Lack of tumor recognition by cytolytic T lymphocyte clones recognizing peptide 195–203 encoded by gene <Emphasis Type="Italic">MAGE-A3</Emphasis> and presented by HLA-A24 molecules

Gene MAGE-A3 encodes tumor-specific antigenic peptides recognized by T cells on many tumors. MAGE-A3 peptides presented by HLA class I molecules have been identified using CD8 lymphocytes stimulated with cells that either expressed gene MAGE-A3 or were pulsed with candidate peptides. One antigen identified with the latter method is peptide MAGE-A3(195-203) IMPKAGLLI, presented by HLA-A24 molecules. It has been used to vaccinate advanced cancer patients. Here, we have used HLA/peptide tetramers to detect T cells recognizing this peptide. Their frequency was estimated to be 2 x 10(-8) of the blood CD8 cells in non-cancerous HLA-A24(+) individuals, which is tenfold lower than the reported frequencies of T cells against other MAGE peptides. In the blood of a patient vaccinated with MAGE-A3, the estimated frequency was 5 x 10(-7). Anti-MAGE-3.A24 cytolytic T cell clones were derived, that lysed peptide-pulsed cells with half-maximal effect at the low concentration of 500 pM. However, these CTL did not recognize a panel of HLA-A24(+) tumor cells that expressed MAGE-A3 at levels similar to those found in HLA-A1(+) tumor cells recognized by anti-MAGE-3.A1 CTLs. Furthermore, 293-EBNA cells transfected with MAGE-A3 and HLA-A24 constructs were hardly recognized by the anti-MAGE-3.A24 CTL clones. These results suggest that peptide MAGE-A3(195-203) is poorly processed and is not an appropriate target for cancer immunotherapy.     

Asymmetry in the recognition of HLA-A3 molecules by virus-specific cytotoxic T cells

Cytotoxic T cells specific for influenza virus A/HK or Epstein-Barr virus were used to study the heterogeneity of the HLA-A3 molecule. Variability of the recognition of HLA-A3 in both systems was observed. The hierarchy was both effector cell and target cell specific. An extreme example of the hierarchy of HLA-A3 recognition is the following. Virus-specific cytotoxic T lymphocytes of a given donor were found to recognize all HLA-A3-matched target cells, including target cells of a donor from whom the virus-specific effector cells did not recognize target cells of that given donor: Donor A recognizes target B but donor B does not recognize target A. Both will recognize a third HLA-A3-matched target cell C. Cold target inhibition studies confirmed that the recognition of target cell B by effector cell A involved the recognition of only HLA-A3. Examples of such asymmetric recognition were found in both influenza A and Epstein-Barr virus-specific cytotoxic T-lymphocyte responses but not one combination was asymmetric in both systems. This suggests that influenza virus A/HK-specific cytotoxic T lymphocytes recognize other HLA-A3 histotopes than do Epstein-Barr virus-specific cytotoxic T lymphocytes.     

Molecular analysis of an HLA-A2 functional variant CLA defined by cytolytic T lymphocytes

By using cytolytic T lymphocytes (CTL), the HLA-A2 serologic specificity may be divided into at least four subtypes designated as A2.1 to A2.4. The HLA-A2.4 antigen expressed by donor CLA is not recognized by allogeneic CTL specific for either A2.1, A2.2, or A2.3, but is indistinguishable from HLA-A2.1 by H-Y-specific, HLA-A2-restricted CTL and by isoelectric focusing. The structure of this HLA-A2.4 antigen was compared with the known structure of the main A2.1 subtype expressed on JY cells to establish the molecular basis for the immunologic differences between the two antigens. Comparative peptide mapping and radiochemical sequence analysis were used to establish that they differed by a single amino acid change: Phe at position 9 in HLA-A2.1 was replaced by Tyr in HLA-A2.4 from donor CLA. This position displays the highest variability score among all polymorphic residues of the class I HLA antigens. But its participation in the specific determinants recognized by CTL has not been previously established, because no other known HLA variant or H-2 mutant has been found to vary at this position. In addition, HLA-A2.4 from CLA is the only HLA-A2 subtype antigen that is identical to A2.1 in the segment spanning residues 147 to 157, a region in which all three A2.1, A2.2, and A2.3 antigens are different.     

Enhancement of cytotoxic T-lymphocyte responses in patients with gastrointestinal malignancies following vaccination with CEA peptide–pulsed dendritic cells

Carcinoembryonic antigen (CEA) is strongly expressed in a vast majority of gastrointestinal carcinomas. Recently, epitope peptides of CEA were identified. We have demonstrated HLA-A24–restricted peptide, CEA652[9] (TYACFVSNL), was capable of eliciting specific cytotoxic T lymphocytes (CTLs) which could lyse tumor cells expressing HLA-A24 and CEA. HLA-A24 is the most applicable MHC class I allele in the Japanese population. In this pilot study, we have used the peptide-pulsed dendritic cells (DCs) generated from peripheral blood mononuclear cells (PBMCs) supplemented with GM-CSF and IL-4 as the source of the vaccine. Eight patients with advanced CEA-expressing gastrointestinal malignancies received subcutaneous injections every 2 or 3 weeks. Immunomonitoring was performed by ELISpot (enzyme-linked immunosorbent spot) assay to measure the precursor frequency of CTLs and their capacity to elicit antitumor CTLs in vitro. Four of seven patients have developed their CTL response after vaccinations. DTH reaction was observed in one of eight patients at the DC-injected site. Skin biopsy at the injected site showed the infiltration of the lymphocytes. Furthermore, A24/CEA peptide tetramer assay revealed an increase in peptide-specific T-cell precursor frequency in vaccinated patients. No significant toxic adverse effects were observed, except for mild diarrhea in one case after three vaccinations. Three patients have shown stabilization of the disease after vaccinations. In conclusion, our results clearly demonstrated that our vaccination protocol was safe and might develop a CEA-specific CTL response in cancer patients.     

Recognition of HLA-A2 mutant and variant target cells by an HLA-A2 allospecific human cytotoxic T lymphocyte line

HLA-A2 specific human cytotoxic T lymphocytes (CTL) cell lines have been developed using T cell growth factor and coculture of peripheral blood lymphocytes with selected allogeneic target cell lines. The CTL-8 line showed specificity for human leukocyte antigens (HLA)-A2 bearing target cells after 5 weeks in culture when tested against a panel of 14 lymphoblastoid cell lines in a 51Chromium (51Cr) release assay. Purified anti-human leukocyte antigens (HLA) monoclonal antibodies W6/32 and PA2.1 inhibited cytolysis by 85% and 60%, respectively. The CTL-8 line lysed non-HLA-A2 target cells in the presence of lectins concanavalin A (Con A) or phytohemagglutinin-P lectin (PHA-P) indicating the specificity of cytolysis was not due to nonspecific resistance of target cells to the CTL-lytic mechanism. The T5-1 HLA-A2 mutant cell series were tested as targets for the CTL-8 line. Cell clones 8.18.1, 8.21.1 and 8.6.1, which express altered HLA-A2 molecules as determined by their decreased reactivity with allospecific monoclonal antibodies, were lysed by the CTL-8 line as efficiently as the T5-1 wild type. These cell lines also acted as efficient cold target competitors for a normal HLA-A2 target cell. The 8.14.1 cell clone expressed a lower amount of HLA-A2 alloantigen and showed a corresponding decreased reactivity with CTL-8 in direct cytolytic and cold target competitive inhibition assays. In contrast, the M7 and DK1 HLA-A2 variant cell lines, which express normal HLA-A2 serological determinants, were inefficiently lysed by CTL-8 and did not act as competitive inhibitors of normal HLA-A2 target cells. These results support the concept that the alloantigenic determinant(s) recognized by T cells and antibodies occur at separate regions on the HLA-A2 molecule.     

Identification of two new HLA-A*1101-restricted tax epitopes recognized by cytotoxic T lymphocytes in an adult T-cell leukemia patient after hematopoietic stem cell transplantation

We previously reported that Tax-specific CD8(+) cytotoxic T lymphocytes (CTLs), directed to single epitopes restricted by HLA-A2 or A24, expanded in vitro and in vivo in peripheral blood mononuclear cells (PBMC) from some adult T-cell leukemia (ATL) patients after but not before allogeneic hematopoietic stem cell transplantation (HSCT). Here, we demonstrated similar Tax-specific CTL expansion in PBMC from another post-HSCT ATL patient without HLA-A2 or A24, whose CTLs equally recognized two newly identified epitopes, Tax88-96 and Tax272-280, restricted by HLA-A11, suggesting that these immunodominant Tax epitopes are present in the ATL patient in vivo.     

Structural identity between HLA-A2 antigens differentially recognized by alloreactive cytotoxic T lymphocytes

Alloreactive CTL raised against HLA-A2 Ag often display heterogeneous recognition of HLA-A2+ target cells. This heterogeneity has been found to reflect structural polymorphism among the corresponding target Ag, thus defining HLA-A2 subtypes. A previous study (van der Poel et al. 1986. Human Immunol. 16:247) established the existence of a new HLA-A2.4 variant, A2-SCHU, that was distinguished from A*0206 (A2.4a) by HLA-A2-specific alloreactive CTL. The same CTL subdivided HLA-A2.1 Ag into two subgroups. In the present study, the molecular basis of this heterogeneity has been examined by double-label comparative peptide mapping analysis of differentially recognized A2.1 and A2.4 Ag. In addition, we have determined the complete sequence of polymerase chain reaction-amplified full length cDNA from A2-SCHU. The results show that: 1) A2-SCHU is indistinguishable from A*0206 by peptide mapping; 2) the cDNA sequence of A2-SCHU is identical to that of A*0206; and 3) two differentially recognized A2.1 Ag are both indistinguishable from A*0201 by comparative peptide mapping. These results indicate that differential recognition by alloreactive CTL can occur among structurally identical class I HLA Ag and suggest that allorecognition by such CTL may involve corecognition of endogenous peptides, presumably derived from polymorphic proteins.     

Structural analysis of HLA-A2.4 functional variant KNE. Implications for the mapping of HLA-A2-specific T-cell epitopes

HLA-A2 antigens are divided into four subtypes, designated A2.1 to A2.4, by the use of cytolytic T lymphocytes (CTL). The A2.4 subtype consists of a functionally heterogeneous group of variants that are not recognized by A2.1-, A2.2-, or A2.3-specific CTL lines while it is indistinguishable from A2.1 by isoelectric focusing. The structure of an A2.4 variant expressed on donor KNE has been established by comparative peptide mapping with A2.1 and radiochemical sequencing. It was found to differ from A2.1 by a single amino acid change of Cys to Tyr at position 99. This position is only moderately polymorphic and has not previously been found to vary in any other HLA or H-2 variants. The nature of the change is compatible with its generation by one-point mutation from A2.1. The only other previously characterized A2.4 variant, CLA, differs from A2.1 by a single amino acid replacement at position 9. Both residues 9 and 99 are located in homologous positions within the 1 and 2 domains, respectively. The results shown contribute to the molecular interpretation of the heterogeneity of CTL recognition within the HLA-A2.4 group of antigens.Abbreviations used in this paper CTL cytotoxic T lymphocytes - HPLC high performance liquid chromatography     

In vitro-isolated human cytotoxic T-lymphocyte clones detect variations in serologically defined HLA antigens

T cells of two donors, JR (HLA-A23, 29; B7,7; G; DRw5) and HG (HLA-A2, 23; B40, w44; Cw4), were stimulated with cells from an HLA homozygous lymphoblastoid cell line JY (HLA-A2, 2; B7,7, C-, DRw4, 6) and cloned by limiting dilution after the third stimulation. Two cytotoxic T-cell (CTL) clones, JR-2-16 (from donor JR) and HG-31 (from donor HG), were used for detailed studies. The results of a panel study using lymphocytes from HLA-typed individuals and a study with two HLA recombinant families indicate that the antigens recognized by the CTL clones JR-2-16 and HG-31 were highly associated with HLA-A2 and HLA-B7, respectively. Blocking studies with a monoclonal antibody recognizing a framework determinant on HLA-A, -B and-C antigens and a monoclonal antibody reacting with HLA-A2 support the notion that JR-2-16 and HG-31 interact with the HLA-A2 and the HLA-B7 antigens per se. However, these clones did not recognize the HLA-A2 and HLA-B7 of all donors typed for these antigens, suggesting that the HLA-A2 and HLA-B7 antigens of these particular donors are variants of the serologically defined HLA antigens. These results indicate that in vitro-derived human CTL clones detect variations in the serologically defined allospecificities and can be used as reagents to elucidate the polymorphism of HLA antigens further.Abbreviations used in this paper: CTL cytotoxic - T lymphocytes - BSA bovine serum albumin - PHA phytohemagglutinin - Con A concanavalin A.     

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.     

Selection and accumulation of an HIV-1 escape mutant by three types of HIV-1-specific cytotoxic T lymphocytes recognizing wild-type and/or escape mutant epitopes

It is known that cytotoxic T lymphocytes (CTLs) recognizing HIV-1 escape mutants are elicited in HIV-1-infected individuals, but their role in the control of HIV-1 replication remains unclear. We investigated the antiviral ability of CTLs recognizing the HLA-A*24:02-restricted Gag28 -36 (KYKLKHIVW) epitope and/or its escape mutant (KYRLKHIVW) elicited in the early and chronic phases of the infection. Wild-type (WT)-epitope-specific CTLs, as well as cross-reactive CTLs recognizing both WT and K30R (3R) epitopes, which were predominantly elicited at early and/or chronic phases in HLA-A*24:02(+) individuals infected with the WT virus, suppressed the replication of the WT virus but failed to suppress that of the 3R virus, indicating that the 3R virus was selected by these 2 types of CTLs. On the other hand, cross-reactive and 3R-specific CTLs, which were elicited in those infected with the 3R virus, did not suppress the replication of either WT or 3R virus, indicating that these CTLs did not contribute to the control of 3R virus replication. High accumulation of the 3R mutation was found in a Japanese population recently recruited. The selection and accumulation of this 3R mutation resulted from the antiviral ability of these Gag28-specific CTLs and high prevalence of HLA-A*24:02 in a Japanese population. The present study highlighted the mechanisms for the roles of cross-reactive and mutant-epitope-specific CTLs, as well as high accumulation of escape mutants, in an HIV-1-infected population.     

Substitution analog peptide derived from HER-2 can efficiently induce HER-2-specific, HLA-A24 restricted CTLs

In order to broaden the possibility for anti-HER-2/neu (HER-2) immune targeting, it is important to identify HLA-A24 restricted peptide epitopes derived from HER-2, since HLA-A24 is one of the most common alleles in Japanese and Asian people. In the present study, we have screened HER-2-derived, HLA-A24 binding peptides for cytotoxic T lymphocyte (CTL) epitopes. A panel of HER-2-derived peptides with HLA-A24 binding motifs and the corresponding analogs designed to enhance HLA-A24 binding affinity were selected. Identification of HER-2-reactive and HLA-A24 restricted CTL epitopes were performed by a reverse immunology approach. To induce HER-2-reactive and HLA-A24 restricted CTLs, PBMCs from healthy donors were repeatedly stimulated with monocytes-derived, mature DCs pulsed with HER-2 peptide. Subsequent peptide-induced T cells were tested for the specificity by enzyme linked immunospot, cytotoxicity and tetramer assays. CTL clones were then obtained from the CTL lines by limiting dilution. Of the peptides containing HLA-A24 binding motifs, 16 peptides (nine mers) including wild type peptides (IC₅₀<1,000 nM) and substituted analog peptides (IC₅₀<50 nM) were selected for the present study. Our studies show that an analog peptide, HER-2(905AA), derived from HER-2(905) could efficiently induce HER-2-reactive and HLA-A24 restricted CTLs. The reactivity of the HER-2(905AA)-induced CTL (CTL905AA) was confirmed by different CTL assays. The CTL905AA clones also were able to lyse HER-2(+), HLA-A24(+) tumor cells and cytotoxicity could be significantly reduced in cold target inhibition assays using cold targets pulsed with the HER-2(905) wild type peptide as well as the inducing HER-2(905AA) analog peptide. A newly identified HER-2(905) peptide epitope is naturally processed and presented as a CTL epitope on HER-2 overexpressing tumor cells, and an MHC anchor-substituted analog, HER-2(905AA), can efficiently induce HER-2-specific, HLA-A24 restricted CTLs.     

Mutations in human lymphocytes studied by an HLA selection system

Human lymphocytes mutated at the HLA-A2 or HLA-A3 alleles were enumerated and studied by primary selection using antibody and complement, followed by limiting dilution cloning and secondary selection using immunofluorescence or antibody and complement. The geometric mean frequency of in vivo mutant lymphocytes was 3.08 X 10(-5) for the HLA-A2 allele and 4.68 X 10(-6) for the HLA-A3 allele. Mutagenesis by X-radiation or mitomycin produced a dose-related increase in mutant frequency. HLA-B phenotyping and Southern Analysis of the HLA-A gene suggested that mutation was frequently due to gene deletion, which was often substantial.     

Specificity of peptide binding by the HLA-A2.1 molecule

The HLA-A2 molecule contains a putative peptide binding site that is bounded by two alpha-helices and a beta-pleated sheet floor. Previous studies have demonstrated that the influenza virus matrix peptide M1 55-73 can sensitize target cells for lysis by HLA-A2.1-restricted virus-immune CTL and can induce CTL that can lyse virus-infected target cells. To assess the specificity of peptide binding by the HLA-A2.1 molecule, we examined the ability of seven variant M1 peptides to be recognized by a panel of M1 55-73 peptide-specific HLA-A2.1-restricted CTL lines. The results demonstrate that five out of the seven variant M1 55-73 peptides could be recognized by A2.1-restricted M1 55-73 peptide-specific CTL lines. The two variant peptides that were not recognized by any CTL could bind to HLA-A2.1 as indicated by their ability to compete for presentation of the M1 55-73 peptide. In addition, 5 of a panel of 24 unrelated peptides tested could also compete for M1 55-73 presentation by HLA-A2.1. One peptide derived from the sequence of a rotavirus protein could sensitize HLA-A2.1+ targets for lysis by M1 55-73 peptide-specific CTL. We conclude from these studies that: 1) the HLA-A2.1 molecule can bind a broad spectrum of peptides; 2) T cells selected for the ability to recognize one peptide plus a class I molecule can actually recognize an unrelated peptide presented by that same class I molecule; and 3) a stretch of three adjacent hydrophobic amino acids may be an important common feature of peptides that can bind to HLA-A2.1.     

Supra-agonist peptides enhance the reactivation of memory CTL responses

Single amino acid substitutions at TCR contacts may transform a natural peptide Ag in CTL ligands with partial agonist, antagonist, or null activity. We obtained peptide variants by changing nonanchor amino acid residues involved in MHC class I binding. These peptides were derived from a subdominant HLA-A2-presented, latent membrane protein 2-derived epitope expressed in EBV-infected cells and in EBV-associated tumors. We found that small structural changes produced ligands with vastly different activities. In particular, the variants that associated more stably to HLA-A2/molecules did not activate any CTL function, behaving as null ligands. Interestingly, T cell stimulations performed with the combination of null ligands and the natural epitope produced significantly higher specific CTL reactivation than reactivation of CTLs induced by the wild-type epitope alone. In addition, these particular variants activated memory CTL responses in the presence of concentrations of natural epitope that per se did not induce T cell responses. We show here that null ligands increased ZAP-70 tyrosine kinase activation induced by the natural epitope. Our results demonstrate for the first time that particular peptide variants, apparently behaving as null ligands, interact with the TCR, showing a supra-agonist activity. These variant peptides did not affect the effector T cell functions activated by the natural epitope. Supra-agonist peptides represent the counterpart of antagonists and may have important applications in the development of therapeutic peptides.     

T cell activity after dendritic cell vaccination is dependent on both the type of antigen and the mode of delivery

Previous work in both human and animal models has shown that CTL responses can be generated against proteins derived from tumors using either peptide-pulsed dendritic cells (DCs) or nucleic acids from the tumor transfected into autologous DCs. Despite the efficacy of this approach for vaccine therapy, many questions remain regarding whether the route of administration, the frequency of administration, or the type of Ag is critical to generating T cell responses to these Ags. We have investigated methods to enhance CTL responses to a peptide derived from the human proto-oncogene HER-2/neu using mice containing a chimeric HLA A2 and H2Kb allele. Changes in amino acids in the anchor positions of the peptide enhanced the binding of the peptide to HLA-A2 in vitro, but did not enhance the immunogenicity of the peptide in vivo. In contrast, when autologous DCs presented peptides, significant CTL activity was induced with the altered, but not the wild-type, peptide. We found that the route of administration affected the anatomic site and the time to onset of CTL activity, but did not impact on the magnitude of the response. To our surprise, we observed that weekly administration of peptide-pulsed DCs led to diminishing CTL activity after 6 wk of treatment. This was not found in animals injected with DCs every 3 wk for six treatments or in animals initially given DCs weekly and then injected weekly with peptide-pulsed C1R-A2 transfectants.     

Comparative structural analysis of HLA-A3 antigens distinguishable by cytotoxic T lymphocytes: variant E1

Influenza-specific cytotoxic T cells restricted by HLA-A3 recognize differences between HLA-A3 antigens that are serologically indistinguishable. To examine whether this differential recognition had a structural basis, we have compared the structures of HLA-A3 molecules from Epstein Barr virus-transformed peripheral blood lymphocytes of two individuals, E1 and M17. M17 was representative of the majority of HLA-A3-bearing individuals, whereas E1 was a variant distinguished by cytotoxic T cells. Peptide map comparisons revealed a small number of differences when particular amino acids were used to radiolabel the A3 molecules. Sequence analysis and comparison of the results with the prototypic HLA-A3 sequence localized the variability to a tryptic peptide spanning residues 147-157. To obtain the complete amino acid sequence for the E1 and M17 A3 molecules in the 147-157 region, the CNBr fragments beginning at residue 139 were isolated and sequenced. Two amino acid differences were detected between the HLA-A3 molecule of the CTL-defined variant E1 and that of M17. At position 152, Glu in donor M17 had changed to Va1 in donor E1, and at position 156, Leu in donor M17 had changed to Gln in donor E1. The finding that A3 molecules from E1 are altered in the region between residues 147 and 157 is consistent with studies on HLA-A2 variants and Kb mutants showing that this region of class I molecules is important for CTL recognition but not for recognition by serologic reagents.     

Comparative structural analysis of HLA-A2 antigens distinguishable by cytotoxic T lymphocytes. II. Variant DK1: evidence for a discrete CTL recognition region

Multiple amino acid sequence differences distinguish individual HLA antigens. Those residues important in immune recognition events have not been defined. Recent studies have identified HLA-A2 structural variants that, although serologically indistinguishable from other HLA-A2 antigens, are recognized poorly, if at all, by HLA-A2-restricted, influenza virus-immune, or HLA-A2-specific alloimmune CTL. In this study we utilize double-label tryptic peptide comparisons performed by both reverse-phase HPLC and cation exchange chromatography, in conjunction with conventional and microsequence analysis, to characterize the HLA-A2 heavy chains derived from variant DK1. We detect a single tryptic peptide that distinguishes DK1 HLA-A2 from the predominant HLA-A2 heavy chain species. This peptide spans residues 147 to 157 in the second heavy chain domain, and carries substitutions at positions 149, 152, and 156. Residues in this segment of the polypeptide are also altered in another HLA-A2 variant, as well as one H-2Kb mutant. Thus, this segment appears to be critical in forming determinants important in CTL recognition of class I antigens in general. On the basis of these and other results, we suggest that in contrast to recognition by alloantibodies, a discrete region of class I antigens may be crucial for CTL recognition.     

Tumor-specific cytotoxic T lymphocyte responses against chondrosarcoma with HLA haplotype loss restricted by the remaining HLA class I allele

Although loss of HLA expression by malignant cells has also been demonstrated, it has not been clarified how the loss of HLA expression observed in vitro actually results in immune escape. We demonstrated two major findings: (i) a part of chromosome 6 coding for HLA haplotypes was deleted from the genome of chondrosarcoma cell line, OUMS-27; furthermore, immunohistostaining for HLA-A11 showed that the original chondrosarcoma tissue lost the expression of HLA-A11, implicating that HLA haplotype loss was already present in the original tumor tissue and (2) HLA class I-restricted and autologous tumor-specific cytotoxic T cells (CTL) were generated from peripheral blood lymphocytes of the patient with chondrosarcoma, from whom OUMS-27 originated. This CTL line was maintained by weekly stimulation with OUMS-27, and lysed OUMS-27 in an HLA-A24 dependent manner but did not either K562 or autologous (EBV)-transformed B cells. These observations indicated that OUMS-27 and its original tumor are still immunogenic and can present antigen peptides with the remaining HLA-A24, even if HLA expression is partially lost. Tumor specific immunotherapy can be applied to the treatment of malignancies, even if HLA expression is partially lost.