Yoshimasa Wada

Identification of human antitumor cytotoxic T lymphocytes epitopes of recoverin, a cancer‐associated retinopathy antigen, possibly related with a better prognosis in a paraneoplastic syndrome

Cancer‐associated retinopathy (CAR) is a rare paraneoplastic syndrome, and the recoverin‐specific autoantibody is suggested to contribute to the pathogenesis of retinopathy, including apoptosis of retinal cells. Because it is known that CAR+ cancer patients have a preferable prognosis, we hypothesized that aberrantly expressed recoverin in cancer cells can become a target of cytotoxic T lymphocytes (CTL). Here we tested nine recoverin‐derived HLA‐A24‐binding peptides for their capacity to elicit antitumor CTL. We observed recoverin‐specific CTL responses in two HLA‐A24+ CAR+ cancer patients. In addition, the CTL responses were obtained from three of ten CAR– cancer patients and two of six healthy individuals. The CTL precursor frequency of CAR+ cancer patients and that of CAR– cancer patients was higher than that of healthy individuals. Of nine recoverin peptides, R49 (QFQSIYAKF), R49.2 (QFQSIYAKFF), and R64 (AYAQHVFRSF) were discovered to induce the peptide‐specific CTL. Taken together, our present data suggest that peripheral activation of recoverin‐specific antitumor CTL is likely to contribute to the preferable prognosis of CAR+ cancer patients. Moreover, in cases other than CAR+ cancer patients, recoverin may offer the opportunity to design epitope‐based immunotherapeutic approaches for treating HLA‐A24+ cancer patients with a recoverin‐expressing tumor.

In vitro proliferation and the cytotoxic specificity of a cryopreserved cytotoxic T cell clone reacting against human autologous tumor cells.

Proliferation and functional maintenance of CTL after cell cryopreservation often proves to be quite difficult. We developed an improved method for proliferating cryopreserved CTL, and for gaining their specific cytotoxic function. T cells were cryopreserved at -180 degrees C in RPMI 1640 containing 50% FCS and 10% DMSO. The cryopreserved T cells were well recovered by culturing in a medium containing the supernatant of primary cultures with TIL and autologous tumor cells, in addition to a high concentration (350 U/ml) of rIL-2. Furthermore, these cells were proliferated more efficiently when MMC-treated autologous tumor cells were used in vitro as a feeder and an antigenic stimulant. However, such a high dose IL-2 cultivation resulted in the loss of cytotoxic reactivity of CTL clone. In contrast, the withdrawal of rIL-2 from in vitro cultivation for 24 h prior to the cytotoxic assays conferred the specificity of cytotoxicity on CTL. By these methods, one can obtain a large number of CTL, and pursue the physiologic detail of the specific cytotoxic mechanism of CTL against autologous human tumor cells.

Induction of cytotoxic T lymphocytes from peripheral blood of human histocompatibility antigen (HLA)-A31+ gastric cancer patients by in vitro stimulation with antigenic peptide of signet ring cell carcinoma

Antigenic peptides have been used as a cancer vaccine in melanoma patients and have led to a drastic regression of metastatic tumors. However, few antigens have been identified in non‐melanoma tumors. We recently purified a new natural antigenic peptide, designated F4.2, by biochemical elution from a human gastric signet cell carcinoma cell line and showed that it is recognized by an autologous human histocompatibility antigen (HLA)‐A31‐restricted cytotoxic T lymphocyte (CTL) clone. Here we describe in vitro induction of F4.2‐specific CTLs from peripheral blood T lymphocytes of HLA‐A31+ gastric cancer patients. The T cells of seven HLA‐A31+ patients with gastric cancers were stimulated in vitro by F4.2‐pulsed autologous dendritic cells which had been induced from peripheral blood of each patient by incubation in the presence of granulocyte macrophage colony‐stimulating factor (GM‐CSF) and IL‐4. We tested the cytotoxicity of the T cells against F4.2‐loaded C1R‐A *31012 by a 6‐h 51Cr release assay after 3 stimulations with F4.2‐pulsed dendritic cells. F4.2‐specific cytotoxicity was detectable in the stimulated T cells from two of the seven HLA‐A31+ patients. Further, both F4.2‐specific CTLs also lysed the gastric cancer cell line, HST‐2, from which F4.2 was derived. These results suggest that F4.2 peptide may be useful as an HLA‐A31‐restricted peptide vaccine in certain patients with gastric cancer.

T-cell receptor gene structures of HLA-A26-restricted cytotoxic T lymphocyte lines against human autologous pancreatic adenocarcinoma

We isolated two cytotoxic T lymphocyte (CTL) lines, which were independently obtained by mixed lymphocyte‐tumor cell culture from tumor‐infiltrating lymphocytes of a patient with pancreatic adenocarcinoma. Both lines behaved identically in all the functional aspects tested and appeared to be HLA‐A26‐restricted. We analyzed their T cell receptor (TCR) gene structures, including V‐(D)‐J junctional sequences, which are unique to each T‐cell clonotype and contribute to TCR diversity. Each line consisted of a clonal T‐cell expressing Vα18 and Vβ7. The α chain gene was composed of Vα18/ JαF/Cα and the, β‐chain gene, of Vβ7.1/Dβ/Jβ1.4/Cβ2. The sequences were all in‐frame and therefore should yield functional transcripts. The junctional sequences were identical between the two lines. These data suggested that the two CTL clones having the same CDR3 had descended from a common precursor lymphocyte. The clonal expansion of CTL lines with the identical CDR3 implies that they are directed against the same tumor antigen, which seemed to be immunologically dominant in the specific interaction between the CTL and the autologous pancreatic adenocarcinoma.

Identification of a Human T Cell Clone with the Cytotoxic T Lymphocyte and Natural Killer-like Cytotoxic Function against Autologous Mammary Carcinoma and K562 Line

Pleural exudative lymphocytes (PLED from a 60‐year‐old female patient showed high cytotoxicity against the autologous mammary tumor line, HMC‐2, and NK‐susceptible K562 cells, although PLEL demonstrated only weak cytotoxic potentials against several allogeneic tumor lines. We successfully obtained seven cytotoxic T cell clones from PLEL bulk populations, and assessed the possibility that these lymphocytes are simply natural killer (NK)‐like cells or have the dual cytotoxic activity of cytotoxic T lymphocytes (CTL) and NK‐like cells. These clones, designated as TcHMC‐2, showed strong cytotoxicity against both HMC‐2 and K562 cells. In contrast, allogeneic human peripheral blood‐derived NK cells could not kill HMC‐2 targets. Furthermore, a blocking study of TcHMC‐2 cytotoxicity using monoclonal antibodies against CD3, CD8 and human MHC class I products showed that all of these antigen molecules were involved in the cytotoxicity of TcHMC.2 clone against autologous HMC‐2 cells, indicating MHC class I recognitive cytotoxicity. These data indicate that the TcHMC‐2 clone may have dual cytotoxicity with CTL‐ and NK‐like activity against autologous HMC‐2 mammary tumor and K562 cells, respectively.

Brefeldin A Blocks the Cytotoxicity of T Cell Receptor α/β and γ/δ Cytotoxic T Lymphocyte Clones Reacting against Human Autologous Cancer Cells

We studied the effector mechanism of T cell receptor (TCR) α/β‐ and γ/δ‐type cytotoxic T lymphocyte (CTL) clones that react with human autologous tumor cells. Treatment of tumor cells with a fungal antibacterial reagent, brefeldin A (BFA), resulted in the inhibition of cytotoxicity of an autologous tumor (HST‐2)‐specific CD8+ TCRα/β‐type CTL, TcHST‐2. Other anti‐metabolites such as chloroquine, cycloheximide and colchicine did not affect the cytotoxicity. The cell‐surface antigen expression, including MHC class I molecules, was not influenced by BFA treatment. Furthermore, BFA did not influence the cytotoxicity of lymphokine‐activated killer cells and natural killer cells. Since BFA blocks the transport of peptides from endoplasmic reticulum to the Golgi apparatus, the above data suggest that BFA could affect washing out of the peptide fragments from the MHC class I groove. Consequently, target tumor cells were protected from killing by CTL, Moreover, we obtained a CD4−, 8−, TCR γ/δ‐type (Vδ1+) CTL clone, TcHOT, that reacts against an autologous ovarial carcinoma, HOT. BFA could also inhibit this cytotoxicity, and it is likely that different presenting molecules other than MHC class I proteins participate in the cytotoxicity of this TCR gm/δ‐ type CTL. These studies suggest that both TCR α/β‐ and γ/δ‐type CTL may require antigenic peptides that are most likely derived from the BFA‐sensitive, intracellular endogenous target proteins.

A gene encoding human gastric signet ring cell carcinoma antigen recognized by HLA-A31-restricted cytotoxic T lymphocytes.

We previously reported acid-extracted natural antigenic peptide (F4.2 [YSWMDISCWI]) of a gastric signet ring cell carcinoma HST-2 cells, recognized by HLA-A*31012-restricted autologous cytotoxic T lymphocytes, TcHST-2 line. In this study, the full-length cDNA (1101 bp), termed c98, predicting a protein composed of 170 amino acids was obtained. Because TcHST-2 cells could lyse the HLA-A31 antigen (+) allogeneic tumor cells that were introduced with c98 gene, this gene was suggested to possess antigenicity. Beginning at N-terminal 61 amino acid, the N-terminal six amino acid sequence that is completely identical to F4.2 was present in c98; however, a sequence of four amino acids in C-terminal was not found. Nevertheless, this peptide, c9861–70, seemed to be immunogenic, because cells pulsed with c9861–70 peptide were lysed in a dose-dependent manner by TcHST-2 cells. The c98 gene was expressed ubiquitously in tumor cells as well as in normal tissues. However, some tumor cells, including HST-2 cells, expressed this antigen in a high content, and such cells were lysed by TcHST-2 cells in the context of HLA-A31 antigen. However, TcHST-2 cells did not lyse cells that expressed lower amounts of c98 than HST-2 cells. These data suggested that c98-gene product and/or c9861–70 peptides could be used as a candidate for tumor vaccines in cancer immunotherapy.

Cellular Stress- and Transformation-associated Cell Surface Antigens Expressed on Human and Rodent Tumor Cells

Stress‐induced proteins may have significant roles in anti‐tumor resistance. To clarify the immunobiological roles of these proteins, we first developed monoclonal antibody (mAb) H1A that detects the HeLa cell‐surface antigens whose expression was enhanced by treatment of the cells with physico‐chemical stressors, such as heat, H2O2 and tumor necrosis factor. H1A (IgM) detects several molecules with mol. wt. 30, 43, 75, 90, 100, 120 and 150 kDa in Western blot analysis of HeLa cell lysates. Although the antigen was constitutively expressed on the HeLa cell surface, the cell‐surface expression of H1A‐defined antigen was rapidly enhanced (within 1 h) after heat treatment of HeLa cells. H1A antigens were also transformation‐associated, since 1) the activated oncogene‐transformed fibroblasts expressed the antigens, but parental nontransformed cells did not, and 2) certain human neoplastic but not normal cells strongly expressed the antigens. Furthermore, H1A mAb also partly blocked the cytotoxicity of purified protein derivatives‐stimulated human T cell receptor γδ‐type T cells towards HeLa cells. Taken together, these data indicate that H1A‐defined stress‐inducible proteins may play a vital role in anti‐tumor resistance by cytotoxic T cells.