Studies on the induction and expression of T-cell-mediated immunity. XIII. Membrane-associated antigens of cytotoxic T lymphocytes involved in cytotoxicity

An xenogeneic rat anti-mouse T-cell serum, designated RAT*, has been shown to block the cytolytic activity of cytotoxic T lymphocytes (CTL) at a postbinding step. RAT* serum or the IgG fraction was extensively absorbed with the target cell, P815, a DBA mastocytoma, and used with or without further absorption to immunoprecipitate specific molecules from radiolabeled membrane extracts of CTL derived from either in vivo-allosensitized mice or from cytotoxic clones maintained in in vitro cultures. Cell surface sialic acid residues were labeled by oxidation with sodium periodate (NaIO4) and reduction with tritiated sodium borohydride ([3H]NaBH4). Alternatively, cell surface proteins were labeled with 125I by lactoperoxidase-catalyzed iodination. Nonidet P-40 (NP-40)-solubilized radiolabeled membranes were then immunoprecipitated with RAT* serum and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Three membrane-associated molecules of 95,000, 140,000 and 180,000 Mr were found by such analysis. The sensitivity of these three molecules to trypsinization and their susceptibility to labeling with [3H]NaBH4 suggested that they are glycoproteins. Moreover, when RAT* serum or the IgG fraction was absorbed with various cell types, its ability to immunoprecipitate the three molecules correlated with its ability to block cytolysis. Adsorption of RAT* serum with CTL, but not with nonimmune thymocytes, significantly reduced the ability of RAT* serum to inhibit cytotoxicity and to immunoprecipitate the 95k, 140k, and 180k molecules. Thus, these findings suggest that one or more of these cell surface molecules of CTL may be involved in the cytolytic process.     

Studies on the induction and expression of T cell-mediated immunity. XIV. Antigen-nonspecific oxidation-dependent cellular cytotoxicity (ODCC) mediated by sodium periodate oxidation of cytotoxic T lymphocytes

Alloimmune murine thymus-derived cytotoxic lymphocytes (CTL) generated in vivo or in vitro are shown to lyse antigen-nonspecific target cells (tumor cells, Con A, and LPS blasts) following treatment of CTL with an oxidizing agent, sodium periodate (NaIO4). It has been shown that NaIO4 oxidizes terminal sialic acid residues of cell surface macromolecules. The presence of reactive aldehyde groups, generated by NaIO4 modification, is required for the expression of antigen-nonspecific cytotoxicity because treatment of modified cells with a reducing agent such as potassium borohydride (KBH4) resulted in the abrogation of cytotoxicity. However, KBH4 treatment of unmodified or NaIO4-modified CTL has no effect on antigen-specific cytotoxicity. The modification of CTL by NaIO4 is sufficient to lead to the formation of lymphocyte-target cell conjugates and lysis of bound targets. Monoclonal antibodies directed against the Lyt-2 antigens of CTL, but not Lyt-1 antigens, in the absence of complement inhibited the nonspecific cytotoxicity resulting from NaIO4 modification of effector lymphocytes. These findings suggest that the mere interaction with or perturbation of appropriate cell surface molecule(s) of effector lymphocytes such as Lyt antigens by receptor-ligand interaction in SCMC or by NaIO4 modification in ODCC may lead to the expression of cytotoxicity. The present studies demonstrate a functional role of surface carbohydrates on CTL in cell-to-cell recognition and interactions. Furthermore, the results suggest that target cell modification is not a requisite for recognition and lysis in an antigen-nonspecific cytotoxic system such as ODCC. However, partial blocking of ODCC by alloantibodies directed against the H-2 of unmodified target cells suggests that NaIO4-modified CTL recognize unrelated target H-2 antigens. The implication of these findings on the molecular mechanism of cell-mediated cytotoxicity is discussed.     

In vitro induction of tumor-specific immunity. IV. Specific adoptive immunotherapy with cytotoxic T cells induced in vitro to plasmacytoma antigens

Summary Specifically activated cytotoxic T cells (CL's) were raised by cocultivation in vitro of normal spleen cells with syngeneic plasmacytoma cells. These CL's were fully active both in vitro in lysing ⁵¹Cr-labelled tumor cells and in in vivo Winn assays, in inhibiting the growth of tumor cells in syngeneic mice when inoculated admixed with the tumor cells, even at ratios of 2 CL's to 1 tumor cell. However the same CL populations were only marginally effective in an immunotherapy model in which CL's were injected intravenously and tumor cells subcutaneously or intramuscularly (at a ratio of up to 100 CL/1 tumor cell). It is suggested that the in vitro conditions alter cell surface properties, thereby interfering with normal cell traffic, and that if this problem can be overcome, in vitro educated CL's may constitute a potential source of activated cells for human tumor immunotherapy.National Health and Medical Research Council postgraduate research scholar     

Studies of H-2 restriction in cell-mediated cytotoxicity and transplantation immunity to Leukemia-associated antigens.

H-2 dependency of T cell-mediated cytotoxicity and transplantation immunity to leukemia-associated antigens has been investigated. Through the use of a 20-hr 125IUdR release assay, it was found that the induction of T cell-mediated cytotoxicity against Friend virus-induced leukemias of different H-2 haplotype orgins could be produced by immunization with both syngeneic and allogeneic tumor cells; the effector cells that were generated by syngeneic immunization could also provide effective killing of allogeneic tumor cells, although the killing of allogeneic targets might require a longer incubation time (20 to 40 hr). Furthermore, in vivo transplantation immunity against Friend virus-induced leukemias also was induced by immunization with both syngeneic and allogeneic tumors and syngeneic immunization could induce specific protection against the challenge with a-logeneic tumor in x-irradiated hosts. These findings clearly indicate that, both at the sensitizing phase and effector phase of the immune response, there is no strict H-2 dependency for T cell-mediated cytotoxicity or in in vivo transplantation imunity to leukemia-associated antigens.     

Studies on the induction and expression of T cell-mediated immunity. VIII. Effector-target junctions and target cell membrane disruption during cytolysis.

Target cell destruction following contact of the target cell by specific alloimmune cytotoxic thymus-derived lymphocytes (CTL) has been examined by time-lapse film (TLF), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Effector-target conjugates of murine CTL with leukemia cells were prepared for use in these studies. TLF shows that contact of the two cells results in tumor cell zeosis involving violent membrane blebbing, and subsequent tumor cell death. TEM of the contact region shows that the CTL-tumor cell junction is extremely adherent. Examination of conjugates incubated at 37 °C to permit tumor cell lysis shows tumor cell membrane stretching and rupture, and tumor cell membrane fragments adhering to CTL. Close examination of the contact region has revealed electron-lucent junctions spanning the gap between the two cell membranes, but no packaging or secretory apparatus was prominent. The results are consistent with the mechanism of cell-mediated cytolysis being a membrane phenomenon involving junctions connecting the CTL and target cell and the initial target cell lesion observable as a stretching and rupture. The shear force of vigorous cell movements is most likely responsible for this target membrane tearing, creating a target cell lesion which results in loss of osmotic integrity and cell death.     

Studies of cloned simian immunodeficiency virus-specific T lymphocytes. gag-specific cytotoxic T lymphocytes exhibit a restricted epitope specificity.

CD8+ CTL inhibit the replication of HIV and simian immunodeficiency virus of macaques (SIVmac) in PBL and, therefore, are likely to play an important role in containing the spread of the AIDS virus in infected individuals. We have generated a series of gag-specific lytic T lymphocyte clones from PBL: of an SIVmac-infected rhesus monkey. These T cell clones are CD3+CD8+ and are MHC class I-restricted in their target specificity. They are, therefore, CTL. Interestingly, all gag-specific CTL clones, as well as the gag-specific lytic activity of PBL of this monkey, demonstrated specificity for a single 25 amino acid fragment of the SIVmac gag protein. Moreover, they were restricted in their lytic function by a single MHC class I allele. These findings illustrate a powerful method for cloning AIDS virus-specific T lymphocytes and demonstrate a remarkably restricted epitope specificity of this AIDS virus-specific CTL response.     

Studies of the mechanisms for the induction of in vivo tumor immunity. I. Induction of primary and secondary cell-mediated cytotoxic responses by adoptive transfer of lymphocytes.

Cell-mediated immunity to FBL-3, a syngeneic Friend virus-induced leukemia in C57BL/6 mice, could be adoptively transferred. Characteristic primary and secondary cytotoxic responses could be induced by adoptive transfer of normal and presensitized lymphocytes, respectively. In vivo tumor immunity could also be produced by adoptive transfer of presensitized lymphocytes. Both the primary and secondary cell-mediated cytotoxic reactions were T-cell dependent. The specificity of these reactions was primarily directed against F (Friend) type-specific antigen and FMR (Friend, Moloney, Rauscher) common antigen. The cytotoxic responses produced by adoptive transfer experiments gave better correlation to in vivo tumor immunity than those generated by in vitro mixed lymphocyte tumor cell culture reactions.     

Heat shock enhances the expression of cytotoxic granule proteins and augments the activities of tumor-associated antigen-specific cytotoxic T lymphocytes

Focal inflammation causes systemic fever. Cancer hyperthermia therapy results in shrinkage of tumors by various mechanisms, including induction of adaptive immune response. However, the physiological meaning of systemic fever and mechanisms of tumor shrinkage by hyperthermia have not been completely understood. In this study, we investigated how heat shock influences the adaptive immune system. We established a cytotoxic T lymphocyte (CTL) clone (#IM29) specific for survivin, one of the tumor-associated antigens (TAAs), from survivin peptide-immunized cancer patients’ peripheral blood, and the CTL activities were investigated in several temperature conditions (37–41 °C). Cytotoxicity and IFN-γ secretion of CTL were greatest under 39 °C condition, whereas they were minimum under 41 °C. To address the molecular mechanisms of this phenomenon, we investigated the apoptosis status of CTLs, expression of CD3, CD8, and TCRαβ by flow cytometry, and expression of perforin, granzyme B, and Fas ligand by western blot analysis. The expression of perforin and granzyme B were upregulated under temperature conditions of 39 and 41 °C. On the other hand, CTL cell death was induced under 41 °C condition with highest Caspase-3 activity. Therefore, the greatest cytotoxicity activity at 39 °C might depend on upregulation of cytotoxic granule proteins including perforin and granzyme B. These results suggest that heat shock enhances effector phase of the adaptive immune system and promotes eradication of microbe and tumor cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-012-0348-0) contains supplementary material, which is available to authorized users.     

Tumor-specific immunity induced by somatic hybrids: IV. Relationship between immunogenicity and expression of surface tumor-associated antigens

Semi-allogeneic hybrid clones were derived by fusion of the TEPC-15 plasmacytoma (H-2^d) and mouse L cells of C3H (H-2^k) origin. Three representative clones were chosen to study the relationship between the expression of different membrane antigens and their immunogenicities leading to protection of recipient mice from the parent TEPC-15 plasmacytoma. The level of surface tumor-specific transplantation antigens (TSTA) was measured by radioimmunoprecipitation with syngeneic anti-TSTA and by inhibition of anti-TSTA binding to the TEPC-15 tumor cells. The most immunogenic hybrid clone (LTC-1) expressed the highest level of TSTA and the weakly immunogenic (LTC-2) and nonimmunogenic (LTC-4) hybrid clones exhibited relatively low levels of TSTA on the surface. Moreover, the strongly immunogenic LTC-1 hybrid cells, but not the parent tumor cells, were effective in priming recipient spleen cells to generate TEPC-15 tumor-specific cytotoxic cells upon subsequent in vitro exposure to the TSTA-bearing cells. Therefore, the level of TSTA on the semi-allogeneic hybrid clones may play an important role in enhancing the immunogenicity of TSTA.     

Demonstration in vitro of cytotoxic T cells with apparent specificity toward tumor-specific transplantation antigens on chemically induced tumors.

Chemically induced tumors of mice exhibit apparently unique antigenicity upon syngeneic transplantation into appropriately immunized hosts. An in vitro counterpart of this pattern in terms of specificity has not been reported. Data are presented that demonstrate that immune peritoneal exudates contain cells cytotoxic for the specific immunogen tumor but with rare exceptions, not toward other syngeneic methylcholanthrene-induced fibrosarcomas. Only tumors highly immunogenic by transplantation criteria induce cytotoxic PEC regularly; nonimmunogenic tumors consistently fail to do so. The effector cell responsible is eliminated by pretreatment with anti-Thy.1 but not anti-Ig plus complement. In concomitant experiments, PEC populations cytotoxic in vitro also conveyed adoptive protection against the specific tumor in syngeneic hosts. This in vitro assay appears to provide a tool for studying T cell-mediated cytotoxicity toward a set of unique surface antigens present on chemically induced tumors.     

Cloned lines of human cytotoxic T lymphocytes with specificity for influenza virus

The generation of human cytotoxic T cell clones with specificity for influenza virus and some of their characteristics are described. The clones were generated by limiting dilution of peripheral blood lymphocytes after two in vitro stimulations with autologous influenza A/USSR virus-infected cells and were grown in T cell growth factor. The majority of the virus-specific clones showed cross-reactivity for different influenza A virus subtypes but did not recognize influenza B virus-infected cells. The HLA specificity of two clones was further analyzed. One clone, LL33, was specific for HLA-Bw60, the other, clone WH5, for HLA-A1. Clone WH5 also seemed to recognize the serologically related HLA-A26 as restriction element for the recognition of the viral antigen. Whereas the virus-specific CTL clones had the OKT3+,4-,8+ phenotype, another clone, WH 49, exhibiting natural killer-like activity, was found to have the OKT3+,4+,8- phenotype.     

Studies of the mechanisms for the induction of in vivo tumor immunity. VI. Induction of specific and nonspecific cell-mediated immunity in tumor-bearing hosts and its correlation with transplantation tumor immunity

Studies were performed to determine the development of cell-mediated cytotoxic response at tumor site in C57BL/6 mice bearing progressively growing FBL-3 ascites leukemia. The effectors isolated from tumor ascites are found to be highly cytotoxic for leukemic target cells. The levels of cytotoxicity obtained with effectors isolated from tumor site are generally higher than those obtained with immune mice. This cytotoxicity is both specific and nonspecific. The specific cytotoxicity against tumor-associated antigen is mainly mediated by T cells and the nonspecific cytotoxicity against unrelated tumor cells is mediated largely by macrophages. The T-cell-enriched preparation did not give significant natural killer activity. When testing the ability of these effectors to produce in vivo immunity against the challenge of FBL-3, it was found that only T cells could confer the transplantation-type immunity, but the immunity was transient. The macrophage-enriched preparation isolated from tumor ascites failed to give in vivo protection. These findings indicate that in FBL-3 system, mice with progressively growing tumors are able to develop immune response against tumor cells. However, this immunity is probably interfered with by a suppressor factor(s) or suppressor cells which restrict their activity to eliminate the tumor cells effectively.