Differential recognition of the serologically defined HLA-A2 antigen by allogeneic cytotoxic T Cells

Human alloimmune cytotoxic T cells, sensitized selectively against the HLA-A2 antigen, were tested on a panel of selected target cells. Five HLA-A2 positive outlier cells could be identified. These outlier cells were only weakly lysed by HLA-A2 specific CTLs, although they were serologically indistinguishable from the other HLA-A2 positive, strongly lysed target cells. Furthermore, it was found that the outlier cells were poor cold target inhibitors in contrast to the other HLA-A2 positive target cells, which showed adequate inhibition of specific lysis of HLA-A2 positive target cells. Population studies indicate that the frequency of such HLA-A2 outlier cells may be approximately 10%.     

Definition of four HLA-A2 subtypes by CML typing and biochemical analysis

The population of HLA-A2-positive individuals, currently considered serologically homogeneous, can be divided into three subtypes on the basis of antigen recognition by various HLA-A2-specific cytotoxic T lymphocytes (CTLs). When these three types of HLA-A2 antigens were analyzed biochemically, they were found to be distinct. Isoelectric focusing (IEF) of HLA antigens digested with neuraminidase (NANAse) suggested that the difference(s) reside in the polypeptide backbone of the HLA-A2 heavy chain. Biochemical analysis distinguishes three distinct categories of HLA-A2 antigens: (1) a major subtype, designated HLA-A2.I, (2) a minor subtype, designated HLA-A2.II, possessing a more basic isoelectric point (IEP) and (3) a minor HLA-A2 subtype more acidic in its IEP than HLA-A2.I, designated HLA-A2.III. A fourth HLA-A2 subtype could be defined by discordance between cell-mediated lympholysis (CML) typing and biochemical analysis. The latter HLA-A2 antigen was defined as a variant by CTL, but was biochemically indistinguishable from the major subtype HLA-A2.I.     

Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells

Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membraneprotein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies,such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma.However,the therapeutic amount ofCTLs is often hampered by the limited supply of antigen-presenting cells.To address this issue,an artificialantigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetramericcomplex,anti-CD28 antibody and CD54 molecule to a cell-sized latex bead,which provided the dual signalsrequired for T cell activation.By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral bloodmononuclear cells from HLA-A2 positive healthy donors,LMP2 antigen-specific CTLs were induced andexpanded in vitro.The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell,the cytotoxicity was inhibited bythe anti-HLA class Ⅰ antibody (W6/32).These results showed that LMP2 antigen-specific CTLs could beinduced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC.Thus,aAPCs coated with an HLA-pLMP2 complex,anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specificCTLs for adoptive immunotherapy.     

The self determinants recognized by human virus-immune T cells can be distinguished from the serologically defined HLA antigens

The self specificity of human influenza virus-immune cytotoxic T cells has been analyzed in order to clarify the relationship between the self antigens that they recognize and the serologically defined HLA-A and -B antigens. Virus-immune effectors from HLA-A2-positive donors were tested on panels of virus-infected target cells from donors who were either HLA-mismatched or matched only for HLA-A2. Virus-immune T cells from 11 out of 11 A2-positive donors lysed all A2-matched virus-infected target cells (and no HLA-mismatched targets), except that each of these effector cells consistently failed to lyse virus-infected target cells from one A2-positive donor (designated M7). Although the A2 specificity of donor M7 could also be distinguished from the A2 antigen of other donors by alloimmune cytotoxic T cells, no differences in the A2 antigen of donor M7 could be defined by extensive serologic analyses. These results indicate that there is a strong but incomplete association between a self antigen recognized by virus-immune T cells and the serologically defined HLA-A2 specificity.     

Analysis of the functional epitopes on different HLA-A2 molecules

Recent studies show that the serologically defined HLA-A2 molecule can be subdivided according to functional and biochemical characteristics. By the use of various HLA-A2-specific cytotoxic T lymphocytes (CTLs) and isoelectric focusing, the serologically homogeneous HLA-A2 molecule can be divided into four subtypes. The polymorphism of the serologically defined HLA-A2 molecule has also been demonstrated by the use of HLA-A2-restricted CTLs. This study was designed to analyze the functional epitopes on different HLA-A2 molecules with special regard to the recognition patterns of different types of HLA-A2-restricted CTLs directed against minor histocompatibility (minor H) antigens. Fifteen so-called HLA-A2 variants belonging to distinct HLA-A2 subtypes were tested as target cells in the cell-mediated lympholysis (CML) assay against (1) HLA-A2-restricted antiminor H-Y CTLs, (2) HLA-A2 and -B7-restricted antiminor H-Y CTLs, and (3) HLA-A2, -Bw62 and -B27-restricted antiminor HA CTLs. We found that those three CTLs recognized only one of those HLA-A2 variants. Furthermore, positive reactions by the antiminor H CTLs were only observed on those variant cells which carried, in addition to the HLA-A2 variant, either another normal HLA-A2 molecule or another required restricting class I molecule necessary for associative recognition. These results indicate that the absence of HLA-A2 normal allotypic target determinant(s) leads to the loss of epitope(s) necessary for recognition of minor H-Y and minor HA transplantation antigens by HLA-restricted CTLs. We can conclude from the present study that HLA-A2-restricted antiminor H CTLs use, in general, the same epitope (or cluster of epitopes) for cellular recognition as alloimmune HLA-A2-specific CTLs.     

烟曲霉抗原Asp f16HLA-A*0201限制性的CD8+CTL抗原表位生物信息学预测与实验室鉴定

目的 预测与鉴定烟曲霉抗原Asp f16的HLA-A *0201限制性CD8+细胞毒性T细胞(CTL)抗原表位.方法 以国人常见的HLA-A*0201位点为靶点,依据生物信息学软件扫描烟曲霉特异性抗原Asp f16的全部427个氨基酸序列.使用HLA-A *0201转基因小鼠制备骨髓来源的树突状细胞(DC)和CTL.流式细胞仪技术检测DC表面MHC Ⅱ类抗原,CD80,CD86和CD11c的表达来验证其是否成熟.ELISPOT试验检测烟曲霉抗原多肽特异性CTL产生的细胞因子IFN-γ.四聚体(Tetramer)试验证实烟曲霉特异性CTL与抗原肽,HLA-A*0201分子复合体的亲和性.结果 根据与MHC I类分子结合的半衰期评分,选择了3个HLA-A*0201限制性抗原表位.流式细胞仪分析示成熟DC高表达HLA Ⅱ类抗原,CD80,CD86和CD11c.Tetramer试验证实烟曲霉特异性T细胞受体与抗原肽,HLA-A*0201分子复合体的高亲和性.ELISPOT实验结果 表明烟曲霉抗原肽体外可以活化CD8+CTL,被负载了抗原肽的DC刺激活化后可以产生IFN-γ.结论 本研究成功鉴定烟曲霉抗原Asp f16的HLA-A*0201限制性CD8+CTL表位,可作为疫苗设计的候选表位,为进一步研发新型抗烟曲霉疫苗提供参考.     

Peptide-specific, allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2

The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from peptide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A426-434) or a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 positive (HLA-A2+ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2+ve PBL co-cultured with the LMP2A426-434pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr369-377 pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen.The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag77-85) was included for control. The cultural bulk of HLA-A2+ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%±3.72% vs 7.01%±0.87%, P＜0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%±0.33% vs 0.05%±0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%±2.58% vs 6.87%±1.01%,P＜0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%±0.3% vs 0.06%±0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the alloreactive lines depends on both the bound peptide and the allotype of MHC. Our observations support the hypothesis that the cumularive effect of T cells specific to each peptide epitope could account for the strength and diversity of the alloresponse. The method using manipulated pMHC and the LTMLC to generate pMHC-specific, alloreactive CTLs is of potential importance for adoptive T-cell immunotherapy.     

Identification of hepatitis C virus (HCV) 2a-derived epitope peptides having the capacity to induce cytotoxic T lymphocytes in human leukocyte antigen-A24+ and HCV2a-infected patients

Since virus-specific cytotoxic T lymphocytes (CTLs) play a critical role in preventing the spread of hepatitis C virus (HCV), vaccine-based HCV-specific CTL induction could be a promising strategy to treat HCV-infected patients. In this study, we tried to identify HCV2a-derived epitopes, which can induce human leukocyte antigen (HLA)-A24-restricted and peptide-specific CTLs. Peripheral blood mononuclear cells of HCV2a-infected patients or healthy donors were stimulated in vitro with HCV2a-derived peptides, which were prepared based on the HLA-A24 binding motif. As a result, three peptides (HCV2a 576-584, HCV2a 627-635, and HCV2a 1085-1094) efficiently induced peptide-specific CTLs from HLA-A24(+) HCV2a-infected patients as well as healthy donors. The cytotoxicity was exhibited by peptide-specific CD8(+) T cells in an HLA-A24-restricted manner. In addition, the HCV2a 627-635 peptide was frequently recognized by immunoglobulin G of HCV2a-infected patients. These results indicate that the identified three HCV2a peptides might be applicable to peptide-based immunotherapy for HLA-A24(+) HCV2a-infected patients.     

Characterization of the HLA-A2.2 subtype: T cell evidence for further heterogeneity

Five blood donors were identified whose HLA-A2 is different from the common HLA-A2. Their A2 molecule (A2.2) had a more basic isoelectric point than normal A2 (A2.1). Cytotoxic T lymphocytes (CTL) restricted. by HLA-A2.1, specific for influenza A and Epstein-Barr viruses, failed to lyse virus-infected target cells with HLA-A2.2. Identical patterns were obtained with both viruses. CTL from four of the A2.2-positive donors recognized target cells prepared from others in the group that shared only the HLA-A2.2 antigen. The A2.2 antigen from one donor seemed to be different in that target cells were not recognized by CTL from donors with the normal A2.1 nor with basic A2.2. There seems, therefore, to be heterogeneity within the HLA-A2.2 subtype.     

Multiple viral ligands naturally presented by different class I molecules in transporter antigen processing-deficient vaccinia virus-infected cells

The transporter associated with antigen processing (TAP) delivers the viral proteolytic products generated by the proteasome in the cytosol to the endoplasmic reticulum lumen that are subsequently recognized by cytotoxic T lymphocytes (CTLs). However, several viral epitopes have been identified in TAP-deficient models. Using mass spectrometry to analyze complex human leukocyte antigen (HLA)-bound peptide pools isolated from large numbers of TAP-deficient vaccinia virus-infected cells, we identified 11 ligands naturally presented by four different HLA-A, HLA-B, and HLA-C class I molecules. Two of these ligands were presented by two different HLA class I alleles, and, as a result, 13 different HLA-peptide complexes were formed simultaneously in the same vaccinia virus-infected cells. In addition to the high-affinity ligands, one low-affinity peptide restricted by each of the HLA-A, HLA-B, and HLA-C class I molecules was identified. Both high- and low-affinity ligands generated long-term memory CTL responses to vaccinia virus in an HLA-A2-transgenic mouse model. The processing and presentation of two vaccinia virus-encoded HLA-A2-restricted antigens took place via proteasomal and nonproteasomal pathways, which were blocked in infected cells with chemical inhibitors specific for different subsets of metalloproteinases. These data have implications for the study of the effectiveness of early empirical vaccination with cowpox virus against smallpox disease.     

Two subgroups of HLA Bw44 defined by cell-mediated lympholysis that differ in Bw44 expression on platelets and in patterns of genetic linkage disequilibrium

Possible immunogenic heterogeneity of the HLA-Bw44 antigen was investigated using cytotoxic T lymphocytes (CTL) generated between donors identical for HLA-A2,3,-B7,w44. Highly discriminatory CTL combinations were identified that defined two subgroups of Bw44, designated 44.1 and 44.2. Out of 47 Bw44-positive donors tested in a population study, 30 were lysed by the CTL defining 44.1, and 19 were lysed by the CTL defining 44.2. All Bw44 cells could be typed as either 44.1 or 44.2, except two Bw44-positive cells that were phenotypically homozygous for the serologically defined Bw44 antigen and were lysed by both CTL. No Bw44-negative donors (zero out of 37) expressed either 44.1 or 44.2, although cold target blocking was required to eliminate a contaminating reactivity of one CTL population on Bw35 and some Bw45 cells. CTL were also raised between responder/stimulator combinations mismatched for Bw44. These CTL lysed all Bw44-positive target cells, indicating a CML antigen shared by all Bw44 cells. But clear discrimination of the 44.1 and 44.2 subgroups was obtained when appropriate cold target blocking cells were added. All donors with 44.2 expressed high levels of serologically detectable Bw44 on their platelets, and all with 44.1 expressed low levels (p less than 0.005). Furthermore, population studies indicate that 44.1 is in positive linkage disequilibrium with HLA-A2 and possibly DR4, whereas 44.2 is in positive linkage disequilibrium with HLA-DR7 and possibly HLA-A23, -A26, and -A29. These data suggest the existence of two genetically and functionally different subgroups of Bw44 antigens.     

Peptide-specific, allogeneic T cell response in vitro induced by a self-peptide binding to HLA-A2

The role of the bound peptide in alloreactive T-cell recognition is controversial, ranging from pep-tide-independent to peptide-specific recognition of alloreactive T-cells. The aim of this study is to find the evidence that there exist peptide/MHC complex (pMHC)-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a viral peptide (LMP2A426-434) or a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 positive (HLA-A2 ve) and 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The HLA-A2 ve PBL co-cultured with the LMP2A426-434 pulsed T2 (T2/LMP) stands for the nominal T-cell response to a viral antigen, and the HLA-A2-ve PBLs co-cultured with the Tyr369-377 pulsed T2 (T2/Tyr) for alloreactive T-cell response to an allogeneic antigen. The specificity of the expanded CTLs after the LTMLC was detected by their specific cytotoxicity and binding ability to specific pMHC-tetramer. An HLA-A2 restricted, HIV peptide (Gag77-85)was included for control. The cultural bulk of HLA-A2 ve PBLs with the T2/LMP showed an elevated specific cytotoxicity against the T2/LMP compared to that against the T2/HIV (26.52%±3.72% vs 7.01%±0.87%, P<0.001), and an increased frequency of binding to LMP-tetramer compared to that binding to HIV-tetramer (0.98%±0.33% vs 0.05%±0.01%, P=0.0014). The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the T2/Tyr than that against T2/HIV (28.07%±2.58% vs 6.87%±0.01 %, P<0.001), and a higher frequency of binding to the Tyr-tetramer than that binding to the HIV-tetramer (0.88%±0.3% vs 0.06%±0.03%, P=0.0018). Our results indicate that the LTMLC is able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs. A relatively large proportion of alloreactive CTLs should be pMHC-specific, i.e., the specificity of the alloreactive lines depends on both the bound peptide and the allotype of MHC. Our observations support the hypothesis that the cumulative effect of T cells specific to each peptide epitope could account for the strength and diversity of the alloresponse. The method using manipulated pMHC and the LTMLC to generate pMHC-specific, alloreactive CTLs is of potential importance for adoptive T-cell immunotherapy.     

The immunogenicity of a novel cytotoxic T lymphocyte epitope from tumor antigen PL2L60 could be enhanced by 4-chlorophenylalanine substitution at position 1

PIWIL2, a member of PIWI/AGO family, is expressed in germline stem cells and precancerous stem cells, but not in adult somatic cells. PIWIL2 plays an important role in tumor development. It is considered as a cancer–testis antigen (CT80). It has been reported that the spliced fragment of PIWIL2, PL2L60, was widely expressed in cancer cell lines. In this study, HLA-A2-restricted epitopes from PL2L60 were predicted by online tools. To improve the activity of the native epitope, a candidate peptide P281 with potent binding affinity was chosen to investigate the modification strategy. A series of aromatic amino acids were introduced to substitute the first residue of P281. Then, we tested the binding affinity and stability of the peptide analogs and their ability to elicit specific immune responses both in vitro and in vivo. Our results indicated that the cytotoxic T lymphocytes (CTLs) induced by [4-Cl-Phe¹]P281 could elicit more potent activities than that of P281 and other analogs. The CTLs induced by this analog could lyze target cells in HLA-A2-restricted and antigen-specific manners. [4-Cl-Phe¹]P281 also showed the best resistance against degradation in human serum. In conclusion, the introduction of the unnatural amino acid, 4-Cl-Phe, into the first position could enhance the activity of the native epitope to induce cytotoxic T lymphocytes. It might be a good strategy to modify other promising native epitopes. The novel epitopes identified in this study could be used as novel candidates to the immunotherapy of HLA-A2 positive patients with tumors expressing PL2L60.     

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 vitro and in vivo identification of a novel cytotoxic T lymphocyte epitope from Rv3425 of Mycobacterium tuberculosis

The identification of novel cytotoxic T lymphocyte (CTL) epitopes is important to analysis of the involvement of CD8(+) T cells in Mycobacterium tuberculosis infection as well as to the development of peptide vaccines. In this study, a novel CTL epitope from region of difference 11 encoded antigen Rv3425 was identified. Epitopes were predicted by the reversal immunology approach. Rv3425-p118 (LIASNVAGV) was identified as having relatively strong binding affinity and stability towards the HLA-A*0201 molecule. Peripheral blood mononuclear cells pulsed by this peptide were able to release interferon-γ in healthy donors (HLA-A*02(+) purified protein derivative(+)). In cytotoxicity assays in vitro and in vivo, Rv3425-p118 induced CTLs to specifically lyse the target cells. Therefore, this epitope could provide a subunit component for designing vaccines against Mycobacterium tuberculosis.     

A new epitope peptide derived from hepatitis C virus 1b possessing the capacity to induce cytotoxic T-lymphocytes in HCV1b-infected patients with HLA-A11, -A31, and -A33

Background Hepatitis C virus (HCV) frequently causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma after long-term persistent infection. Among various genotypes of HCV, HCV1b is resistant to standard interferon therapy, and thus the development of new treatment modality is needed. Results To provide a scientific basis for specific immunotherapy for HCV1b, we investigated HCV1b-derived epitope peptides recognized by human leukocyte antigen (HLA)-A11, -A31, or -A33-restricted cytotoxic T-lymphocytes (CTLs), and report here three novel vaccine candidate peptides selected by both antibody screening and CTL-inducing capacity from among 46 peptides of conserved regions of HCV1b sequences with binding motifs to HLA-A11, -A31, and -A33. Significant levels of IgG reactive to each of the three peptides were detected in the plasma of more than 50% of the HCV1b⁺ patients. One peptide at positions 30–39 of the core protein induced peptide-specific CTLs from peripheral blood mononuclear cells (PBMCs) of HLA-A11⁺, -A31⁺, and -A33⁺ patients. The other two peptides at positions 35–43 of the core protein and at positions 918–926 of the non-structural protein 2 also induced peptide-specific CTLs from the PBMCs of HLA-A11⁺ and -A33⁺ patients. Conclusion Therefore, the peptide at positions 30–39 of the core protein could be an appropriate target molecule of specific immunotherapy for all HLA-A11⁺, -A31⁺, and -A33⁺ patients with HCV1b-related diseases.     

Screening and identification of severe acute respiratory syndrome-associated coronavirus-specific CTL epitopes

Severe acute respiratory syndrome (SARS) is a highly contagious and life-threatening disease that emerged in China in November 2002. A novel SARS-associated coronavirus was identified as its principal etiologic agent; however, the immunopathogenesis of SARS and the role of special CTLs in virus clearance are still largely uncharacterized. In this study, potential HLA-A*0201-restricted spike (S) and nucleocapsid protein-derived peptides were selected from an online database and screened for potential CTL epitopes by in vitro refolding and T2 cell-stabilization assays. The antigenicity of nine peptides which could refold with HLA-A*0201 molecules was assessed with an IFN-gamma ELISPOT assay to determine the capacity to stimulate CTLs from PBMCs of HLA-A2(+) SARS-recovered donors. A novel HLA-A*0201-restricted decameric epitope P15 (S411-420, KLPDDFMGCV) derived from the S protein was identified and found to localize within the angiotensin-converting enzyme 2 receptor-binding region of the S1 domain. P15 could significantly enhance the expression of HLA-A*0201 molecules on the T2 cell surface, stimulate IFN-gamma-producing CTLs from the PBMCs of former SARS patients, and induce specific CTLs from P15-immunized HLA-A2.1 transgenic mice in vivo. Furthermore, significant P15-specific CTLs were induced from HLA-A2.1-transgenic mice immunized by a DNA vaccine encoding the S protein; suggesting that P15 was a naturally processed epitope. Thus, P15 may be a novel SARS-associated coronavirus-specific CTL epitope and a potential target for characterization of virus control mechanisms and evaluation of candidate SARS vaccines.     

Factors affecting the presentation of exogenous hepatitis B virus core antigen

Hepatitis B virus core antigen (HBcAg) plays a critical role in terminating acute Hepatitis B virus infection and may be used as a potential vaccine candidate. The cell surface major histocompatibility complex (MHC) class 1 molecules are thought to be involved in the presentation of HBcAg. Surface MHC class 1 HLA A2 heavy chain (HC) and trimeric molecules were characterized on transfected Hela cells used as antigen presenting cells (APC) for the presentation of HBcAg. The results show that antibodies against HC HLA A2 and trimeric HLA-A2 molecules resulted in increased activation of HBcAg 18-27 minimal peptide specific cytotoxic T lymphocytes (CTLs), while the addition of exogenous beta2-microglobulin decreased the activation of HBcAg specific CTLs. Further, specific CD8+ T cells were activated only when Hela cells as APCs were primed with HBcAg (peptide, soluble or embedded on virosomes) at pH 6.5.     

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.