Identification of a new HLA-A*0201-restricted cytotoxic T lymphocyte epitope from CML28

Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. CML28, a recently discovered cancer-testis (CT) antigen from chronic myelogenous leukemia, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A*0201 is one of the most common histocompatibility molecule in Chinese, we aim at identifying CML28 peptides presented by HLA-A*0201. A panel of CML28-derived antigenic peptides was predicted using a computer-based program. Four peptides with highest predicted score were synthesized and tested for their binding affinities to HLA-A*0201 molecule. Then these peptides were assessed for their immunogenicity to elicit specific immune responses mediated by CTLs both in vitro, from PBMCs sourced from four healthy HLA-A*0201⁺ donors, and in vivo, in HLA-A*0201 transgenic mice. One of the tested peptides, CML28_(173–181), induced peptide-specific CTLs in vitro as well as in vivo, which could specifically secrete IFN-γ and lyse major histocompatibility complex (MHC)-matched tumor cell lines endogenously expressing CML28 antigen and CML28_(173–181) pulsed Jurkat-A2/Kb cells, respectively. These results demonstrate that CML28_(173–181) is a naturally processed and presented CTL epitope with HLA-A*0201 motif and has a promising immunogenicity both in vitro and in vivo. As CML28 is expressed in a large variety of histological tumors besides chronic myelogenous leukemia, we propose that the newly identified epitope, CML28_(173–181), would be of potential use in peptide-based, cancer-specific immunotherapy against a broad spectrum of tumors.     

Identification of a novel HLA-A2-restricted cytotoxic T lymphocyte epitope from cancer-testis antigen PLAC1 in breast cancer

Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor antigens is essential for the development of peptide vaccines against tumor immunotherapy. Among all the tumor antigens, the caner-testis (CT) antigens are the most widely studied and promising targets. PLAC1 (placenta-specific 1, CT92) was considered as a novel member of caner-testis antigen, which expressed in a wide range of human malignancies, most frequently in breast cancer. In this study, three native peptides and their analogues derived from PLAC1 were predicted by T cell epitope prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Binding affinity and stability assays in T2 cells showed that two native peptides, p28 and p31, and their analogues (p28-1Y9?V, p31-1Y2L) had more potent binding activity towards HLA-A*0201 molecule. In ELISPOT assay, the CTLs induced by these four peptides could release IFN-γ. The CTLs induced by these four peptides from the peripheral blood mononuclear cells (PBMCs) of HLA-A*02⁺ healthy donor could lyse MCF-7 breast cancer cells (HLA-A*0201⁺, PLAC1⁺) in vitro. When immunized in HLA-A2.1/K^b transgenic mice, the peptide p28 could induce the most potent peptide-specific CTLs among these peptides. Therefore, our results indicated that the peptide p28 (VLCSIDWFM) could serve as a novel candidate epitope for the development of peptide vaccines against PLAC1-positive breast cancer.     

Melanomas with concordant loss of multiple melanocytic differentiation proteins: immune escape that may be overcome by targeting unique or undefined antigens

Melanoma-reactive HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pmel17/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A*0201⁺ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pmel17/gp100, gp75/trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLA-A*0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A*0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A*0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP. Received: 31 October 1997 / Accepted: 4 August 1999     

Identification of an HLA-A*0201-restricted CD8+ T-cell epitope encoded within Leptospiral immunoglobulin-like protein A

Leptospirosis is an important zoonosis in humans. Immunity against leptospiral infection was thought to be primarily humoral, and limited studies have addressed the role of CD8⁺ T cells. Leptospiral immunoglobulin-like protein A (LigA) is an important protective antigen of Leptospira and a potential target for Leptospira-specific cell-mediated immunity. In this study, twenty LigA-derived peptides were tested their binding affinity and stability for the HLA-A*0201 molecule. Peptides with high binding affinity and stability for HLA-A*0201 were then assessed their capacity to elicit specific cytotoxic T-lymphocyte (CTL) responses using cytotoxicity, ELISPOT assays for IFN-γ and HLA-A*0201-peptide tetramer assays. We identified a HLA-A*0201-restricted epitope, LigA_305–313 KLIVTPAAL in Leptospira LigA. CTLs specific for LigA_305–313 were elicited both in HLA-A2.1/K^b transgenic mice and in patients with a clinical and/or laboratory diagnosis of leptospirosis. Staining of the HLA-A*0201–LigA_305–313 tetramer revealed the presence of LigA_305–313-specific CTLs in peripheral blood mononuclear cells (PBMCs) sourced from five patients infected with three different serovars of Leptospira. In conclusion, we report the existence of specific cytotoxic CD8⁺ T cells in patients with leptospirosis and we suggest that the newly identified epitope, LigA_305–313, will be helpful in enhancing the understanding of the mechanism of immunity to leptospirosis.     

An HLA-A3-binding prostate acid phosphatase-derived peptide can induce CTLs restricted to HLA-A2 and -A24 alleles

We previously reported peptide vaccine candidates for HLA-A3 supertype (-A3, -A11, -A31, -A33)-positive cancer patients. In the present study, we examined whether those peptides can also induce cytotoxic T lymphocyte (CTL) activity restricted to HLA-A2, HLA-A24, and HLA-A26 alleles. Fourteen peptides were screened for their binding activity to HLA-A*0201, -A*0206, -A*0207, -A*2402, and -A*2601 molecules and then tested for their ability to induce CTL activity in peripheral blood mononuclear cells (PBMCs) from prostate cancer patients. Among these peptides, one from the prostate acid phosphatase protein exhibited binding activity to HLA-A*0201, -A*0206, and -A*2402 molecules. In addition, PBMCs stimulated with this peptide showed that HLA-A2 or HLA-A24 restricted CTL activity. Their cytotoxicity toward cancer cells was ascribed to peptide-specific and CD8⁺ T cells. These results suggest that this peptide could be widely applicable as a peptide vaccine for HLA-A3 supertype-, HLA-A2-, and -A24-positive cancer patients.     

In silico analysis of six known Leishmania major antigens and in vitro evaluation of specific epitopes eliciting HLA-A2 restricted CD8 T cell response

Background

As a potent CD8⁺ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population.

Methods and Findings

Six Leishmania (L.) major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3) were screened for potential CD8⁺ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele). Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2⁺ individuals recovered from L. major. HLA-A2⁻ individuals recovered from L. major and HLA-A2⁺ healthy donors were included as control groups. Individual response of HLA-A2⁺ recovered volunteers as percent of CD8⁺/IFN-γ⁺ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2⁻ recovered individuals. Based on cutoff scores calculated from the response of HLA-A2⁻ recovered individuals, 31.6% and 13.3% of HLA-A2⁺ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2⁻ recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2⁺ recovered individuals.

Conclusion

Using in silico prediction we demonstrated specific response to LmsTI-1 (pool II) and LPG-3- (pool IV) related peptides specifically presented in HLA-A*0201 context. This is among the very few reports mapping L. major epitopes for human HLA types. Studies like this will speed up polytope vaccine idea towards leishmaniasis.     

Assessment of promoter elements of the germ cell-specific proacrosin gene

Presence of the simian virus 40 (SV40) has recently been demonstrated in a relatively high percentage of human mesotheliomas and it is associated with the development of these malignancies in pleural cavities. Therefore, we have initiated a study to identify candidate peptides presented by the human HLA-A*0201 molecule for vaccination approaches against SV40 and monitoring of SV40 directed human immune responses. Initial screening of SV40 large T (Tag) domains required for transformation of cells for HLA-A*0201 binding motifs revealed ten possible binding peptides. Screening of these candidate peptides showed that seven of the ten peptides could bind and stabilize HLA-A*0201 molecules. In an in vitro immunization assay the two peptides with the highest binding affinity for HLA-A*0201, Tag aa 396-405 and aa 577-585, were tested for their ability to induce peptide specific cytotoxic T cells in two healthy donors. One donor developed cytotoxic T cells against Tag aa 396-405 and in T cell cultures of both donors Tag aa 577-585 specific T cells were initiated. The T cells against Tag aa 577-585 not only recognized and killed peptide pulsed cells, but, most importantly, SV40 transformed human mesothelial cells. This is the first demonstration of the induction of SV40 specific human cytotoxic T lymphocytes that recognize endogenously processed peptides from SV40. This peptide identification study opens the possibility to investigate immune responses against SV40 in mesothelioma patients and in individuals exposed to SV40.     

CD8 T cell responses to myelin oligodendrocyte glycoprotein-derived peptides in humanized HLA-A*0201-transgenic mice

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the CNS. Though originally believed to be CD4-mediated, additional immune effector mechanisms, including myelin-specific CD8(+) T cells, are now proposed to participate in the pathophysiology of MS. To study the immunologic and encephalitogenic behavior of HLA-A*0201-binding myelin-derived epitopes in vivo, we used a humanized HLA-A*0201-transgenic mouse model. Eight HLA-A*0201-binding peptides derived from myelin oligodendrocyte glycoprotein (MOG), an immunodominant myelin self-Ag, were identified in silico. After establishing their relative affinity for HLA-A*0201 and their capacity to form stable complexes with HLA-A*0201 in vitro, their immunological characteristics were studied in HLA-A*0201-transgenic mice. Five MOG peptides, which bound stably to HLA-A*0201 exhibited strong immunogenicity by inducing a sizeable MOG-specific HLA-A*0201-restricted CD8(+) T cell response in vivo. Of these five candidate epitopes, four were processed by MOG-transfected RMA target cells and two peptides proved immunodominant in vivo in response to a plasmid-encoding native full-length MOG. One of the immunodominant MOG peptides (MOG(181)) generated a cytotoxic CD8(+) T cell response able to aggravate CD4(+)-mediated EAE. Therefore, this detailed in vivo characterization provides a hierarchy of candidate epitopes for MOG-specific CD8(+) T cell responses in HLA-A*0201 MS patients identifying the encephalitogenic MOG(181) epitope as a primary candidate.     

A Conserved E7-derived Cytotoxic T Lymphocyte Epitope Expressed on Human Papillomavirus 16-transformed HLA-A2+ Epithelial Cancers

Human Papillomavirus 16 (HPV-16) has been identified as the causative agent of 50% of cervical cancers and many other HPV-associated tumors. The transforming potential/tumor maintenance capacity of this high risk HPV is mediated by two viral oncoproteins, E6 and E7, making them attractive targets for therapeutic vaccines. Of 21 E6 and E7 peptides computed to bind HLA-A*0201, 10 were confirmed through TAP-deficient T2 cell HLA stabilization assay. Those scoring positive were investigated to ascertain which were naturally processed and presented by surface HLA molecules for CTL recognition. Because IFNγ ELISpot frequencies from healthy HPV-exposed blood donors against HLA-A*0201-binding peptides were unable to identify specificities for tumor targeting, their physical presence among peptides eluted from HPV-16-transformed epithelial tumor HLA-A*0201 immunoprecipitates was analyzed by MS³ Poisson detection mass spectrometry. Only one epitope (E7_11–19) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E7_11–20), previously used as a vaccine candidate, was neither detected by MS³ on HPV-transformed tumor cells nor effectively recognized by 9-mer specific CTL. These data underscore the importance of precisely defining CTL epitopes on tumor cells and offer a paradigm for T cell-based vaccine design.     

Immuno-informatics based approaches to identify CD8+ T cell epitopes within the Leishmania donovani 3-ectonucleotidase in cured visceral leishmaniasis subjects

Leishmaniases are vector-borne diseases for which no vaccine exists. These diseases are caused by the Leishmania species complex. Activation of the CD8⁺ T cell is crucial for protection against intracellular pathogens, and peptide antigens are attractive strategies for the precise activation of CD8⁺ T in vaccine development against intracellular infections. The traditional approach to mine the epitopes is an arduous task. However, with the advent of immunoinformatics, in silico epitope prediction tools are available to expedite epitope identification. In this study, we employ different immunoinformatics tools to predict CD8⁺ T cell specific 9 mer epitopes presented by HLA-A*02 and HLA-B40 within the highly conserved 3′-ectonucleotidase of Leishmania donovani. We identify five promiscuous epitopes, which have no homologs in humans, theoretically cover 85% of the world's population and are highly conserved (100%) among Leishmania species. Presentation of selected peptides was confirmed by T2 cell line based HLA-stabilization assay, and three of them were found to be strong binders. The in vitro peptide stimulation of peripheral blood mononuclear cells (PBMC) from cured HLA-A02⁺ visceral leishmaniasis (VL) subjects produced significantly higher IFN-γ, IL-2 and IL-12 compared to no peptide control healthy subjects. Further, CD8⁺ cells from treated VL subjects produced significantly higher intracellular IFN-γ, lymphocyte proliferation and cytotoxic activity against selected peptides from the PBMCs of treated HLA-A02⁺ VL subjects. Thus, the CD8⁺ T cell specific epitopes shown in this study will speed up the development of polytope vaccines for leishmaniasis.     

A CTL-based liposomal vaccine capable of inducing protection against heterosubtypic influenza viruses in HLA-A*0201 transgenic mice

The current vaccination strategy against influenza is to induce the production of antibodies directed against surface antigens of viruses. However, the frequent changes in the surface antigens of influenza viruses allow the viruses to avoid antibody-mediated immunity. On the other hand, it is known that cytotoxic T-lymphocyte (CTL) populations directed against internal antigens of influenza A virus are broadly cross-reactive to influenza virus subtypes. In the present study, liposomal conjugates with CTL epitope peptides derived from highly conserved internal antigens of influenza viruses were evaluated for their ability to protect against infection with influenza viruses. Liposomal conjugates with peptide M1 58-66, an HLA-A*0201-binding CTL epitope present within the amino-acid sequence of the M1 coding region, successfully induced antigen-specific CD8⁺ T-cells and CTLs in HLA-A*0201-transgenic mice. Moreover, after nasal infection with either the H1N1 or H3N2 virus, viral replication in the lung was significantly inhibited in the immunized mice. These protective activities lasted at least 6 months after the immunization. Thus, these results suggest that liposome-coupled CTL epitope peptides derived from highly conserved internal antigens of influenza viruses might be applicable to the development of vaccines that induce protection against infection with heterosubtypic influenza viruses.     

Cytotoxic T-lymphocyte responses to human papillomavirus type 16 E5 and E7 proteins and HLA-A*0201-restricted T-cell peptides in cervical cancer patients

Previously, we found that human papillomavirus type 16 (HPV-16) E5 protein is a tumor rejection antigen and can induce cytotoxic T-lymphocyte (CTL) activity. Therefore, in this study, human leukocyte antigen A*0201 (HLA-A*0201)-restricted human CTL epitopes of HPV-16 E5 protein were identified using a bioinformatics approach, and the abilities of these predicted peptides to induce an immune response in HLA-A*0201 transgenic mice were confirmed by assaying E5-specific CTLs and in vitro-generated CTLs from normal peripheral blood T lymphocytes of HLA-A2-positive human donors. Second, the CTL responses to HLA-A*0201 CTL epitopes (E5 63-71 and E7 11-20) were examined in HPV-16-infected patients with HLA-A2. Third, the effect of HLA-A-type alleles on CTL activities in response to the entire E5 and E7 proteins was examined in cervical cancer patients. E5 and E7 peptides (but not the whole proteins) stimulated E5- and E7-specific CTL recall responses in HPV-16- and HLA-A2-positive cervical cancer patients, and HPV-16 E5 and E7 proteins stimulated na?ve T cells in HPV-16-negative cervical cancer patients with HLA-A11 and -A24 haplotypes. In summary, this is the first demonstration that E5 63-71 is an HLA-A*0201-restricted T-cell epitope of HPV-16 E5.     

In vivo hierarchy of immunodominant and subdominant HLA-A*0201-restricted T-cell epitopes of HBx antigen of hepatitis B virus

A polyepitopic CD8+ T-cell response is critical for the control of hepatitis B virus (HBV) infection. The HBV X protein (HBx) is a multifunctional protein that is important for the viral life cycle and for host-virus interactions. The aim of this study was to analyze the immunogenicity and dominance of various HLA-A*0201-restricted HBx-derived epitopes. For this purpose, we immunized HLA-A*0201-transgenic mice with HBx-derived peptides and DNA. This is a powerful model for studying the induction of HLA-A*0201-restricted immune responses in vivo, as these mice possess a cytotoxic T lymphocyte (CTL) repertoire representative of HLA-A2.1 individuals. We used cytotoxic tests and enzyme-linked immunosorbent spot (ELISPOT) assays to study the induction of specific cytotoxic and interferon (IFN)-gamma-secreting T cells. This allowed us to classify the HBx epitopes according to their T-cell activation capacity. After endogenous processing of the antigen synthesized in vivo after DNA-based immunization, we found that the HBx-specific T-cell response is targeted against one immunodominant epitope. Furthermore, following peptide immunization, we identified six additional novel subdominant T-cell epitopes. Inclusion of well-characterized epitopic sequences of HBx in a new vaccine for chronic HBV infections could help to broaden the T-cell response.     

Differential Phenotypic and Functional Profiles of TcCA-2 -Specific Cytotoxic CD8+ T Cells in the Asymptomatic versus Cardiac Phase in Chagasic Patients

It has been reported that the immune response mediated by T CD8⁺ lymphocytes plays a critical role in the control of Trypanosoma cruzi infection and that the clinical symptoms of Chagas disease appear to be related to the competence of the CD8⁺ T immune response against the parasite. Herewith, in silico prediction and binding assays on TAP-deficient T2 cells were used to identify potential HLA-A*02:01 ligands in the T. cruzi TcCA-2 protein. The TcCA-2-specific CD8⁺ T cells were functionality evaluated by Granzyme B and cytokine production in peripheral blood mononuclear cells (PBMC) from Chagas disease patients stimulated with the identified HLA-A*02:01 peptides. The specific cells were phenotypically characterized by flow cytometry using several surface markers and HLA-A*02:01 APC-labeled dextramer loaded with the peptides. In the T. cruzi TcCA-2 protein four T CD8⁺ epitopes were identified which are processed and presented during Chagas disease. Interestingly, a differential cellular phenotypic profile could be correlated with the severity of the disease. The TcCA-2-specific T CD8⁺ cells from patients with cardiac symptoms are mainly effector memory cells (T_EM and T_EMRA) while, those present in the asymptomatic phase are predominantly naive cells (T_NAIVE). Moreover, in patients with cardiac symptoms the percentage of cells with senescence features is significantly higher than in patients at the asymptomatic phase of the disease. We consider that the identification of these new class I-restricted epitopes are helpful for designing biomarkers of sickness pathology as well as the development of immunotherapies against T. cruzi infection.     

Generation of human tumor-specific CTLs in HLA-A2.1–transgenic mice using unfractionated peptides from eluates of human primary breast and ovarian tumors

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with malignant transformation and aggressive disease. Due to its overexpression in tumor cells and because it has been shown to be immunogenic, this protein represents an excellent target for T-cell immunotherapy. Peptide extracts derived from primary HLA-A*0201-positive (+) HER-2/neu⁺ human tumors by acid elution (acid cell extracts (ACEs)) were tested for their capacity to elicit in HLA-A*0201 transgenic mice, cytotoxic T lymphocytes (CTLs) lysing HLA-A*0201⁺ HER-2/neu⁺ tumor cells. Injections of ACE in transgenic mice induced CTLs capable of specifically lysing HER-2/neu⁺ tumor cell lines (also including the original HER-2/neu⁺ primary tumor cells from which the ACEs were derived) in an HLA-A*0201–restricted fashion. Adoptive transfer of ACE-induced CTLs was sufficient to significantly prolong survival of SCID mice inoculated with HLA-A*0201⁺ HER-2/neu⁺ human tumor cell lines. Cytotoxicity of such ACE-induced CTL lines was directed, at least as detected herein, also against the HER-2/neu peptides HER-2 (9₃₆₉) and HER-2 (9₄₃₅) demonstrating the immunodominance of these epitopes. HER-2 peptide–specific CTLs generated in the HLA-A*0201–transgenic mice, upon peptide immunization, lysed in vitro HER-2/neu⁺ human tumor cell lines in an HLA-A*0201–restricted manner and, when adoptively transferred, conferred sufficient protection in SCID mice inoculated with the same human tumor cell lines as above. However, CTLs induced by ACEs displayed enhanced efficacy in the therapy of xenografted SCID mice compared with the HER-2 peptide–specific CTLs (i.e., HER-2 [9₃₆₉] or HER-2 [9₄₃₅]). Even by administering mixtures of CTLs specific for each of these peptides, the prolongation of survival achieved was still inferior compared with that obtained with ACE-induced CTLs. This suggested that additional epitopes may contribute to the immunogenicity of such tumor-derived ACEs. Thus, immunization with ACEs from HER-2/neu⁺ primary tumor cells appears to be an effective approach to generate multiple and potent CTL-mediated immune responses against HER-2/neu⁺ tumors expressing the appropriate HLA allele(s). By screening ACE-induced CTL lines with synthetic peptides encompassing the HER-2/neu sequence, it is feasible to identify immunodominant epitopes which may be used in mixtures as vaccines with enhanced efficacy in both the prevention and therapy of HER-2/neu⁺ malignancies.This work was supported by grants from the Regional Operational Program Attika (No. 20, MIS code 59605GR) to M.P., and from the GSRT Program (No. PENED 01ED55) to C.N.B.     

STEAP, a prostate tumor antigen, is a target of human CD8+ T cells

STEAP is a recently identified protein shown to be particularly overexpressed in prostate cancer and also present in numerous human cancer cell lines from prostate, pancreas, colon, breast, testicular, cervical, bladder and ovarian carcinoma, acute lymphocytic leukemia and Ewing sarcoma. This expression profile renders STEAP an appealing candidate for broad cancer immunotherapy. In order to investigate if STEAP is a tumor antigen that can be targeted by specific CD8⁺ T cells, we identified two high affinity HLA-A*0201 restricted peptides (STEAP_86–94 and STEAP_262–270). These peptides were immunogenic in vivo in HLA-A*0201 transgenic HHD mice. Peptide specific murine CD8 T cells recognized COS-7 cells co-transfected with HHD (HLA-A*0201) and STEAP cDNA constructs and also HLA-A*0201⁺ STEAP⁺ human tumor cells. Furthermore, STEAP_86–94 and STEAP_262–270 stimulated specific CD8⁺ T cells from HLA-A*0201⁺ healthy donors, and these peptide specific CD8⁺ T cells recognized STEAP positive human tumor cells in an HLA-A*0201-restricted manner. Importantly, STEAP_86–94-specific T cells were detected and reactive in the peripheral blood mononuclear cells in NSCLC and prostate cancer patients ex vivo. These results show that STEAP can be a target of anti-tumor CD8⁺ T cells and that STEAP peptides can be used for a broad-spectrum-tumor immunotherapy.     

Fluorescence labeling of anchor-modified Mart-1 peptide for increasing its affinity for HLA-A*0201: Hit two targets with one arrow

One of the most successful strategies in designing peptide-based cancer vaccines is modifying natural epitope peptides to increase their binding strength to human leukocyte antigens (HLAs). Anchor-modified Mart-1 peptide (ELAGIGILTV) is among the artificial epitope peptides with the highest binding affinity for HLA-A*0201. In this study, by fluorescence labeling of its either C- or N-terminus with N^ε-(5-carboxyfluorescein)-l -lysine, we not only made it traceable but also drastically increased its binding strength to HLA-A*0201. HLA streptamer, for the first time, is introduced for measuring the binding constants (K_a) of the labeled peptides. The affinity of the labeled peptides for the HLA-A*201 of the MCF-7 cells was extraordinarily high and co-incubating them with the highest possible amount of the unlabeled peptide, as a competitor, did not significantly prohibit them from binding to the HLA. The reproducibility of the obtained results was confirmed by using the T2 cell line. The HLA-deficient K562 cell line was used as the negative control. With in silico simulations, we found two hydrophobic pockets on both sides of HLA-A*0201 for anchoring the C- or N-terminal 5-carboxyfluorescein probe, which can explain the extraordinary affinity of the labeled peptides for the HLA-A*0201.     

HLA-A*0201 T-cell epitopes in severe acute respiratory syndrome (SARS) coronavirus nucleocapsid and spike proteins

The immunogenicity of HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) peptide in severe acute respiratory syndrome coronavirus (SARS-CoV) nuclear capsid (N) and spike (S) proteins was determined by testing the proteins' ability to elicit a specific cellular immune response after immunization of HLA-A2.1 transgenic mice and in vitro vaccination of HLA-A2.1 positive human peripheral blood mononuclearcytes (PBMCs). First, we screened SARS N and S amino acid sequences for allele-specific motif matching those in human HLA-A2.1 MHC-I molecules. From HLA peptide binding predictions (http://thr.cit.nih.gov/molbio/hla_bind/), ten each potential N- and S-specific HLA-A2.1-binding peptides were synthesized. The high affinity HLA-A2.1 peptides were validated by T2-cell stabilization assays, with immunogenicity assays revealing peptides N223-231, N227-235, and N317-325 to be the first identified HLA-A*0201-restricted CTL epitopes of SARS-CoV N protein. In addition, previous reports identified three HLA-A*0201-restricted CTL epitopes of S protein (S978-986, S1203-1211, and S1167-1175), here we found two novel peptides S787-795 and S1042-1050 as S-specific CTL epitopes. Moreover, our identified N317-325 and S1042-1050 CTL epitopes could induce recall responses when IFN-gamma stimulation of blood CD8+ T-cells revealed significant difference between normal healthy donors and SARS-recovered patients after those PBMCs were in vitro vaccinated with their cognate antigen. Our results would provide a new insight into the development of therapeutic vaccine in SARS.     

A long peptide from MELOE-1 contains multiple HLA class II T cell epitopes in addition to the HLA-A*0201 epitope: an attractive candidate for melanoma vaccination

CD4⁺ T cells contribute importantly to the antitumor T cell response, and thus, long peptides comprising CD4 and CD8 epitopes may be efficient cancer vaccines. We have previously identified an overexpressed antigen in melanoma, MELOE-1, presenting a CD8⁺ T cell epitope, MELOE-1_36–44, in the HLA-A*0201 context. A T cell repertoire against this epitope is present in HLA-A*0201+ healthy subjects and melanoma patients and the adjuvant injection of TIL containing MELOE-1 specific CD8⁺ T cells to melanoma patients was shown to be beneficial. In this study, we looked for CD4⁺ T cell epitopes in the vicinity of the HLA-A*0201 epitope. Stimulation of PBMC from healthy subjects with MELOE-1_26–46 revealed CD4 responses in multiple HLA contexts and by cloning responsive CD4⁺ T cells, we identified one HLA-DRβ1*1101-restricted and one HLA-DQβ1*0603-restricted epitope. We showed that the two epitopes could be efficiently presented to CD4⁺ T cells by MELOE-1-loaded dendritic cells but not by MELOE-1+ melanoma cell-lines. Finally, we showed that the long peptide MELOE-1_22–46, containing the two optimal class II epitopes and the HLA-A*0201 epitope, was efficiently processed by DC to stimulate CD4⁺ and CD8⁺ T cell responses in vitro, making it a potential candidate for melanoma vaccination.