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We have applied our advanced computational and experimental methodologies to investigate the complex structure and binding mechanism of a modified Wilms' Tumor 1 (mWT1) protein epitope to the understudied Asian-dominant allele HLA-A*24:02 (HLA-A24) in aqueous solution. We have applied our developed multicanonical molecular dynamics (McMD)-based dynamic docking method to analyze the binding pathway and mechanism, which we verified by comparing the highest probability structures from simulation with our experimentally solved x-ray crystal structure. Subsequent path sampling MD simulations elucidated the atomic details of the binding process and indicated that first an encounter complex is formed between the N-terminal's positive charge of the 9-residue mWT1 fragment peptide and a cluster of negative residues on the surface of HLA-A24, with the major histocompatibility complex (MHC) molecule preferring a predominantly closed conformation. The peptide first binds to this closed MHC conformation, forming an encounter complex, after which the binding site opens due to increased entropy of the binding site, allowing the peptide to bind to form the native complex structure. Further sequence and structure analyses also suggest that although the peptide loading complex would help with stabilizing the MHC molecule, the binding depends in a large part on the intrinsic affinity between the MHC molecule and the antigen peptide. Finally, our computational tools and analyses can be of great benefit to study the binding mechanism of different MHC types to their antigens, where it could also be useful in the development of higher affinity variant peptides and for personalized medicine., (© 2023 The Protein Society.)

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 e x 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.

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., (© 2021. The Author(s).)

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.

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., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

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., (Published by Elsevier Inc.)

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&#95;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 (K D  = 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.

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 PPI 5-12 -specific transitional memory CD8 + T cells compared to non-diabetic, age- and HLA-matched subjects. Furthermore, PPI 5-12 -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 InsB 15-24 -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., (© 2019 British Society for Immunology.)

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 p53 125-134 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.

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., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Although treatment of chronic myeloid leukemia (CML) has improved with the development of tyrosine kinase inhibitors (TKIs), patients develop fatal blast crisis (BC) whilst receiving TKI treatment. Alternative treatments for cases resistant to TKIs are required. A serine/threonine protein kinase, T‑lymphokine‑activated killer cell‑originated protein kinase (TOPK), is highly expressed in various malignant tumors. Binding of peptides to human leukocyte antigen was assessed via mass spectrometry in K562 CML cells. TOPK expression was assessed in various CML cell lines and in clinical samples obtained from patients with CML using reverse transcription‑quantitative polymerase chain reaction and western blot assays. It was observed that TOPK was expressed abundantly in BCR/ABL‑positive cell lines and at significantly higher levels in CML clinical samples compared with healthy donor samples. Overexpression of BCR/ABL or the presence of its inhibitor imatinib upregulated and downregulated TOPK expression, respectively, indicating that TOPK may be a target of BCR/ABL. TOPK inhibitor OTS514 suppressed proliferation of BCR/ABL‑positive cell lines and colony formation of CD34‑positive cells from patients with CML compared with lymphoma patients without bone marrow involvement. Furthermore, phosphorylation of TOPK was increased by protein phosphatase 2A (PP2A) inhibitor okadaic acid and was decreased in the presence of PP2A activator FTY720 compared with untreated samples. As constitutive BCR/ABL activity and inhibition of PP2A are key mechanisms of CML development, TOPK may be a crucial signaling molecule for this disease. Inhibition of TOPK may control disease status of CML, even in cases resistant to TKIs.

Background: We conducted a phase III trial of personalized peptide vaccination (PPV) for human leukocyte antigen (HLA)-A24+ recurrent glioblastoma to develop a new treatment modality., Methods: We randomly assigned 88 recurrent glioblastoma patients to receive PPV (n = 58) or the placebo (n = 30) at a 2-to-1 ratio. Four of 12 warehouse peptides selected based on preexisting peptide-specific immunoglobulin G levels or the corresponding placebos were injected 1×/week for 12 weeks., Results: Our trial met neither the primary (overall survival [OS]) nor secondary endpoints. Unfavorable factors for OS of 58 PPV patients compared with 30 placebo patients were SART2-93 peptide selection (n = 13 vs 8, hazard ratio [HR]: 15.9), ≥70 years old (4 vs 4, 7.87), >70 kg body weight (10 vs 7, 4.11), and performance status (PS)3 (8 vs 2, 2.82), respectively. Consequently, the median OS for PPV patients without SART2-93 selection plus one of these 3 favorable factors (<70 y old, ≤70 kg, or PS0-2) was significantly longer than that for the corresponding placebo patients (HR: 0.49, 0.44, and 0.51), respectively. Preexisting immunity against both all 12 warehouse peptides besides SART2-93 and the other cytotoxic T lymphocyte epitope peptides was significantly depressed in the patients with SART2-93 selection (n = 21) compared with that of the patients without SART2-93 selection (n = 67). Biomarkers correlative for favorable OS of the PPV patients were a lower percentage of CD11b+CD14+HLA-DRlow immunosuppressive monocytes and a higher percentage of CD4+CD45RA- activated T cells, the intermediate levels of chemokine C-C ligand 2 (CCL2), vascular endothelial growth factor, interleukin (IL)-6, IL-17, or haptoglobin, respectively., Conclusion: This phase III trial met neither the primary nor secondary endpoints., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Visceral leishmaniasis is a tropical and neglected disease with an estimated 200 000-400 000 cases and 60 000 deaths worldwide each year. Currently, no clinically valid vaccine is available for this disease. In this study, we formulated DNA and protein vaccines encoding HLA-A2, HLA-A24 and HLA-DR1 restricted epitopes of CaNA2 against visceral leishmaniasis. We predicted the secondary and tertiary structures, surface properties, subcellular localization, potential binding sites and HLA-A2, HLA-A24 and HLA-DR1 restricted epitopes of CaNA2. The best candidate CpG ODN (2395, M362, D-SL03 or 685) was screened out as a DNA vaccine adjuvant. We also prepared Kmp-11 and Kmp-11/CaNA2 DNA and protein vaccines, respectively, for comparison. BALB/c mice were immunized with a DNA prime-protein boost immunization strategy and challenged with a newly isolated Leishmania strain from an individual with visceral leishmaniasis. The IgG antibody titers showed that our vaccine had strong immunogenicity with a long duration, especially cellular immunity. The spleen parasite burden of each group demonstrated that the CaNA2 vaccine had a certain immune protective effect on visceral leishmaniasis in BALB/c mice, and the amastigote reduction rate reached 76%. Preliminary safety tests confirmed the safety of the vaccine. Our work demonstrates that the HLA-A2, HLA-A24 and HLA-DR1 restricted epitope CaNA2 DNA prime-protein boost vaccine may be a safe and effective epitope vaccine candidate against visceral leishmaniasis., (© 2018 John Wiley & Sons Ltd.)

X-box binding protein 1 (XBP1), CD138 (Syndecan-1) and CS1 (SLAMF7) are highly expressed antigens in cancers including multiple myeloma (MM). Here, we identify and characterize immunogenic HLA-A24 peptides derived from these antigens for potential vaccination therapy of HLA-A24+ patients with MM. The identified immunogenic HLA-A24-specific XBP1 unspliced (UN) 185-193 (I S P W I L A V L), XBP1 spliced (SP) 223-231 (V Y P E G P S S L), CD138 265-273 (I F A V C L V G F) and CS1 240-248 (L F V L G L F L W) peptides induced antigen-specific CTL with anti-MM activity in an HLA-A24 restricted manner. Furthermore, a cocktail containing the four HLA-A24 peptides evoked MM-specific CTL with distinct phenotypic profiles (CD28, CD40L, 41BB, CD38, CD69) and anti-tumor activities, evidenced by perforin upregulation, CD107a degranulation (cytotoxicity) and Th1-type cytokines (IFN-γ/IL-2/TNF-α) production in response to HLA-A24 + MM cells. The multipeptide-specific CTL included antigen-specific memory CD8 + T cells expressing both T-cell activation (CD38, CD69) and immune checkpoints antigens (CTLA, PD-1, LAG-3, TIM-3). These results provide the framework for a multipeptide vaccination therapy to induce tumor-specific CTL in HLA-A24-positive patients with myeloma and other cancers expressing these antigens.

Hypoxic tumor microenvironment makes cancer cells to be therapy-resistant and hypoxia-inducing factors (HIFs) play a central role in hypoxic adaptation. Especially, renal cell carcinoma (RCC) is often associated with von Hippel-Lindau (VHL) gene mutations, leading to up-regulation of HIFs. However, from a different point of view, this suggests the possibility that HIFs could be promising targets in anti-cancer therapy. In this study, we searched for HIF-1α-derived peptides that are able to induce RCC-reactive cytotoxic T lymphocytes (CTLs) from HLA-A24 + RCC patients. Among five peptides derived from HIF-1α, which were prepared based on the binding motif to the HLA-A24 allele, a HIF-1α 278-287 peptide induced peptide-specific CTLs from peripheral blood mononuclear cells of HLA-A24 + RCC patients most effectively. In immunoblot assays, the expression of HIF-1α was lowly detected in whole and nuclear lysates of RCC cell lines even under normoxia (20% O 2 ), and their expression in whole lysates was increased under hypoxia (1% O 2 ). Additionally, HIF-1α 278-287 peptide-stimulated T cells showed a higher cytotoxicity against HLA-A24 + HIF-1α-expressing RCC cells than against HLA-A24 - HIF-1α-expressing RCC cells. The cytotoxicity was inhibited by the addition of HIF-1α 278-287 peptide-pulsed cold target cells. Altogether, these results indicate that the HIF-1α 278-287 peptide could be a candidate for peptide-based anti-cancer vaccines for HLA-A24 + RCC patients., (Copyright © 2017. Published by Elsevier B.V.)

Programmed death-ligand 1 (PD-L1) binds to programmed death-1 (PD-1) on activated T cells and contributes to T-cell exhaustion. PD-L1 expressed on antigen-presenting cells (APCs) could be thought to inhibit the induction of Ag-specific cytotoxic T lymphocytes (CTLs) by transducing negative signal into T cells; however, the roles of PD-L1 on APCs have not yet been well examined. Therefore, we evaluated the roles of PD-L1 on APCs in the induction of Ag-specific CTLs. CD3 T cells isolated from cytomegalovirus (CMV)-seropositive healthy donors were stimulated with mature dendritic cells pulsed with CMV pp65-derived HLA-restricted peptides in the presence of anti-PD-L1 blocking antibody. Unexpectedly, PD-L1 blockade resulted in a less efficient induction of CMV-specific CTLs, suggesting that PD-L1 play a positive role in the induction of Ag-specific CTLs. For further evaluations and application to adoptive immunotherapy, we generated K562-based artificial APCs, which were retrovirally transduced with HLA class I molecules and various combinations of CD80/86 and PD-L1. K562/HLA+CD80/86+PD-L1 cells produced significantly higher induction of CMV-specific CTLs than K562/HLA or K562/HLA+CD80/86 cells without causing excessive differentiation or functional exhaustion of the induced CTLs, whereas PD-L1 itself did not have a stimulatory effect. Furthermore, only K562/HLA+CD80/86+PD-L1 cells pulsed with HLA-A*24:02-restricted Wilms tumor 1 (WT1) peptide clearly expanded WT1-specific CTLs from healthy donors. Our findings presumed that PD-L1 expressed on APCs along with CD80/86 enhanced the induction of Ag-specific CTLs probably depending on fine-tuning excessive stimulation of CD80/86, and that K562/HLA+CD80/86+PD-L1 cells has therapeutic potential as a novel type of artificial APCs for adoptive immunotherapy.

The αβ T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D Kd of ∼5,000 molecules/μm(2) This value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination.

Induction of specific immune response against therapy-resistant tumor cells can potentially improve clinical outcomes in malignancies. To optimize immunotherapy in the clinic, we aimed to create an in vivo model enabling us to analyze human cytotoxic T-lymphocyte (CTL) responses against human malignancies. To this end, we developed NOD/SCID/IL2rgKO (NSG) mice expressing the HLA class I molecules HLA-A*0201 and A*2402. In the bone marrow (BM) and spleen of HLA class I transgenic (Tg) NSG mice transplanted with cord blood hematopoietic stem cells (HSCs), we found human memory CD8(+) T cells and antigen-presenting cells. To evaluate antigen-specific human CTL responses, we immunized HLA class I Tg NSG mice using polyinosinic:polycytidylic acid mixed Wilms tumor 1 (WT1) peptides, with or without WT1 peptide-loaded autologous dendritic cells. After immunization, the frequencies of HLA-restricted WT1-specific CTLs increased significantly in the spleen. Next, we transplanted the WT1-specific T-cell receptor (WT1-TCR) gene-transduced human HSCs into HLA class I Tg NSG newborn mice. WT1 tetramer-positive CD8(+) T cells differentiated from WT1-TCR-transduced HSCs in the recipients' BM, spleen, and thymus. Upon stimulation with WT1 peptide in vitro, these CTLs produced interferon-γ and showed lytic activity against leukemia cells in an antigen-specific, HLA-restricted manner. HLA class I Tg NSG xenografts may serve as a preclinical model to develop effective immunotherapy against human malignancies., (© 2016 by The American Society of Hematology.)

Adoptive transfer of T cells redirected by a high-affinity antitumor T-cell receptor (TCR) is a promising treatment modality for cancer patients. Safety and efficacy depend on the selection of a TCR that induces minimal toxicity and elicits sufficient antitumor reactivity. Many, if not all, TCRs possess cross-reactivity to unrelated MHC molecules in addition to reactivity to target self-MHC/peptide complexes. Some TCRs display chain centricity, in which recognition of MHC/peptide complexes is dominated by one of the TCR hemi-chains. In this study, we comprehensively studied how TCR chain centricity affects reactivity to target self-MHC/peptide complexes and alloreactivity using the TCR, clone TAK1, which is specific for human leukocyte antigen-A*24:02/Wilms tumor 1(235-243) (A24/WT1(235)) and cross-reactive with B*57:01 (B57). The TAK1β, but not the TAK1α, hemi-chain possessed chain centricity. When paired with multiple clonotypic TCRα counter-chains encoding TRAV12-2, 20, 36, or 38-2, the de novo TAK1β-containing TCRs showed enhanced, weakened, or absent reactivity to A24/WT1(235) and/or to B57. T cells reconstituted with these TCRα genes along with TAK1β possessed a very broad range (>3 log orders) of functional and structural avidities. These results suggest that TCR chain centricity can be exploited to enhance desired antitumor TCR reactivity and eliminate unwanted TCR cross-reactivity. TCR reactivity to target MHC/peptide complexes and cross-reactivity to unrelated MHC molecules are not inextricably linked and are separable at the TCR sequence level. However, it is still mandatory to carefully monitor for possible harmful toxicities caused by adoptive transfer of T cells redirected by thymically unselected TCRs., (©2015 American Association for Cancer Research.)

Background: Cytomegalovirus (CMV)-specific CD8(+) cytotoxic T lymphocytes (CMV-CTLs) play a crucial role in preventing CMV disease. However, the actual in vivo dynamics of CMV-CTL clones after allogeneic hematopoietic stem cell transplantation (alloHCT) are still unclear., Methods: Using a single-cell T-cell receptor repertoire analysis, we monitored clones and chimerism of CMV-CTLs in 3 CMV-seropositive alloHCT recipients from CMV-seronegative donors, with or without CMV reactivation., Results: Nearly all of the CMV-CTLs during follow-up were CD45RA(-) CCR7(-) effector memory/CD45RA(+) CCR7(-) effector T cells, and were highly matured. In each case, the use of BV gene families was restricted, especially in BV5, 7, 28, and 29. Although no common predominant CMV-CTL clones were found, several shared motifs of complementarity-determining region-3 were identified among the 3 cases; QGA in all, TGE and TDT in Case 1 and Case 2, and RDRG in Case 2 and Case 3. In all cases, CMV-CTL clones that were detected for the first time after alloHCT persisted as the dominant clones. In Case 1, without CMV reactivation, recipient-derived CMV-CTLs exclusively persisted as a dominant clone, while all CMV-CTLs in the other 2 cases, with CMV reactivation, were donor derived., Conclusion: Clone monitoring and chimerism analyses should help to further clarify novel aspects of immuno-reconstitution after alloHCT., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Human TCRαβ(+) CD4(-)CD8(-) double-negative (DN) T cells represent a minor subset in peripheral blood, yet are important in infectious diseases and autoimmune responses. We examined the frequency of DN T cells in 17 patients after allogeneic hematopoietic stem cell transplantation (aHSCT) at 1, 2, 3, 6, and 12 months post-aHSCT and show that these cells increase early after aHSCT and decrease with time after aHSCT. DN T cells reside in the terminally differentiated effector (CD45RA(+)CCR7(-)) T-cell population and are polyclonal, determined by T-cell receptor Vβ CDR3 analysis. Gene expression analysis of ex vivo sorted DN T cells showed a distinct set of gene expression, including interleukin-8, as compared with CD4(+) or CD8(+) T cells. DN T cells contributed to MHC class I-restricted EBV-directed immune responses, defined by antigen-specific cytokine production and by detection of HLA-A*02:01-restricted EBV BMLF-1 (GLCTLVAML), LMP-2A (CLGGLLTMV), and HLA-A*24:02-restricted EBV BRLF-1 (DYCNVLNKEF) and EBNA3 (RYSIFFDY)-specific T cells. We created retroviral-transfected Jurkat cell lines with a Melan-A/MART-1-specific TCR(+) and the CD8α chain to study TCR(+) DN T cells in response to their nominal MHC class I/peptide ligand. We show that DN T cells exhibit increased TCRζ chain phosphorylation as compared with the TCR(+)CD8(+) transgenic T-cell line. DN T cells contribute to antigen-specific T-cell responses and represent an effector T-cell population that may be explored in immunotherapeutic approaches against viral infections or transformed cells.

Cancer immunotherapy is a promising new approach to cancer treatment. It has been demonstrated that a high number of tumor-specific cytotoxic T cells (CTLs) is associated with increased disease-specific survival in lung cancer patients. Identification of superior CTL epitopes from tumor antigens is essential for the development of immunotherapy for malignant tumors. The EML4-ALK fusion gene was recently identified in a subset of non-small cell lung cancers (NSCLCs). In this study we searched for HLA-A 02:01- and HLA-A 24:02‑restricted epitopes derived from EML4-ALK by screening predicted EML4-ALK‑derived candidate peptides for the induction of tumor‑reactive CTLs. Nine EML4-ALK‑derived peptides were selected by a computer algorithm based on a permissive HLA-A 02:01 or HLA-A 24:02 binding motif. One of the nine peptides induced peptide-specific CTLs from human peripheral blood mononuclear cells. We were able to generate a peptide‑specific CTL clone. This CTL clone specifically recognized peptide‑pulsed T2 cells and H2228 cells expressing HLA-A 02:01 and EML4-ALK that had been treated with IFN-γ 48 h prior to examination. CTL activity was inhibited by an anti-HLA‑class I monoclonal antibody (W6/32), consistent with a class I-restricted mechanism of cytotoxicity. These results suggest that this peptide (RLSALESRV) is a novel HLA-A 02:01-restricted CTL epitope and that it may be a new target for antigen-specific immunotherapy against EML4‑ALK-positive cancers.

We previously reported that heat shock protein 105 (HSP105) is overexpressed in a variety of human cancers, including colorectal, pancreatic and esophageal cancer and has proven to be a novel biomarker for the immunohistochemical detection of these cancers. In the present study, we used HLA-transgenic mice (Tgm) and the peripheral blood mononuclear cells (PBMCs) of colorectal cancer patients to identify HLA-A2 and HLA-A24-restricted HSP105 epitopes, as a means of expanding the application of HSP105-based immunotherapy to HLA-A2- or HLA-A24-positive cancer patients. In addition, we investigated by ex vivo IFN-γ ELISPOT assay whether the HSP105-derived peptide of cytotoxic T cells (CTLs) exists in PBMCs of pre-surgical colorectal cancer patients. We found that four peptides, HSP105 A2-7 (RLMNDMTAV), HSP105 A2-12 (KLMSSNSTDL), HSP105 A24-1 (NYGIYKQDL) and HSP105 A24-7 (EYVYEFRDKL), are potential HLA-A2 or HLA-A24-restricted CTL HSP105-derived epitopes. HSP105-specific IFN-γ-secreting T cells were detected in 14 of 21 pre-surgical patients with colorectal cancer in response to stimulation with these four peptides. Our study raises the possibility that these HSP105 peptides are applicable to cancer immunotherapy in patients with HSP105-expressing cancer, particularly colorectal cancer.

Wilms tumor gene (WT1) protein is an attractive target for cancer immunotherapy. We aimed to investigate the feasibility of a combination therapy consisting of gemcitabine and WT1 peptide-based vaccine for patients with advanced pancreatic cancer and to make initial assessments of its clinical efficacy and immunologic response. Thirty-two HLA-A*24:02 patients with advanced pancreatic cancer were enrolled. Patients received HLA-A*24:02-restricted, modified 9-mer WT1 peptide (3 mg/body) emulsified with Montanide ISA51 adjuvant (WT1 vaccine) intradermally biweekly and gemcitabine (1000 mg/m) on days 1, 8, and 15 of a 28-day cycle. This combination therapy was well tolerated. The frequencies of grade 3-4 adverse events for this combination therapy were similar to those for gemcitabine alone. Objective response rate was 20.0% (6/30 evaluable patients). Median survival time and 1-year survival rate were 8.1 months and 29%, respectively. The association between longer survival and positive delayed-type hypersensitivity to WT1 peptide was statistically significant, and longer survivors featured a higher frequency of memory-phenotype WT1-specific cytotoxic T lymphocytes both before and after treatment. WT1 vaccine in combination with gemcitabine was well tolerated for patients with advanced pancreatic cancer. Delayed-type hypersensitivity-positivity to WT1 peptide and a higher frequency of memory-phenotype WT1-specific cytotoxic T lymphocytes could be useful prognostic markers for survival in the combination therapy with gemcitabine and WT1 vaccine. Further clinical investigation is warranted to determine the effectiveness of this combination therapy.

Because WT1 is expressed in leukemia cells, the development of cancer immunotherapy targeting WT1 has been an attractive translational research topic. However, concern of this therapy still remains, since WT1 is abundantly expressed in renal glomerular podocytes. In the present study, we clearly showed that WT1-specific cytotoxic T lymphocytes (CTLs) certainly exerted cytotoxicity against podocytes in vitro; however, they did not damage podocytes in vivo. This might be due to the anatomical localization of podocytes, being structurally separated from circulating CTLs in glomerular capillaries by an exceptionally thick basement membrane.

While many HLA class I molecules interact directly with the peptide loading complex (PLC) for conventional loading of peptides certain class I molecules are able to present peptides in a way that circumvents the PLC components. We investigated micropolymorphisms at position 156 of HLA-A(*)24 allotypes and their effects on PLC dependence for assembly and peptide binding specificities. HLA-A(*)24:06(156Trp) and HLA-A(*)24:13(156Leu) showed high levels of cell surface expression while HLA-A(*)24:02(156Gln) was expressed at low levels in tapasin deficient cells. Peptides presented by these allelic variants showed distinct differences in features and repertoire. Immunoprecipitation experiments demonstrated all the HLA-A(*)24/156 variants to associate at similar levels with tapasin when present. Structurally, HLA-A(*)24:02 contains the residue triad Met97/His114/Gln156 and a Trp156 or Leu156 polymorphism provides tapasin independence by stabilizing these triad residues, thus generating an energetically stable and a more peptide receptive environment. Micropolymorphisms at position 156 can influence the generic peptide loading pathway for HLA-A(*)24 by altering their tapasin dependence for peptide selection. The trade-off for this tapasin independence could be the presentation of unusual ligands by these alleles, imposing significant risk following hematopoietic stem cell transplantation (HSCT).

Cellular immunity is important for the control of CMV infection after allogeneic hematopoietic cell transplantation (Allo-HCT). However, the actual in vivo dynamics of CMV-specific cytotoxic T cell (CMV-CTL) clones are still unclear. We conducted clone monitoring of tetramer(+) CMV-CTLs in HLA-A*2402-positive donor-patient pairs, using a direct single-cell analysis that enabled the simultaneous identification and quantification of CTL clones. Clone dynamics were assessed in three cases with or without CMV reactivation. In Case-1 without CMV reactivation, despite the long-term use of systemic steroid, dominant clones of Donor-1 persisted and remained dominant. The CMV-CTLs at 1 year after Allo-HCT included a high proportion of CD45RA(+)CCR7(-) effector and CD27(-)CD57(+)mature T cells. On the other hand, in Cases-2 and -3 with CMV reactivation, novel clones appeared and became dominant during the follow-up. Their CMV-CTLs included more CD27(+) immature T cells at 1 year after Allo-HCT. With regard to clonotypes, HLA-A*2402-restricted CMV-CTLs tended to select BV7 and BJ1-1 genes for complementarity-determining region 3 (CDR3) of T-cell receptor (TCR)-β. Specific amino-acid sequences of CDR3 of TCR-β were found in each case. Patterns of clone reconstitution and phenotype would be different according to CMV reactivation. In vivo clone monitoring of CMV-CTLs could provide insight into the mechanism of immunological reconstitution following Allo-HCT.

Subgroup B adenovirus serotype 11 (Ad11) occasionally causes fatal infections in immunocompromised patients. The present study describes a novel Ad11 epitope presented by HLA-A*24:02 that could be used for adoptive immunotherapy. Ten synthetic Ad11 hexon protein-derived nonamer peptides that bound to HLA-A*24:02 were selected by a computer algorithm and MHC stabilization assay. Stimulation of peripheral blood mononuclear cells from HLA-A*24:02+ donors with each of these synthetic peptides induced peptide-specific CD8(+) T-cells for three peptides. Testing the reactivity of these peptide-specific CD8(+) T-cells against various target cells confirmed that peptide TYFNLGNKF is naturally processed in Ad11-infected cells and is presented by HLA-A*24:02. Emergence of TYFNLGNKF-specific CD8(+) T-cells coincided with the clearance of adenoviruses in a patient with Ad11 disease. Importantly, TYFNLGNKF-specific CD8(+) T-cells were suggested to be not serotype cross-reactive. The novel HLA-A*24:02-restricted Ad11 epitope could be used for anti-Ad11 adoptive immunotherapy and to monitor immunity to Ad11 using MHC tetramers., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In an attempt to induce cytotoxic T lymphocytes (CTLs) that react to ovarian cancer cells, we isolated a CTL clone that specifically recognizes claudin-1 in an HLA-A*24:02-restricted manner. Naïve CD8(+) T lymphocytes were obtained from a healthy adult donor and stimulated twice in vitro with HLA-modified TOV21G cells that were originally derived from an ovarian clear-cell carcinoma line. The TOV21G modification involved RNAi-mediated gene silencing of intrinsic HLA molecules and lentiviral transduction of a synonymously mutated HLA-A*24:02. Then, cDNA library construction using mRNA extracted from the parental TOV21G cells and subsequent expression cloning were conducted. These experiments revealed that a CTL clone obtained from the bulk culture recognized a minimal epitope peptide RYEFGQALF, which was derived from an autoantigen claudin-1 presented by HLA-A*24:02 molecules. This clone exhibited cytolytic activities against three ovarian cancer cell lines and normal bronchial epithelial cells in an HLA-A*24:02-restricted manner. Our data indicate that HLA-modified cancer cells can be used as an artificial antigen-presenting cell to generate antigen-specific CTLs in a manner restricted by an HLA allele of interest., (Copyright © 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Although adult T-cell leukemia (ATL) has a poor prognosis, successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) in some cases suggests that a cellular immune-mediated strategy can be effective. So far, however, no effective target for anti-ATL immunotherapy has been defined. Here we demonstrated for the first time that human telomerase reverse transcriptase (hTERT) is a promising therapeutic target for ATL, and we developed a novel redirected T-cell-based immunotherapy targeting hTERT. hTERT messenger RNA was produced abundantly in ATL tumor cells but not in steady-state normal cells. Rearranged human leukocyte antigen-A*24:02 (HLA-A*24:02) -restricted and hTERT461-469 nonameric peptide-specific T-cell receptor (TCR) α/β genes were cloned from our previously established cytotoxic T lymphocyte clone (K3-1) and inserted into a novel retroviral TCR expression vector encoding small interfering RNAs for endogenous TCR genes in redirected T cells (hTERT-siTCR vector). Consequently, allogeneic or autologous gene-modified CD8(+) T cells prepared using the hTERT-siTCR vector successfully killed ATL tumor cells, but not normal cells including steady-state hematopoietic progenitors, in an HLA-A*24:02-restricted manner both in vitro and in vivo. Our experimental observations support the development of a novel hTERT-targeting redirected T-cell-based adoptive immunotherapy for ATL patients, especially those for whom suitable allo-HSCT donors are lacking.

We investigated whether HLA-A*24 typing complements screening for HLA-DQ and for antibodies (Abs) against insulin, GAD, IA-2 (IA-2A), and zinc transporter-8 (ZnT8A) for prediction of rapid progression to type 1 diabetes (T1D). Persistently Ab(+) siblings/offspring (n = 288; aged 0-39 years) of T1D patients were genotyped for HLA-DQA1-DQB1 and HLA-A*24 and monitored for development of diabetes within 5 years of first Ab(+). HLA-A*24 (P = 0.009), HLA-DQ2/DQ8 (P = 0.001), and positivity for IA-2A ± ZnT8A (P < 0.001) were associated with development of T1D in multivariate analysis. The 5-year risk increased with the number of the above three markers present (n = 0: 6%; n = 1: 18%; n = 2: 46%; n = 3: 100%). Positivity for one or more markers identified a subgroup of 171 (59%) containing 88% of rapid progressors. The combined presence of HLA-A*24 and IA-2A(+) ± ZnT8A(+) defined a subgroup of 18 (6%) with an 82% diabetes risk. Among IA-2A(+) ± ZnT8A(+) relatives, identification of HLA-A*24 carriers in addition to HLA-DQ2/DQ8 carriers increased screening sensitivity for relatives at high Ab- and HLA-inferred risk (64% progression; P = 0.002). In conclusion, HLA-A*24 independently predicts rapid progression to T1D in Ab(+) relatives and complements IA-2A, ZnT8A, and HLA-DQ2/DQ8 for identifying participants in immunointervention trials.

The mechanisms of MHC allele associations with paroxysmal nocturnal hemoglobinuria (PNH) and its aplastic anemia subtype (AA/PNH) remain unclear. It might be dependent on MHC molecule functional properties, such as a scope and frequency of antigen sampling and presentation. For documented PNH-associated MHC alleles we analyzed current reference databases on MHC molecule-eluted peptide presentation repertoires and searched for a range of presented peptides. MHC class II expression was measured on CD34+ cells and appeared to be increased in PNH patients. Two class I alleles (HLA-A*24:02 and B*18:01) have been previously confirmed to associate with protection and increased risk of AA/PNH, respectively. Their product molecules presented immunodominant epitopes derived from proapoptotic (serine/threonine-protein phosphatase) and antiapoptotic (phospholipase D), respectively, intracellular enzymes dependent on phosphoinositide (PI) content. For total PNH and non-aplastic PNH (n/PNH) subtype-associated DRB1*15:01 and DRB1*04:01 class II molecules presentation of exceptionally broad arrays of their own peptide fragments has been found. We conclude that self antigen peptides presented with high frequency in the context of MHC molecules of increased expression may be involved in the immune recognition and the regulation of HSC in the periphery. The block in the normal plasma membrane PI production due to the PIG-A mutation can help explain the differences in the activation of intracellular regulatory pathways observed between PNH and normal HSC. This is evident in the variation in MHC association patterns and peptide presentation repertoires between these two groups of patients.

Many cancer-testis antigen genes have been identified; however, few human leukocyte antigen (HLA)-A24-restricted cytotoxic T cell (CTL) epitope peptides are available for clinical immunotherapy. To solve this problem, novel tools increasing the efficacy and accuracy of CTL epitope detection are needed. In the present study, we utilized a highly active dendritic cell (DC)-culture method and an in silico HLA-A24 peptide-docking simulation assay to identify novel CTL epitopes from MAGE-A6 and MAGE-A12 antigens. The highly active DCs, called α-type-1 DCs, were prepared using a combination of maturation reagents to produce a large amount of interleukin-12. Meanwhile, our HLA-A24 peptide-docking simulation assay was previously demonstrated to have an obvious advantage of accuracy over the conventional prediction tool, bioinformatics and molecular analysis section. For CTL induction assays, peripheral blood mononuclear cells derived from six cases of HLA-A24(+) melanoma were used. Through CTL induction against melanoma cell lines and peptide-docking simulation assays, two peptides (IFGDPKKLL from MAGE-A6 and IFSKASEYL from MAGE-A12) were identified as novel CTL epitope candidates. Finally, we verified that the combination of the highly active DC-culture method and HLA-A24 peptide-docking simulation assay might be tools for predicting CTL epitopes against cancer antigens.

Targeting of tumor angiogenesis with vaccines is a potentially valuable approach to cancer treatment. Elpamotide is an immunogenic peptide derived from vascular endothelial growth factor receptor 2, which is expressed at a high level in vascular endothelial cells. We have now carried out a phase I study to evaluate safety, the maximum tolerated dose, and potential pharmacodynamic biomarkers for this vaccine. Ten HLA-A*24:02-positive patients with advanced refractory solid tumors received elpamotide s.c. at dose levels of 0.5, 1.0, or 2.0 mg once a week on a 28-day cycle. Five patients experienced an injection site reaction of grade 1 and 2, which was the most frequent adverse event. In the 1.0 mg cohort, one patient experienced proteinuria of grade 1 and another patient developed both hypertension and proteinuria of grade 1. No adverse events of grade 3 or higher were observed, and the maximum tolerated dose was therefore not achieved. The serum concentration of soluble vascular endothelial growth factor receptor 2 decreased significantly after elpamotide vaccination. Microarray analysis of gene expression in PBMCs indicated that several pathways related to T cell function and angiogenesis were affected by elpamotide vaccination, supporting the notion that this peptide induces an immune response that targets angiogenesis in the clinical setting. In conclusion, elpamotide is well tolerated and our biomarker analysis indicates that this anti-angiogenic vaccine is biologically active. Clinical trial registration no. UMIN000008336., (© 2012 Japanese Cancer Association.)

Human breast cancer-associated antigen, mammaglobin-A (Mam-A), potentially offers a novel therapeutic target as a breast cancer vaccine. In this study, we define the CD8(+) cytotoxic T lymphocyte (CTL) response to Mam-A-derived candidate epitopes presented in the context of HLA-A24 (A*2402). HLA-A24 has a frequency of 72% in Japanese, 27% in Asian Indian, and 18% in Caucasian populations. Using a human leukocyte antigen (HLA)-binding prediction algorithm we identified 7 HLA-A24-restricted Mam-A-derived candidate epitopes (MAA24.1-7). Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-A2402 (T2.A24) indicated that MAA24.2 (CYAGSGCPL) and MAA24.4 (ETLSNVEVF) have the highest HLA-A24 binding affinity. Further, 2 CD8(+) CTL cell lines generated in vitro against T2.A24 cells individually loaded with Mam-A-derived candidate epitopes demonstrated significant cytotoxic activity against MAA24.2 and MAA24.4. In addition, the same CD8(+) CTL lines lysed the HLA-A24(+)/Mam-A(+) stable transfected human breast cancer cell lines AU565 and MDA-MB-361. However, these CTLs had no cytotoxicity against HLA-A24(-)/Mam-A(+) and HLA-A24(+)/Mam-A(-) breast cancer cell lines. In summary, our results define HLA-A24-restricted, Mam-A-derived, CD8(+) CTL epitopes that can potentially be employed for Mam-A-based breast cancer vaccine therapy to breast cancer patients with HLA-A24 phenotype., (Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Human T-lymphotropic virus-1 (HTLV-1) causes HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia-lymphoma in individuals with dysfunctional immune responses. In this study, to characterize the HTLV-1-specific cytotoxic T lymphocyte (CTL) populations in asymptomatic HTLV-1 carriers (ACs), HAM/TSP patients, and carriers with autoimmune disorders (CAIDs), we examined the role of programmed death-1 and its ligand (PD-1/PD-L1) in HTLV-1-specific CTL functions using an HTLV-1 Tax/HLA-A*0201 tetramer and an HTLV-1 Tax/HLA-A*2402 tetramer. Interestingly, the percentage of HTLV-1 Tax301-309/HLA-A*2402 tetramer(+)CD8(+) cells expressing PD-1 in ACs was significantly higher than the percentage of HTLV-1 Tax11-19/HLA-A*0201 tetramer(+)CD8(+) cells expressing PD-1. PD-1 expression was significantly downregulated on HTLV-1-specific CTLs in HAM/TSP compared with ACs. PD-L1 expression was observed in a small proportion of unstimulated lymphocytes from ACs and was greater in ACs than in HAM/TSP and CAIDs after short-term culture. Furthermore, CTL degranulation was impaired in HAM/TSP, whereas anti-PD-L1 blockade significantly increased CTL function in ACs. Downregulation of PD-1 on HTLV-1-specific CTLs and loss of PD-L1 expression in HAM/TSP and CAIDs, along with impaired function of HTLV-1-specific CTLs in HAM/TSP, may underlie the apparently dysfunctional immune response against HTLV-1., (Copyright © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)