Masanobu Nakao

Identification of a Gene Coding for a New Squamous Cell Carcinoma Antigen Recognized by the CTL

Peptide-based specific immunotherapy has resulted in tumor regression in some melanoma patients. However, tumor Ags and peptides for specific immunotherapy, except for treatment of melanomas, have not yet been well identified. In this study, we report a gene encoding a new squamous cell carcinoma (SCC) Ag recognized by cells of the HLA-A24-restricted and tumor-specific CTL line. This gene with 3958-bp length was transcribed from the chromosome 6q22 with six exons, and its mRNA was ubiquitously expressed in both SCCs and normal tissues, and partly expressed in adenocarcinomas. The deduced 958-aa sequence encoded by this gene showed no similarity to any known amino acid sequences. This gene product had a characteristic of an endoplasmic reticulum-resident protein. A 100-kDa protein was detected in the vast majority of SCCs from various tissues, in majority of renal cell carcinomas and brain tumors, and in about one-third of melanomas and adenocarcinomas from various organs other than the breast. In contrast, it was not expressed at all in any of the normal cells or tissues tested, including the testis and fetal liver. Three different peptides at positions 93–101, 161–169, and 899–907 of this Ag were recognized by this CTL line, and all of them induced HLA-A24-restricted and tumor-specific CTLs from PBMCs of SCC patients. Therefore, these peptides may be useful for peptide-based specific immunotherapy of HLA-A24+ patients with SCC in various organs, as well as for treatment of other cancer.

Identification of a SART‐1‐derived peptide capable of inducing HLA‐A24‐restricted and tumor‐specific cytotoxic T lymphocytes

We have described the SART‐1 gene–encoding peptides recognized by HLA‐A2601‐restricted and tumor‐specific cytotoxic T lymphocytes (CTLs). We now have investigated whether SART‐1 encodes peptides capable of inducing the HLA‐A24‐restricted CTLs. Among the 18 different peptides with HLA‐A24‐binding motifs, the SART‐1690–698 peptide (EYRGFTQDF) was most strongly recognized by the HLA‐A24‐restricted and tumor‐specific CTLs established from an esophageal cancer patient. After a third stimulation in vitro, this peptide induced HLA‐A24‐restricted CTLs recognizing the SART‐1259+ tumor cells in PBMCs of all HLA‐A24 homozygous and the majority of HLA‐A24 heterozygous cancer patients and healthy donors tested. A similar activity, induction of CTLs from PBMCs, was observed in the Saccharomyces cerevisiae–derived nonapeptide (EYRGFTPMF) that shares 7 amino acids with the SART‐1690–698 peptide. The SART‐1690–698 peptide–induced CTL activity was significantly higher in PBMCs of HLA‐A24 homozygotes than in HLA‐A24 heterozygotes. The CTL precursor frequency in PBMCs after a third stimulation in vitro with the SART‐1690–698 peptide was high (>1/200) in both cancer patients and healthy donors. The SART‐1690–698 peptide could thus be useful for specific immunotherapy of HLA‐A24+ cancer patients. Int. J. Cancer 81:459–466, 1999.

Histocompatibility leukocyte antigen-A2402-restricted cytotoxic T lymphocytes recognizing adenocarcinoma in tumor-infiltrating lymphocytes of patients with colon cancer

To cast light on T cell‐mediated specific immunity at the tumor site of colon cancer, we investigated whether interleukin‐2 (IL‐2)‐activated tumor‐infiltrating lymphocytes (TIL) from colon cancer show histocompatibility leukocyte antigen (HLA)‐class I‐restricted cytotoxicity against adenocarcinoma. IL 2‐activated TIL from all four HLA‐A24 patients examined lysed HLA‐A2402+ adenocarcinomas, but not HLA‐A2402 tumors. Those of two of the four cases also lysed HLA‐A2402+ squamous cell carcinomas. CD8+ cytotoxic T lymphocyte (CTL) clones recognizing HLA‐A2402+ adenocarcinomas were established from one CTL line. This CTL line produced IFN‐γ upon recognition of an HLA‐A2402‐ adenocarcinoma transfected with HLA‐A2402 cDNA. These results suggest the presence of HLA‐A2402‐restricted CTL recognizing adenocarcinoma at the tumor site of colon cancer. Furthermore, HLA‐A31‐restricted CTL activity was found in IL‐2‐activated TIL from one of two HLA‐A31+ patients, suggesting the existence of HLA‐class I‐restricted CTL involving an allele other than A24

Higher susceptibility of erythropoietin-producing renal cell carcinomas to lysis by lymphokine-activated killer cells

Erythropoietin production by renal cell carcinoma (RCC) is reported to be a potential marker for interleukin-2/interferon-alpha-responding tumor. We have investigated whether erythropoietin of RCC cells is involved in the immune recognition by lymphokine-activated killer (LAK) cells. Cells from primary culture of RCC cells expressing erythropoietin-mRNA or producing erythropoietin were more susceptible to lysis by LAK cells than those not expressing or producing it, respectively. RCC cells transfected with erythropoietin-cDNA became more susceptible to lysis by LAK cells than their erythropoietin-negative parental cells. These results indicate higher susceptibility of erythropoietin-producing RCC cells to lysis by LAK cells, suggesting that erythropoietin of RCC cells is involved in the immune recognition by LAK cells.

Induction of human leukocyte antigen-A26-restricted and tumor-specific cytotoxic T lymphocytes by a single peptide of the SART1 antigen in patients with cancer with different A26 subtypes.

Peptide antigens available for use in specific immunotherapy of patients with cancer have not been fully determined. Although the authors have reported the SART1 gene encoding epitopes recognized by HLA-A2601–restricted and tumor-specific cytotoxic T lymphocytes (CTLs), the HLA-A26 allele is mainly subdivided into A2601, A2602, and A2603 subtypes. In this study, the authors attempted to determine whether the SART1-derived peptide at position 736-744 (KGSGKMKTE) is suitable to induce HLA-A26–restricted and tumor-specific CTLs in patients with cancer who have these subtypes. This peptide induced the HLA-A26 subtype-restricted and tumor-specific CTLs in HLA-A2601+ or HLA-A2603+ peripheral blood mononuclear cells, respectively. It also induced the HLA-A26–restricted CTL activity in HLA-A2602+ peripheral blood mononuclear cells. Therefore, this peptide could be useful for specific immunotherapy of patients with cancer who have any of the three HLA-A26 subtypes.

Cytokines Required for Induction of Histocompatibility Leukocyte Antigen‐class I‐restricted and Tumor‐specific Cytotoxic T Lymphocytes by a SART1‐derived Peptide

Although there have been several reports on peptides of human tumor‐rejection antigens capable of inducing histocompatibility leukocyte antigen (HLA)‐class I‐restricted and tumor‐specific cytotoxic T lymphocytes (CTLs), it is not yet clear which cytokines are required for CTL induction. This study has investigated the cytokine combinations required for optimal induction of CTLs by SARTI690–698 peptide, which is capable of inducing HLA‐A24‐restricted and tumor‐specific CTLs in peripheral blood mononuclear cells (PBMCs). Pretreatment of PBMCs as a source of antigen‐presenting cells (APCs) with interferon (IFN)‐γ, or to some extent with IFN‐α, but not with any of the other cytokines tested, augmented the peptide‐induced CTL activity in HLA‐A24 heterozygotes, but not in HLA‐A24 homozygotes. This IFN‐γ ‐mediated augmentation was inhibited by either interleukin (IL)‐4 or IL‐10. IL‐2 alone in culture, along with weekly stimulation by peptide‐pulsed APCs, was sufficient for the differentiation and proliferation of CTLs for the initial several weeks of culture. This IL‐2‐mediated activation of CTLs was inhibited by the addition of IFN‐γ, IL‐4, or IL‐10 to the IL‐2 culture. For further expansion of the CTLs, dendritic cells (DCs) induced from PBMCs with IL‐4 and granulocyte macrophage colony‐stimulating factor (GM‐CSF) were required as APCs. These results indicate that IFN‐γ and IL‐2 are important in the activation of APCs and CTLs, respectively, while GM‐CSF and IL‐4 are needed for the induction of DCs, which in turn are required for further expansion of mature CTLs. These results are important in allowing for a better understanding of the cellular and molecular basis of tumor‐specific immunity, and also for the development of peptide‐based specific immunotherapy.

Histocompatibility Leukocyte Antigen‐A2‐restricted and Tumor‐specific Cytotoxic T Lymphocytes from Tumor‐infiltrating Lymphocytes of a Patient with Testicular Embryonal Cancer

T lymphocytes play an important role in tumor rejection. To understand T cell‐mediated specific immunity at the tumor site of testicular embryonal cancer, we investigated whether interleukin‐2 (IL‐2)‐activated tumor‐infiltrating lymphocytes (TIL) of a patient with testicular embryonal cancer show histocompatibility leukocyte antigen (HLA)‐class I‐restricted and tumor‐specific cytotoxicity. We established a CD3+CD4‐CD8+ cytotoxic T lymphocyte (CTL) line from the IL‐2‐activated TIL of a 37‐year‐old patient with testicular embryonal cancer. A 6 h 51Cr‐release assay was performed to measure the cytotoxicity of the CTL. The CD3+CD4‐CD8+ CTL line showed cytotoxicity against HLA‐A2+ tumor cells, including freshly isolated autologous tumor cells, adenocarcinoma cell lines from various organs (lung, breast, pancreas, colon and kidney) and squamous cell carcinomas (esophagus and oral cavity). No other cell lines examined, including an autologous tumor cell line and HLA‐A2” tumor cell lines, were lysed by this CTL line. These results suggest the existence of HLA‐A2‐restricted and tumor‐specific CTL at the tumor site of testicular embryonal cancer.

T-Lymphocyte Response in Renal Cell Carcinoma

Several important immunological properties of tumor-infiltrating lymphocytes (TIL) from renal cell carcinoma (RCC) have been reported: (a) the magnitude of lymphocyte infiltration in RCC was higher than that in the other cancers (1); (b) the autologous tumor cell lysis in IL-2-activated TIL from RCC was higher than that in the other cancers except metastatic melanoma (1–4); (c) non-T cells, including CD3-CD56 + or CD3-CD16+ natural killer cells, were primarily responsible for autologous tumor cell lysis (1–5); (d) T cells were enriched with memory T cell markers (6); and (e) IL-2-activated TIL showed oligoclonal expansion (7–9). Therefore, TIL in RCC appeared to be more involved in host tumor-interaction than PBMC. However, there are few findings, to our knowledge, supporting the idea that RCC-specific T cells exist in TIL, PBMC, or the other immune organs in RCC patients. (10) Neither RCC TIL nor PBMC cultured with IL-2 alone displayed tumor-specific cytotoxicity. (1–8) These results suggest that RCC TIL have little or no CTL precursors, possibly because of the lower immunogeneity of untreated RCC cells. Poorly immunogenic animal tumor cells can be converted into highly immunogenic tumors by several techniques: (a) viral infection of tumor cells (11,12), (b) treatment of tumor cells with mutagenic agents such as 1-methyl-3-nitro-l-nitrosoguanidine (13, 14), (c) treatment of tumor cells with ultraviolet-B (UV) radiation or the others (15–19), (d) transfection of tumor cells with MHC class I antigens (20,21), and (e) transfection of tumor cells with cytokine genes (22, 23).