Generation of human tumor-reactive cytotoxic T cells against peptides presented by non-self HLA class I molecules

The cyclin-D1 protein, which was found to be overexpressed in various human tumors, promotes cell cycle progression from the G1 into the S phase. It is normally expressed at low levels in several tissues and is likely to induce immunological tolerance. We have recently shown in a murine system that T cell tolerance to a widely expressed protein was circumvented by raising cytotoxic T lymphocytes (CTL) from major histocompatability complex mismatched donors. In this study, we tested whether it is possible to raise human allo-restricted CTL against the cyclin-D1 protein. The human cell line T2 is deficient in the genes encoding the transporter associated with antigen processing (TAP), resulting in inefficient loading of HLA-A2 class I molecules with endogenous peptides. Thus, a large number of A2 molecules can bind exogenously supplied synthetic peptides. Peripheral blood mononu clear cells from HLA-A2-negative donors were stimulated with T2 cells presenting cyclin-D1-derived synthetic peptides. Cloning of bulk cultures revealed that a large proportion of CTL clones were peptide specific. One peptide induced CTL which lysed cyclin-D1-expressing breast cancer cells, but not control Epstein-Barr virus-transformed B lymphoid cells. The results show that HLA-A2-negative donors can be used to isolate tumor-reactive CTL spe cific for cyclin-D1 peptides presented by HLA-A2 class I molecules.     

Extensive T cell receptor cross-reactivity on structurally diverse haptenated peptides presented by HLA-A2

Previous studies have shown that individual TCRs are able to effectively recognize multiple peptide/MHC complexes that have varying degrees of structural diversity. These TCR cross-reactivities have usually been demonstrated by using peptides that have different amino acid sequences. To further examine the extent to which TCRs can accommodate structurally diverse ligands, we analyzed human TCR cross-reactivity to eight structurally distinct haptens that are coupled to the HLA-A2-binding Tax peptide with a lysine substitution at position 5 (Tax-5K, LLFG[K-hapten]PVYV). The results demonstrate that 71% percent of the haptenated-peptide-induced CTL lines could cross-react on at least one other hapten. We compared the effects of HLA-A2 mutants with substitutions at known TCR contact sites for recognition by hapten-cross-reactive CTL. Recognition of the A2 mutants was remarkably similar whether they were presenting the immunizing or the cross-reactive peptide, indicating that similar amino acid contacts are made by the TCR during recognition of both complexes. Thus, hapten cross-reactivity is apparently accomplished without major adjustments to the interaction between the TCR and the surface of the HLA-A2 molecule. Collectively, these results suggest that TCRs possess the molecular flexibility to accommodate very structurally diverse ligands while retaining conserved interactions with the surface of the MHC molecule.     

Naturally occurring peptides associated with HLA-A2 in ovarian cancer cell lines identified by mass spectrometry are targets of HLA-A2-restricted cytotoxic T cells

Identifying naturally occurring peptides bound to HLA class I molecules recognized by HLA-restricted cytotoxic T lymphocytes (CTL) is both relevant and central to the development of effective immunotherapeutic strategies against cancer. Several cancer-related genes have been reported for ovarian cancer, but very few are known to be naturally processed T cell epitopes. In the present study we used mass spectrometry to identify 16 novel HLA-A2-bound peptides from HLA-A2(+) ovarian cancer cell lines. All 16 peptides are derived from source proteins with diverse functions and marked homology to known proteins found in public databases. Synthetic peptide analogues of identified sequences were found to stabilize HLA-A2.1, albeit with varying affinities. The peptides were found to be antigenic in that a primary CD8(+) CTL response could be elicited from normal donor blood. The CTL generated were not only peptide specific, but failed to recognize targets pulsed with control peptides. In addition, recognition of shared HLA-A2-restricted epitopes by these CTL is suggested by their reactivity with a subset of HLA-A2(+) tumor lines and freshly isolated cancer cells or cell lines established from peritoneal ascites. These results were further corroborated by competitive inhibition of lysis of an otherwise susceptible cell line in the presence of cold peptide-pulsed targets. Furthermore, lack of recognition of several HLA-A2(+) control cell lines or cells isolated from normal ovaries suggests that these peptides are cancer related. These findings broaden the list of CTL-defined antigens that could lead to the development of multi-epitope vaccines for the treatment of ovarian cancer.     

A MAGE-A4 peptide presented by HLA-A2 is recognized by cytolytic T lymphocytes.

The MAGE-encoded antigens that are recognized by cytolytic T lymphocytes (CTL) are shared by many tumors and are strictly tumor specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes. We have used a method to identify CTL epitopes, which selects naturally processed peptides. CD8(+) T cells, obtained from individuals without cancer, were stimulated with autologous dendritic cells infected with a recombinant adenovirus containing the MAGE-A4 coding sequence. Responder cell microcultures that specifically lysed autologous EBV-transformed B cells infected with vaccinia-MAGE-A4 were cloned using autologous stimulator cells infected with a Yersinia enterocolitica carrying the MAGE-A4 sequence. An anti-MAGE-A4 CTL clone was obtained and the epitope was found to be decapeptide GVYDGREHTV (amino acids 230-239) presented by HLA-A2 molecules. The CTL clone lysed HLA-A2 tumor cells expressing MAGE-A4. This is the first reported antigenic peptide encoded by MAGE-A4. It may be valuable for cancer immunotherapy because MAGE-A4 is expressed in 51% of lung carcinomas and 63% of esophageal carcinomas, whereas about 50% of Caucasians and Asians express HLA-A2.     

An SV40 VP1-derived epitope recognized by CD8+ T cells is naturally processed and presented by HLA-A*0201 and cross-reactive with human polyomavirus determinants

The CD8+ T cell responses directed toward the VP1 antigens of human polyomaviruses JC and BK recently were shown to be cross-reactive. Two HLA-A0201-restricted determinants from each virus have been defined and include JCp100-108 (ILMWEAVTL) and BKp108-116 (LLMWEAVTV) as well as JCp36-44 (SITEVECFL) and BKp44-52 (AITEVECFL). We asked whether VP1 from the related SV40 contains similar HLA-A0201-restricted determinants. In this study, we demonstrate that CD8+ T cells specific for SV40 VP1 p110-118 (ILMWEAVTV), but not p46-54 (SFTEVECFL), can be induced in HLA-A0201-transgenic mice and that these CD8+ T cells cross-react with the corresponding determinants from JC and BK virus. The SV40 p110 determinant was found to be processed and presented in SV40-infected cells. These results indicate that the JCp36/BKp44 determinants are distinctive for the human polyomaviruses while the JCp100/BKp108/SVp110 determinants are shared by all three viruses, providing a target for CD8+ T cell cross-reactivity.     

A peptide encoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE-3

The human MAGE-3 gene is expressed in many tumors of several histological types but it is silent in normal tissues, with the exception of testis. Antigens encoded by MAGE-3 may, therefore, be useful targets for specific anti-tumor immunization of cancer patients. We reported previously that MAGE-3 codes for an antigenic peptide recognized on a melanoma cell line by autologous cytolytic T lymphocytes (CTL) restricted by HLA-A1. Here we report that the MAGE-3 gene also codes for another antigenic peptide that is recognized by CTL restricted by HLA-A2. MAGE-3 peptides bearing consensus anchor residues for HLA-A2 were synthesized and tested for binding. T lymphocytes from normal individuals were stimulated with autologous irradiated lymphoblasts pulsed with each of three peptides that showed strong binding to HLA-A2. Peptide FLWGPRALV was able to induce CTL. We obtained CTL clones that recognized not only HLA-A2 cells pulsed with this peptide but also HLA-A2 tumor cell lines expressing the MAGE-3 gene. The proportion of melanoma tumors expressing this antigen should be approximately 32 % in Caucasian populations, since 49 % of individuals carry the HLA-A2 allele and 65 % of melanomas express MAGE-3.     

A MAGE-A1 peptide presented to cytolytic T lymphocytes by both HLA-B35 and HLA-A1 molecules

Antigens encoded by melanoma antigen (MAGE) genes are of particular interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. Antigenic peptide EADPTGHSY encoded by MAGE-A1 and known to be presented by HLA-A1 is currently being used in therapeutic vaccination trials. We report here that a cytotoxic T-lymphocyte (CTL) clone, which is restricted by HLA-B35, recognizes the same peptide and, importantly, lyses HLA-B35 tumor cells expressing MAGE-A1. This peptide can be presented to CTL by both HLA-B*3501 and HLA-B*3503 molecules, which are expressed by approximately 19% of Caucasians. These results infer that the current clinical use of peptide EADPTGHSY can now be extended to HLA-B35 patients.     

Identification of HLA-A2 binding peptides from cytomegalovirus and its recognition by cytotoxic T lymphocytes..

The human pathogen CMV, is a major cause of mortality in the case of immunocompromised recipients of allogeneic bone marrow transplants. The CD8⁺ class I restricted response to CMV plays a crucial role in the control of CMV infection in asymptomatic immunocompetent hosts; however, the viral antigen recognised by CD8⁺ CTLs are not well characterised. The lower matrix 65 kD phosphoprotein is a prime candidate for production of CMV antigenic peptides and it been has shown that it can act as target for CTL's. We have used an in vitro assay to investigate potential viral antigens recognised by HLA-A2 restricted CTLs. Synthetic peptides were designed using the published pp65 protein sequence to contain the consensus binding motif for HLA-A2. These peptides were used in a standard T2 binding assay. T2 cells (HLA - A2, B5) were incubated overnight in the presence of the synthetic peptides. The positive control HLA-A2-binding influenza matrix peptide, AE41, resulted in a 3 fold increase in cell surface HLA-A2 expression. Incubation with the designed CMV pp65 peptides resulted in varying degrees of HLA-A2 expression. In particular, peptide AE45 showed a two-fold increase in expression. The aim of our project is to define CMV specific epitopes recognised by cytotoxic T cells (CTL). Using the T2 binding assay we have identified certain CMV pp65 synthetic peptides that bind specifically to the HLA-A2 molecule. We are now in the process of analysing the recognition of such pp65 peptides by CTL's especific for the pp65 protein. Further definition of CMV specific peptide epitopes presented by particular Class I molecules will allow studies of the CTL response to CMV in infected patients with defined HLA haplotypes.     

Activation-induced changes in peptides presented by T cells.

We compared the sets of peptides presented via HLA Class I in a CD4 + T cell line (Jurkat) before and after activation with PMA plus PHA.

We found that cell activation resulted in the de n vo presentation of some peptides.

Sequence analysis revealed that one of the newly presented peptides derived from IL-1 receptor antagonist (IL-1ra).

Since IL-1ra was not known to be expressed by lymphocytes, we also investigated ist pattern of expression in lymphocytes of the T lineage.

RT-PCR analysis allowed us to demonstrate that IL-1ra is expressed upon activation in Jurkat cells, in CD4+ lymphocytes from peripheral blood, but not in CD8+ ones, and in thymocytes.

This suggests that activation in CD4+ T cells is followed by the novo presentation of peptides der ved from proteins expressed only upon activation. Interestingly, of the two forms of IL-1ra expre sed in different cell lineages, the intracellular one and the secreted one, only the former is expre sed in activated CD4+ cells.     

The Th1 immune response against HIV-1 Gag p24-derived peptides in mice expressing HLA-A02.01 and HLA-DR1

Using HLA-DR1-transgenic H-2 class II knockout mice, we identified two new HLA-DR1-restricted HIV-1 Gag p24-derived epitopes (Gag(321-340 )and Gag(331-350)) and confirmed the immunogenicity of seven that have been previously described. The human relevance was confirmed for the two new ones (Gag(321-340 )and Gag(331-350)) assaying peripheral blood mononuclear cells from HLA-DR1(+) HIV-1-infected long-term asymptomatic subjects and showing that Gag(331-350) could prime CD4(+) T cells from two HLA-DR1(+) HIV-1 seronegative donors in vitro. Seven of these epitopes, structurally conserved among HIV-1 clade B isolates, were selected for a comparative evaluation of their Th1 helper potential by immunizing HLA-A02.01/HLA-DR1-transgenic, H-2 class I/class II knockout mice with recombinant mouse invariant chain constructs in which each helper epitope was inserted in association with two reporter HIV-1-derived HLA-A02.01-restricted CD8(+) T cell epitopes. A T helper effect was demonstrated in all cases, and was particularly strong with epitopes Gag(301-320),Gag(321-340 )and Gag(271-290), which should, therefore, be considered in the design of new vaccines.     

An allo-specific peptide derived from HLA-A2 is presented by HLA-DQ7

The peptide motifs of two HLA class II molecules, DR11 and DQ7, were determined from natural peptides. The EBV transformed B cells JVM (HLA-A2-DRB1^*1102-DQA1^*0501-DQB1^*0301) were cultured to a final yield of 2 10¹⁰ cells. DR11 and DQ7 molecules were immunopurified and peptides were extracted after acid elution and separated by reverse-phase HPLC. Five peptides from DR11 and five from DQ7 were sequenced using Edman degradation and other peptides were analysed by pool sequencing. Peptides were from 11 to 15 amino acids in length and P often occurred at the second residue for both DR5 and DQ7. The peptide motif for DR11 was I at position i and K or R at position i + 4. For DQ7 the most significant signal was A in the middle of the peptide as also described by Falk et al. The source of one peptide eluted from DQ7 was a polymorphic part of the HLA-A2 heavy chain (from 56th to 69th amino-acid). It was also exactly the same peptide than the synthetic peptide used by Krensky et al in the 10th international workshop to modulate lysis by HLA-A2-specific cytotoxic T lymphocytes. The biochemical characterisation of this peptide from HLA-DQ7 strongly supports functional tests showing an indirect presentation of alloantigens by MHC molecules.     

液-质联用分析肝癌细胞HLA-A2类分子递呈的抗原肽

目的：寻找MHCI类分子递呈的、T淋巴细胞识别的肝癌抗原肽。方法：人肝癌细胞系hHCC以枸橼酸－磷酸盐（pH3.3)液洗脱,洗脱物经粗分离,得到相对分子质量（Mr)＜5000的各种多肽组分混合液;取各组分多肽分离液分别与抗原加工缺陷细胞（T2,HLA－A2）进行细胞表位重建,加入特异性细胞毒T淋巴细胞（CTL),^51Cr释放法测杀伤活性;选活性值高的抗原肽组分进行反相高效液相色谱－质谱（RP－HPLC－MS）检测,对活性峰手工读谱分析其一级结构;最后进行氨基酸同源性分析。结果：酸洗10^10hHCC细胞,样品蛋白质浓度2mg/ml;通过RP－HPLC－MS检测,其质谱图经手工解析,氨基酸序列为SXXVHXNEV,X＝1或L;氨基酸同源性分析证明SXXVHXNEV为SLIVHLNEV,是肝细胞生长因子受体的第1075－1083肽段,第1080位点发生突变,由F变为L,由HLA－A2分子递呈。结论：细胞膜温和酸洗技术结合RP－HPLC－MS联用技术,是寻找肝癌抗原肽的有效方法,尚未见相同研究报道。发现的肝癌抗原肽SLIVHLNEV是肝细胞生长因子受体的突变物,可能具有重要生物学意义。     

HLA-A2限制性Survivin点突变肽诱导特异性抗肝癌CTL反应

目的 探讨HLA-A2限制性Survivin点突变抗原肽体外诱导CTLs的抗肝癌作用.方法 生物信息学软件BIMAS和SYFPEITHI用来鉴定点突变的HLA-A2限制性Survivin抗原九肽;流式细胞术检测突变肽与HLA-A2分子结合效率;肽体外刺激肝癌患者腹水来源的肿瘤浸润淋巴细胞(TILs)诱导反应性CTLs,用流式细胞术及ELISA检测反应性CTLs释放IFN-γ情况;肽诱导的CTLs与肝癌细胞系HepG2及BEL-7402共孵育,CytoTox 96(R)非放射性细胞毒性检测法检测对肿瘤细胞的裂解情况,倒置相差显微镜观察肝癌细胞形态学变化.结果 生物信息学分析筛选出与MHC分子结合效率评分显著提高的点突变Survivin抗原九肽Sur79M2 (KMSSGCAFL),流式细胞术检测证实负载Sur79M2的T2细胞,HLA-A2表达率显著提高,经Sur79M2体外刺激诱导的CTLs与靶细胞共孵育后能释放较高水平的IFN-γ,Sur79M2诱导的CTLs能通过HLA-A2限制性机制有效杀伤肝癌细胞系HepG2,对HLA-A2阴性的BEL-7402细胞无明显杀伤作用.结论 点突变肽Sur79M2能在体外诱导反应性CTLs产生,该CTLs能以HLA-A2限制性方式有效杀伤肝癌细胞系.     

Identification and design of p53-derived HLA-A2-binding peptides with increased CTL immunogenicity

The replacement of a suboptimal amino acid in a primary anchor position with an optimal residue improves human leucocyte antigen (HLA) binding and immunogenicity, while maintaining cytotoxic T lymphocyte (CTL) specificity. Using a neural network capable of performing quantitative predictions of peptide binding to HLA-A2 molecules, we identified three p53 protein-derived nonamer peptides with intermediate binding owing to suboptimal amino acids in the P2 anchor position. These peptides were synthesized along with the corresponding analogs, where the natural P2 residue had been replaced with the optimal leucine residue. All three modified peptides bound to and more efficiently stabilized HLA-A2 molecules than the corresponding nonmodified peptides. The HLA-A2 transgenic mice were used for immunization. Two of the epitopes were more immunogenic in their modified than in their natural versions. The CTLs raised against the modified peptides efficiently killed the target cells pulsed with the corresponding native peptide. In terms of sensitizing the targets cells for the CTL killing, the modified peptides were more efficient than native peptides. Finally, the CTLs induced by modified peptide killed HLA-A2 transgenic mouse fibrosarcoma cells transfected with human p53 DNA. The data suggest that modified self-peptides derived from overexpressed tumour-associated proteins can be used in vaccine development against cancer, and that quantitative predictions of HLA binding is of value in the rational selection and improvement of target epitopes recognized by CTLs.     

Salivary detection of HIV-1 antibodies using recombinant HIV-1 peptides

Salivary antibodies may play a role in the absence of HIV-1 transmission by saliva. We evaluated the presence of salivary IgG antibodies to HIV-1 using a recombinant ELISA. Whole saliva was collected from 21 HIV-1-seropositive individuals and assayed in an ELISA, ASQ (Beckman Instruments, Brea, CA), consisting of a panel of six HIV-1 recombinant peptides. Saliva samples from 20 individuals demonstrated IgG to one or more peptides and 18 to two or more peptides. Samples from 20 seropositive individuals were reactive with the gp41 peptide, whereas only 12 were reactive with the two gp120 peptides. Nineteen of twenty salivas also had detectable IgG antibodies to HIV-1 by Western blotting. The results indicate that viral-specific IgG antibodies are present in the saliva of a high percentage of HIV-infected individuals and that a recombinant peptide ELISA for saliva might be useful for the detection of HIV-1 infection.     

Identification and characterization of HIV-1 epitopes presented by HLA-A*2603: comparison between HIV-1 epitopes presented by A*2601 and A*2603

Human leukocyte antigen (HLA)-A*26 is one of the alleles associated with a slow progression to AIDS. Identification and characterization of HIV-1-specific epitopes presented by this allele are necessary for studies on the immunopathogenesis of AIDS and vaccine development in Asia, where three HLA-A*26 subtypes are frequently found. In the present study, we sought to identify HLA-A*2603-restricted HIV-1 epitopes by using reverse immunogenetics and to compare them with HLA-A*2601-restricted ones recently identified. We found that 31 of 110 HIV-1 peptides bound to HLA-A*2603 and that only two peptides (Gag169-177 and Env63-72) induced specific CD8+T cells by stimulating peripheral blood mononuclear leukocytes from HIV-1-infected individuals carrying HLA-A*2603. The specific cytotoxic T lymphocyte clones killed HIV-1 recombinant vaccinia-infected cells, indicating that these two peptides were naturally occurring peptides presented by HLA-A*2603. Gag169-177-specific CD8+T cells were frequently detected in both HLA-A*2601+ and -A*2603+ individuals with chronic HIV-1 infection, whereas Env63-72-specific ones were frequently detected only in the HLA-A*2603+ individuals. Gag169-177 peptide bound equally to both HLA-A*26 antigens, whereas Env63-72 peptide bound to A*2603 much more strongly than to A*2601. These findings suggest that the relative affinity of these peptides for the HLA-A*26 subtypes determines whether these peptides are recognized as epitopes in HIV-1-infected individuals carrying these alleles.     

An endogenously synthesized decamer peptide efficiently primes cytotoxic T cells specific for the HIV-1 envelope glycoprotein

The immunodominant H-2D^d-restricted cytotoxic T lymphocyte (CTL) response to the HIV-1 gp160 envelope glycoprotein maps to a single determinant in the V3 loop, designated p18. Using a series of peptides synthesized on pins we have determined that the minimal core sequence of this determinant required for CTL recognition comprises 8 amino acids (residues 320-327). However, 9mer and l0mer peptides containing this core sequence were more effective than the 8mer peptide at sensitizing D^d-expressing target cells. To analyze the antigenicity of endogenously synthesized p18, minigenes encoding a 10-amino acid determinant (residues 318-327) and a 67-amino acid peptide (residues 281-348; containing the V3 loop) were expressed using vaccinia virus (Vac) recombinants. Both peptides were as effective as wild-type gpl60 in their ability to sensitize target cells for lysis by gpl60-specific CTL. Immunization of BALB/c mice with Vac recombinants encoding both gp160 peptides elicited gp160-specific CTL. These data demonstrate that both the V3 loop itself and a 10-residue epitope are sufficient to prime CTL in vivo and strongly support the potential use of minigene-encoded CTL epitopes for recombinant vaccines designed to induce protective T cell-mediated immunity against HIV-1.     

Soluble HLA-A2.1 restricted peptides that are recognized by influenza virus specific cytotoxic T lymphocytes

The influenza A virus matrix protein derived peptide with amino acids 57-68 (Lys-Gly-Ileu-Leu-Gly-Phe-Val-Phe-Thr-Leu-Thr-Val) is recognized by influenza virus HLA-A2 restricted CTL. Because of the large number of hydrophobic residues this peptide is very insoluble. Substitution with a number of polar amino acids resulted in a soluble peptide (Lys-Lys-Ala-Leu-Gly-Phe-Val-Phe-Thr-Leu-Asp-Lys) that was very effective in sensitizing HLA-A2 positive target cells. Further substitution of threonine in position 65 with lysine resulted in a soluble antagonist peptide that inhibited sensitization. Both agonist and antagonist peptides retained 20% of their biological activity when tyrosine was added at the N terminus. Soluble radio-iodinated peptides can now be prepared that will be useful reagents to study the interaction of peptides and class I molecules.