Yumiko Mizushima

Separation of the Tumor Rejection Antigen of Rous Sarcoma Virus-induced Murine Fibrosarcoma

The tumor antigen capable of inducing tumor resistance (tumor rejection antigen; TRA) was separated and some of its physicochemical properties were characterized. Cytosol and plasma membrane fractions were separated from Rous sarcoma virus (RSV)‐induced CSA1M tumor cells. Immunization with membrane but not cytosol fraction of these tumor cells together with complete Freunds adjuvant resulted in complete protection against subsequent challenge with viable CSA1M cells. The TRA activity contained in the membrane fraction was recovered in the sodium dodecyl sulfate (SDS)‐solubilized fraction after the SDS‐extraction of CSA1M membranes. This CSA1M SDS‐solubilized preparation gave protection against syngeneic RSV‐induced CSA9F tumor cells as well as the homologous tumor cell type, but failed to induce resistance to RSV‐unrelated tumor cells. The membrane or SDS‐solubilized fraction from RSV‐unrelated tumor cells was unable to generate anti‐CSA1M protective immunity. Physicochemical analyses have demonstrated that TRA activity in the SDS‐solubilized fraction was completely abolished by treatment with proteinase K but was only marginally affected after treatment with glycosidase mixture. When the SDS‐solubilized preparation was applied to a Sephacryl S‐300 superfine column, TRA activity was recovered in the range of molecular weight of 50‐90 kD. Further fractionation of this TRA‐positive fraction by SDS‐polyacrylamide gel electrophoresis revealed that the molecular size of TRA is 56–68 kD. These results indicate that membrane proteins which were isolated from CSA1M tumor cells and have a molecular size of about 60 kD are capable of inducing RSV‐induced tumor‐specific in vivo protective immunity.

Thymic stroma-derived T cell growth factor (TSTGF): III. Its ability to promote T cell proliferation without stimulating interleukin 2- or 4-dependent autocrine mechanism.

A thymic stroma‐derived cell clone, MRL104.8a produced a T cell growth factor designated as thymic stroma‐derived T cell growth factor (TSTGF). This factor that is distinct from previously described T cell growth factors such as interleukin (IL) 2 or 4 was capable of promoting the growth of IL2‐dependent, antigen‐specific helper T cell clones. While such growth promotion was induced without requirement of the relevant antigen and exogenous IL2, we further investigated whether it depended on activation of an IL2‐ or IL4‐dependent autocrine mechanism. Helper T cell clones, 8‐E and 8‐5, were able to proliferate in response to stimulation with either antigen or TSTGF. 8‐E and 8‐5 produced IL2 and IL4, respectively, in cultures following antigenic stimulation, whereas neither IL2 nor IL4 activity was detected in cultures during TSTGF‐induced proliferation. The proliferation of these helper T cell clones by antigenic stimulation was almost completely inhibited when anti‐IL2 receptor or anti‐IL4 antibody was added to the cultures. The addition of cyclosporin A (CsA) to cultures of 8‐E and 8‐5 clones together with antigen also resulted in the complete inhibition of cellular proliferation in association with the suppression of IL2 and IL4 production. In contrast, TSTGF‐induced proliferation was not affected by addition of either type of antibody or CsA. These results indicate that TSTGF is a novel T cell growth factor that can exert its own growth‐promoting effect without depending on an IL2‐ or IL4‐operating autocrine mechanism.

Thymic stroma-derived T-cell growth factor (TSTGF): II. Biochemical and functional characterization.

The culture supernatant (SN) from a cloned line of thymic stroma‐derived cells in fibroblastic form (TSCF) contained a factor capable of supporting the growth of the interleukln (IL) 2‐dependent, antigen‐specific helper T cell (Th) clone 9‐16 without requiring IL2 and antigen. This active substance, designated as thymic stroma‐derived T‐cell growth factor (TSTGF), was partially purified through DEAE‐Sephacel chromatography and PBE 94 chromatofocusing. The original SN did not contain IL1, IL2, IL3, IL4, or interferon activities; but an appreciable magnitude of colony‐stimulating factor (CSF) activity in addition to TSTGF was present, whereas the partially purified preparation of TSTGF was depleted of any type of CSF activity. The elution profile of TSTGF activity on the chromatofocusing has revealed that TSTGF has an isoelectric point (pI) of about 6.0. When a purified TSTGF sample was applied to Sephacryl S‐200 column chromatography and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis, TSTGF activity was eluted in a single peak around an apparent molecular weight of about 25,000. The activity of TSTGF also was shown to be relatively stable with heat treatment and in the wide range of pH, but it was abolished by treatment with either trypsin or dithiothreitol. These results indicate that TSTGF, a novel T‐cell growth factor, is the protein that has an apparent molecular weight of about 25,000 and a pI of 6.0, and in the intact molecule, it contains the disulfide bond(s) required to maintain and/or express its biologic activity.

Application of T Cell—T Cell Interaction to Enhanced Tumor-Specific Immunity Capable of Eradicating Tumor Cells in Vivo

Investigations have attempted to delineate the consequences of malignant transformation of cells by the appearance of new cell surface structures [tumor-associated antigens (TAA) or tumor-associated transplantation antigens (TATA)] that could be identified by specific antiserum or by their ability to induce a specific cellular immune response. Considerable efforts have been undertaken to establish the significance of these tumor cell surface structures by correlating their cell surface expression with changes that take place during the course of neoplastic disease. The most compelling evidence for the existence of TATA comes from the study of chemically induced tumors of inbred rodents. These tumors express neoantigens capable of immunizing syngeneic or autochthonous hosts against subsequent challenge with the same tumor.(1–4)

Role of L3T4 Antigen in the Activation of Various Functions of Lyt-1+2- T Cells against Vaccinia Virus

The present study defines assay systems for vaccinia virus‐reactive Lyt‐1+2‐ T cells mediating various functions and investigates the positivity of L3T4 antigen on these Lyt‐1+2‐ T cells as well as the role of L3T4 antigen in the activation of these T cells with respect to their functions. C3H/He mice were immunized against vaccinia virus by inoculating viable virus intraperitoneally (i.p.). Antivaccinia virus reactivity in lymphoid cells from these immunized mice was assessed by a) proliferative response, b) helper T cell activities involved in cytotoxic T lymphocyte (CTL) and B cell (antibody) responses, c) delayed type‐hypersensitivity (DTH) response, and d) production of lymphokines such as interleukin 2 (IL2) and macrophage‐activating factor (MAF). The results demonstrate that all of the above anti‐vaccinia virus responses were mediated by Lyt‐1+2‐ T cells and that these Lyt‐1+2‐ T cells expressed L3T4 antigens on their cell surfaces. Moreover, such anti‐vaccinia Lyt‐1+2‐ T cell responses were inhibited in the presence of anti‐L3T4 antigen antibody. These results indicate that there is a reciprocal relationship between Lyt‐2 and L3T4 markers, and that L3T4 antigen is closely related to the activation of various functions of anti‐vaccinia virus Lyt‐1+2‐ T cells.