Impact of MHC class I alleles on the M. tuberculosis antigen-specific CD8+ T-cell response in patients with pulmonary tuberculosis

Challenged by scattered understanding of protective immunity to Mycobacterium tuberculosis (MTB), we have mapped peptide epitopes to human leukocyte antigen (HLA)-A*0101, A*0201, A*1101, A*2402, B*0702, B*0801 and B*1501 of the secreted mycobacterial antigen Ag85B, a vaccine candidate that may be associated with immune protection. Affinity (ED(50)) and half-life (t(1/2), off-rate) analysis for individual peptide species on HLA-A and HLA-B molecules revealed binding ranges between 10(-3) and 10(-7) M. After selection of the best matches, major histocompatibility complex class I/peptide tetramer complexes were constructed to measure the CD8+ T-cell responses directly ex vivo in peripheral blood mononuclear cells (PBMC) derived from 57 patients with acute pulmonary tuberculosis. Three patterns of (allele-) specific CD8+ recognition were identified: (a). Focus on one dominant epitope with additional recognition of several subdominant T-cell epitopes (HLA-A*0301, A*2402, B*0801 and B*1501); (b). Co-dominant recognition of two distinct groups of peptides presented by HLA-B*0702; and (c). Diverse and broad recognition of peptides presented by HLA-A*0201. Peptides that bound with slow off-rates to class I alleles, that is HLA-A*0201, were associated with low frequency of CD8+ T cells in PBMCs from patients with tuberculosis. HLA-B alleles showed fast off-rates in peptide binding and restricted high numbers (up to 6%) of antigen-specific CD8+ T cells in patients with pulmonary tuberculosis.     

Identification of an HLA-A*0201-restricted T-cell epitope on the MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis, by MPT51 overlapping peptide screening

CD8+ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8+ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-gamma) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-gamma by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-gamma and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells confirmed that this epitope binds to HLA-A*0201. The T cells were capable of lysing MPT51 p53-62 peptide-pulsed T2 cells. In addition, MPT51 p53-62-specific memory CD8+ T cells were found in tuberculin skin test-positive HLA-A*0201+ healthy individuals. Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible.     

A simplified PCR-SSP method for HLA-A2 subtype in a population of Wuhan, China

HLA-A2 is the most frequent HLA-A allele in all ethnic populations, and an important restriction element for peptide presentation to T cells in infectious disease and cancer. However, the HLA-A2 supertype consisting of up to 75 subtypes, mutation studies and analyses using cytotoxic T lymphocytes suggest the functional relevance of subtype-specific differences in HLA-A2 molecules for peptide binding and T-cell recognition. Therefore, it is necessary for T-cell response study to discriminate the HLA-A2 subtypes and to understand the profile of HLA-A2 allellc distribution in a given population. In this study, we developed a simple, robust approach based on the nested polymerase chain reaction using sequence-specific primers （PCR-SSP） to discriminate 17 HLA-A2 subtypes which cover the most HLA-A2 alleles （〉 99% allele frequency） reported in Chinese, using 15 combinations of 19 allelic specific primers. In the first round of PCR, 3 combinations of 5 primers were used to determine whether the tested sample was HLA-A2 positive, meanwhile the subtypes of HLA-A＊0209 and HLA-A＊0215N were determined for the variant position of these two subtypes is in exon 4 instead of exon 2, 3. Samples of HLA-A2 positive were subtyped in the second round of PCR, using PCR products of the first round as templates. This strategy was applied to test the samples of 78 random HLA-A2 positive individuals for their HLA-A2 subtypes. Those samples were screened for HLA-A2 positive by the first round PCR-SSP from 154 healthy blood donors in Wuhan, China. The subtyping results were verified by using flow cytometric analysis （FCM） with HLA-A2 specific monoclonal antibody BB7.2 and DNA sequencing. The typing results of the samples show 50.7% random individuals in the population carry HLA-A2, HLA-A＊0201 ranks the first （allele frequency = 15.5%）, followed by A＊0207 （5.8%）, A＊0206 （4.7%）, A＊0203 （2.6%）, A＊0210 （0.7%）, and these 5 alleles account for 99.0% HLA-A2 subtypes of allele frequency. Our study indicates that the developed typing method is simple and reliable for HLA-A2 subtyping in Chinese, and the profile of allelic distribution of HLA-A2 subtypes is revealed in the population of Wuhan, China.     

Direct measurement of CD4+ and CD8+ T-cell responses to CMV in HIV-1-infected subjects

Data from murine models of chronic viral infection suggest that CD4+ T-cell responses to viral pathogens are important in sustaining the number and/or function of CD8+ cytotoxic T-cell (CTL) effectors. In this study, we used cytokine flow cytometry (CFC), staining with HLA-A*0201-peptide tetramers, and peptide stimulation with epitopic peptides to study functional CD4+ and CD8+ T-cell responses to cytomegalovirus (CMV) in human subjects coinfected with CMV and the human immunodeficiency virus, type 1 (HIV-1). We show that strong CD4+ and CD8+ T-cell responses to CMV antigens are sustained over time in HIV-1-infected individuals. Those who maintain a strong CD4+ T-cell response to CMV are also likely to maintain higher frequencies of CD8+ T cells capable of binding to HLA-A*0201-CMV pp65 (A2-pp65) tetramers as well as responses to pp65 peptide stimulation with effector cytokine production. These data support the hypothesis that declines in frequencies of CD4+ T-cell responses to CMV are associated with an inability to sustain high levels of CMV-specific CD8+ T-cell responses in HIV-1-infected subjects. These declines may precede the onset of CMV-associated end organ disease.     

Preparation and Identification of HLA-A＊1101 Tetramer Loading with Human Cytomegalovirus pp65 Antigen Peptide

MHC/peptide tetramer technology has been widely used to study antigen-specific T cells, especially for identifying virus-specific CD8^＋ T cells in humans. The tetramer molecule is composed of HLA heavy chain, β2-microglobulin （β2m）, an antigenic peptide, and fluorescent-labeled streptavidin. To further investigate the HLA-A＊1101-restricted CD8^＋ T cell responses against human cytomegalovirus （HCMV）, we established an approach to prepare HLA-A＊1101 tetramer complexed with a peptide from HCMV. The cDNA encoding HLA-A＊1101 heavy chain was cloned and the prokaryotic expression vector for the ectodomain of HLA-A＊1101 fused with a BirA substrate peptide （HLA-A＊1101-BSP） at its carboxyl terminus was constructed. The fusion protein was highly expressed as inclusion bodies under optimized conditions in Escherichla coli. Moreover, HLA-A＊1101-BSP protein was refolded in the presence of β2m and an HCMV peptide pp6516.24 （GPISGHVLK, GPI）. Soluble HLA-A＊1101-GPI monomer was biotinylated and purified to a purity of 95%, which was subsequently combined with streptavidin to form tetramers at a yield of 〉 80%. The HLA-A＊1101-GPI tetramers could bind to virus-specific CD8^＋ T cells, suggesting soluble HLA-A＊1101-GPI tetramers were biologically functional. This study provides the basis for further evaluation of HLA-A＊1101-restricted CD8^＋ T cell responses against HCMV infection.     

HLA-A*0201-restricted cytotoxic T-cell epitopes in three PE/PPE family proteins of Mycobacterium tuberculosis

CD8⁺ T cells are thought to play an important role in protective immunity against tuberculosis. We report the identification of three peptides derived from Rv1818c, Rv3812 and Rv3018c proteins of Mycobacterium tuberculosis that bound to HLA-A*0201 molecules and their ability to induce in vitro T-cell response in peripheral blood lymphocytes from HLA-A*0201-positive healthy individuals (PPD+) and patients with TB. The peptide-specific cytotoxic T lymphocytes (CTL) generated were capable of recognizing peptide pulsed targets. Three 9-mer peptides bound with high affinity to HLA-A*0201 and displayed low dissociation rates of the bound peptide from HLA. Epitope-specific recognition was demonstrated by the release of perforin and γ-interferon. Overall, our results demonstrate the presence of HLA class I-restricted CD8⁺ CTL against proteins from PE and PPE proteins of M. tuberculosis and identify epitopes that are strongly recognized by HLA-A*0201-restricted CD8⁺ T cells in humans. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

HLA-A*0206-BSP和-A*0207-BSP融合基因的构建与表达

采用RT-PCR技术从HLA-A*0206和-A*0207阳性个体的PBMC中分别克隆出HLA-A*0206和-A*0207基因的全长cDNA序列,构建HLA-A*0206和-A*0207克隆载体。再利用PCR技术从构建的克隆载体中扩增HLA-A*0206和-A*0207的α链(重链)胞外段序列,分别经双酶切置换本室保存的HLA-A*0201-BSP重组体中的HLA-A*0201胞外段序列,使HLA-A*0206和-A*0207与BirA酶底物肽(BirA substrate peptide,BSP)序列融合,构建HLA-A*0206-BSP和-A*0207-BSP融合基因的表达载体,经限制性酶切和DNA测序证实。然后将该表达载体转化E.coliBL21(DE3)后获得表达产物,通过体外稀释复性,初步纯化的表达产物通过ELISA和Western blot检测证明能够与β2微球蛋白(HLA I类分子轻链)及HLA-A2限制性抗原肽(HBV core 18-27)折叠形成具有HLA I类分子天然构象的抗原肽/HLA-A2复合物单体。为进一步构建HLA-A*0206和-A*0207四聚体,探讨相应HLA-A2亚型的功能特点提供了物质基础。     

Heterogeneity of HLA-A2 in Asia-Oceanic populations

HLA-A2 is one of the most common yet most diversified HLA antigens with 17 subtypes so far identified at the molecular level. A2 subtyping may have significant impact on clinical medicines. We developed a PCR/SSO-based comprehensive typing protocol for HLA-A and investigated the distribution of A2 alleles in regional ethnic groups. A2 was detected with high frequencies in most study populations. A total of 480 A2+ samples were identified and subtyped. The gene frequencies of A2 ranged from 34% in Chinese, 29% in Australian Caucasoids, 21% in Polynesians, 14% in Javanese and 13% in Australian Aborigines. However, in Melanesians and Micronesians A2 was absent. Six A2 alleles were found in the present experiments including A^*0201, 0203, 0205, 0206, 0207 and 0210. In Aborigines all the A2+ donors were typed as 0201. In Caucasoids A^*0201 accounted for 95% of A2+ samples though other three subtypes A^*0203, 0205 and 0207 were also detected. Extraordinary A2 heterogeneity was observed in Asia-Pacific populations where A^*0201 has become a minority. In Chinese all the six A2 alleles were discovered with A^*0201, 0203, 0206 and 0207 as the four major ones. In Javanese A2 was equally divided into A^*0201, 0203 and 0206 while in Polynesians A2 is overwhelmingly dominated by the oriental A^*0206 (71%). Our study also showed that comprehensive DNA matching for A2 would eliminate most A mismatches in the unrelated-donor transplantation in study populations.     

Identification of an HLA-A*0201-restrictive CTL epitope from MUC4 for applicable vaccine therapy

Recent research has indicated that MUC4 plays an important role in the development of many tumors and may prove useful as a novel cancer immunotherapy target. We aimed to identify HLA-A*0201-restrictive cytotoxic T lymphocyte (CTL) epitopes of the cancer-associated antigen MUC4. The MUC4 sequence was scanned for immunogenic peptides using HLA-binding prediction software. Dendritic cells (DCs) from peripheral blood mononuclear cells (PBMCs) were induced by cytokines. Five possible CTL epitopes were selected by software analysis, synthesized, and used to pulse mature DCs. The CD8⁺ T cells from PBMCs from an HLA-A*0201 healthy donor were stimulated with autologous MUC4-peptide-loaded DCs and expanded in vitro. T cell activation was assessed by ELISPOT, and cytotoxicity was determined by ⁵¹chromium (⁵¹Cr)-release assays. Our results show that CTLs induced by peptide P01204 could lyse T2 cells pulsed with peptide P01204 and HCT-116 cells (MUC4⁺, HLA-A2⁺). Compared with a control peptide, P01204 increased the number of IFN-γ producing T cells. Overall, these results suggest that P01204 is a novel HLA-A*0201-restrictive CTL epitope of the cancer-associated antigen MUC4. This will provide a foundation for the development of tumor-specific peptide vaccines.     

Cytotoxic T-lymphocyte clones, established by stimulation with the HLA-A2 binding p5365-73 wild type peptide loaded on dendritic cells In vitro, specifically recognize and lyse HLA-A2 tumour cells overexpressing the p53 protein

Mutations in the tumour suppressor gene p53 are among the most frequent genetic alterations in human malignancies, often associated with an accumulation of the p53 protein in the cytoplasm. We have generated a number of cytotoxic T lymphocyte (CTL) clones that specifically recognize the HLA-A*0201 p53 wild type peptide RMPEAAPPV [65-73], designated R9V, by the in vitro stimulation of CD8 enriched peripheral blood lymphocytes from a healthy HLA-A*0201 donor using peptide loaded autologous dendritic cells. A total of 22 CTL clones were generated from the same bulk culture and demonstrated to carry identical T-cell receptors. The CTL clone, 2D9, was shown to specifically lyse the HLA-A*0201+ squamous carcinoma cell line SCC9 and the breast cancer cell line MDA-MB-468. Our data demonstrate that human peripheral blood lymphocytes from normal healthy individuals comprise T cells capable of recognizing p53 derived wild type (self) peptides. Furthermore, the capacity of R9V specific T cell clones to exert HLA restricted cytotoxicity, argues that the R9V peptide is naturally presented on certain cancer cells. This supports the view that p53 derived wild type peptides might serve as candidate target antigens for the immunotherapeutic treatment of cancer.     

Identification of cytotoxic T cell epitopes within Epstein-Barr virus (EBV) oncogene latent membrane protein 1 (LMP1): evidence for HLA A2 supertype-restricted immune recognition of EBV-infected cells by LMP1-specific cytotoxic T lymphocytes

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) and latent membrane proteins (LMP) are the only antigens consistently expressed in malignancies such as nasopharyngeal carcinoma (NPC) and Hodgkin's disease (HD). Since EBNA1 is not recognized by EBV-specific cytotoxic T lymphocytes (CTL), there is increasing interest in the identification of the potential target epitopes within LMP1. Although LMP1-specific CTL have been isolated from seropositive individuals, earlier attempts to identify the peptide epitopes recognized by these T cells have been unsuccessful. In the present report we used a novel protocol to identify CTL epitopes within LMP1 which can be recognized by both polyclonal and clonal CTL. Firstly, a computer-based program was employed to identify the potential HLA-binding peptides within LMP1. Polyclonal CD8+ CTL were then isolated from seropositive donors that recognized the peptide epitopes YLLEMLWRL and YLQQNWWTL from LMP1 in association with HLA A2. Limiting dilution analysis of the memory CTL response revealed that the LMP1-specific CTL response constitutes a minor component of the CTL response in healthy virus carriers. Interestingly, analysis of YLLEMLWRL-specific CTL revealed that these CTL were able to lyse EBV-infected B cells expressing different HLA A2 supertype alleles including A*0201, A*0202, A*0203, A*0204, A*0206, A*6802 and A*6901. These data strongly support the notion that HLA class I supertype-restricted CTL may be of significant use in the development of peptide-based immunotherapeutics against EBV-associated malignancies in different ethnic populations.     

CTL识别的HLA-A2限制性人卵巢癌相关抗原OVA66表位的鉴定

目的：鉴定CTL识别的HLA—A2限制性人卵巢癌相关抗原OVA66表位。方法：以细胞因子从外周血单个核细胞(PBMC)中诱导树突状细胞(DC)，通过形态学观察和流式细胞术进行鉴定。用表位预测法选取并合成两种肽分子，分别脉冲成熟的DC，并刺激HLA—A2^ 健康人自体CD8^ T细胞，1wk后，用脉冲肽的自体PBMC以每7d的间隔刺激该CD8^ T细胞3次。以共接受4次抗原肽刺激的T细胞作为CTL，用乳酸脱氢酶(LDH)释放试验，检测CTL对靶细胞的杀伤效应。用酶联免疫斑点法(ELISPOT)．检测CTL中抗原特异性分泌IFN-γ的T细胞数。结果：形态学和流式细胞术的结果显示．PBMC可诱生成熟的DC。肽1235(FLPDHINIV)诱导的CTL．可特异性杀伤1235脉冲的T2细胞和OVA66^ 、HLA—A2^ 的SW480细胞，且L235诱导的特异性分泌IFN-γ的T细胞数增加。结论：卵巢癌相关抗原OVA66的HLA—A2限制性CTL表位1235．能激发对肿瘤抗原的特异性免疫应答，为制备肿瘤特异性肽疫苗奠定了实验基础。     

Differential binding of viral peptides to HLA-A2 alleles. Implications for human papillomavirus type 16 E7 peptide-based vaccination against cervical carcinoma

Several cancer immune intervention protocols aim at inducing T cell immunity against antigens presented by HLA-A2, the most common human MHC class I molecule. In the context of HLA-A*0201, we previously identified two cytotoxic T lymphocyte epitopes (E7(11-20) and E7(86-93)) encoded by the human papillomavirus type 16 E7 (HPV16 E7) oncoprotein, which is a tumor-specific antigen for cervical carcinoma. This study reports that the two HPV16 epitopes and a control hepatitis B virus epitope bind equally well to five HLA-A2 alleles (A*0201, A*0202, A*0203, A*0204, and A*0209). These HLA-A2 variants display comparable binding characteristics in accordance with the A2 supertype (M. F. Del Guercio et al., J. Immunol. 1995. 154: 685-693). Cervical carcinoma patients expressing these alleles may benefit from vaccination with the two HPV16 E7 peptides. In contrast, none of the peptides tested bound to A*0207 or A*0208, whereas heterogeneous binding was observed for A*0205 and A*0206. Therefore, the amino acid substitutions that discriminate these HLA-A2 variants from A*0201 affect antigen presentation. Taken together, our findings have implications for application of the A2 supertype concept and for vaccination with A*0201-binding peptides, in particular HPV16 E7 peptides.     

Identification of a new HLA-A*0201-restricted CD8+ T cell epitope from hepatocellular carcinoma-associated antigen HCA587

For the development of peptide-based cancer immunotherapies, we aimed to identify specific HLA-A*0201-restricted CTL epitopes in hepatocellular carcinoma (HCC) associated antigen HCA587, which has been identified as a member of the cancer/testis (CT) antigens highly expressed in HCC. We first combined the use of an HLA-A*0201/peptide binding algorithm and T2 binding assays with the induction of specific CD8(+) T cell lines from normal donors by in vitro priming with high-affinity peptides, then IFN-gamma release and cytotoxicity assays were employed to identify the specific HLA-A*0201 CD8(+) T cell epitope using peptide-loaded T2 cells or the HCA587 protein(+) HCC cell line HepG2. In the six candidate synthesized peptides, two peptides showed higher binding ability in T2 binding assays. No. 2 peptide, encompassing amino acid residues FLAKLNNTV (HCA587(317-325)), was able to activate a HCA587-specific CD8(+) T-cell response in human lymphocyte cultures from two normal donors and two HCC patients, and these HCA587-specific CD8(+) T cells recognized peptide-pulsed T2 cells as well as the HCA587 protein(+) HCC cell line HepG2 in IFN-gamma release and cytotoxicity assays. The results indicate that no. 2 peptide is a new HLA-A*0201-restricted CTL epitope capable of inducing HCA587-specific CTLs. Our data suggest that identification of this new HCA587/HLA-A*0201 peptide FLAKLNNTV may facilitate the design of peptide-based immunotherapies for the treatment of HCA587-bearing HCC patients.     

Preparation and Characterization of HLA-A＊0201 Tetramer Loaded with IE-1316.324 Antigenic Peptide of Human Cytomegalovirus

Major histocompatibility complex （MHC） class I tetramer technology has become the central technique for analyzing antigen-specific CD8^＋ T cell responses and it has been widely used to explore the differentiation and formation of memory CD8^＋ T cells. Previously, a simplified and efficient procedure for preparing high quality HLA-A＊0201 tetramers has been established in our lab and the tetramers loaded with HCMV peptide pp6549s.50a has been successfully applied to investigate HCMV-specific CD8^＋ T cells in Chinese populations. Using similar procedure we reported here the construction of HLA-A＊0201 tetramer loaded with another dominant epitope derived from immediate early （IE）-1 316.324 （VLEETSVML, VLE） of HCMV （A2-VLE） and characterization of this tetramer. After A2-VLE monomer was prepared and purified, its tetramer was then formed at a yield of 83%. The optimized amount of A2-VLE tetramer for staining 100 μl whole blood was 0.5 μg with incubation at 4℃ for 1 h. Furthermore, the dissociation constant of the tetramer binding to the specific CD8^＋ T cells of one HLA-A2^＋ donor was estimated to be 32.7 nmol/L, which is markedly higher than that of MHC monomer. The construction of A2-VLE tetramer provides an alternative choice for investigating HCMV-specific CD8^＋ T cell responses and will deepen our understanding of the differentiation and formation of HCMV-specific memory CD8^＋ T cells. Cellular ＆ Molecular Immunology.     

High frequencies of functionally impaired cytokeratin 18-specific CD8+ T cells in healthy HLA-A2+ donors

Combining cell surface phenotyping with functional analysis, human CD8+ T cells have been divided into several subsets which are being studied extensively in diverse physiological situations, such as viral infection, cancer and ageing. In particular, so-called terminally differentiated effector cells possess a CD45RA+ CCR7- CD27- CD28- phenotype, contain perforin and, in different models, have been shown to exert direct ex vivo killing and to release interleukins upon both antigen-nonspecific and -specific stimulation. Using HLA class I multimers, we have identified a high frequency of peripheral CD8+ T cells that recognize a peptide derived from the self protein cytokeratin 18 presented by the HLA-A*0201 molecule. These cells can be detected in approximately 15% of the HLA-A2-positive healthy donors tested. A detailed analysis revealed that they must have divided extensively in vivo, have an effector cell phenotype and express various natural killer cell-associated receptors. Interestingly, however, they remained unresponsive to antigen-specific stimulation in vitro in terms of cytotoxicity and cytokine secretion. Thus, cytokeratin 18-specific cells constitute a frequently encountered, new CD8+ T lymphocyte subpopulation without classical effector status and with so far unknown function.     

The use of HLA-A*0201-transfected K562 as standard antigen-presenting cells for CD8(+) T lymphocytes in IFN-gamma ELISPOT assays.

ELISPOT assays are increasingly used for a direct detection and quantification of single antigen-specific T cells in freshly isolated peripheral blood mononuclear cells (PBMC). They are particularly attractive for the monitoring of specific T lymphocyte responses in clinical trials assessing antigen-specific immunizations in patients with cancer or chronic viral infections. However, one major limitation for the broad clinical implementation of ELISPOT assays is the lack of an inexhaustible source of suitable HLA-matched antigen-presenting cells (APC). Currently available allogeneic or xenogeneic APC (such as the human lymphoid hybrid T2 or HLA-transfected insect cells) can either lead to strong background spot production by APC-reactive T lymphocytes or have a low antigen presentation capability. Both phenomena can prevent the detection of low frequency T cell responses in PBMC. In search of alternative APC for ELISPOT assays, the human chronic myelogenous leukemia cell line K562 that per se does not express HLA class I and class II molecules on the cell surface was transfected with the HLA-A*0201 gene. Clonal HLA-A*0201-expressing K562 (K562/A*0201) cells were able to process and present endogenously expressed and exogenously loaded melanoma peptide antigens to HLA-A*0201-restricted cytolytic T lymphocyte clones in cytotoxicity and IFN-gamma ELISPOT assays. K562/A*0201 cells were then used as APC in IFN-gamma spot assays to detect ex vivo CD8(+) T lymphocytes responsive to known HLA-A*0201-binding peptide epitopes derived from cytomegalovirus, Epstein-Barr virus, influenza virus and melanoma in PBMC from HLA-A*0201-positive donors. In the majority of cases, peptide-pulsed K562/A*0201 cells were similarly efficient in the ability to visualize single antigen-specific CD8(+) T lymphocytes when compared to T2 cells. However, in contrast to T2, background reactivity of CD8(+) T cells responsive to unpulsed K562/A*0201 was regularly found to be negligible, thereby enhancing the sensitivity of the ELISPOT assay, particularly in donors with strong anti-T2 reactivity. K562 cells transfected with HLA-A*0201 or other HLA genes can serve as standard APC for monitoring T lymphocyte responses against tumor and viral peptide antigens.     

Prediction of HLA class I-restricted T-cell epitopes of islet autoantigen combined with binding and dissociation assays

Identification of cognate peptides recognized by human leucocyte antigen (HLA)/T cell receptor (TCR) complex provides insight into the pathogenic process of type 1 diabetes (T1D). We hypothesize that HLA-binding assays alone are inadequate metrics for the affinity of peptides. Zinc transporter-8 (ZnT8) has emerged in recent years as a novel, major, human autoantigen. Therefore, we aim to identify the HLA-A2-restricted ZnT8 epitopes using both binding and dissociation assays. HLA class I peptide affinity algorithms were used to predict candidate ZnT8 peptides that bind to HLA-A2. We analyzed 15 reported epitopes of seven β-cell candidate autoantigens and eight predicted candidate ZnT8 peptides using binding and dissociation assays. Using IFN-γ ELISpot assay, we tested peripheral blood mononuclear cells (PBMCs) from recent-onset T1D patients and healthy controls for reactivity to seven reported epitopes and eight candidate ZnT8 peptides directly ex vivo. We found five of seven recently reported epitopes in Chinese T1D patients. Of the eight predicted ZnT8 peptides, ZnT8(153-161) had a strong binding affinity and the lowest dissociation rate to HLA-A*0201. We identified it as a novel HLA-A*0201-restricted T-cell epitope in three of eight T1D patients. We conclude that ZnT8(153-161) is a novel HLA-A*0201-restricted T-cell epitope. We did not observe a significant correlation (P = 0.3, R = - 0.5) between cytotoxic T cell (CTL) response and peptide/HLA*0201 complex stability. However, selection of peptides based on affinity and their dissociation rate may be helpful for the identification of candidate CTL epitopes. Thus, we can minimize the number of experiments for the identification of T-cell epitopes from interesting antigens.     

Dendritic Cells Engineered to Express Defined Allo-HLA Peptide Complexes Induce Antigen-specific Cytotoxic T Cells Efficiently Killing Tumour Cells

Most tumour-associated antigens (TAA) are non-mutated self-antigens. The peripheral T cell repertoire is devoid of high-avidity TAA-specific cytotoxic T lymphocytes (CTL) due to self-tolerance. As tolerance is major histocompatibility complex-restricted, T cells may be immunized against TAA presented by a non-self human leucocyte antigen (HLA) molecule and transferred to cancer patients expressing that HLA molecule. Obtaining allo-restricted CTL of high-avidity and low cross-reactivity has, however, proven difficult. Here, we show that dendritic cells transfected with mRNA encoding HLA-A*0201, efficiently present externally loaded peptides from the antigen, Melan-A/MART-1 to T cells from HLA-A*0201-negative donors. CD8⁺ T cells binding HLA-A*0201/MART-1 pentamers were detected already after 12 days of co-culture in 11/11 donors. The majority of cells from pentamer⁺ cell lines were CTL and efficiently killed HLA-A*0201⁺ melanoma cells, whilst sparing HLA-A*0201⁺ B-cells. Allo-restricted CTL specific for peptides from the leukaemia-associated antigens CD33 and CD19 were obtained with comparable efficiency. Collectively, the results show that dendritic cells engineered to express defined allo-HLA peptide complexes are highly efficient in generating CTL specifically reacting with tumour-associated antigens.     

Lack of tumor recognition by hTERT peptide 540-548-specific CD8(+) T cells from melanoma patients reveals inefficient antigen processing

Telomerase is a ribonucleoprotein complex responsible for the maintenance of the length of the telomeres during cell division, which is active in germ-line cells as well as in the vast majority of tumors but not in most normal tissues. The wide expression of the human telomerase catalytic subunit (hTERT) in tumors makes it an interesting candidate vaccine for cancer. hTERT-derived peptide 540-548 (hTERT(540)) has been recently shown to be recognized in an HLA-A*0201-restricted fashion by T cell lines derived from peptide-stimulated peripheral blood mononuclear cells (PBMC) from healthy donors. As a first step to the inclusion of this peptide in immunotherapy clinical trials, it is crucial to assess hTERT(540)-specific T cell reactivity in cancer patients as well as the ability of hTERT-specific CD8(+) T lymphocytes to recognize and lyse hTERT-expressing target cells. Here, we have analyzed the CD8(+) T cell response to peptide hTERT(540) in HLA-A*0201 melanoma patients by using fluorescent HLA-A*0201/hTERT(540) peptide tetramers. HLA-A*0201/hTERT(540) tetramer(+) CD8(+) T cells were readily detected in peptide-stimulated PBMC from a significant proportion of patients and could be isolated by tetramer-guided cell sorting. hTERT(540)-specific CD8(+) T cells were able to specifically recognize HLA-A*0201 cells either pulsed with peptide or transiently transfected with a minigene encoding the minimal epitope. In contrast, they failed to recognize hTERT-expressing HLA-A*0201(+) target cells. Furthermore, in vitro proteasome digestion studies revealed inadequate hTERT processing. Altogether, these results raise questions on the use of hTERT(540) peptide for cancer immunotherapy.