Shigeyoshi Fujimoto

Cellular interaction between cytotoxic and suppressor t cells against syngeneic tumors in the mouse.

Abstract Splenic T cells from animals bearing growing syngeneic tumors specifically inhibited the effector process of tumor cell lysis by the cytotoxic T cell which had been activated in vitro by mitomycin C treated homologous tumor. The suppression was strictly specific for the individual tumor by which suppressor cells were generated, whereas in some cases cytotoxic T cells generated by two closely related sarcomas showed a certain degree of crossreactivity. This suggests that suppressor and cytotoxic T cells recognize different antigenic moieties on tumor cells; one unique to the individual tumor and the other shared by related tumor cell lines. The suppressor T cell from tumor bearing animals possessed Ia antigen controlled by a gene in I-J subregion of H-2 major histocompatibility complex. Cytotoxic T cells generated by some but not all syngeneic tumors were also killed by anti-Ia and complement; however, the Ia antigen on such cytotoxic T cells was found to be controlled by a locus in I-A subregion. In general, the cytotoxic T cells generated by newly established tumor cell lines had Ia antigen, whereas some old cell lines, which were capable of growing across the H-2 barrier, activated the Ia negative cytotoxic T cell. These results collectively indicate that the immunological resistance against tumors is dependent on the balance of activations of the cytotoxic and suppressor T cells with different specificities and phenotypic expressions.

Tumor-specific CD4(+) suppressor T-cell clone capable of inhibiting rejection of syngeneic sarcoma in A/J mice.

Elimination of CD4+ T cells by anti‐CD4 antibody caused regression of a methylcholanthrene‐induced S713a sarcoma growing in syngeneic A/J mice, and the tumor regression was essentially required for CD8+ T cells. A CD4+ T‐cell clone, designated T595B1, was established to elucidate the characteristics of CD4+ suppressor T cells. T595B1 expressed CD3, T‐cell receptor (TCR)β, TCR‐Vβ2, CD4, CD25, CD45RB, CD44, LFA‐1, and ICAM‐1 molecules on its cell surface and showed MHC class II I‐Ek–restricted tumor antigen‐specific proliferation. T595B1 cells specifically suppressed in vitro CTL induction of S713a in a dose‐dependent manner. Furthermore, culture supernatant of T595B1 cells also suppressed in vitro CTL induction, but its suppressive activity was not specific. Cytokine analyses revealed that T595B1 cells secreted IL‐4, IL‐5, IL‐6, and IL‐10 but not IFN‐γ, IL‐2, TNF, or TGFβ, indicating that this clone belongs to the so‐called T helper 2 (Th2) type. However, the suppressive activity of the culture supernatant to the in vitro CTL induction was not abrogated by any neutralizing antibody to IL‐4, IL‐5, IL‐6, IL‐10, or TGF‐β. Repeated adoptive transfer of T595B1 cells into syngeneic immune mice entirely impaired their capacity to reject S713a sarcoma, resulting in progressive tumor growth in these mice. Int. J. Cancer 87:680–687, 2000.

The generation of interleukin‐2‐dependent suppressor T‐cells from patients with systemic metastasis of gastric carcinoma and the phenotypic characterization of the cells defined by monoclonal antibodies

Suppressor cells, which might be activated in patients with gastric carcinoma, were successfully enriched by the use of interleukin‐2 (IL‐2) prepared from human tonsils and spleens. That is, peripheral blood lymphocytes cultured for 3 or 4 weeks with IL‐2 strongly inhibited the patients own lymphocyte‐proliferative responses to alloantigen or phytohemagglutinin (PHA). Quantitative fluorescence measurement for immunologic analysis of phenotypic characterization of the cells was made on FACS‐IV with monoclonal antibodies anti‐Leu‐1 anti‐Leu‐2a, anti‐Leu‐3a, anti‐Leu‐4, anti Leu‐5, anti‐Leu‐7, and anti‐HLA‐DR and goat anti‐human immunoglobulin (Ig). Functional suppressor T‐cells expanded with IL‐2 showed the following phenotype: Leu‐1+ Leu‐2a+, Leu‐3a−, Leu‐4+, Leu‐5+, Leu‐7−, HLA‐DR+, human Ig−. The IL‐2‐dependent suppressor T‐cells could be obtained only when the cells were derived from patients with systemic metastasis of gastric carcinoma. These findings suggest that generation of IL‐2‐dependent suppressor T‐cells is the result of large tumor burdens; this may exert negative cellular control in the immune responses, thus inducing the status of the lower cell‐mediated antitumor immunity, and may promote cancer progression in gastric cancer patients.

Decrease in the level of poly(ADP-ribose) synthetase during nerve growth factor-promoted neurite outgrowth in rat pheochromocytoma PC12 cells

We have studied the changes in the levels of the enzyme molecule and mRNA for poly(ADP-ribose) synthetase during nerve growth factor-promoted neurite outgrowth in rat pheochromocytoma PC12 cells. When the PC12 cells were cultured in the presence of nerve growth factor, the content of enzyme molecules decreased along with neurite outgrowth to 50% of the original amounts in 2 days and the content of mRNA for the enzyme also decreased to approximately 50% in 2 days. These results suggest that the decrease of the enzyme molecule may be due to depression of expression of the gene for synthetase during the process. Taken together with previous observations, the decrease of the synthetase seems to be required for some cellular differentiation.

Morphological characterization of a new human epithelioid sarcoma cell line, ES020488, in vitro and in vivo

SummaryA new human epithelioid sarcoma cell line (ES020488) was established from a cutaneous metastasis in 26-year-old man, and was morphologically characterized in vitro and in vivo by comparison with the original tumor. The ES020488 cells showed a male karyotype ranging from 39 to 83 chromosomes, with various abnormalities but no specific pattern. The cells were round, polygonal or spindle-shaped with abundant cytoplasm and round nuclei containing prominent nucleoli; they proliferated in a sheet-like pattern. Tumors transplanted into nude mice revealed essentially the same features as the original tumor. Both in vitro and in vivo, the cells immunohistochemically expressed vimentin, cytokeratin, and EMA, but not desmin and S-100 protein. Ultrastructural study revealed irregular or round nuclei containing abundant euchromatin and prominent nucleoli, many intermediate filaments running irregularly or around the nucleus, and a number of filopodia-like processes. ES020488 cells were thus proven to retain and exhibit the unique morphological characteristics of an epithelioid sarcoma both in vitro and in vivo. These cells are possibly derived from synovioblastic mesenchyme.

Augmentation of Human Cytotoxic T Lymphocytes against Autologous Tumor by a Factor Released from Human Monocytic Leukemia Cell Line

A human acute monocytic leukemia cell line, THP‐1, releases a factor which activates human cytotoxic (killer) T lymphocytes (CTL) against autologous tumor in vitro. The factor, named cytotoxic (killer) T cell activating factor (KAF), is an acidic protein of 70,000 to 100,000 dalton molecular size. Peripheral blood leukocytes from two patients, bearing epithelioid sarcoma or malignant schwannoma, were cultured for 7 days with individual autologous tumor to induce CTL directed to the corresponding tumor. Monocyte‐depleted peripheral leukocytes generated lesser CTL activity than the monocyte‐containing leukocyte population. However, the KAF was able to replace the monocyte function. The KAF acted at the CTL generation phase as well as the effector phase. The KAF‐activated killer cells possessed CD4‐8+ surface phenotype. The CTL killed autologous tumor or other unrelated tumor cell lines only when they shared some of the HLA class I antigens. It was also demonstrated that the KAF does not activate killer cells without proper antigenic stimuli, because the KAF‐augmented CTL possess specificity against autologous tumor or other HLA‐A or ‐B matched tumor cell lines. The therapeutic applicability of human KAF for anti‐tumor CTL therapy against autologous tumor is discussed.

Generation of T cell growth factor (TCGF)-dependent splenic lymphoid cell line with cell-mediated immunosuppressive reactivity against syngeneic murine tumor☆

Splenic T cells obtained from tumor-bearing mice could be cultured with T cell growth factor (TCGF) for over 12 months. The TCGF-dependent lymphoid cell line strongly inhibited cell-mediated anti-tumor immunity directed against syngeneic tumor. However, the suppression was non-specific for the given tumor. The cell line expanded with TCGF expressed a phenotypic characterization of T cells defined by monoclonal anti-Thy-1.2 antibody.

Induction of Specific Cytotoxic T Lymphocytes against Autologous Brain Tumor by Crossreactive Allo‐tumor Cell Stimulation

Cytotoxic T lymphocytes (CTL) against autologous malignant brain tumor were generated in peripheral blood lymphoid cells (PBL) prepared from a patient with a malignant brain tumor by stimulation of the cultured PBL for 7 days with attenuated Crossreactive malignant melanoma (MM2) cells pretreated with mitomycin C. The Crossreactive MM2 cells were effective for antigen stimulation for CTL induction in place of autologous glioblastoma cells, which are difficult to expand in culture. The optimal ratio between nylon wool‐passed T lymphocytes and nylon wool‐adherent accessory cells to induce CTL in the patients PBL was found to be 25 to 1. In vitro ‐activated CTLs induced by MM2 were cytotoxic not only to MM2, but also to the autologous tumor cells in an HLA class I‐restricted manner, and their surface phenotype was found to be CD3+ and CD8+. CTL therapy using cross‐reactive allogeneic tumor cells as the stimulator could be clinically valuable to treat malignant brain tumors.

Specific immunosuppression of human anti-murine antibody responses in hu-PBL-SCID mice

Severe combined immunodeficient (SCID) mice were reconstituted with normal human peripheral blood leukocytes (PBLs) and were shown to produce a human anti-mouse immunoglobulin antibody response on immunization with heat-treated murine monoclonal IgG1 antibody to ovalbumin, referred to as ha-Mab-2. The human anti-mouse antibody response was proportional tot the number of B cells and mononuclear cells transferred from a given batch of PBL. However, pretreatment of hu-PBL-SCID mice with a tolerogenic covalent conjugate of monomethoxypolyethylene glycol (mPEG) and Mab-2 suppressed this response on subsequent injections of ha-Mab-2, and this suppression was shown to be antigen-specific, i.e., it did not suppress the antibody response to ovalbumin and did not affect the level of production of human immunoglobulin of hu-PBL-SCID mice. The suppression was due to the generation of human suppressor CD8+ T (Ts) cells, which down regulated CD4+ helper T cells in an antigen and HLA class I specific manner, i.e., these findings were in accord with the previously shown immunosuppressive effect of tolerogenic mPEG conjugates in normal mice.

Fluctuation of gene expression for poly(ADP-ribose) syntetase during hemin-induced erythroid differentiation of human leukemia K562 cells and its reversion process

We have studied the regulation of gene expression for poly(ADP-ribose) synthetase during erythroid differentiation and its reversion process. When human leukemia K562 cells were incubated in the presence of 80 microM hemin, benzidine-positive cells appeared at day 2 and 90% of the cells became positive at day 6. However, RNA blot analysis reveals that mRNA for gamma-globin was already abundant in untreated K562 cells and the level of the message was slightly increased by hemin-treatment. Spectroscopic analysis and polyacrylamide gel electrophoresis of the induced cell extracts indicate that hemoglobin molecules were not detected in untreated cells, and increased successively up to day 6. The hemin-induced cells were thoroughly washed, and then recultured in the absence of hemin. The benzidine-positive cells mostly disappeared 3 days after the elimination of the inducer. During the hemin-induced erythroid differentiation, the activity and mRNA for poly(ADP-ribose) synthetase decreased to 50% and 20% of the initial level at day 3 and a low level of the gene expression was maintained afterwards, whereas the activity and mRNA returned to the initial value 1 day after hemin elimination. The results indicate that the hemin-induced erythroid differentiation of K562 cells is a reversible process and depression of the synthetase may be involved in the progress of differentiation.