Release of L-alanine by tumor cells

Culture supernatants from the weakly immunogenic T cell lymphoma L5178Y ESb were found to contain substantial amounts of alanine and lactate at a ratio of about 1:10. Supernatants from cells of the highly immunogenic mutant line ESb-D also contained lactate but only minute amounts of alanine. Moreover, ESb cells converted 14C-labeled glucose or pyruvate into labeled alanine and lactate at a ratio of about 1:10, whereas ESb-D cells yielded only labeled lactate and no detectable alanine. The injection of L-alanine in combination with L-lactate into mice strongly suppressed the capacity of their spleen cells to generate cytotoxic responses. The injection of L-alanine also suppressed the immunogenicity of ESb-D cells, as demonstrated by the generation of cytotoxic activity in vivo and by the in vivo immunization (priming) for secondary cytotoxic responses against ESb-D cells in vitro. Taken together, these experiments suggest the possibility i) that the ESb cells prevent the induction of cytotoxic responses by releasing immunosuppressive alanine, and ii) that the immunogenic mutant ESb-D may have gained immunogenicity by losing this immunosuppressive property.     

Transfer of long-lasting tumor immunity by immune T cells from MHC congenic mice: Migration,survival and tumor-protectivity of cytotoxic donor cells

Immunocompetent B10.D2 (H-2^d) mice are able to reject the highly malignant lymphoma ESb of DBA/2 (H-2^d) origin very effectively. Seven days after intravenous injection of the ESb tumor cells, B10.D2 mice developed a strong tumor-rejection response which was associated with the generation of anti-tumor T cells in their spleens with direct cytotoxic activity. Most of the cytotoxic potential was directed against the minor histocompatibility differences as demonstrated by the lysis of unrelated DBA/2 derived Eb tumor cells and normal DBA/2 but no B10.D2 derived ConA lymphoblasts. A previously performed clonal analysis, however, revealed a minority population of CTL clones which specifically recognized the ESb specific transplantation antigen (ESb-TATA). When transferred systemically into DBA/2 mice, the B10.D2 anti-ESb immune spleen cells could delay the outgrowth of s.c. transplanted ESb tumor cells. When the ESb tumor cells were experimentally distributed in a s.c. implanted sponge-matrix, the i.v. injected B10.D2 immune cells could confer complete protective immunity against the metastatic tumor, provided the recipients were pre-treated with 5 Gy to allow a better take of the allogeneic cells. The distribution of intravenously injected B 10D2 donor spleen cells was assessed in the recipients up to 50 days by cytotoxicity testing and assaying for the expression of the ₂ microglobulin allelic form b ( ₂m^b). These tests revealed a high propensity of donor cells to populate the spleen and lymph nodes of the DBA/2 recipients. Again this was particularly marked in sublethally irradiated mice where a long-lasting lymphoid chimerism was established.     

Expression of increased immunogenicity by thiol-releasing tumor variants.

Even moderate variations of the extracellular cysteine concentration were previously shown to affect T cell functions in vitro despite high concentrations of cystine. We therefore analyzed the membrane transport activities of T cells for cysteine and cystine, and the role of low molecular weight thiol in T cell-mediated host responses against a T cell tumor in vivo. A series of T cell clones and tumors including the highly malignant lymphoma L5178Y ESb and its strongly immunogenic variant ESb-D was found to express extremely weak transport activity for cystine but strong transport activity for cysteine. However, not all cells showed the expected requirement for cysteine (or 2-mercaptoethanol (2-ME)) in the culture medium. One group of clones and tumors including the malignant ESb-lymphoma did not respond to changes of extracellular cystine concentrations and was strongly thiol dependent. This group released only little acid soluble thiol (cysteine) if grown in cystine-containing cultures. The other T cell lines, in contrast, were able to maintain high intracellular GSH levels and DNA synthesis activity in cystine-containing culture medium without cystein or 2-ME and released substantial amounts of thiol. This group included the immunogenic ESb-D line. Additional thiol-releasing ESb variants were obtained by culturing large numbers of L5178Y ESb tumor cells in cultures without cysteine or 2-ME. All of these ESb variants showed a significantly decreased tumorigenicity and some of them induced cytotoxic and protective host responses even against the malignant ESb parent tumor. Taken together, our experiments suggest that the host response against a tumor may be limited in certain cases by the failure of the stimulator (i.e., the tumor) cell to deliver sufficient amounts of cysteine to the responding T cells.     

New antigens presented on tumor cells can cause immune rejection without influencing the frequency of tumor-specific cytolytic T cells

The specific immune response against syngeneic tumors by T cells is dependent on the existence of tumor-associated transplantation antigens (TATA). In the case of the chemically induced DBA/2-derived lymphoma Eb and its highly metastatic variant ESb the immunogenicity of these antigens is not sufficient to prevent tumor growth. Therefore we tested in two systems the influence of additional antigens as possible helper determinants for the generation of tumor-specific immune responses. In the Eb tumor system additional antigens were induced by mutagenization. The frequency of cytotoxic T lymphocytes (CTL) in response to mutagenized Eb cells was higher than that in response to untreated Eb cells. Fine specificity analysis revealed there there was no increase in the CTL response against the original TATA, but an activation of additional CTL clones responding to mutagen-induced antigens. In the ESb tumor system we tested the effect of additional recognition of minor histocompatibility antigens on the frequency of TATA-specific CTL. Transplantation of ESb tumor cells into B10.D2 mice, which are H-2-identical but differ in minor antigens, results in strong tumor rejection responses. In a limiting dilution mixed-leukocyte-tumor microculture system it was found that the minor antigens are recognized at the clonal level as independent antigens. The overall frequency of anti-tumor CTL in ESb-immunized B10.D2 mice was about 1/3000. Among these, the frequency of TATA-specific CTL was 1/16,709 and thus not significantly different from that of syngeneic DBA/2 mice. Thus neither minor antigens nor mutagen-induced antigens acted in the Eb/ESb tumor system as helper determinants and did not increase the frequency of tumor-specific CTLs.     

Cell-mediated immune responses in vitro. II. Simultaneous generation of cytotoxic lymphocyte responses to two sets of alloantigens of limited cross-reactivity.

The conditions for generation of simultaneous and independent cytotoxic lymphocyte (CL) responses to each of two sets of alloantigens of limited cross-reactivity by mouse spleen cells in vitro have been investigated. Responder spleen cells were incubated with mitomycin C-treated C57BL/6 (H-2b) or DBA/2 (H-2d) stimulator spleen cells and day 5 CL responses were assayed with 51Cr-labeled EL-4 leukemia (H-2b) and P815 mastocytoma (H-2d) as target cells. Spleen cells from mice of the various H-2 haplotypes tested differed greatly in their ability to develop specific CL responses against alloantigens on the stimulator spleen cells and in the degree of cross-reactive cytotoxic activity against target cells bearing alloantigens not present on the stimulator spleen cells. In contrast to the other strains examined, DBA/1 (H-2q) spleen cells developed specific CL responses to either H-2b or H-2d alloantigens without exhibiting significant cross-reactive activity on the inappropriate target cell. The CL responses to H-2b and H-2d alloantigens by DBA/1 spleen cells were comparable in magnitude and had similar stimulator cell-dose requirements. Further, DBA/1 spleen cells developed CL responses of normal magnitude simultaneously against both target cells when incubated with both mitomycin C-treated C57BL/6 and DBA/2 stimulator cells.     

Effect of levamisole on cytotoxic T-cell-mediated immune resistance to L1210 murine leukemia in hyperimmune mice

Summary The effect of levamisole (LMS) on T-cell-mediated antitumor immunity was examined in adult and aged mice hyperimmune to L1210 leukemia. The immune resistance of aged mice was depressed compared with that of adult mice, which almost completely rejected 5×10⁷ L1210 cells inoculated IP. A significant level of tumor-specific cytotoxicity was detected in the spleen cells of adult hyperimmune mice by the ⁵¹Cr-release assay after in vitro sensitization with mitomycin C-treated L1210 cells. This was mediated by cytotoxic T cells, since in vivo administration of antithymocyte serum or in vitro treatment of the spleen cells with anti-Thy 1.2 antibody and complement abrogated the cytotoxicity completely. In aged mice, however, cytotoxic T-cell activity was lower although the animals were immune to L1210.Administration of LMS (0.38 mg/kg) restored the depressed cytotoxicity of aged mice to the level seen in adult mice. Furthermore, in adult hyperimmune mice LMS augmented T-cell-mediated cytotoxic activity and restored the reduced cytotoxicity caused by in vivo administration of antithymocyte serum. These results indicate that LMS was effective in augmenting T-cell-mediated tumor immunity in immunologically competent or deficient hosts.     

Recruitment and activation of tumor-specific immune T cells in situ. CD8+ cells predominate the secondary response in sponge matrices and exert both delayed-type hypersensitivity-like and cytotoxic T lymphocyte activity

This study analyzes the involvement of CD4+ and CD8+ T cells in a secondary cellular immune response to the highly metastatic murine lymphoma ESb in situ. This tumor line expresses tumor-associated transplantation Ag which can induce protective immunity in vivo and specific CTL in vitro. In tumor-immune mice the injection of a tumor vaccine (x-irradiated ESb tumor cells) into s.c. implanted vascularized sponges resulted in the generation of a specific secondary immune response characterized by massive leukocyte recruitment and generation of strong CTL activity at the restimulation site. During the antitumor immune response the CD4+:CD8+ T cell ratio decreased significantly and specifically in the restimulated sponges. Depletion of CD8+ but not CD4+ T cells from the tumor immune mice before restimulation significantly reduced the delayed-type hypersensitivity-like response and totally blocked the generation of tumor-specific CTL activity in situ. Only a minority of the CD8+ immune T cells which predominated the secondary response in situ expressed IL-2R and lymph node homing receptors as detected by the mAb MEL-14.     

Augmentation of the generation of cytotoxic T lymphocytes against syngeneic tumor cells by recombinant human tumor necrosis factor

In order to clarify the effect of recombinant human tumor necrosis factor (rHu-TNF) on the antitumor T cell immune response, we examined the effect of rHu-TNF on the generation of cytotoxic T cells (CTL) against syngeneic tumor cells. Spleen cells from X5563 plasmacytoma-transplanted mice were stimulated in vitro with mitomycin C-treated X5563 cells in the presence or absence of rHu-TNF. The generation of CTL was augmented in a dose-dependent manner by the addition of rHu-TNF. The augmenting effect of rHu-TNF was more marked when indomethacin was added to the culture. The augmenting effect was observed only when rHu-TNF was added at the early stage of the generation of CTL. The cell surface phenotype of CTL generated was L3T4- and Lyt2+. The augmentation was shown not only by the chromium-51 release assay but also by the Winn assay. As to the specificity, the augmentation of CTL generation was observed by the addition of rHu-TNF when responder-primed spleen cells were stimulated with the tumor cells in vitro. On the other hand, augmentation was not observed when responder spleen cells were not stimulated with the tumor cells in vitro, or when responder spleen cells were obtained from normal mice. The CTL generated was not cytotoxic against other tumor cells of the same haplotype. Thus, rHu-TNF augmented the generation of CTL against syngeneic tumor cells in an antigen-specific manner. The in vivo effect of rHu-TNF was examined by administering rHu-TNF into X5563-bearing mice. The spleen cells of rHu-TNF-injected mice generated a much higher CTL activity against X5563 cells in vitro than did the spleen cells of uninjected mice. From these results, a possibility can be considered that in some cases, rHu-TNF may exert its antitumor activity by stimulating the immune system.     

Specific eradication of micrometastases by transfer of tumour-immune T cells from major-histocompatibility-complex congenic mice

Summary DBA/2 (H-2^d) mice bearing a transplanted highly metastatic lymphoma (ESb) in a state of widely disseminated disease could be successfully treated by a combination of surgery (removal of the local tumour), irradiation (5 Gy) and adoptive immunotherapy. The immunotherapy was achieved by transfer of anti-ESb-immune spleen cells from B10.D2 mice, which express the same major histocompatibility complex (MHC) molecules as DBA/2. In contrast, anti-ESb-immune cells from MHC-disparate C57BL/6 mice did not confer protective immunity. The B10.D2 anti-ESb-immune T cells contain two types of cytolytic specificity as detected by limiting-dilution analysis: (1) clones with specificity for the ESb-tumour-associated transplantation antigen (TATA) (at low frequency), and (b) clones with specificity for minor DBA/2 histocompatibility (H) antigens (at high frequency). Immune B10.D2 cells raised against different tumour lines or against TATA^– ESb tumour variants did not confer the 100% protection seen with immune cells against ESb TATA⁺ cells. Finally we demonstrate that the allogeneic immune cells are more potent in terms of protective immunity than corresponding syngeneic immune cells. The data suggest that the strong graft-versus-leukemia effect with immune T cells from allogeneic MHC-identical but not from MHC-disparate mice was due to T cells with MHC-restricted specificity for an ESb-associated TATA. A graft-versus-host reactivity that developed much later and could not be prevented was most likely due to T cells sensitized against normal minor H antigens of the host. Our results are of potential relevance for allogeneic bone marrow transplantation and adoptive immunotherapy protocols.     

The involvement of antigen-presenting cells and suppressor cells in the ultraviolet radiation-induced inhibition of secondary cytotoxic T cell sensitization

When mice were treated with ultraviolet (UV) radiation before immunization with the skin sensitizers, trinitro-chlorobenzene or dinitrofluorobenzene, they showed greatly depressed levels of priming for a secondary in vitro cytotoxic response against haptenated cells. Because the irradiation and skin painting were done on separate sites, these results suggest that the UV radiation had a systemic effect. The priming response of irradiated mice to allogeneic stimulation was normal, indicating some antigen selectivity to the inhibitory effect of UV radiation. A defect in antigen-presenting cells, previously demonstrated in UV-irradiated mice, was found to be largely responsible for the depressed priming response observed in these animals. In addition, the UV-irradiated, immunized mice possessed suppressor cells that were capable of blocking priming for cytotoxic responses against haptenated cells in normal mice.     

Studies on the recovery from tolerance to tumor antigens

Summary The present study investigates the potential of bone marrow cells from mice tolerant to tumor antigens to repopulate tumor-specific effector T cells. C3H/He mice were inoculated i.v. with 10⁶ 10000 R X-irradiated syngeneic X5563 plasmacytoma tumor cells three times at 4-day intervals. This regimen abrogated the ability of spleen cells from these mice to develop anti-X5563 cytotoxic and in vivo protective (tumor-neutralizing) T cell-mediated immunity as induced by i.d. inoculation of viable X5563 cells followed by surgical resection of the tumor. Since such suppression was induced in a tumor-specific way, this represented a state of antitumor tolerance. When bone marrow cells from normal or X5563-tolerant mice were transferred i.v. into 950 R X-irradiated syngeneic C3H/He mice, both groups of recipient mice generated anti-X5563 tumor immunity over a similar time course and to almost the same degree. Anti-X5563 tumor immunity induced in (C3H/He×C57BL/6) F1 mice which had been transferred with bone marrow cells from normal or X5563-tolerant C3H/He mice were mediated by T cells expressing the Ly phenotype of C3H/He, but not of C57BL/6, excluding the possibility that the antitumor effector cells were derived from recipient mice. It was also demonstrated that C3H/He mice which had been reconstituted with normal marrow were rendered tolerant when the tolerance regimen was started 7 weeks, but not 1 week after the bone marrow reconstitution. These results indicate that bone marrow cells from antitumor tolerant mice are not rendered tolerant to the tumor but can provide the potential to repopulate antitumor CTL and in vivo protective effector T cells.This work was supported by the Special Project Cancer-Bioscience from the Ministry of Education, Science and Culture, Japan Abbreviations used: MHC, major histocompatibility complex; CTL, cytotoxic T lymphocytes; TNP, trinitrophenyl; C, complement; TNBS; trinitrobenzene sulfonate; MMC, mitomycin C     

Augmentation of delayed-type hypersensitivity and resistance against allogeneic or syngeneic methylcholanthrene-induced tumors in mice preimmunized with the tumor extracts

Delayed-type hypersensitivity (DTH) responses against methylcholanthrene-induced fibrosarcomas in C3H/He and BDF1 mice were developed in BDF1 mice by sc injection of the respective mitomycin C-treated tumor cells. The DTH responses to the allogeneic and the syngeneic tumor cells were accelerated and enhanced tumor-specifically by priming 7 days previously with KCl extracts of the respective tumors. The ability in the mice primed with the tumor extracts enhancing the DTH response against the tumor cells could be transferred to recipient mice by the spleen cells, but not by the T-cell-depleted spleen cells. Rejection of allogeneic tumor was accelerated under the development of accelerated and enhanced DTH response against the allogeneic tumor antigens. Moreover, resistance to syngeneic tumor growth increased significantly with the development of accelerated and enhanced DTH response against the syngeneic tumor antigens. Thus, the augmentation of DTH response by preimmunization with tumor extracts was accompanied by the increased resistance to tumor growth, suggesting that T cells involved in the augmentation of tumor-specific DTH response play some role in increasing the resistance to tumor growth.     

Self-reactive T cell receptor-reactive CD8+ T cells inhibit T cell lymphoma growth in vivo

Syngenic C57BL/6 mice (H-2(b)) vaccinated with mitomycin C-treated L12R4 T lymphoma cells develop protective immunity toward the MHC class II-negative tumor cells. In the present study, we characterize the nature, mode of function, and specificity of the effector cells in this immunity. These cells are TCR-specific CD8(+) T lymphocytes with effector function in vitro as well as in vivo upon transfer to naive mice. They produce high levels of IFN-gamma and TNF-alpha, but little or no IL-4. By means of TCRbeta-negative variant L12R4 cells, P3.3, and TCR-Vbeta2 cDNA-transfected and TCR-Vbeta2-expressing P3.3 lymphoma cells, we found that a significant part of the effector T cells are specific for the Vbeta12 region. The growth inhibition of L12R4 cells in vitro was inhibited by anti-H-2, anti-K(b), and anti-D(b) mAb. Furthermore, vaccination with Vbeta12 peptide p67-78, which binds to both K(b) and D(b) MHC class I molecules, induces partial protection against L12R4 T lymphoma cells. Thus, self-reactive TCR-Vbeta-specific, K(b)-, or D(b)-restricted CD8(+) T cells mediate inhibition of T cell lymphoma growth in vitro and in vivo.     

Asialo-GM1-positive T killer cells are generated in F1 mice injected with parental spleen cells

(C57BL/6 x DBA/2)F1 mice transplanted with parental C57BL/6 spleen cells become splenic chimeras, show donor antihost cytotoxic T cell activity, and lose their T cell-mediated, humoral, and natural immunity. Injection of anti-asialo-GM1 (ASGM1) into transplanted mice strongly suppresses splenic cytotoxic activity and causes a significant reduction of spleen cells expressing ASGM1, Thy-1, and Lyt-2. In vitro treatment of spleen cells from transplanted mice with antibody and complement shows that the cytotoxic effector cells are ASGM1+, Thy-1+, Lyt-2+, L3T4-, NK1.1-, and H-2d-, hence of donor origin. The cytotoxic effector cells are specific for H-2d targets and lack NK activity. In an attempt to explore whether in vivo elimination of the cytotoxic effector cells has any influence on splenic chimerism or humoral immunity, F1 mice injected with parental splenocytes were treated with anti-ASGM 1. Results show that this treatment eliminates a substantial proportion of cytotoxic effector cells but has no effect on splenic chimerism or restoration of humoral immunity. It therefore appears that cytotoxic effector cells are not primarily responsible for induction of chimerism or suppression of humoral immunity. In support of this injection of parental spleen cells with the nu/nu mutation induces killer cells in F1 mice but fails to induce splenic chimerism or immunosuppression. In contrast, injection of parental spleen cells with the bg/bg mutation generates both splenic chimerism and suppression of humoral immunity although their ability to generate cytotoxic effector cells in F1 hosts is seriously impaired and comparable to the cytotoxic potential of C57BL/6 nu/nu cells. It is concluded that the ASGM1 + cytotoxic T cells are not primarily responsible for splenic chimerism and suppression of humoral immunity and that the two effects are likely caused by parental cells with a different phenotype and function.     

The role of host lymphocytes and host macrophages in antitumor reactions after injection of sensitized lymphocytes and tumor target cells into naive mice

Summary DBA/2 mice were immunized i.p. against syngeneic SL2 lymphosarcoma cells. At various days after the last immunization peritoneal and spleen lymphocytes were collected. The lymphocyte suspensions were enriched for T-cells by nylon wool filtration.The peritoneal T-cells from immunized mice (a) expressed direct specific antitumor cytotoxicity in vitro, (b) induced macrophage cytotoxicity in vitro, and (c) exerted tumor neutralization measured in a Winn-type assay. Spleen T-cells from these immunized mice (a) expressed no direct specific antitumor cytotoxicity in vitro, (b) only induced moderate macrophage cytotoxicity in vitro, but (c) exerted tumor neutralization in a Winn assay.For effective tumor neutralization in vivo effector target cell ratios of 1000:1 were required. When the effector/target ratio of 1000:1 was maintained but the absolute numbers of effector and target cells were lowered from 10⁶ to 10⁵ lymphocytes and 10³ to 10² target cells respectively, no tumor neutralization was obtained.The major effect of the sensitized-transferred T-lymphocytes seemed to be the induction of cytotoxic macrophages in the (naive) recipient mice, as the peritoneal macrophages collected from the recipient mice 7 days after i.p. injection of a mixture of sensitized T-cells and tumor cells were cytotoxic. Purified peritoneal T-lymphocytes collected from these recipient mice were able to induce macrophage cytotoxicity in vitro but expressed no cytotoxic T-cell activity.In conclusion, our results show that in the tumor system used, tumor neutralization after transfer of sensitized lymphocytes is not dependent on the presence of cytotoxic T-lymphocytes. Lymphocytes with the strongest potency to render macrophages cytotoxic (in vitro and in vivo) also induce the best tumor neutralization in vivo, suggesting an important role for host macrophages as antitumor effector cells.     

Mitomycin C-treated or irradiated concanavalin A-activated T cells augment the activation of cytotoxic T cells in vivo

The activation of cytotoxic T lymphocytes (CTL) in vivo after immunization of normal or cyclophosphamide-treated mice with allogeneic cells was strongly augmented by the administration of mitomycin C-treated or irradiated concanavalin A-activated spleen cells (Con A-spl). This effect of the Con A-spl was abrogated by treatment with Anti-Thy 1 antibody plus complement, and was therefore presumably mediated by activated "helper" T cells. (The term "helper" cell is only operationally defined in this context and indicates that the augmenting irradiation resistant T cells are obviously not CTL precursor cells). These observations indicated (i) that even the cytotoxic response against allogeneic stimulator cells suffers in vivo from insufficient "helper" T cell activity, and (ii) that the injection of Con A-spl may serve as a simple procedure to apply this "helper" activity in vivo. This procedure was at least as effective as the repeated injection of interleukin 2 (IL-2)-containing cell supernatants with up to four 30-unit doses of IL-2 per mouse. IL-2-containing cell supernatants were found to mediate similar effects only if injected into the footpads but not intravenously. This was in line with the reported observation that IL-2 has an extremely short half-life in vivo. The injection of Con A-spl was also found to augment the proliferative response in the regional lymph nodes.     

Neutralizing Antibodies Protect against Lethal Flavivirus Challenge but Allow for the Development of Active Humoral Immunity to a Nonstructural Virus Protein

Antibody-mediated neutralization of viruses has been extensively studied in vitro, but the precise mechanisms that account for antibody-mediated protection against viral infection in vivo still remain largely uncharacterized. The two points under discussion are antibodies conferring sterilizing immunity by neutralizing the virus inoculum or protection against the development of disease without complete inhibition of virus replication. For tick-borne encephalitis virus (TBEV), a flavivirus, transfer of neutralizing antibodies specific for envelope glycoprotein E protected mice from subsequent TBEV challenge. Nevertheless, short-term, low-level virus replication was detected in these mice. Furthermore, mice that were exposed to replicating but not to inactivated virus while passively protected developed active immunity to TBEV rechallenge. Despite the priming of TBEV-specific cytotoxic T cells, adoptive transfer of serum but not of T cells conferred immunity upon naive recipient mice. These transferred sera were not neutralizing and were predominantly specific for NS1, a nonstructural TBEV protein which is expressed in and on infected cells and which is also secreted from these cells. Results of these experiments showed that despite passive protection by neutralizing antibodies, limited virus replication occurs, indicating protection from disease rather than sterilizing immunity. The protective immunity induced by replicating virus is surprisingly not T-cell mediated but is due to antibodies against a nonstructural virus protein absent from the virion.     

The role of the BCG dose and the mouse strain in the inhibition of development of neoplasms susceptible and nonsusceptible to the action of natural cytotoxic cells

The susceptibility of Ehrlich Ascites Carcinoma (EAC) cells to the action of natural cytotoxic cells of DBA/2 and Balb/c mice in vitro was established. Leukaemia L 1210 cells proved insensitive to the in vitro action of natural cytotoxic cells of DBA/2 mice, but not to those of Balb/c ones. BCG, one of the inductors of cytotoxic NK cells, when administered to DBA/2 or Balb/c mice before introduction of EAC cells inhibited the growth of this tumour but did not retard the development of leukaemia L 1210 in DBA/2 mice. The change in the number of peritoneal exsudate cells (PEC) in DBA/2 mice after intraperitoneal injection of BCG was demonstrated to be dependent on the dose and the time elapsed after bacilli introduction. The antitumour action of BCG does not depend on changes in the number of PEC caused by the bacilli. Both large (3.0 mg) and small (0.02 mg) doses of BCG inhibit the development of EAC in Balb/c mice ("sensitive" to BCG), notwithstanding the time of administration of the bacilli. In DBA/2 mice ("resistant" to BCG) development of EAC can be inhibited only by the large dose of BCG since small one is sometimes ineffective.     

Lymphocytes generated by in vivo priming and in vitro sensitization demonstrate therapeutic efficacy against a murine tumor that lacks apparent immunogenicity

The adoptive transfer of sensitized lymphocytes is an effective means to mediate the regression of established tumors. However, successful therapy can only be demonstrated in animal models where tumors are intrinsically immunogenic, capable of eliciting systemic immunity. To explore the potential of this therapeutic approach to tumors of less immunogenicity, we have selected and used a murine tumor, MCA 102, for the current study because all attempts to immunize syngeneic mice failed. We report here that inoculation of mice with a mixture of tumor cells and a bacterial adjuvant, Corynebacterium parvum led to the production of sensitized, but not fully functional, lymphocytes in the draining lymph nodes (LN). These cells, termed pre-effector cells, could nevertheless further differentiate to acquire full immunologic function by an established in vitro sensitization culture method. In adoptive immunotherapy experiments, transfer of as few as 1.5 X 10(7) in vitro sensitized cells not only reduced established pulmonary MCA 102 metastases but also prolonged survival and cured tumors in a majority of the treated animals. In order to elicit pre-effector cells in vivo, inoculation with both tumor cells and C. parvum was essential. Although a broad range of numbers of MCA 102 tumor cells appeared to be effective, generation of pre-effector cells was dependent on the dose of C. parvum. We have found that a C. parvum dose of 25 micrograms was optimal, whereas higher doses of the adjuvant had suppressive effects. Analysis of the kinetics of their appearance revealed that the generation of pre-effector cells was transient. They were detectable 7 days after in vivo priming followed by a rapid decline. Furthermore, pre-effector cells were detected only in the regional draining LN. No reactivity was demonstrable in the spleen, mesenteric LN, PBL, or bone marrow. Taken together, these results expand the scope of immunotherapy by demonstrating the feasibility of manipulating a limited and obscure immune response to the MCA 102 tumor for therapeutic efficacy.     

Induction of oncofetal antigen-specific suppressor pathways, involving Thy-1+ cells, during the early stages of tumor progression

The early stages of tumor progression were modelled by intraperitoneally injecting BALB/c mice daily with exponentially increasing numbers of mitomycin C-treated, syngeneic MPC-11 tumor cells. At various stages of this regime, mesenteric lymph node (MLN) and spleen cells were assessed for regulatory activity on the induction of cytotoxic T lymphocytes (CTL) in vitro. Cells present in both MLN and spleens of mice whose daily tumor dose had reached 102,400 MPC-11 cells impaired the generation of CTL specific for MPC-11 and specific for oncofetal antigen(s) shared between MPC-11 and Day 14-15 syngeneic fetal liver cells. Depletion of Thy-1+ cells from the regulatory cell populations removed the suppressive activity. The regulatory cells did not affect the induction of CTL specific for H-2b antigens in the context of H-2d (i.e., BALB/c) class I MHC.