Analysis of individual specific cytotoxic T lymphocytes for two MAGE-3-derived epitopes presented by HLA-A24.

BACKGROUND: The human MAGE-3 gene encodes tumor-specific antigens that are recognized by cytotoxic T lymphocytes (CTLs) and expressed in a high percentage of various malignant tumors. Of the five MAGE-3-derived CTL epitopes identified to date, two nonapeptides (TFPDLESEF and IMPKAGLLI, designated MAGE-3.A24a and MAGE-3.A24b, respectively) can be expressed on the tumor surface by binding to the HLA-A24 molecule, which is the most frequent HLA class I molecule in Asian populations. To compare the immunogenecities of the two peptides, individual specific CTL lines were generated for each peptide (MAGE-3.A24a and MAGE-3.A24b). METHODS: Peripheral blood mononuclear cells (PBMCs) from four HLA-A24+ healthy donors were stimulated in vitro with autologous dendritic cells pulsed with MAGE-3.A24a, MAGE-3.A24b or both and were subsequently cultivated with a cytokine combination including interleukin-2. RESULTS: We succeeded in generating peptide-specific CTL lines in two of the four donors. The two CTL lines showed similar cytolytic levels against three MAGE-3+/HLA-A24+ cancer cell lines and also target cells pulsed with the corresponding peptide. Cytolytic activities were blocked by either anti-CD8 or anti-HLA-A24 monoclonal antibodies. CONCLUSIONS: The results suggest that MAGE-3.A24a and MAGE-3.A24b peptides have equal potential in inducing MAGE-3-specific and HLA-A24-restricted CTLs.

A newly identified MAGE-3-derived epitope recognized by HLA-A24-restricted cytotoxic T lymphocytes.

Five MAGE-3-derived peptides carrying an HLA-A24-binding motif were synthesized. Binding capacity of these peptides was analyzed by an HLA-class-I stabilization assay. Two of the 5 peptides bound to HLA-A*2402 molecule with high affinity, and 3 peptides with low affinity. Peripheral-blood mononuclear cells (PBMC) depleted of CD4+T cells were stimulated with the peptides to determine whether these peptides would induce cytotoxic T lymphocytes (CTL) from PBMCs obtained from 7 healthy HLA-A*2402+ donors. Peptide M3-p97 (TFPDLESEF; corresponding to amino-acid residues 97-105 of MAGE-3), with high binding capacity to the HLA-A*2402 molecule, elicited the peptide-specific and HLA-A24-restricted CD8+CTL lines in 2 of the 7 donors, while none of the 4 other peptides induced CTL specific for the corresponding peptide in any of the donors. CTL lines induced by stimulation with peptide M3-p97 exhibited cytolytic activities against HLA-A*2402 transfectant cell lines (C1R-A*2402) in the presence of peptide M3-p97, but not in unloaded or irrelevant peptide-pulsed C1R-A*2402 cells. The CTL lines and a cloned CD8+CTL isolated from one of the bulk populations by limiting dilution could lyse MAGE-3+/HLA-A*2402+ squamous-cell-carcinoma(SCC) lines but neither MAGE-3-/HLA-A*2402+ nor MAGE-3+/HLA-A*2402- SCC lines, indicating that M3-p97 can be naturally processed and presented on the tumor-cell surface in association with HLA-A*2402 molecules. Combined with the 4 currently reported CTL epitopes derived from MAGE-3 and presented by HLA-A1, HLA-A2, HLA-A24 or HLA-B44, identification of this CTL epitope presented by the HLA-A*2402 molecule will extend the application of MAGE-3-derived peptides for immunotherapy for cancer patients.

Frequency of HLA-A alleles in Japanese patients with head and neck cancer.

BACKGROUND: Association between certain human leukocyte antigen (HLA) types such as HLA-A1 and -A3 and squamous cell carcinoma of the head and neck (SCCHN) has been demonstrated in the Caucasian population. HLA typings in these studies were performed by conventional serological methods. However, recent comparison studies between serological and molecular typings have revealed that the former are often inaccurate. METHODS: The frequency of HLA-A alleles in 100 Japanese patients with SCCHN and 100 control subjects was determined by the polymerase chain reaction, with primers specific for the HLA-A locus, in combination with dot-blot hybridization with 31 sequence-specific oligonucleotides. RESULTS: The frequencies of HLA-A*2602 and HLA-A*3303 were higher and those of HLA-A*2603 and HLA-A*3101 were lower in the patients with SCCHN than in healthy controls, but these differences were not statistically significant. In the 39 male patients with laryngeal carcinoma, the most common malignancies in Japanese patients with SCCHN, the frequency of HLA-A*2402 was significantly lower than that in the 80 male controls; however, after correction of the P value, statistical significance was not confirmed. In oral carcinoma patients, the frequency of HLA-A*2402 was significantly higher than that in healthy controls. CONCLUSIONS: These results suggest that the contribution of certain HLA-A alleles to susceptibility to SCCHN may differ between sites in the head and neck regions, despite these cancers being of an identical histological type, and that HLA-A*2402 may influence the development of oral carcinoma in Japanese patients.

Molecular analyses of HLA class II-associated susceptibility to subtypes of autoimmune diseases unique to Asians.

It is well known that individuals positive for particular HLA-class II alleles show high risks for the development of Takayasu arteritis and other diseases caused by immunological disorders such as autoimmune diseases and allergies. HLA class II molecules present antigenic peptides to CD4+ T cells. Their extensive polymorphism affects the structures of peptides bound to HLA class II molecules to create individual differences in immune responses to antigenic peptides. To better understand the mechanisms for association between HLA class II alleles and susceptibility to autoimmune diseases, it is important to identify self-peptides presented by disease-susceptible HLA class II molecules and triggering disease-causative T cells. Many autoimmune diseases are observed in all ethnic groups, whereas the incidences of diseases, clinical manifestations and disease-susceptible HLA class II alleles are different among various ethnic groups for some autoimmune diseases. These phenomena suggest that differences in autoimmune self-peptide(s) in the context of disease-susceptible HLA class II molecules may cause these differences. Therefore, comparisons among disease-susceptible HLA class II alleles, autoimmune self-peptides and clinical manifestations of autoimmune diseases in different ethnic groups would be helpful in determining the pathogenesis of the diseases. In this paper, we describe our recent findings on: (1) the uniqueness of both clinical manifestations and HLA-linked genetic background of Asian-type (optico-spinal form) multiple sclerosis; (2) the structural characteristics of peptides bound to HLA-DQ molecules susceptible to insulin-dependent diabetes mellitus; (3) the identification of a disease-related autoantigenic peptide presented by disease-susceptible HLA-DQ molecules in Asians-specific infant onset myasthenia gravis; and (4) a manipulation of human T cell response by altered peptide ligands, as a possible candidate for new and antigen-specific immuno-suppressive therapy against autoimmune diseases.

Peptide-based molecular analyses of HLA class II-associated susceptibility to autoimmune diseases.

Recent advances in knowledge of crystal structures of MHC class II molecules has advanced understanding of the molecular basis for interactions between peptides and HLA class II molecules. Polymorphism of HLA class II molecules influences structures of peptides bound to HLA class II molecules. To better understand mechanisms related to particular HLA class II alleles and autoimmune diseases, it is important to identify self-peptides presented by disease-susceptible HLA class II molecules and triggering disease-causative autoreactive T cells. Autoimmune diseases occur in Caucasians, Blacks and Asians, albeit with a different incidence. In some autoimmune diseases, disease-susceptible HLA class II alleles are closely related but different, and clinical manifestations of diseases differ among ethnic groups. These phenomena strongly suggest that difference in autoimmune self-peptide(s) in the context of disease-susceptible HLA class II molecules may explain the different clinical manifestations of diseases. Therefore, a comparison among disease-susceptible HLA class II alleles, autoimmune self-peptides and clinical manifestations of autoimmune diseases in different ethnic groups would be instructive. We directed efforts to determining: (1) HLA-class II alleles specific to Asian populations and which are associated with susceptibility to autoimmune diseases, (2) binding-peptide motifs for these HLA class II molecules, and (3) self-peptides presented by susceptible HLA class II molecules to stimulate autoreactive T cells related to the development of autoimmune diseases in Asians. In this review, our related recent investigations are described and the uniqueness of HLA class II-associated autoimmune diseases in Asians is given emphasis.

Differential binding of peptides substituted at putative C-terminal anchor residue to HLA-DQ8 and DQ9 differing only at beta 57.

HLA-DQ8 (DQA1*0302-DQB1*0302: DQ beta 57 Ala) and (DQA1*0302-DQB1*0303: DQ beta 57 Asp) differ only at beta 57, at which polymorphism reportedly confers distinct susceptibility to insulin-dependent diabetes mellitus (IDDM). To identify the differential peptide binding affected by beta 57, we determined DQ9-binding peptides by affinity-based selection of a phage random peptide library using the biotinylated DQ9 complex. Nonconservative single-residue substitution of high-affinity DQ8- and DQ9-binding peptide (1KLPDYVLWSSSTVVGLGAAGA21) at the underlined residues significantly decreased the peptide binding to DQ8 and DQ9. Affinities of the wild-type 21-mer K4DYVLWSSSTV13 and K4AYAAWAAATA13 to DQ8 and DQ9 were practically the same. The K4DYVLWSSSTV13-based analogue peptides with substitutions at 12T showed that residues R, K, H, E, D, Q, N, T, S, V, L, I, F, M, W, and Y permitted binding to DQ8, whereas only R, T, V, L, I, F, M, W, and Y did so to DQ9. Thus, significant differences exist between DQ9 and DQ8, in that the majority of polar residues, regardless of their static charges at the residue 12, permitted binding to IDDM-susceptible DQ8, which is not the case for DQ9. The affinities of K4DYVLWSSSXV13 AND K4AYAAWAAAAX13 (where X is T, A, K, D, or I) were almost equal to DQ8 and DQ9, suggesting the DQ8- and DQ9-binding peptide motifs could accept both the 8-mer and 9-mer frames depending on intervening sequences between N- and C-terminal anchor residues. The biochemical basis of peptide-HLA interactions determined by DQ beta 57 is discussed.

HLA-DQ-binding peptide motifs. 1. Comparative binding analysis of type II collagen-derived peptides to DR and DQ molecules of rheumatoid arthritis-susceptible and non-susceptible haplotypes.

The frequency of the HLA-DR4-DQ4 haplotype (DRB1(*)0405-DQA1(*)0302-DQB1(*)0401) is significantly increased in Japanese patients with rheumatoid arthritis (RA) and DRB1(*)0405-binding peptide motifs were identified in our previous studies. To clarify the DQ4-binding peptide motifs, the primary structure of DQ4-binding peptides was determined by affinity-based selection of a phage random peptide library. Analog peptides of a high-affinity DQ4 binder revealed that two major anchors (VxxxxxxxR; where x is any amino acid) play an essential role in binding to DQ4. The affinity of synthetic VAAAAAAAR-based analog peptides showed that substituting V to W, G, L, I, M, P, F, Y or A and R to H, M, L, I or V allows binding. The involvement of the ninth residue of the peptides, especially Arg, was critical for high-affinity binding. In comparison with other class II-binding peptide motifs reported to date, peptide motifs for DQ4 were unique, in that Gly and Pro are allowed as low-affinity N-terminal anchors. Interestingly, 94 putative DQ4-binding motifs were detected in the human type II collagen molecule, since it is composed of (Gly-X1-X2)n and is rich in R and P at positions X2. However, no significant differences were observed between the affinities of the collagen-derived peptides with DR or DQ molecules of RA-susceptible DR4-DQ4 and with those of non-susceptible DR4-DQ8 (DRB1(*)0406-DQA1(*)0301-DQB1(*)0302) haplotypes, indicating that the susceptibility to RA is not a simple immune response gene phenomenon specific to collagen. The immunogenetic implications of the unique peptide motifs for DQ are discussed.