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.     

Cell-mediated immunity to Friend virus-induced leukemia. III. Characteristics of secondary cell-mediated cytotoxic response.

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 immunity to Friend virus-induced leukemia. II. Characteristics of primary cell-mediated cytotoxic response.

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.     

Antigen-driven T cell clones can proliferate in vivo, eradicate disseminated leukemia, and provide specific immunologic memory

The aim of the current study was to determine the ability of antigen-driven cloned helper cell independent cytotoxic T lymphocytes (HITc) to proliferate and to survive in vivo and to mediate tumor therapy. The HITc clone utilized (denoted 1.B6) was specifically cytolytic to FBL-3, a syngeneic Friend virus-induced murine leukemia. Activation in vitro (48 hr) with FBL-3 induced secretion of interleukin 2 (IL 2), expression of IL 2 receptors (IL 2R), and in vitro proliferation. These cells could be "rested" for several weeks without stimulation, which resulted in reduced expression of IL 2R; however, restimulation with antigen resulted in reinduction of IL 2R and proliferation. The ability of cloned HITc to proliferate and to survive in vivo was examined in cyclophosphamide (CY) pretreated donor mice congenic for the Thy-1 gene. Adoptively transferred cloned HITc could be found in large numbers, and were widely distributed in vivo 1 wk after transfer. In tumor therapy, 1.B6 cells when injected into a site of tumor (i.p.) and used as an adjunct to CY were effective against disseminated FBL-3. In this circumstance, cloned 1.B6 cells could be recovered from cured mice 125 days after transfer and were shown to specifically lyse tumor and proliferate in vitro in response to FBL-3. Thus as an adjunct to CY, tumor-specific cloned HITc are capable of eradicating disseminated leukemia, persisting long-term in vivo, and providing specific immunologic memory.     

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.     

Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors.

FBL-3 is a highly immunogenic murine leukemia of C57BL/6 origin induced by Friend murine leukemia virus (MuLV). Immunization of C57BL/6 mice with FBL-3 readily elicits CD8+ cytotoxic T lymphocytes (CTL) capable of lysing FBL-3 as well as syngeneic leukemias induced by Moloney and Rauscher MuLV. The aim of this current study was to identify the immunogenic epitope(s) recognized by the FBL-3-specific CD8+ CTL. A series of FBL-3-specific CD8+ CTL clones were generated from C57BL/6 mice immunized to FBL-3. The majority of CTL clones (32 of 38) were specific for F-MuLV gag-encoded antigen. By using a series of recombinant vaccinia viruses expressing full-length and truncated F-MuLV gag genes, the antigenic epitope recognized by the FBL-3 gag-specific CTL clones, as well as by bulk-cultured CTL from spleens of mice immune to FBL-3, was localized to the leader sequence of gPr80gag protein. The precise amino acid sequence of the CTL epitope in the leader sequence was identified as CCLCLTVFL (positions 85-93) by examining lysis of targets incubated with a series of synthetic leader sequence peptides. No evidence of other CTL epitopes in the gPr80gag or Pr65gag core virion structural polyproteins was found. The identity of CCLCLTVFL as the target peptide was validated by showing that immunization with the peptide elicited CTL that lysed FBL-3. The CTL elicited by the Gag peptide also specifically lysed syngeneic leukemia cells induced by Moloney and Rauscher MuLV (MBL-2 and RBL-5). The transmembrane peptide was shown to be the major gag-encoded antigenic epitope recognized by bulk-cultured CTL derived from C57BL/6 mice immunized to MBL-2 or RBL-5. Thus, the CTL epitope of FBL-3 is localized to the transmembrane anchor domain of the nonstructural Gag polyprotein and is shared by leukemia/lymphoma cell lines induced by Friend, Moloney, and Rauscher MuLV.     

Tumor gangliosides inhibit the tumor-specific immune response.

Tumor gangliosides are highly immunosuppressive membrane glycosphingolipids that are shed into the tumor cell microenvironment. We directly tested the impact of shed gangliosides on the in vivo antitumor immune response in a syngeneic fully autochthonous system (FBL-3 erythroleukemia cells, C57BL/6 mice, and highly purified FBL-3 cell gangliosides). The major FBL-3 ganglioside was identified as GM1b by mass spectrometry. Substantial ganglioside shedding (90 pmol/108 cells/h), a requisite for their inhibition of the immune function of tumor-infiltrating leukocytes, was detected. Immunosuppression by FBL-3 gangliosides was potent; 5-20 microM inhibited the tumor-specific secondary proliferative response (80-100%) and suppressed the generation of tumor-specific CTLs (97% reduction of FBL-3 cell lysis at an E:T ratio of 100:1). In vivo, coinjection of 10 nmol of FBL-3 gangliosides with a primary FBL-3 cell immunization led to a reduced response to a secondary challenge (the increase in the draining popliteal lymph node mass, cell number, and lymphocyte thymidine incorporation were lowered by 70, 69, and 72%, respectively). Coinjection of gangliosides with a secondary tumor challenge led to a 61, 74, and 42% reduction of the increase in lymph node mass, cell number, and thymidine uptake and a 63-74% inhibition of the increase of draining lymph node T cells (CD3+), B cells (CD19+), and dendritic cells/macrophages (Mac-3+). Overall, the clear conclusion that tumor-derived gangliosides inhibit syngeneic antitumor immune responses implicates these molecules as a potent factor in promoting tumor formation and progression.     

Regulation of the In Vitro Secondary Cell-Mediated Cytotoxic Response against Syngeneic FBL-3 Leukemia by Macrophages

Depletion of macrophages from immune spleen cells by treatment with carbonyl iron and magnet or by in vivo treatment with carrageenan enhanced the in vitro secondary cell-mediated cytotoxic response against a syngeneic Friend virus-induced leukemia, FBL-3 cells of C57BL/6 mice. However, further depletion of macrophages by passing the carbonyl iron-treated immune spleen cells through a nylon wool column abrogated the cytotoxic response. The addition of splenic macrophage-enriched preparations from either FBL-3-immune or normal mice suppressed the cytotoxic response of immune spleen cells treated with carbonyl iron and magnet. This suppressive effect of splenic macrophages presented a marked contrast with the enhancing effect of normal peritoneal macrophages on the same cell-mediated cytotoxic response, indicating regulation of the generation of killer T cells against a syngeneic tumor by functionally distinct macrophages. The suppressed cell-mediated cytotoxic response against FBL-3 cells by immune spleen cells was augmented by the addition of indomethacin to the culture medium, and this augmentation with indomethacin was greatly decreased by depletion of phagocytic cells from the immune spleen by treatment with carbonyl iron and magnet. The mechanisms of regulation of the cell-mediated cytotoxic response with soluble factors released from macrophages are discussed.     

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.     

Idiotype vaccination against murine B cell lymphoma. Humoral and cellular requirements for the full expression of antitumor immunity

The roles of humoral and cellular antitumor immune responses induced by immunization with tumor-derived idiotypic IgM were studied in a syngeneic, transplantable B cell lymphoma (38C13) of C3H mice. Id vaccination with keyhole limpet hemocyanin-conjugated Id induced protection against a subsequent lethal tumor challenge. Such immunizations elicited anti-idiotypic antibodies that were cytotoxic in in vitro antibody-dependent cellular cytotoxicity assays as well as in vivo passive transfer experiments. L3T4+ T cells, which proliferated in vitro in response to the specific Id protein, were also induced. However, cells mediating direct cytotoxicity, either in vitro or in vivo, were not observed in the lymph nodes, spleens, or peritoneal cavity of immune mice or at the site of tumor regression as demonstrated by using a tumor sponge implantation model. In addition, in vitro sensitization of immune lymphocytes against 38C13 tumor cells failed to induce cytotoxicity. Immunization with lipid conjugated Id also elicited a T cell proliferative response but failed to induce anti-idiotypic antibodies and did not confer resistance to tumor growth. These results suggest that anti-idiotypic antibodies play the major role in the destruction of 38C13 tumor cells. However, in vivo depletion of L3T4+ or Lyt-2+ cells from 38C-Id-keyhole limpet hemocyanin-immunized mice resulted in diminished protection against a tumor challenge. Thus, although humoral responses appear to play the predominant part in tumor destruction, cellular responses are also required for the full expression of antitumor immunity in this system.     

Specificity of adoptive chemoimmunotherapy of established syngeneic tumors

To examine the specificity of adoptive chemoimmunotherapy (ACIT) of established syngeneic tumors, two noncross-reactive C57BL/6 tumors were studied: a Friend virus-induced tumor (FBL-3) and a chemically induced virus-negative tumor EL-4(G-). In vitro studies confirmed that these tumors are antigenically distinct by demonstrating that the cytotoxic responses of spleen cells from mice immunized in vivo and reexposed to tumor in vitro are immunologically specific. Studies of ACIT with cells from mice immunized in vivo demonstrated similar specificity. Mice receiving 5 x 10(6) FBL-3 on day 0 all died by day 13. Treatment on day 5 with cyclophosphamide (CY), 180 mg/kg, prolonged the median survival time (MST) to day 23. Treatment on day 5 with CY plus 2 x 10(7) normal nonimmune C57BL/6 cells or CY plus cells sensitized to EL-4(G-) had no additional effect on survival whereas 2 x 10(7) C57BL/6 cells sensitized to FBL-3 in vivo prolonged MST to day 64 and cured 13 of 32 mice. Similarly, mice given 2 x 10(5) EL-4(G-) on day 0 all died by day 16, and CY on day 5 prolonged the MST to day 22. As an adjunct to CY, 2 X 10(7) normal cells or cells sensitized to FBL-3 had a modest effect, prolonging the MST to days 37 and 36, respectively. However, treatment with CY plus 2 x 10(7) cells immune to EL-4(G-) cured 22 of 32 mice. The results demonstrate the immunologic specificity of ACIT of syngeneic tumors treated with immune syngeneic cells.     

Eradication of disseminated murine leukemia by treatment with high-dose interleukin 2

Interleukin 2 (IL 2) in high concentration induces lymphocytes to become nonspecifically cytolytic to a wide variety of tumor targets. We evaluated the therapeutic potential of such lymphokine-activated killer (LAK) cells in vivo and high-dose II 2 in vivo against disseminated murine leukemia. To quantitate the potential anti-leukemia effect of LAK cells in vivo, B6 mice were injected i.p. with graded doses of FBL-3 leukemia cells followed by LAK cells. In this Winn-type assay, 1 X 10(7) LAK cells were able to prevent the outgrowth of 1 X 10(2) FBL-3 cells in only 50% of mice and did not prevent the outgrowth of 1 X 10(6) tumor cells. Thus LAK cells, highly cytolytic to FBL-3 in vitro, mediated only a limited anti-tumor effect when applied directly to leukemia cells in vivo. LAK cells used as an adjunct to chemotherapy induced a small but non-curative effect against FBL-3, however. In this circumstance, LAK cells were markedly less effective than were immune spleen cells from mice previously sensitized to FBL-3. To test the anti-leukemia effect of high-dose IL 2 in vivo, B6 mice were inoculated with 5 X 10(6) FBL-3 cells followed by repeated doses of IL 2 at dose levels shown to induce LAK in vivo. "LAK-inducing" IL 2 doses on days 5 to 9 after FBL-3 inoculation, when tumor was disseminated, cured 50% of the mice. Treatment on days 5 to 9 was far more effective than on days 0 to 4, implying that the evolution of a host-tumor interaction was essential for the therapeutic effect of IL 2. Mice cured of FBL-3 by high-dose IL 2 were found to be immune to FBL-3, suggesting that tumor eradication resulted from a collaboration between LAK activity and tumor-specific immunity.     

Analysis of protective and cytotoxic immune responses in vivo against metabolically inactivated and untreated cells of a mutagenized tumor line (requirements for tumor immunogenicity)

The immunogenicity of a mutagenized subline (ESb-D) of the weakly immunogenic T-cell lymphoma L 5178 Y ESb has been characterized. The injection of 10(6) ESb-D cells ip did not establish lethal tumors in untreated DBA/2 mice but established tumors in sublethally irradiated mice. Injection of ESb-D cells into otherwise untreated DBA/2 mice established also a state of protective immunity against the subsequent injection of otherwise lethal doses of ESb tumor cells. Protection was only obtained after injection of intact but not UV-irradiated or mitomycin-C-treated ESb-D cells. A direct T-cell-mediated cytotoxic activity was also demonstrable in the spleen cells of DBA/2 mice after injection of ESb-D cells but not ESb cells. The cytotoxic activity was variant specific for ESb-D target cells, and it was induced only with intact but not UV-irradiated or mitomycin C-treated ESb-D cells. This suggested that the induction of protective and cytotoxic immunity may require the persistence of the antigen or unusually high antigen doses. The in vivo priming for a secondary in vitro cytotoxic response, in contrast, was achieved with intact and also with mitomycin C-treated ESb-D cells but again not with UV-irradiated ESb-D cells. This indicated that the metabolic activity was a minimal requirement for the in vivo immunogenicity of the ESb-D tumor line. The secondary cytotoxic activity was demonstrable on ESb-D and ESb target cells and could be restimulated in vitro about equally well with ESb-D and ESb cells. But the in vivo priming was again only obtained with ESb-D cells and not with ESb cells. These experiments thus demonstrated that the requirements for immunogenicity are more stringent in vivo than in vitro, and more stringent for the induction of direct cytotoxic and protective immunity in vivo than for the in vivo priming for secondary in vitro responses.     

Generation of cytotoxic lymphocytes by SV40-induced antigens

In order to study the correlation of in vivo tumor transplantation immunity and in vitro immunologic assays, cell-mediated cytotoxicity against SV40-transformed cells was studied in AL/N strain mice by using 51Cr-release assay. Killing of SV40-transformed AL/N fibroblast cells was observed by spleen cells of AL/N mice immunized with syngeneic SV40-transformed cells. Immunization with the solubilized SV40 tumor-specific transplantation antigen (TSTA) that induced transplantation immunity in vivo did not elicit cytotoxic spleen cells in vitro. However, the spleen cells from mice immunized with solubilized TSTA and then sensitized in vitro with SV40-transformed cells became cytotoxic against SV40-transformed fibroblasts. Similarly, SV40 TSTA (T antigen) purified by immunoprecipitation was able to prime the lymphocytes in AL/N mice: the primed lymphocytes could differentiate into cytotoxic lymphocytes upon in vitro stimulation by SV40-transformed cells. These data indicate that SV40 TSTA (T antigen) plays a role in the induction of cytotoxic lymphocytes.     

Distinct proliferative T cell clonotypes are generated in response to a murine retrovirus-induced syngeneic T cell leukemia: viral gp70 antigen-specific MT4+ clones and Lyt-2+ cytolytic clones which recognize a tumor-specific cell surface antigen

After immunization of B6 mice with the syngeneic retrovirus-induced T cell leukemia/lymphoma FBL-3, two major tumor-specific proliferative T cell clonotypes were derived. T cell clones derived from long-term lines propagated by in vitro culture with irradiated tumor cells and syngeneic spleen cells were exclusively of the Lyt-2+ phenotype. Such clones were cytolytic, retained their proliferative phenotype indefinitely when expanded by repeated cycles of reactivation and rest, and recognized a tumor-specific cell surface antigen in association with class I MHC molecules. This tumor cell antigen was not present on nontransformed virus-infected cells. Class II MHC-restricted MT4+ clones specific for the viral antigen gp70 were derived from lymph node T cells of FBL-3 tumor-immune mice only by in vitro culture with purified Friend virus in the presence of syngeneic splenic APC. Once derived, however, such clones could be stimulated in the presence of FBL-3 tumor cells and syngeneic spleen cells, demonstrating the reprocessing of tumor-derived gp70 antigen by APC in the spleen cell population. In contrast, no reprocessing of the tumor cell surface antigen by splenic APC for presentation to the class I MHC-restricted T cell clones could be demonstrated. Evidence is presented that FBL-3 T leukemia/lymphoma cells function as APC for Lyt-2+ class I MHC-restricted clones, and that no concomitant recognition of Ia molecules is required to activate these clones. Both Lyt-2+ and MT4+ clones were induced to proliferate in the presence of exogenous IL2 alone, but this stimulus failed to result in significant release of immune interferon. In contrast, antigen stimulation of both clones resulted in proliferation as well as significant immune interferon release. Immune interferon production is not required for the generation of MHC-restricted cell-mediated cytolytic function.     

Adoptively transferred antigen-specific T cells can be grown and maintained in large numbers in vivo for extended periods of time by intermittent restimulation with specific antigen plus IL-2

The aim of the current study was to determine whether cultured tumor Ag-specific T cells could be induced to grow and maintained functional in large numbers in vivo by intermittent restimulation in vivo with specific Ag plus IL-2. T cells derived from spleens of B6 mice (Thy-1.2) immune to FBL-3, a Friend virus-induced leukemia, were activated by in vitro stimulation with irradiated FBL-3 and expanded by culture for 14 days with low concentrations of IL-2. The resultant FBL-3-specific T cell lines were adoptively transferred into cyclophosphamide pretreated congenic hosts (B6/Thy-1.1), and restimulated every 14 days by an injection of irradiated FBL-3 plus a 7-day course of IL-2. Donor T cells residing in the host were identified and quantified by use of antibody to the Thy-1.2 allele. The results confirmed that stimulation with FBL-3 on the day of transfer (day 0) plus IL-2 on days 0 to 6 induced rapid growth of donor T cells to approximately an 11-fold increase in total donor T cell number recoverable from host ascites and spleen by day 7. However, prolonging the course of IL-2 administration to 35 days did not maintain the number or the specific cytolytic function of donor T cells. By contrast, intermittent restimulation with specific Ag plus IL-2 induced intermittent regrowth of donor T cells in vivo, maintained the number of donor T cells in vivo at greater than the number input for longer than 1 mo, and allowed detection of substantially augmented donor T cell-mediated specific antitumor function over that period of time.     

Effector phenotypes and mechanisms of antitumor immune reactivity of tumor-immunized and tumor-bearing mice in two syngeneic tumors.

By using two different syngeneic tumors, Meth A sarcoma and RL male 1 lymphoma of BALB/c origin, the present study was designed to investigate the subset(s) of T cells mediating in vivo antitumor immune responses and some of the effector mechanisms of in vivo protective immunity in BALB/c mice immunized against tumor or bearing tumor. Spleen cells from the mice immunized against Meth A tumor or bearing Meth A tumor inhibited the growth of Meth A tumor in the Winn assay. In the Meth A-immunized mice, L3T4+ (CD4+) cells played a major role in mediating the inhibitory activity against Meth A tumor growth, whereas in the Meth A-bearing mice, the antitumor protective immunity was mediated by both L3T4+ and Lyt-2+ (CD8+) cells. Spleen cells from the Meth A-immunized or Meth A-bearing mice were not able to generate cytotoxic T lymphocytes (CTL) directed against Meth A tumor after the in vitro restimulation of spleen cells with mitomycin C (MMC)-treated Meth A cells, while fresh spleen cells from the Meth A-immunized or Meth A-bearing mice were able to induce the strong delayed-type hypersensitivity (DTH) responses to Meth A tumor. The DTH response to Meth A tumor was mediated by L3T4+ cells in the Meth A-immunized mice and by both L3T4+ and Lyt-2+ cells in the Meth A-bearing mice. In the similar experiments performed in the RL male 1 lymphoma, the antitumor activity in spleen cells from the RL male 1-immunized or RL male 1-bearing mice depended on Lyt-2+ but not L3T4+ cells in the Winn assay. When spleen cells from the RL male 1-immunized or RL male 1-bearing mice were cultured with MMC-treated RL male 1 cells for 5 days, an appreciable CTL response to RL male 1 tumor was induced. These results suggest that the nature of tumor and/or tumor antigens determines which T cell subset is required to exhibit the protective immunity against tumor and thus the different effector mechanisms could be induced in the different tumor models. Furthermore, these data support the conclusion that antitumor T cell responses are affected by the immune state of host to tumor.     

CD4+ T lymphocytes are critical mediators of tumor immunity to simian virus 40 large tumor antigen induced by vaccination with plasmid DNA

A mechanistic analysis of tumor immunity directed toward the viral oncoprotein simian virus 40 (SV40) large tumor antigen (Tag) has previously been described by our laboratory for scenarios of recombinant Tag immunization in BALB/c mice. In the present study, we performed a preliminary characterization of the immune components necessary for systemic tumor immunity induced upon immunization with plasmid DNA encoding SV40 Tag as a transgene (pCMV-Tag). Antibody responses to SV40 Tag were observed via indirect enzyme-linked immunosorbent assay following three intramuscular (i.m.) injections of pCMV-Tag and were typified by a mixed Th1/Th2 response. Complete tumor immunity within a murine model of pulmonary metastasis was achieved upon two i.m. injections of pCMV-Tag, as assessed by examination of tumor foci in mouse lungs, without a detectable antibody response to SV40 Tag. Induction-phase and effector-phase depletions of T cell subsets were performed in vivo via administration of depleting rat monoclonal antibodies, and these experiments demonstrated that CD4(+) T lymphocytes are required in both phases of the adaptive immune response. Conversely, depletion of CD8(+) T lymphocytes did not impair tumor immunity in either immune phase and resulted in the premature production of antibodies to SV40 Tag. Our findings are unique in that a dominant role could be ascribed to CD4(+) T lymphocytes within a model of DNA vaccine-induced tumor immunity to Tag-expressing tumor cells. Additionally, our findings provide insight into the general mechanisms of vaccine-induced tumor immunity directed toward tumors bearing distinct tumor-associated antigens.     

The role of tumor-specific Lyt-1+2- T cells in eradicating tumor cells in vivo. I. Lyt-1+2- T cells do not necessarily require recruitment of host's cytotoxic T cell precursors for implementation of in vivo immunity

The present study determines the Ly phenotype of T cells mediating tumor cell rejection in vivo and investigates some of cellular mechanisms involved in the in vivo protective immunity. C3H/HeN mice were immunized to syngeneic X5563 plasmacytoma by intradermal (i.d.) inoculation of viable X5563 tumor cells, followed by the surgical resection of the tumor. Spleen cells from these immune mice were fractionated by treatment with anti-Lyt antibodies plus complement, and each Lyt subpopulation was tested for the reconstituting potential of in vivo protective immunity in syngeneic T cell-depleted mice (B cell mice). When C3H/HeN B cell mice were adoptively transferred with Lyt-1-2+ T cells from the above tumor-immunized mice, these B cell mice exhibited an appreciable cytotoxic T lymphocyte (CTL) response to the X5563 tumor, whereas they failed to resist the i.d. challenge of X5563 tumor cells. In contrast, the adoptive transfer of Lyt-1+2- anti-X5563 immune T cells into B cell mice produced complete protection against the subsequent tumor cell challenge. Although no CTL or antibody response against X5563 tumors was detected in the above tumor-resistant B cell mice, these mice were able to retain Lyt-1+2- T cell-mediated delayed-type hypersensitivity (DTH) responses to the X5563 tumor. These results indicate that Lyt-1+2- T cells depleted of the Lyt-2+ T cell subpopulation containing CTL or CTL precursors are effective in in vivo protective immunity, and that these Lyt-1+2- T cells implement their in vivo anti-tumor activity without inducing CTL or antibody responses. The mechanism(s) by which Lyt-1+2- T cells function in vivo for the implementation of tumor-specific immunity is discussed in the context of DTH responses to the tumor-associated antigens and its related Lyt-1+2- T cell-mediated lymphokine production.     

Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells

The important role of tumor-specific cytotoxic CD8⁺ T cells is well defined in the immune control of the tumors, but the role of effector CD4⁺ T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4⁺ T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4⁺ T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8⁺ T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4⁺ T cells and increases FV-specific CD4⁺ T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4⁺ T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4⁺ T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.