood DNA of 50 preeclampsia pregnant women and 48 normal pregnant women and umbilical cord blood DNA of their children of Han nationality in China. The frequencies and distributions of alleles and genotypes among the mothers and their children were compared between the two groups. The differences of frequencies and distributions of genotypes were compared between the two groups according to the mothers’ genotype compatibility. Results Twenty HLA-A alleles were detected in preeclampsia pregnant women and normal pregnant women; 21 HLA-A alleles were found in preeclampsia group fetuses and 22 HLA-A alleles in control group fetuses. There was no statistical difference in the HLA-A genes’ frequency between the two groups of pregnant women and their fetuses. When the sharing antigen was 1, the number of maternal-fetal pairs in the preeclampsia group was more than that in the control group; the difference was statistically significant (P P P = 0.0148); there is no significant difference in pregnant women’s genes homozygosity between early-onset preeclampsia group and the control group. Conclusions HLA-A * 24: 02 may be a susceptibility gene for early preeclampsia.

Background Peptide-vaccination therapy targeting tumour-associated antigens can elicit immune responses, but cannot be used to eliminate large tumour burden. In this study, we developed a therapeutic single-chain variable-fragment (scFv) antibody that recognises the cancer stem-like cell/cancer-initiating cell (CSC/CIC) antigen, DNAJB8. Methods We screened scFv clones reacting with HLA-A24:20/DNAJB8-derived peptide (DNAJB8_143) complex using naive scFv phage-display libraries. Reactivity and affinity of scFv clones against the cognate antigen were quantified using FACS and surface plasmon resonance. Candidate scFv clones were engineered to human IgG1 (hIgG1) and T-cell-engaging bispecific antibody (CD3xJB8). Complement-dependent cytotoxicity (CDC) and bispecific antibody-dependent cellular cytotoxicity (BADCC) were assessed. Results scFv clones A10 and B10 were isolated after bio-panning. Both A10-hIgG1 and B10-hIgG1 reacted with DNAJB8-143 peptide-pulsed antigen-presenting cells and HLA-A24(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. A10-hIgG1 and B10-hIgG1 showed strong affinity with the cognate HLA/peptide complex (KD = 2.96 × 10−9 M and 5.04 × 10−9 M, respectively). A10-hIgG1 and B10-hIgG1 showed CDC against HLA-A24(+)/DNAJB8(+) cell lines. B10-(CD3xJB8) showed superior BADCC to A10-(CD3xJB8). Conclusion We isolated artificial scFv antibodies reactive to CSC/CIC antigen DNAJB8-derived peptide naturally present on renal cell carcinoma and sarcoma. Immunotherapy using these engineered antibodies could be promising.

Cancer vaccines induce cancer-specific T-cells capable of eradicating cancer cells. The impact of cancer peptide vaccines (CPV) on the tumor microenvironment (TME) remains unclear. S-588410 is a CPV comprising five human leukocyte antigen (HLA)-A*24:02-restricted peptides derived from five cancer testis antigens, DEPDC1, MPHOSPH1, URLC10, CDCA1 and KOC1, which are overexpressed in esophageal cancer. This exploratory study investigated the immunologic mechanism of action of subcutaneous S-588410 emulsified with MONTANIDE ISA51VG adjuvant (median: 5 doses) by analyzing the expression of immune-related molecules, cytotoxic T-lymphocyte (CTL) response and T-lymphocytes bearing peptide-specific T-cell receptor (TCR) sequencing in tumor tissue or blood samples from 15 participants with HLA-A*24:02-positive esophageal cancer. Densities of CD8+, CD8+ Granzyme B+, CD8+ programmed death-1-positive (PD-1+) and programmed death-ligand 1-positive (PD-L1+) cells were higher in post- versus pre-vaccination tumor tissue. CTL response was induced in all patients for at least one of five peptides. The same sequences of peptide-specific TCRs were identified in post-vaccination T-lymphocytes derived from both tumor tissue and blood, suggesting that functional peptide-specific CTLs infiltrate tumor tissue after vaccination. Twelve (80%) participants had treatment-related adverse events (AEs). Injection site reaction was the most frequently reported AE (grade 1, n = 1; grade 2, n = 11). In conclusion, S-588410 induces a tumor immune response in esophageal cancer. Induction of CD8+ PD-1+ tumor-infiltrating lymphocytes and PD-L1 expression in the TME by vaccination suggests S-588410 in combination with anti-PD-(L)1 antibodies may offer a clinically useful therapy. Trial registration UMIN-CTR registration identifier: UMIN000023324. Electronic supplementary material The online version of this article (10.1007/s00262-020-02619-3) contains supplementary material, which is available to authorized users.

BackgroundHuman 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.MethodsIn 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.ResultsTherapeutic 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/Dd) 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.ConclusionsWe 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.

Previous studies have demonstrated that human leukocyte antigen (HLA)-A*24:02 is a common genetic risk factor for antiepileptic drug-induced skin rash, while HLA-B*15:02 is a specific risk factor for carbamazepine (CBZ)-induced Stevens Johnson syndrome and toxin epidermal necrolysis. The HLA-B*15:02 allele can alter the repertoire of endogenous peptides to trigger CBZ-induced hypersensitivity. However, it is uncertain whether HLA-A*24:02 could produce alterations in the peptide repertoire during treatment with antiepileptic drugs.We generated stable HMy2.C1R cells expressing HLA-A*24:02 and HLA-B*15:02, clarified into 4 groups according to with or without CBZ treatment. We employed LC/MSto detect the HLA-bound peptides in 4 groups. Furthermore, we conducted in silico analysis to seek th differential expressed genes (DEGs) associated with HLA-A*24:02 and HLA-B*15:02. Finally, we verify the DEGs via qRT-PCR and Western blotting.A total of 134 peptides were identified from the four groups, mainly comprising15 mer peptides. In CBZ-treated groups, 29 and 30 peptides showed significantly increased respectively in HLA-A*24:02 and HLA-B*15:02 positive cells comprising Lysine in PΩ, but the sources of these lysine peptides are different. Three peptides were exclusively detected in the HLA-A*24:02 positive cells treated with CBZ, of which 'SRQVVRSSK' was derived from the immune associated protein coronin 1A (CORO1A). CORO1A and its mRNA were significantly expressed in HLA-A*24:02 positive cells treated with CBZ. Additionally, this significantly high expression was identified in HLA-A*24:02 positive cells that were treated with lamotrigine (LTG). Nonetheless, CORO1A were not decreased in HLA-B*15:02 positive cells with or without CBZ or LTG treatment.These findings confirmed that the alteration in the endogenous peptidome was a general mechanism of HLA-linked skin rashes and suggests that CORO1A is involved in HLA-A*24:02-associated skin rash.

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.

COVID-19 vaccines are currently being administrated worldwide and playing a critical role in controlling the pandemic. They have been designed to elicit neutralizing antibodies against Spike protein of the original SARS-CoV-2, and hence they are less effective against SARS-CoV-2 variants with mutated Spike than the original virus. It is possible that novel variants with abilities of enhanced transmissibility and/or immunoevasion will appear in the near future and perfectly escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Several lines of evidence suggest the contribution of CTLs on the viral control in COVID-19, and CTLs target a wide range of proteins involving comparatively conserved non-structural proteins. Here, we identified twenty-two HLA-A*24:02-restricted CTL candidate epitopes derived from the non-structural polyprotein 1a (pp1a) of SARS-CoV-2 using computational algorithms, HLA-A*24:02 transgenic mice and the peptide-encapsulated liposomes. We focused on pp1a and HLA-A*24:02 because pp1a is relatively conserved and HLA-A*24:02 is predominant in East Asians such as Japanese. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by a number of mutations in the Sequence Read Archive database of SARS-CoV-2 variants. The information of such conserved epitopes might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any SARS-CoV-2 variants by the induction of both anti-Spike neutralizing antibodies and CTLs specific for conserved epitopes.ImportanceCOVID-19 vaccines have been designed to elicit neutralizing antibodies against the Spike protein of the original SARS-CoV-2, and hence they are less effective against variants. It is possible that novel variants will appear and escape from vaccine-elicited immunity. Therefore, the current vaccines may need to be improved to compensate for the viral evolution. For this purpose, it may be beneficial to take advantage of CD8+ cytotoxic T lymphocytes (CTLs). Here, we identified twenty-two HLA-A*24:02-restricted CTL candidate epitopes derived from the non-structural polyprotein 1a (pp1a) of SARS-CoV-2. We focused on pp1a and HLA-A*24:02 because pp1a is conserved and HLA-A*24:02 is predominant in East Asians. The conservation analysis revealed that the amino acid sequences of 7 out of the 22 epitopes were hardly affected by mutations in the database of SARS-CoV-2 variants. The information might be useful for designing the next-generation COVID-19 vaccine that is universally effective against any variants.

Although mutant-specific T cells are elicited in some individuals infected with HIV-1 mutant viruses, the detailed characteristics of these T cells remain unknown. A recent study showed that the accumulation of strains expressing Nef135F, which were selected by HLA-A*24:02-restricted T cells, was associated with poor outcomes in individuals with the detrimental HLA-B*35:01 allele and that HLA-B*35:01-restricted NefYF9 (Nef135-143)-specific T cells failed to recognize target cells infected with Nef135F mutant viruses. Here, we investigated HLA-B*35:01-restricted T cells specific for the NefFF9 epitope incorporating the Nef135F mutation. Longitudinal T-cell receptor (TCR) clonotype analysis demonstrated that 3 types of HLA-B*35:01-restricted T cells (wild-type [WT] specific, mutant specific, and cross-reactive) with different T cell repertoires were elicited during the clinical course. HLA-B*35:01+ individuals possessing wild-type-specific T cells had a significantly lower plasma viral load (pVL) than those with mutant-specific and/or cross-reactive T cells, even though the latter T cells effectively recognized the mutant virus-infected cells. These results suggest that mutant-specific and cross-reactive T cells could only partially suppress HIV-1 replication in vivo. An ex vivo analysis of the T cells showed higher expression of PD-1 on cross-reactive T cells and lower expression of CD160/2B4 on the mutant-specific T cells than other T cells, implying that these inhibitory and stimulatory molecules are key to the reduced function of these T cells. In the present study, we demonstrate that mutant-specific and cross-reactive T cells do not contribute to the suppression of HIV-1 replication in HIV-1-infected individuals, even though they have the capacity to recognize mutant virus-infected cells. Thus, the collaboration of HLA-A*24:02 with the detrimental allele HLA-B*35:01 resulted in the coevolution of HIV-1 alongside virus-specific T cells, leading to poorer clinical outcomes. IMPORTANCE HIV-1 escape mutations are selected under pressure from HIV-1-specific CD8+ T cells. Accumulation of these mutations in circulating viruses impairs the control of HIV-1 by HIV-1-specific T cells. Although it is known that HIV-1-specific T cells recognizing mutant virus were elicited in some individuals infected with a mutant virus, the role of these T cells remains unclear. Accumulation of phenylalanine at HIV-1 Nef135 (Nef135F), which is selected by HLA-A*24:02-restricted T cells, led to poor clinical outcome in individuals carrying the detrimental HLA-B*35:01 allele. In the present study, we found that HLA-B*35:01-restricted mutant-specific and cross-reactive T cells were elicited in HLA-B*35:01+ individuals infected with the Nef135F mutant virus. These T cells could not effectively suppress HIV-1 replication in vivo even though they could recognize mutant virus-infected cells in vitro. Mutant-specific and cross-reactive T cells expressed lower levels of stimulatory molecules and higher levels of inhibitory molecules, respectively, suggesting a potential mechanism whereby these T cells fail to suppress HIV-1 replication in HIV-1-infected individuals.

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_CD8α. Adoptive transfer of TEG011_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_CD8α cells, as well as the total number of TEG011_CD8α cells per mice, was significantly improved over time, mainly due to a dominance of CD4+CD8+ double-positive TEG011_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_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.

Proteins of the major histocompatibility complex (MHC) class I, or human leukocyte antigen (HLA) in humans interact with endogenous peptides and present them to T cell receptors (TCR), which in turn tune the immune system to recognize and discriminate between self and foreign (non-self) peptides. Of especial importance are peptides derived from tumor-associated antigens. T cells recognizing these peptides are found in cancer patients, but not in cancer-free individuals. What stimulates this recognition, which is vital for the success of checkpoint based therapy? A peptide derived from the protein p53 (residues 161-169 or p161) was reported to show this behavior. T cells recognizing this unmodified peptide could be further stimulated in vitro to create effective cancer killing CTLs (cytotoxic T lymphocytes). We hypothesize that the underlying difference may arise from post-translational glycosylation of p161 in normal individuals, likely masking it against recognition by TCR. Defects in glycosylation in cancer cells may allow the presentation of the native peptide. We investigate the structural consequences of such peptide glycosylation by investigating the associated structural dynamics. Agency for Science, Technology and Research (A*STAR) Published version This research was funded by A*STAR.

Metronidazole, a widely used drug for the treatment of infections with anaerobic and facultative anaerobic bacteria and protozoa, can frequently cause metronidazole-induced cutaneous adverse reactions (McADRs). The aim of the present study was to investigate the association between human leucocyte antigen (HLA) alleles and McADRs in a Chinese Han population. The frequency of HLA-B*24:02 carriers among the McADR patients was 73.3%, which was significantly higher than that of the population controls (32.16%, OR = 5.80, 95% CI = [1.80-18.72], Pc = 0.004) and of the metronidazole-tolerant patients (26.67%, OR = 7.56, 95% CI = [2.02-28.35], Pc = 0.004). Molecular docking showed that metronidazole and one of its major metabolites had the potential to bind in the HLA groove and that there was a relatively stable binding state of the HLA-B*24:02-metronidazole/the metabolite complex. The CDR3 repertoires of both T cell receptor (TCR)Vα and Vβ of the patients showed a significantly skewed or an oligoclonal distribution. The TCRVβ CDR3 of the patients shared a similar motif, "CASSxxxxxxQxF." The current study demonstrated that both the HLA-A*24:02 allele and TCR are involved in the pathogenesis of McADRs.

Heat shock protein 105 (HSP105) is overexpressed in many cancers, including colorectal cancer (CRC) and esophageal cancer (EC). We carried out a phase I clinical trial of HLA‐A24‐ and HLA‐A2‐restricted HSP105 peptide vaccines in patients with CRC or EC. In this additional study of the trial, we examined the immunological efficacy of the novel vaccine. Thirty patients with advanced CRC or EC underwent HSP105 peptide vaccination. Immunological responses were evaluated by ex vivo and in vitro γ‐interferon enzyme‐linked immunospot assays and their correlation with patients’ prognosis was analyzed. The HSP105 peptide vaccines induced peptide‐specific CTLs in 15 of 30 patients. Among HLA‐A24 patients (n = 15), 7 showed induction of CTLs only ex vivo, whereas among HLA‐A2 patients (n = 15), 4 showed the induction ex vivo and 6 in vitro. Heat shock protein 105‐specific CTL induction correlated with suppression of cancer progression and was revealed as a potential predictive biomarker for progression‐free survival (P = .008; hazard ratio = 3.03; 95% confidence interval, 1.34‐6.85) and overall survival (P = .025; hazard ratio = 2.72; 95% confidence interval, 1.13‐6.52). Production of cytokines by HSP105 peptide‐specific CTLs was observed at the injection sites (skin) and tumor tissues, suggesting that HSP105‐specific CTLs not only accumulated at vaccination sites but also infiltrated tumors. Furthermore, we established 2 HSP105 peptide‐specific CTL clones, which showed HSP105‐specific cytokine secretion and cytotoxicity. Our results suggest that the HSP105 peptide vaccine could induce immunological effects in cancer patients and improve their prognosis.

We describe a unique ABO compatible and 9/10 HLA-matched case of successful allogeneic hematopoietic cell transplantation (HCT) after effective desensitization of a strong anti-HLA-A24 donor-specific antibody (DSA) with mean fluorescence intensity of approximately 18 000. Due to absence of a suitable matched unrelated donor the patient sibling was considered the best available donor, and it was decided to desensitize patient prior to transplant. The strength of HLA-A24 DSA slowly decreased over the course of treatment, necessitating a total of 23 sessions of therapeutic plasma exchange in order to bring the DSA strength to undetectable levels, followed by a successful transplant. In summary, the outcome of this case shows effective application of desensitization treatment to remove strong DSA in HCT patients.

Recent advances in next-generation sequencing have enabled rapid and efficient evaluation of the mutational landscape of cancers. As a result, many cancer-specific neoantigens, which can generate antitumor cytotoxic T-cells inside tumors, have been identified. Previously, we reported a metastatic melanoma case with high tumor mutation burden, who obtained complete remission after anti-PD-1 therapy and surgical resection. The rib metastatic lesion, which was used for whole-exome sequencing and gene expression profiling in the HOPE project, showed upregulated expression of PD-L1 mRNA and a high single-nucleotide variants number of 2712. In the current study, we focused on a metastatic melanoma case and candidate epitopes among nonsynonymous mutant neoantigens of 1348 variants were investigated using a peptide-HLA binding algorithm, in vitro cytotoxic T-cell induction assay and HLA tetramer staining. Specifically, from mutant neoantigen data, a total of 21,066 9-mer mutant epitope candidates including a mutated amino acid anywhere in the sequence were applied to the NetMHC binding prediction algorithm. From in silico data, we identified the top 26 mutant epitopes with strong-binding capacity. A cytotoxic T-cell induction assay using 5 cancer patient-derived PBMCs revealed that the mutant ARMT1 peptide sequence (FYGKTILWF) with HLA-A*2402 restriction was an efficient neoantigen, which was detected at a frequency of approximately 0.04% in the HLA-A24 tetramer stain. The present success in identifying a novel mutant antigen epitope might be applied to clinical neoantigen screening in the context of an NGS-equipped medical facility for the development of the next-generation neoantigen cancer vaccines.

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 plasmodium species that infect human. 2-) Aligned sequences in the respective organisms' proteomes are searched with blastp. 3-) Binding predictions of the identified SARS-CoV-2 peptide to MHC class I supertype representatives are performed. 4-) Blastp searches of predicted-epitopes that bind strongly to the identified HLA allele are performed by limiting searches to human and to the plasmodium species. 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 human, for peptides sourced by limiting searches to plasmodium species at step-4. 8-) Step-5 and 6 are performed with results of 7. Results: CFLGYFCTCYFGLFC peptide of SARS-CoV-2 has the highest identity to P. vivax. Its GYFCTCYFGLF and YFCTCYFGLF parts are predicted to bind strongly to HLA-A*24:02. Results obtained only for peptides homologous to YFCTCYFGLF, as follows: YYCARRFGLF, YYCHCPFGVF, and YYCQQYFFLF are potential HLA-A*24:02 epitopes in the human proteome. Among FFYTFYFELF, YFVACLFILF, and YFPTITFHLF peptides in the plasmodium species' proteomes with strong binding affinity to HLA-A*24:02, only FFYTFYFELF of P. falciparum is homologous to the potential HLA-A*24:02 epitope YFYLFSLELF in the human proteome. Conclusion: Immune responses to the identified-peptides with similar sequences and strong binding affinities to HLA-A*24:02 may lead to autoimmune response risk in individuals with HLA-A*24:02 serotypes, upon getting infected with SARS-CoV-2 or P. falciparum., Comment: 37 pages, 4 figures

Genomic full-length sequence of HLA-A*24:02:61 was identified by group-specific sequencing in a Chinese individual.

HER2-targeted therapy has not benefited patients with low levels of HER2 expression; however, combination therapy may be effective. Primary analysis of a phase IIb trial investigating the HER2-derived vaccine nelipepimut-S (NPS) did not benefit the intention-to-treat population, but subset analysis showed a benefit in triple-negative breast cancer (TNBC) patients. The subset analysis of this multicenter, randomized, single-blind, phase IIb trial identified significant improvement in 36-month disease-free survival (DFS) between NPS (n = 55) and placebo (n = 44) in TNBC (HR 0.25, p = 0.01) and those who express HLA-A24 (HR 0.41, p = 0.05). The TNBC cohort demonstrated improved 36-month DFS in those with HER2 1+ expression (HR 0.17, p = 0.01), HLA-A24 positivity (HR 0.08, p 0.01), or in those who received neoadjuvant chemotherapy (HR 0.21, p 0.01). NPS vaccination with trastuzumab was associated with improved 36-month DFS among patients with TNBC. The observed benefit to this high-risk subgroup warrants confirmation in a phase III trial.

Cancer peptide vaccines are a promising cancer immunotherapy that can induce cancer-specific cytotoxic T lymphocytes (CTLs) in tumors. However, recent clinical trials of cancer vaccines have revealed that the efficacy of the vaccines is limited. Targeting single antigens and vaccination with short peptides are partly the cause of the poor clinical outcomes. We synthesized a novel multi-epitope long peptide, TAS0314, which induced multiple epitope-specific CTLs in HLA knock-in mice. It also showed superior epitope-specific CTL induction and antitumor activity. We also established a combination treatment model of vaccination with PD-1/PD-L1 blockade in HLA-A*2402 knock-in mice, and it showed a synergistic antitumor effect with TAS0314. Thus, our data indicated that TAS0314 treatment, especially in combination with PD-1/PD-L1 blockade, is a promising therapeutic candidate for cancer immunotherapy.

Indigenous people worldwide are at high risk of developing severe influenza disease. HLA-A*24:02 allele, highly prevalent in Indigenous populations, is associated with influenza-induced mortality, although the basis for this association is unclear. Here, we define CD8+ T-cell immune landscapes against influenza A (IAV) and B (IBV) viruses in HLA-A*24:02-expressing Indigenous and non-Indigenous individuals, human tissues, influenza-infected patients and HLA-A*24:02-transgenic mice. We identify immunodominant protective CD8+ T-cell epitopes, one towards IAV and six towards IBV, with A24/PB2550–558-specific CD8+ T cells being cross-reactive between IAV and IBV. Memory CD8+ T cells towards these specificities are present in blood (CD27+CD45RA− phenotype) and tissues (CD103+CD69+ phenotype) of healthy individuals, and effector CD27−CD45RA−PD-1+CD38+CD8+ T cells in IAV/IBV patients. Our data show influenza-specific CD8+ T-cell responses in Indigenous Australians, and advocate for T-cell-mediated vaccines that target and boost the breadth of IAV/IBV-specific CD8+ T cells to protect high-risk HLA-A*24:02-expressing Indigenous and non-Indigenous populations from severe influenza disease., The immunology of Indigenous populations is generally understudied outside the context of diseases that are prevalent in these communities. Here the authors identify prevalence of influenza CD8+ T cell epitopes in an Indigenous Australian population expressing the susceptibility allomorph HLA A*24:02 and validate immunodominance of some of these epitopes in mice.

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

First‑line chemotherapy for men with metastatic castration‑resistant prostate cancer (mCRPC) has been employed to improve overall survival (OS) and progression‑free survival (PFS). However, several new agents for CRPC after first‑line chemotherapy prolonged survival by only a few months. To develop a new treatment modality, we conducted a phase III randomized trial of personalized peptide vaccination (PPV) for human leukocyte antigen (HLA)‑A24‑positive patients with castration‑resistant prostate cancer (CRPC) for whom docetaxel chemotherapy failed. This randomized, double‑blind, placebo‑controlled, phase III trial was carried out at 68 medical centers in Japan. Patients were randomly assigned at a 2:1 ratio to receive PPV or placebo. Four of 12 warehouse peptides selected based on pre‑existing peptide‑specific immunoglobulin G levels or the corresponding placebo were subcutaneously injected in 6 doses weekly and then bi‑weekly following the maximum of 30 doses until disease progression. The primary end‑point was overall survival (OS). Efficacy analyses were performed by the full analysis set. Between August 2013 and April 2016, 310 patients were randomly assigned, and 306 patients were analyzed. Baseline characteristics were balanced between groups. The estimated median OS was 16.1 months [95% confidence interval (CI), 13‑18.2] with PPV and 16.9 months (95% CI, 13.1‑20.4) with placebo [hazard ratio (HR), 1.04, 95% CI, 0.80‑1.37; P=0.77]. Grade ≥3 adverse events were observed in 41% of both groups. The analysis of treatment arm effects among subgroups revealed lower HRs for OS in favor of the PPV arm in patients with

One nucleotide replacement in codon 233 of HLA-A*24:02:01:01 results in a novel allele, HLA-A*24:02:31.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer with limited treatments. Asia has the highest HCC incidence rates; China accounts for over 50% of all HCC cases worldwide. T‐cell receptor (TCR) ‐engineered T‐cell immunotherapies specific for human leukocyte antigen (HLA) ‐A*02:01‐restricted α‐fetoprotein (AFP) peptide have shown encouraging results in clinics. HLA‐A*24:02 is more common than HLA‐A*02:01 in Asian countries, including China. Here we identified a novel HLA‐A*24:02‐restricted peptide KWVESIFLIF (AFP(2–11)) located in AFP signal peptide domain by mass spectrometric analysis of HLA‐bound peptides from HepG2 cells. A TCR (KWV3.1) specific for AFP(2–11)‐HLA‐A*24:02 was isolated from peripheral blood mononuclear cells of a healthy donor. The binding affinity of soluble KWV3.1 to its antigen was determined to be ~55 μm, within the affinity range of native TCRs for self‐antigens. KWV3.1‐transfected T cells could specifically activate and kill AFP(2–11) pulsed T2‐A24 cells and AFP(+) HLA‐A*24:02(+) tumor cell lines, demonstrating that AFP(2–11) can be naturally presented on the surface of AFP(+) tumor cell lines. The newly identified antigenic peptide can provide a novel target for immunotherapeutic strategies for patients with AFP(+) HLA‐A*24:02(+) HCC.

BackgroundHLA-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.MethodsPost-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 ResultsWe found 78 and 18 patients with HIV-1 DNA ≥100 copies/106PBMCs and with HIV-1 DNA 6PBMCs, 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 6PBMCs (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 6PBMCs (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 6PBMCs did not have HLA-A24.ConclusionsIn 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.

For efficacy of peptide vaccination immunotherapy for patients with cancer, endogenous expression of the target peptide/human leukocyte antigen (HLA) on cancer cells is required. However, it is difficult to evaluate the expression status of a peptide/HLA complex because of the lack of a soluble T‐cell receptor (TCR) that reacts with tumor‐associated antigen (TAA) with high avidity. In the present study, we developed two soluble TCR‐multimers that were each directed to TAA, survivin‐2B (SVN‐2B) and PBF in the context of HLA‐A24 (SVN‐2B TCR‐multimer and PBF TCR‐multimer, respectively), from CTL clones that were established from peptide‐vaccinated patients. Both TCR multimers could recognize cognate peptide‐pulsed antigen‐presenting cells, C1R‐A24 cells, in a CD8‐independent method. Moreover, the PBF TCR‐multimer successfully recognized a PBF peptide naturally presented on HLA‐A24+ PBF + osteosarcoma cells. Taken together, the results indicated that a TCR‐multimer might be useful for detection of a TAA‐derived peptide presented by HLA in patients receiving immunotherapy.

Background HLA-B*35 is an HLA allele associated with rapid progression to AIDS. However, a mechanism underlying the detrimental effect of HLA-B*35 on disease outcome remains unknown. Recent studies demonstrated that most prevalent subtype HLA-B*35:01 is a detrimental allele in HIV-1 clade B-infected individuals. We here investigated the effect of mutations within the epitopes on HLA-B*35:01-restricted CD8+ T cells having abilities to suppress HIV-1 replication. Methods We analyzed 16 HLA-B*35:01-restricted epitope-specific T cells in 63 HIV-1 clade B-infected Japanese B*35:01+ individuals and identified HLA-B*35:01-restricted CD8+ T cells having abilities to suppress HIV-1 replication. We further analyzed the effect of HLA-associated mutations on the ability of these T cells. Findings The breadth of T cell responses to 4 epitopes was inversely associated with plasma viral load (pVL). However, the accumulation of an Y135F mutation in NefYF9 out of the 4 epitopes, which is selected by HLA-A*24:02-restricted T cells, affected the ability of YF9-specific T cells to suppress HIV-1 replication. HLA-B*35:01+ individuals harboring this mutation had much higher pVL than those without it. YF9-specific T cells failed to suppress replication of the Y135F mutant in vitro. These results indicate that this mutation impairs suppression of HIV-1 replication by YF9-specific T cells. Interpretation These findings indicate that the Y135F mutation is a key factor underlying the detrimental effect of HLA-B*35:01 on disease outcomes in HIV-1 clade B-infected individuals. Fund Grants-in-aid for AIDS Research from AMED and for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan., Highlights • T cells specific for 4 HLA-B*35:01-restricted epitopes have abilities to suppress HIV-1 replication in vivo. • An Y135F mutation selected by HLA-A*24:02-restricted T cells affected HIV-1 control by NefYF9-specific T cells in vivo. • The NefY135F mutation impaired suppression of HIV-1 replication by NefYF9-specific T cells in vitro.

Aims/hypothesis: HLA-A*24 carriership hampers achievement of insulin independence in islet allograft recipients. However, less than half of those who fail to achieve insulin independence carry the allele. We investigated whether genetic polymorphism at the recipients’ zinc transporter 8-encoding SLC30A8 gene (rs13266634) could complement their HLA-A*24 status in predicting functional graft outcome. Methods: We retrospectively analysed data of a hospital-based patient cohort followed for 18 months post transplantation. Forty C-peptide-negative type 1 diabetic individuals who received >2 million beta cells (>4000 islet equivalents) per kg body weight in one or two intraportal implantations under similar immunosuppression were genotyped for SLC30A8. Outcome measurements included achievement and maintenance of graft function. Metabolic benefit was defined as 331 pmol/l C-peptide, in addition to achievement of insulin independence and maintenance of C-peptide positivity. Results: In multivariate analysis, HLA-A*24 positivity, presence of SLC30A8 CT or TT genotypes and BMI more than or equal to the group median (23.9 kg/m 2) were independently associated with failure to achieve insulin independence (p = 0.015–0.046). The risk increased with the number of factors present (p < 0.001). High BMI interacted with SLC30A8 T allele carriership to independently predict difficulty in achieving graft function with metabolic benefit (p = 0.015). Maintenance of C-peptide positivity was mainly associated with older age at the time of implantation. Only HLA-A*24 carriership independently predicted failure to maintain acceptable graft function once achieved (p = 0.012). Conclusions/interpretation: HLA-A*24, the SLC30A8 T allele and high BMI are associated with poor graft outcome and should be considered in the interpretation of future transplantation trials. Trial registration: ClinicalTrials.gov NCT00798785 and NCT00623610.

Background We conducted a phase I/Ib, open-label, single-arm trial to assess the safety, tolerability and optimal scheduling regimen of OTSGC-A24 cancer vaccine in patients with advanced gastric cancer. Methods Patients with advanced gastric cancer with HLA-A*24:02 haplotype were included in this study. OTSGC-A24 was administered at 1 mg in 3-weekly (3w), 2-weekly (2w), and weekly (1w) cohorts to evaluate the safety, immunological response and schedule. Based on the highest specific cytotoxic T lymphocyte (CTL) induction rate at 4 weeks, using the ELISPOT test, cohorts were expanded to define the optimal dosing schedule for OTSGC-A24. Results In this study, 24 advanced gastric cancer patients with HLA-A*24:02 haplotype were enrolled and treated in 3 cohorts (3w cohort: 3; 2w cohort: 11 and 1w cohort: 10 patients). The most common adverse events were decreased appetite (29%), diarrhea (21%), myalgia (25%). The most common treatment-related adverse event was injection site erythema (25%). No dose-limiting toxicities were observed in any cohort and OTSGC-A24 was well tolerated. Positive CTL responses after vaccination were observed in 15 patients (75%) at 4 weeks: 3w cohort (33%), 2w cohort (88%), 1w cohort (78%). At 12 weeks, 18 patients had responded (90%); 3w cohort (100%), 2w cohort (100%), 1w cohort (78%). The best radiological was stable disease (40%). Median progression free survival was 1.7 months (95% CI: 1.4 to 3.5) and median overall survival was 5.7 months (95% CI 3.8 to 8.6). Conclusions OTSGC-A24 combined peptide cancer vaccine was well tolerated. Significant responses in CTL were observed and the recommended phase 2 dose is 1 mg OTSGC-A24 sub-cutaneous, every 2 weeks. Although no radiological response was observed, a respectable overall survival was achieved, consistent with other immunotherapy agents being investigated in gastric cancer. Trial registration ClinicalTrials.gov Identifier: NCT01227772, Date registered: 21 Oct 2010.

Cytotoxic T lymphocyte (CTL)–based immunotherapies have had remarkable success at generating objective clinical responses in patients with advanced metastatic melanoma. Although the melanocyte differentiation antigens (MDA) MART-1, PMEL, and tyrosinase were among the first melanoma tumor-associated antigens identified and targeted with immunotherapy, expression within normal melanocytes of the eye and inner ear can elicit serious autoimmune side effects, thus limiting their clinical potential as CTL targets. Using a tandem mass spectrometry (MS) approach to analyze the immunopeptidomes of 55 melanoma patient–derived cell lines, we identified a number of shared HLA class I–bound peptides derived from the melanocyte-specific transporter protein SLC45A2. Antigen-specific CTLs generated against HLA-A*0201- and HLA-A*2402–restricted SLC45A2 peptides effectively killed a majority of HLA-matched cutaneous, uveal, and mucosal melanoma cell lines tested (18/25). CTLs specific for SLC45A2 showed significantly reduced recognition of HLA-matched primary melanocytes that were, conversely, robustly killed by MART1- and PMEL-specific T cells. Transcriptome analysis revealed that SLC45A2 mRNA expression in normal melanocytes was less than 2% that of other MDAs, therefore providing a more favorable melanoma-to-melanocyte expression ratio. Expression of SLC45A2 and CTL sensitivity could be further upregulated in BRAF(V600E)-mutant melanoma cells upon treatment with BRAF or MEK inhibitors, similarly to other MDAs. Taken together, our study demonstrates the feasibility of using tandem MS as a means of discovering shared immunogenic tumor-associated epitopes and identifies SLC45A2 as a promising immunotherapeutic target for melanoma with high tumor selectivity and reduced potential for autoimmune toxicity. Cancer Immunol Res; 5(8); 618–29. ©2017 AACR.

We screened cell division associated 1 (CDCA1) as an oncogene that is overexpressed on several cancers, including prostate cancer. We also identified a highly immunogenic HLA-A*2402-restricted epitope peptide corresponding to part of the CDCA1 protein. We conducted a phase I clinical trial 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, 6 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 This article is protected by copyright. All rights reserved.

Background S-288310, a cancer peptide vaccine composed of two HLA-A*24:02-restricted peptides derived from two oncoantigens, DEP domain-containing 1 (DEPDC1) and M-phase phosphoprotein 1 (MPHOSPH1), was investigated in urothelial carcinoma (UC) of the bladder. Patients and methods Thirty eight HLA-A*24:02-positive patients with progressive UC were enrolled in this study. In the phase I part of the study, three patients each were treated with S-288310 at 1 mg or 2 mg/peptide subcutaneously once a week to evaluate safety and tolerability. In the phase II, 32 patients were randomized to receive either 1 mg or 2 mg to evaluate the difference in cytotoxic T lymphocytes (CTL) induction and safety. Results S-288310 was safe and well tolerated in the phase I. Of 27 patients evaluable for immune responses in the phase II, there was no difference in CTL induction rate between the 1 mg (100%) and 2 mg (80.0%) groups. Of 32 patients receiving S-288310 in the phase II, the most frequent drug-related AE was the injection site reaction that was observed in 29 patients (90.6%), but none of the patients discontinued administration due to these reactions and no dose relationship in the frequency and severity was observed. The objective response rate of the 32 patients was 6.3% and the disease control rate was 56.3%. The median overall survival (OS) rates for patients vaccinated with S-288310 after one regimen of chemotherapy, 2 regimens, or 3 or more were 14.4, 9.1 and 3.7 months, respectively, and 32.2% of patients post first-line treatment were alive at 2 years. OS of patients who showed CTL induction to both peptides was longer than that of those with CTL induction to no or one peptide. Conclusion S-288310 was well-tolerated and effectively induced peptide-specific CTLs, which were correlated with longer survival for patients with UC of the bladder. Trial registration ID JapicCTI-090980

Accumulation of mutant p53 proteins is frequently found in a wide range of cancers. While conventional antibodies fail to target intracellular proteins, proteosomal degradation results in the presentation of p53-derived peptides on the tumour cell surface by class I molecules of the major histocompatibility complex (MHC). Elevated levels of such p53-derived peptide-MHCs on tumour cells potentially differentiate them from healthy tissues. Here, we report the engineering of an affinity-matured human antibody, P1C1TM, specific for the unmutated p53125-134 peptide in complex with the HLA-A24 class I MHC molecule. We show that P1C1TM distinguishes between mutant and wild-type p53 expressing HLA-A24+ cells, and mediates antibody dependent cellular cytotoxicity of mutant p53 expressing cells in vitro. Furthermore, we show that cytotoxic PNU-159682-P1C1TM drug conjugates specifically inhibit growth of mutant p53 expressing cells in vitro and in vivo. Hence, p53-associated peptide-MHCs are attractive targets for the immunotherapy against mutant p53 expressing tumours., Several cancers harbour mutant p53 and express higher levels of p53-derived peptide-MHCs. Here, the authors report affinity matured human antibody, P1C1TM, specific for the p53125-134 peptide in complex with the HLA-A24 class I MHC molecule and show its efficacy and specificity for mutant p53 expressing tumours.

Summary Understanding how peptide selection is controlled on different major histocompatibility complex class I (MHC I) molecules is pivotal for determining how variations in these proteins influence our predisposition to infectious diseases, cancer, and autoinflammatory conditions. Although the intracellular chaperone TAPBPR edits MHC I peptides, it is unclear which allotypes are subjected to TAPBPR-mediated peptide editing. Here, we examine the ability of 97 different human leukocyte antigen (HLA) class I allotypes to interact with TAPBPR. We reveal a striking preference of TAPBPR for HLA-A, particularly for supertypes A2 and A24, over HLA-B and -C molecules. We demonstrate that the increased propensity of these HLA-A molecules to undergo TAPBPR-mediated peptide editing is determined by molecular features of the HLA-A F pocket, specifically residues H114 and Y116. This work reveals that specific polymorphisms in MHC I strongly influence their susceptibility to chaperone-mediated peptide editing, which may play a significant role in disease predisposition., Graphical Abstract, Highlights • TAPBPR exhibits a binding preference for HLA-A molecules over HLA-B and -C • HLA-A2 and -A24 superfamily members are the strongest TAPBPR binders • F pocket architecture impacts MHC I susceptibility to TAPBPR-mediated peptide editing, Ilca et al. explore which human leukocyte antigen class I allotypes are subjected to TAPBPR-mediated peptide editing, revealing TAPBPR has preference for HLA-A, particularly for supertypes A2 and A24, over HLA-B and -C molecules.

Summary In type 1 diabetes (T1D), autoreactive cytotoxic CD8+ T cells are implicated in the destruction of insulin‐producing β cells. The HLA‐B*3906 and HLA‐A*2402 class I genes confer increased risk and promote early disease onset, suggesting that CD8+ T cells that recognize peptides presented by these class I molecules on pancreatic β cells play a pivotal role in the autoimmune response. We examined the frequency and phenotype of circulating preproinsulin (PPI)‐specific and insulin B (InsB)‐specific CD8+ T cells in HLA‐B*3906 + children newly diagnosed with T1D and in high‐risk HLA‐A*2402 + children before the appearance of disease‐specific autoantibodies and before diagnosis of T1D. Antigen‐specific CD8+ T cells were detected using human leucocyte antigen (HLA) class I tetramers and flow cytometry was used to assess memory status. In HLA‐B*3906 + children with T1D, we observed an increase in PPI5–12‐specific transitional memory CD8+ T cells compared to non‐diabetic, age‐ and HLA‐matched subjects. Furthermore, PPI5–12‐specific CD8+ T cells in HLA‐B*3906 + children with T1D showed a significantly more antigen‐experienced phenotype compared to polyclonal CD8+ T cells. In longitudinal samples from high‐risk HLA‐A*2402 + children, the percentage of terminal effector cells within the InsB15–24‐specific CD8+ T cells was increased before diagnosis relative to samples taken before the appearance of autoantibodies. This is the first study, to our knowledge, to report HLA‐B*3906‐restricted autoreactive CD8+ T cells in T1D. Collectively, our results provide evidence that β cell‐reactive CD8+ T cells restricted by disease‐associated HLA class I molecules display an antigen‐experienced phenotype and acquire enhanced effector function during the period leading to clinical diagnosis, implicating these cells in driving disease., In this study, we characterised circulating autoreactive CD8+ T cells in children with type 1 diabetes with HLA-B*3906+ and HLA-A*2402+ genotypes which are associated with increased risk and early onset of disease. In HLA-B*3906+ children with newly diagnosed type 1 diabetes, we observed an increase in PPI5–12‐specific transitional memory CD8+ T cells compared to non‐diabetic, age‐ and HLA‐matched subjects. Furthermore, PPI5–12‐specific CD8+ T cells in HLA‐B*3906+ children with T1D showed a significantly more antigen‐experienced phenotype compared to polyclonal CD8+ T cells. Further, in longitudinal samples from high-risk HLA-A*2402+ children, the percentage of terminal effector cells within the InsB15–24‐specific CD8+ T cell population was increased before diagnosis relative to samples taken before the appearance of autoantibodies.

HLA-A*24:02:78 differs from HLA-A*24:02:01:01 in exon 3 by a single nucleotide.

Seven new HLA alleles were characterized by next generation sequencing in the Spanish population.

Colorectal cancer is the third commonest malignancy in Asia including Malaysia. The immunogenic cancer-testis antigens, which are expressed in a variety of cancers but with limited expression in normal tissues except the testis, represent an attractive approach to improve treatment options for colorectal cancer. We aimed to validate four PASD1 peptides as the immunotherapeutic targets in colorectal cancer. First, PASD1 mRNA and protein expression were determined via real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. The PASD1 peptides specific to HLA-A*24:02 were investigated using IFN-y-ELISpot assay, followed by the cytolytic and granzyme-B-ELISpot assays to analyze the cytolytic effects of CD8+ T cells. Gene and protein expressions of PASD1 were detected in 20% and 17.3% of colorectal cancer samples, respectively. PASD1(4) peptide was shown to be immunogenic in colorectal cancer samples. CD8+ T cells raised against PASD1(4) peptide were able to lyze HLA-A*24:02+ PASD1+ cells. Our results reveal that PASD1(4) peptide represents a potential target for colorectal cancer.

Identification of the novel HLA-A*24:02:131 allele that differs from HLA-A*24:02:01:01 in exon 4.

T-cell receptors possess the unique ability to survey and respond to their permanently modified ligands, self HLA-I molecules bound to non-self peptides of various origin. This highly specific immune function is impaired following hematopoietic stem cell transplantation (HSCT) for a timespan of several months needed for the maturation of T-cells. Especially, the progression of HCMV disease in immunocompromised patients induces life-threatening situations. Therefore, the need for a new immune system that delivers vital and potent CD8+ T-cells carrying TCRs that recognize even one human cytomegalovirus (HCMV) peptide/HLA molecule and clear the viral infection long term becomes obvious. The transcription and translation of HCMV proteins in the lytic cycle is a precisely regulated cascade of processes, therefore, it is a highly sensitive challenge to adjust the exact time point of HCMV-peptide recruitment over self-peptides. We utilized soluble HLA technology in HCMV-infected fibroblasts and sequenced naturally sHLA-A*24:02 presented HCMV-derived peptides. One peptide of 14 AAs length derived from the IE2 antigen induced the strongest T-cell responses; this peptide can be detected with a low ranking score in general peptide prediction databanks. These results highlight the need for elaborate and HLA-allele specific peptide selection.

HLA-A*24:460 differs from HLA-A*24:02 by one nucleotide in codon 285.

Intracellular tumor-associated antigens are targeted by antibodies known as T-cell receptor mimic antibodies (TCRm-Abs), which recognize T-cell epitopes with better stabilities and higher affinities than T-cell receptors. However, TCRm-Abs have been proven difficult to produce using conventional techniques. Here, we developed TCRm-Abs that recognize the survivin-2B-derived nonamer peptide, AYACNTSTL (SV2B80-88), presented on HLA-A*24 (SV2B80-88/HLA-A*24) from immunized mice by using a fluorescence-activated cell sorting-based antigen-specific plasma cells isolation method combined with a high-throughput single-cell-based immunoglobulin-gene-cloning technology. This approach yielded a remarkable efficiency in generating candidate antibody clones that recognize SV2B80-88/HLA-A*24. The screening of the antibody clones for their affinity and ability to bind key amino-acid residues within the target peptide revealed that one clone, #21-3, specifically recognized SV2B80-88/HLA-A*24 on T2 cells. The specificity of #21-3 was further established through survivin-2B-positive tumor cell lines that exogenously or endogenously express HLA-A*24. A bispecific T-cell engager comprised of #21-3 and anti-CD3 showed specific cytotoxicity towards cells bearing SV2B80-88/HLA-A*24 by recruiting and activating T-cells in vitro. The efficient development of TCRm-Ab overcomes the limitations that hamper antibody-based immunotherapeutic approaches and enables the targeting of intracellular tumor-associated antigens.

We previously conducted a phase I clinical trial combining the HLA-A*2402-restricted KIF20A-derived peptide vaccine with gemcitabine for advanced pancreatic cancer (PC) and confirmed its safety and immunogenicity in cancer patients. In this study, we conducted a multicenter, single-armed, phase II trial using two antiangiogenic cancer vaccines targeting VEGFR1 and VEGFR2 in addition to the KIF20A peptide. We attempted to evaluate the clinical benefit of the cancer vaccination in combination with gemcitabine. Chemotherapy naïve PC patients were enrolled to evaluate primarily the 1-year survival rate, and secondarily overall survival (OS), progression free survival (PFS), response rate (RR), disease control rate (DCR) and the peptide-specific immune responses. All enrolled patients received therapy without the HLA-A information, and the HLA genotypes were used for classification of the patients. Between June 2012 and May 2013, a total of 68 patients were enrolled. No severe systemic adverse effects of Grade 3 or higher related to these three peptides were observed. The 1-year survival rates between the HLA-A*2402-matched and -unmatched groups were not significantly different. In the HLA-A*2402 matched group, patients showing peptide-specific CTL induction for KIF20A or VEGFR1 showed a better prognosis compared to those without such induction (P = 0.023, P = 0.009, respectively). In the HLA-A*2402-matched group, the patients who showed a strong injection site reaction had a better survival rate (P = 0.017) compared to those with a weak or no injection site reaction. This phase II study demonstrated that this therapeutic peptide cocktail might be effective in patients who demonstrate peptide-specific immune reactions although predictive biomarkers are needed for patient selection in its further clinical application.