Biomedical subjects
Publications and source records attributed to C C Ting.
A humoral factor (molecular weight less than 60,000) that was present in the ascitic fluid of mice bearing intraperitoneal tumors and in pleural effusions from human cancer patients was found to promote the growth of a murine tumor and to suppress cell-mediated tumor immunity. However, the hosts that had recovered from the immunosuppressive state produced a serum factor that could neutralize the immunosuppressive effect.
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We compared three ddifferent isotopic assays of cell-mediated cytotoxicity under identical test conditions: 51Cr, 125iododeoxyuridine, and [3H]proline release assays. We made these comparisons both in a syngeneic mouse tumor system and an allogeneic system. It was found that several parameters could affect considerably the results obtained with these tests, such as: baseline controls, preparation of target cells, and methods of calculation. In comparison of three different baseline controls, the normal control gave the most consistent results, whereas the other two controls (autologous and medium controls) gave varying results depending upon the condition of the target cells. For use as target cells, established tissue culture cells were usually superior because of a much lower spontaneous release of the isotope, when compared to fresh ascites tumor cells or short-term tissue culture cells. The advantage of established culture cells was particularly noted in isotopic assays with prolonged incubation (20-40 hr). In addition, the methods used for calculation were also shown to affect the apparent outcome of the results.
Three assays of cell-mediated cytotoxicity in mice, involving release of either 51Cr (CRA), 125iododeoxyuridine (IRA), or [3H]proline (PRA), were compared under identical test conditions. Experiments were performed with effector cells from mice immunized with FBL-3 tumor cells, a syngeneic Friend virus-induced leukemia, or with allogeneic normal spleen cells. With established tissue culture cells as targets, similar results were obtained in all three assays. The cytotoxicity produced by cells from in vivo-immunized mice and the induction of cytotoxicity in vitro were T-cell-dependent. When short-term culture target cells were used, the IRA gave a more selective pattern of cytotoxicity than did the other two assays. However, when remaining target cells at the end of the assay were treated with trypsin, higher levels of 125iododeoxyuridine (125IUDR) release were seen, and the results were then comparable to those in the CRA and PRA. These results indicated that 125IUDR, a nuclear label, could only be released after lysis of cells. In contrast, 51Cr or [3H]proline, which are cytoplasmic labels, could also be released from damaged but unlysed cells. These fundamental differences could give different results in these assays, which could determine their correlation with in vivo transplantation immunity.
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.
The humoral antibody response to virally induced tumors insyngeneic hosts has been studied. The tumors include an SV40 tumor SVT2, the Friend virus-induced leukemias FBL-3 and FLC; and Moloney sarcoma virus-induced tumors. It was found that antitumor antibodies could be detected by the isotopic antiglobulin technique in these tumor systems at a relatively early stage of tumor growth. The kinetics of the antibody response in relation to the status of tumor growth varied between different tumors. In geneumor growth than in the regressors of tumor-free hosts. Reinoculation of tumor cells or recurrence of tumor growth produced elevation of antibody levels (secondary response). The specificity of the antibody reactions also varied in different tumor systems: some antibodies were truly tumor-specific and thus might produce a biological effect on in vivo tumor immunity, whereas others were not. These studies indicated that a sensitive antibody assay could be used for early detection of tumor growth. However, its usefulness in evaluation of the status of tumor growth should be carefully studied in each tumor system.
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Immune responses to fetal antigen immunization were studied in C57BL/6 (B6), C3H/HeN (C3H), and BALB/c (BALB) mice. In tests by the isotopic antiglobulin technique, the mice could be grouped in three classes according to their antibody responses: good responders (B6), poor responders (C3H), and nonresponders (BALB). In tumor transplantation experiments, protection against syngeneic tumor-cell challenge, after fetal tissue immunization, was observed only with B6 mice. In addition, experiments on B6 mice showed that resistance to tumor challenge after fetal tissue immunization depended on the quantitative expression of fetal antigens by the tumor cells. Our results indicated that, in addition to other well-known factors, the effectiveness of fetal tissue immunization in tumor-challenge resistance depended on both host responsiveness and the amount of fetal antigen expressed by the tumor cells.
Heparin influenced the induction (afferent interference) and effector (efferent interference) phases of cell-mediated cytotoxic reactions against FBL-3 cells, a syngeneic Friend virus-induced leukemia in inbred C57BL/6 mice. Heparin was cytotoxic at high concentrations (greater than or equal to 100 U/ml) and inhibitory for cell-mediated cytotoxic response at lower concentrations (less than or equal to 50 U/ml).
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The primary cell-mediated cytotoxic response to a Friend virus-induced leukemia, FBL-3, in C57BL/6 mice was measured by the 125IUdR release assay. Intraperitoneal (i.p.) inoculation of 1 x 10(1) FBL-3 cells produced progressive tumor growth (progressors); subcutaneous (s.c.) inoculation of as many as 5 x 10(6) FBL-3 cells produced only transient tumor growth (regressors), and these mice would subsequently resist i.p. challenge of FBL-3 cells at 3 days after s.c. inoculation. The kinetics of the primary cell-mediated cytotoxic response of regressors was biphasic. Significant cytotoxicity could be detected at 3 to 5 days after s.c. inoculation of 5 x 10(6) FBL-3 cells peaked at days 10 to 14, declined to a very low level or became undetectable around days 20 to 30; then the reactivity reappeared and persisted at least up to 60 days. In progressors, the kinetics of the cell-mediated cytotoxic response was similar to the regressors, but the reactivity was much lower. The cytotoxic response was found to be T cell dependent, during both the first peak (days 10 to 14) and the second peak (days 40 to 60). In adoptive transfer experiments, lymphocytes from regressors gave 90% protection against i.p. challenge of FBL-3; lymphocytes from progressors only gave 40% protection.
By employing the 125IUdR release cytotoxicity assay, we have been able to measure the primary and secondary cell-mediated cytotoxic response of C57BL/6 mice to FBL-3 cells, a syngeneic Friend virus-induced leukemia. It was found that the secondary cell-mediated cytotoxic response occurred more rapidly after challenge (within 3 days) than the primary response, and the levels of reactivity were considerably higher. As in the primary response, the secondary cytotoxic reactivity of spleen cells was T cell dependent, being eliminated by pretreatment with anti-theta antibody plus complement. However, the secondary reactivity of pertioneal exudate (PE) cells was not entirely T-cell dependent. The specificity of the secondary cytotoxic response was analyzed by primary or secondary immunization with various tumor cells and by testing of cytotoxic lymphocytes against a variety of target cells. When spleen cells were used for testing, only tumor cells induced by Friend, Moloney, or Rauscher (FMR) leukemia viruses could produce secondary cell-mediated cytotoxic responses against FBL-3 cells. This correlated well with the specificity observed in the in vivo tumor transplantation protection studies. Similarly, spleen cells immune to FBL-3 had appreciable cytotoxicity against tumor cells induced by FMR viruses. The FBL-3 immune mice also gave significant protection against the challenge of FMR leukemias. When PE cells were used for testing, they gave higher levels of cytotoxicity against tumor cells induced by FMR viruses, but also gave less, but appreciable, cytotoxicity against non-FMR tumors. The latter reactivity might be related to the antigens induced by the murine endogenous type C viruses.
Cell-mediated immune reactions appear to play an important role in resistance against growth of leukemia cells in mice. Possible mechanisms for in vivo protection in two tumor systems are discussed. These tumor models, which are a Friend leukemia virus-induced transplantable tumor, FBL-3, and primary murine sarcoma virus (MSV) -induced tumors, are strongly antigenic; under some conditions, tumors regress completely. In mice with regressing FBL-3 tumors, cell-mediated cytotoxicity was measured by release of [125I]iododeoxyuridine. The response was biphasic, with an initial peak at 10 days and a 2nd peak after 30 days. A boost in reactivity could be elicited by later challenge with tumor cells. All of the reactivity was dependent on T-cells, being eliminated by treatment with anti-theta plus complement. The specificity of the reactions was not completely defined, but it was consistent with Friend type-specific antigen plus broader, common antigens. In mice with regressing MSV tumors, strong cell-mediated cytotoxicity, measured mainly by release of 51Cr, was seen against RBL-5, a Rauscher virus-induced leukemia. A single peak of response occurred at about 14 days after virus inoculation. Upon later challenge with RBL-5 cells, a vigorous and rapid secondary response was elicited, mainly in the region of tumor challenge. This cytotoxic reactivity and in vivo resistance to leukemia.lso was completely dependent on T-cells. In addition, macrophage-mediated inhibition of leukemia cell growth in vitro was seen in this system at the time of peak tumor development. The 51Cr release cytotoxicity was specific and directed primarily against an antigen, MEV-SA1, associated with mouse endogenous C-type viruses. The macrophage-induced growth inhibition appeared to be nonspecific. In both the FBL-3 and MSV tumor systems, protection against tumor growth could be adoptively transferred by immune lymphoid cells. In addition to induction of cell-mediated immunity by tumor cell or virus inoculation, cell-mediated cytotoxic reactivity was found to occur naturally in most young mice. This natural killer activity was quite distinct from the experimentally elicited reactions, being mediated by N-cells, a subpopulation of lymphoid cells with no clearly identifiable cell surface markers. The natural cytotoxicity was also directed against antigenic specificities different from those recognized by the MSV-immune cells. The central issue in all of these studies has been to determine the relationships between the in vitro-detected cell-mediated reactivity and in vivo resistance to leukemia.
Primary and secondary cell-mediated cytotoxic responses to FBL-3 cells, a syngeneic Friend virus-induced leukemia in C57BL/6 mice, could be generated by in vitro techniques as tested by the 125IUdR release assay. The specificity of the cytotoxic reactions appeared to be directed against the Friend type-specific antigen and the FMR (Friend, Moloney, Rauscher) antigen which were also the major antigens for transplantation immunity to FBL-3. In comparison to the primary cytotoxic response, the secondary cytotoxic response was accelerated (detected at an earlier time after sensitization), enhanced (gave much higher levels of cytotoxicity), was also longer lasting, and could be induced by a wide dose range of tumor cells. The secondary response could only be induced with lymphocytes obtained from regressors that were resistant to FBL-3 challenge; lymphocytes from mice with progressive tumor growth had no detectable secondary response. It was found that both induction phase and the effector phase of cytotoxic responses were T cell dependent. The characteristics of these reactions were thus very similar to those obtained with in vivo immunization or challenge, providing a good correlation with in vivo tumor immunity.
Inoculation i.p. of C57BL/6 mice with FBL-3 cells, a syngeneic Friend virus-induced leukemia, results in progressive growth of ascitic tumors; in contrast, s.c. inoculation of FBL-3 cells produces transient, localized tumor growth; the recipients are then subsequently resistant to further i.p. challenge of this tumor. Experiments were performed to study the effects of humoral factors that might be present in the ascitic fluid and that could affect the growth of the tumors and the host immune response. It was found that ascitic fluids obtained from various murine tumors could indeed promote the s.c. growth of FBL-3 cells. Furthermore, administration of these ascitic fluids was found to suppress the induction of both the primary and secondary cell-mediated cytotoxic responses to FBL-3 cells in vivo and in vitro and to inhibit the effector phase of these cell-mediated cytotoxic reactions in vitro. These studies indicate that the ascitic fluids obtained from tumor-bearing hosts contain humoral factors that can promote tumor growth and suppress immune responses.
Immunoregulatory alpha-globulin (IRA) derived from normal human plasma decreased cytotoxic reactivity as measured by an in vitro 5-iodo-2'-deoxyridine release assay of immune mouse lymphocytes against the syngeneic Friend virus-induced leukemia, FBL-3. This inhibitory effect depended on the dose of IRA used and was not due to the cytotoxic effects of IRA on the effector cells or target tumor cells. We also found elevated levels of serum alpha-gloubins in FBL-3 tumor-bearing mice as compared to normal mice. These data and the demonstration of decreased specific cytotoxic reactivity in FBL-3 tumor-bearing mice suggest that IRA functions in the suppression of the host's immune response against tumors.
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