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.     

Enhanced Antibody-Dependent Lymphocyte-Mediated Cytotoxic activity in mice cured from an ascitic tumor by a single tumor aspiration

Summary Aspiration of approximately 50% of the tumor volume in the syngenic lethal C3H-L1 ascites tumor system at day 12 after intraperitoneal injection of 2×10⁷ tumor cells cured about 40% of the animals from the ascites tumor. The recovering mice showed strong immunity to a second challenge with tumor cells and transfer of spleen cells from recovering mice protected normal mice to tumor development. Complement-dependent serum-mediated cytotoxicity (CDSC) against tumor cells in vitro appeared both in non-recovering and recovering mice being most pronounced in completely cured animals. Antibody-dependent, lymphocyte-mediated cytotoxicity (ADLC) was absent in tumor-bearing and dying mice but appeared early after recovery in cured mice. No direct lymphocyte-mediated cytotoxicity (DLC) could be demonstrated either in non-recovering or in recovering mice.     

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.     

Specificity of the generation and expression of enhanced anti-plasmacytoma immunity by spleen cells from melphalan-treated MOPC-315 tumor bearers

Summary We have shown previously that low-dose melphalan (L-PAM) therapy of mice bearing a large MOPC-315 plasmacytoma enables their hitherto immunosuppressed spleen cells to exert potent anti-MOPC-315 cytotoxicity following in vitro immunization with MOPC-315 tumor cells [3]. Here we show that, following in vitro immunization with MOPC-315 tumor cells, spleen cells from such L-PAM-treated MOPC-315 tumor bearers exhibited enhanced T-cell-mediated cytotoxicity not only against the MOPC-315 tumor, but also against another plasmacytoma (MOPC-104E) possessing surface immunoglobulin (SIg) of a different idiotype than the MOPC-315 cells, as well as against a variant of the MOPC-315 tumor which does not produce nor possess SIg (SIg^–MOPC-315). The enhanced cytotoxicity was directed against target antigens which are not expressed on the surface of the syngeneic WEHI 22.1 thymoma or the natural killer-sensitive YAC-1 cells. Plasmacytoma shared antigens, other than immunoglobulins, were able to stimulate spleen cells from L-PAM-cured MOPC-315 tumor bearers to generate in vitro a secondary type anti-plasmacytoma cytotoxic response. L-PAM-cured MOPC-315 tumor bearers exhibited in vivo immunity against SIg^–MOPC-315 tumor cells, which was sufficiently triggered by the SIg^– cells to bring about the rejection of a challenge of at least 100-fold the minimal lethal tumor dose of the SIg^–MOPC-315 cells. Thus, SIg^– MOPC-315 tumor cells present among SIg⁺ tumor cells in the parental MOPC-315 tumor inoculum [9, 26] can be eradicated in the L-PAM-treated MOPC-315 tumor bearers by the immune response to SIg⁺ tumor cells as well as by the immune response to SIg^– tumor cells themselves.Supported by Research Grant CA-35761 from the National Cancer Institute and Research Grant IM-435 from the American Cancer SocietyIn partial fulfillment of the requirements for the Doctor of Philosophy degree.     

Identification of a novel population of highly cytotoxic c‐Met‐expressing CD8+ T lymphocytes

CD8⁺ cytotoxic T lymphocytes (CTLs) are critical mediators of anti‐tumor immunity, and controlling the mechanisms that govern CTL functions could be crucial for enhancing patient outcome. Previously, we reported that hepatocyte growth factor (HGF) limits effective murine CTL responses via antigen‐presenting cells. Here, we show that a fraction of murine effector CTLs expresses the HGF receptor c‐Met (c‐Met⁺ CTLs). Phenotypic and functional analysis of c‐Met⁺ CTLs reveals that they display enhanced cytolytic capacities compared to their c‐Met^? CTL counterparts. Furthermore, HGF directly restrains the cytolytic function of c‐Met⁺ CTLs in cell‐mediated cytotoxicity reactions in vitro and in vivo and abrogates T‐cell responses against metastatic melanoma in vivo. Finally, we establish in three murine tumor settings and in human melanoma tissues that c‐Met⁺ CTLs are a naturally occurring CD8⁺ T‐cell population. Together, our findings suggest that the HGF/c‐Met pathway could be exploited to control CD8⁺ T‐cell‐mediated anti‐tumor immunity.     

Successful tumor eradication was achieved by collaboration of augmented cytotoxic activity and anti-angiogenic effects following therapeutic vaccines containing helper-activating analog-loaded dendritic cells and tumor antigen DNA

We reported previously that pigeon cytochrome c-derived peptides (Pan-IA), which bind broad ranges of MHC class II molecules efficiently, activate T helper (Th) function in mice. In an experimental model, Pan-IA DNA vaccines augmented antitumor immunity in tumor antigen-immunized mice. To elicit more potent antitumor immunity and to eradicate tumors in a therapeutic setting, Pan-IA-loaded dendritic cells (DCs) were inoculated in combination with vaccines including ovalbumin (OVA) antigen DNA in tumor-bearing mice. Seventy percent of the immunized mice survived tumor-free for at least 4 months after treatment. In contrast, mice vaccinated with OVA DNA, either with or without naïve DCs, did not eliminate the tumors and died within 5 weeks. Only in mice vaccinated with OVA DNA and Pan-IA-loaded DCs were both cytotoxic and helper responses specific for OVA induced at the spleen and tumor sites as well as at the vaccination sites. Furthermore, accumulation of OVA-specific CD4⁺ and CD8⁺ T lymphocytes and interferon-gamma-mediated anti-angiogenesis were observed in the tumors of these mice. Thus, the combined vaccination primed both tumor-specific cytotoxicity and helper immunity resulting in augmented tumor lysis ability and anti-angiogenic effects. This is the first report to show that most established tumors were successfully eradicated by collaboration of potent antitumor immunity and anti-angiogenic effects by vaccination with tumor antigens and helper-activating analogs. This novel vaccination strategy is broadly applicable, regardless of identifying helper epitopes in target molecules, and contributes to the development of therapeutic cancer vaccines.     

Gamma delta T cells from HIV+ donors can be expanded in vitro by zoledronate/interleukin-2 to become cytotoxic effectors for antibody-dependent cellular cytotoxicity

Background aimsImmunotherapy using γδ T cells capable of mediating antibody-dependent cellular cytotoxicity (ADCC) is a promising anti-human immunodeficiency virus (HIV) strategy. Approved aminobispohsphonate drugs, for example zoledronate (Zometa), stimulate γδ T cells in cancer patients, where they may promote direct tumor killing. Knowing that γδ T cells are modulated during HIV disease, documenting their responses and potential for controlling HIV is important. We investigated whether zoledronate/interleukin (IL)-2 could expand cytotoxic Vδ2 cells from HIV⁺ donors and whether these cells functioned in ADCC.MethodsPeripheral blood mononuclear cells from uninfected controls and HIV⁺ individuals receiving anti-retroviral therapy were treated with isopentenyl pyrophosphate (IPP) or zoledronate plus IL-2 to expand the Vδ2⁺ subset. Immunophenotyping and functional analyzes (cytotoxicity or cytokine expression) allowed us to compare cell properties from individual donors and to compare the responses to each stimulating agent.ResultsZoledronate stimulated a greater expansion of Vδ2 cells in HIV⁺ individuals compared with phosphoantigen IPP, and these cells expressed CD16. CD56 expression (a marker for cytotoxic cells) was lower on zoledronate-expanded cells, consistent with significantly lower cytotoxicity against the Daudi tumor cell line. Cells expanded with either zoledronate or IPP were active in ADCC, were similar in terms of interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression, and degranulated in response to Fc receptor cross-linking.ConclusionsZoledronate causes ex vivo expansion of Vδ2 cells from HIV⁺ individuals. Despite lower expression of CD56 and decreased direct cytotoxicity, these effectors were potent in ADCC. Zoledronate/IL-2- expanded cells have potential for immunotherapy to activate Vδ2 cells in HIV patients and enhance ADCC.     

Th17 and Th17-stimulated CD8<Superscript>+</Superscript> T cells play a distinct role in Th17-induced preventive and therapeutic antitumor immunity

CD4⁺ Th17 cells induce antitumor immunity leading to the eradication of established tumors. However, the mechanism of antitumour immunity and CTL activation by Th17 cells and the distinct role of Th17 and Th17-activated CTLs in antitumor immunity are still elusive. In this study, we generated ovalbumin (OVA)-specific Th17 cells by cultivating OVA-pulsed dendritic cells with CD4⁺ T cells derived from transgenic OTII mice in the presence of IL-6, IL-23, TGF-β, and anti-IFN-γ antibody. We demonstrated that Th17 cells acquired major histocompatibility complex/peptide (pMHC)-I and expressed RORγt, IL-17, and IL-2. Th17 cells did not have any direct in vitro tumor cell–killing activity. However, Th17 cells were able to stimulate CD8⁺ CTL responses via IL-2 and pMHC I, but not IL-17 signaling, which play a major role in Th17-induced preventive immunity against OVA-expressing B16 melanoma. Th17 cells stimulated the expression of CCL2 and CCL20 in lung tumor microenvironments promoting the recruitment of various inflammatory leukocytes (DCs, CD4⁺, and CD8⁺ T cells) stimulating more pronounced therapeutic immunity for early-stage (5-day lung metastases or 3 mm, s.c.) tumor than for well-established (6 mm, s.c.) tumor. The therapeutic effect of Th17 cells is associated with IL-17 and is mediated by Th17-stimulated CD8⁺ CTLs and other inflammatory leukocytes recruited into B16 melanoma via Th17-stimulated CCL20 chemoattraction. Taken together, our data elucidate a distinct role of Th17 and Th17-stimulated CD8⁺ CTLs in the induction of preventive and therapeutic antitumor immunity, which may greatly impact the development of Th17-based cancer immunotherapy.     

Induction of HLA-unrestricted and HLA-class-II-restricted cytotoxic T lymphocytes against MUC-1 from patients with colorectal carcinomas using recombinant MUC-1 vaccinia virus

We recently reported that immunization with a recombinant MUC-1 vaccinia virus (rVMUC-1) protected C57BL/6 mice from challenge with DF3/MUC-1-positive syngeneic tumors. To elucidate whether anti-MUC-1 tumor immunity, especially MUC-1-specific cytotoxic T lymphocytes (CTI), can be induced in cancer patients by rVMUC-1, we stimulated the peripheral blood lymphocytes from patients with DF3/MUC-1⁺ or DF3/MUC-1 colon carcinomas using the autologous monocytes infected with rVMUC-1 (rVAMN). The stimulated T lymphocytes from two patients with DF3/MUC-1-positive colorectal carcinomas (rVPY+T and rVPW+T) demonstrated HLA-unrestricted cytotoxicity against MUC-1, whereas those from the patient with DF3/MUC-1-negative colon carcinoma (rVPA-T) did not. The HLA-unrestricted cytotoxicity was demonstrated by the CD8⁺ T cells possibly recognizing an epitope present on the tandem repeats. Adoptive immunotherapy who performed three times with patient PY, at 4-week intervals. The adoptive transfer of the first stimulated lymphocytes, demonstrating a high level of HLA-unrestricted cytotoxicity against MUC-1, resulted in the significant reduction of the liver metastasis of patient PY. However, HLA-unrestricted cytotoxicity against MUC-1 was extremely reduced at the second transfer and finally eliminated at the third, whereas the CD4⁺ T cells demonstrating HLA-class-II-restricted cytotoxicity against MUC-1 predominantly proliferated at the third adoptive immunotherapy treatment. The liver metastasis and the serum levels of tumor markers (carcinoembryonic antigen CA19-9) demonstrated a rapid and marked increment after the second transfer and especially after the third. These results suggest that the HLA-unrestricted cytotoxic CD8⁺ T cells against MUC-1, induced in patients with DF3/MUC-1⁺ colorectal carcinomas using rVMUC-1, correlate with the antitumor activity in vivo. Received: 22 October 1997 / Accepted: 24 April 1998     

Adjuvant Activity of Mycobacterial Fractions

A quantitative assay and characterization of oil-attached cell wall of Mycobacterium bovis BCG (BCG-CWS) which stimulates cell-mediated immunity of spleen cells to alloantigens in mice were carried out by an in vitro cell-mediated cytotoxicity test using ⁵¹Cr-labeled target cells. C57BL/6J mice (H-2^b) were immunized intraperitoneally with mastocytoma cells (H-2^d) with or without oil-attached BCG-CWS. The cytotoxicity, comparable to that of spleen cells from mice immunized with mastocytoma cells (3 × 10⁷), could be induced in spleens of mice immunized with a mixture of mastocytoma cells (10⁴) and oil-attached BCG-CWS. The enhancing effect persisted for 55 days or more after the alloantigenic immunization. Oil-attached BCG-CWS enhanced cell-mediated cytotoxicity of T cells in the spleen and the mesenteric lymph node, but not in the thymus. The cytotoxicity showed specificity toward the alloantigen used for immunization. In addition to BCG-CWS, the cell walls of Nocardia rubra and Corynebacterium diphtheriae PW8 and the peptidoglycolipids of Mycobacterium tuberculosis Aoyama B were found to be potent stimulants of cell-mediated cytotoxicity in mice. Oil-attached BCG-CWS did not enhance humoral response to mastocytoma cells but enhanced cell-mediated cytotoxicity when viable mastocytoma cells were used as antigen. The above result was supported by the fact that anti-hapten antibody response induced by viable trinitrophenyl (TNP)-mastocytoma cells (10⁴) plus oil-attached BCG-CWS did not increase to the maximum level as was observed in mice immunized with a larger number of mastocytoma cells (3 × 10⁷) alone, while cell-mediated cytotoxicity induced by the same treatment increased to the maximum level obtained by immunization with mastocytoma cells (3 × 10⁷) alone.     

Participation of three lymphoid cell types in the in vitro activation of cell-mediated immunity to a syngeneic gross virus-induced lymphoma in rats.

W/Fu rats inoculated with the syngeneic Gross-virus induced lymphoma, (C58NT)D, had transient lymphocyte-mediated specific cytotoxicity against the tumor cells at 7–15 days after tumor injection. Spleen cells 40 days after immunization (spleen 40) were unreactive by a direct 4-hr ⁵¹Cr release assay, but activity appeared after in vitro culture of these spleen eclls by themselves for 18–24 hr. The nature of the cells involved in the activation of cytotoxicity and the characteristics of the effector cells themselves were studied. Significant differences were seen in the cell types involved in the activation phase and the effector phase. Activation appeared to require the cooperation of three cell types. Induction of activity was lost by treatment of cells with ATS plus complement, by passage over an EAC-column, or by treatment with carbonyl iron. Thus, T cells, CRL and macrophages were necessary for full activation of cytotoxicity in spleen 40. In contrast, after activation, only CRL seemed to be required for cytotoxicity, and treatment wih ATS or carbonyl iron had little effect. The effector cell detected after in vitro activation was quite distinct from that seen in the direct cytotoxicity assay with spleen cells at 10 days after tumor cell inoculation. The early, direct cytotoxic reactivity was dependent on T cells, being eliminated by treatment with ATS and complement but not by EAC columns or carbonyl iron. It appears therefore that the in vitro activation is a separate mechanism for cytotoxicity against tumor cells, rather than a simple recovery of T cells from in vivo inhibition.     

Oncolytic Adenovirus Expressing IL-23 and p35 Elicits IFN-γ- and TNF-α-Co-Producing T Cell-Mediated Antitumor Immunity

Cytokine immunogene therapy is a promising strategy for cancer treatment. Interleukin (IL)-12 boosts potent antitumor immunity by inducing T helper 1 cell differentiation and stimulating cytotoxic T lymphocyte and natural killer cell cytotoxicity. IL-23 has been proposed to have similar but not overlapping functions with IL-12 in inducing Th1 cell differentiation and antitumor immunity. However, the therapeutic effects of intratumoral co-expression of IL-12 and IL-23 in a cancer model have yet to be investigated. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral inoculation of oncolytic adenovirus co-expressing IL-23 and p35, RdB/IL23/p35. Intratumoral administration of RdB/IL23/p35 elicited strong antitumor effects and increased survival in a murine B16-F10 syngeneic tumor model. The levels of IL-12, IL-23, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were elevated in RdB/IL23/p35-treated tumors. Moreover, the proportion of regulatory T cells was markedly decreased in mice treated with RdB/IL23/p35. Consistent with these data, mice injected with RdB/IL23/p35 showed massive infiltration of CD4⁺ and CD8⁺ T cells into the tumor as well as enhanced induction of tumor-specific immunity. Importantly, therapeutic mechanism of antitumor immunity mediated by RdB/IL23/p35 is associated with the generation and recruitment of IFN-γ- and TNF-α-co-producing T cells in tumor microenvironment. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-23 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.     

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.     

Melphalan-induced enhancement of tumor cell immunostimulatory capacity as a mechanism for the appearance of potent antitumor immunity in the spleen of mice bearing a large metastatic MOPC-315 tumor

Summary Exposure of MOPC-315 cells from the primary tumor nodule to a low concentration (0.5 nmol/ml) of melphalan (L-phenylalanine mustard; L-PAM) rendered the tumor cells capable of bringing about the generation of a potent primary antitumor cytotoxic response. Accordingly, the level of antitumor cytotoxicity generated by normal spleen cells immunized in vitro with L-PAM-treated tumor cells was at least five-fold greater than the level generated in response to untreated tumor cells. The marked superiority of L-PAM-treated tumor cells over untreated tumor cells in bringing about the generation of antitumor cytotoxicity was evident over a wide range of responder to stimulator cell ratios. The higher level of antitumor cytotoxicity exhibited by normal spleen cells immunized with L-PAM-treated tumor cells as compared with untreated tumor cells was not merely the result of direct drug-mediated tumoricidal activity, thereby reducing the number of tumor cells present which can act as cold target cell inhibitors during the ⁵¹Cr release assay. This is apparent from the observation that the level of antitumor cytotoxicity generated in response to a given percentage of stimulator tumor cells pretreated with 0.5 nmol L-PAM/ml, a drug concentration associated with retention of 60% tumor cell proliferative capacity, is substantially greater than that generated in response to less than half that percentage of untreated stimulator tumor cells. Moreover, stimulator tumor cells exposed to a fully antiproliferative concentration of L-PAM brought about the generation of a higher level of antitumor cytotoxicity than stimulator tumor cells exposed to mitomycin C at a concentration which inhibited the proliferation of the tumor cells to the same extent as the L-PAM. A low concentration of L-PAM which was effective in rendering isolated tumor cells from the primary tumor nodule capable of bringing about the generation of antitumor cytotoxicity was also effective in inducing the appearance of potent antitumor immune potential in tumor bearer splenic cells containing metastatic tumor cells. Thus, metastatic tumor cells within the spleen of tumor-bearing mice may have the potential to act as in situ stimulators for the generation of potent antitumor immunity which brings about the eradication of a large, metastatic tumorigenic load remaining after clearance of the drug from the circulation.Supported by United States Public Health Service Research Grants CA-30088 and CA-35761 from the National Cancer InstituteIn partial fulfillment of the requirements of the Graduate College for the Doctor of Philosophy degree for RCBSBE was visiting Fulbright Professor from the Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel, and was supported by the Council for International Exchange of Scholars     

Tumor Immunity against a Simian Virus 40 Oncoprotein Requires CD8+ T Lymphocytes in the Effector Immune Phase

The required activities of CD4⁺ T cells and antibody against the virally encoded oncoprotein simian virus 40 (SV40) Tag have previously been demonstrated by our laboratory to be mediators in achieving antitumor responses and tumor protection through antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we further characterize the necessary immune cell components that lead to systemic tumor immunity within an experimental pulmonary metastatic model as the result of SV40 Tag immunization and antibody production. Immunized animals depleted of CD8⁺ T cells at the onset of experimental tumor cell challenge developed lung tumor foci and had an overall decreased survival due to lung tumor burden, suggesting a role for CD8⁺ T cells in the effector phase of the immune response. Lymphocytes and splenocytes harvested from SV40 Tag-immunized mice experimentally inoculated with tumor cells synthesized increased in vitro levels of the Th1 cytokine gamma interferon (IFN-γ), as assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays. CD8⁺ T-cell activity was also heightened in SV40 Tag-immunized and tumor cell-challenged mice, based upon intracellular production of perforin, confirming the cytolytic properties of CD8⁺ T cells against tumor cell challenge. Altogether, these data point to the role of recombinant SV40 Tag protein immunization in initiating a cytotoxic T-lymphocyte (CTL) response during tumor cell dissemination and growth. The downstream activity of CD8⁺ T cells within this model is likely initiated from SV40 Tag-specific antibody mediating ADCC tumor cell destruction.Determining the immunologic mechanisms involved in antitumor responses can provide valuable insight into developing and formulating appropriate immunotherapeutic strategies against a range of human cancers (25). Cell-mediated immunity involving CD8⁺ T lymphocytes is generally regarded as the primary response to utilize due to its potent and efficient cytotoxicity against tumor cell targets in vitro and in animal models (10). Indeed, the proof of concept of this approach is best characterized by specialized conditioning protocols that involve autologous transfer of tumor infiltrating lymphocytes (TILs) in metastatic melanoma patients, with objective responses that approximate 70% (8). However, the efficacy of TILs harvested from additional cancer types have been less than effective, and additional strategies, such as genetic modification of peripheral blood mononuclear cells, are being explored to improve and extend the approach of cytotoxic T-lymphocyte (CTL) immunotherapy clinically (33, 46).The roles of immune components such as CD4⁺ T cells and antibody have been given less attention within the context of promoting tumor immunity against a range of tumor antigens. For example, the ability of CD4⁺ T cells to activate humoral immunity can lead to antitumor responses that involve antibody-dependent cell-mediated cytotoxicity (ADCC) (17). In this scenario, antibody binds its targeted antigen and effectors such as natural killer (NK) cells lyse tumorigenic cells through interaction with the Fc region of the bound antibody. The efficacy of ADCC has been realized in scenarios involving breast cancer and non-Hodgkin''s lymphoma, for example, and to date, the only FDA-approved immunologic treatments against these malignancies involve antibody-based therapies (5).The concurrent roles of antibody—with specific emphasis on ADCC—and CD8⁺ T-cell immunity within the context of tumor immunity have not been widely reported. Several recent studies have commented on the ability of antibody-bound tumor cells, particularly as a whole tumor cell-dendritic cell (DC) vaccination approach, to initiate CTL activity by engaging DCs through Fc receptors (9, 19, 34). However, to our knowledge, the mechanistic aspects of ADCC (e.g., NK-mediated lysis) promoting CD8⁺ T-cell activity have been explored in relatively few studies (27, 41). From an immunotherapeutic standpoint, it may be preferable in certain settings to induce both the humoral and cell-mediated arms of the immune system to offset the progression of tumor cell growth and dissemination. Namely, these strategies could include active or passive approaches to first effectively induce ADCC in response to a tumor antigen, which would promote CTL activity against additional tumor targets through cross-presentation.Our laboratory has been involved in determining the immunologic mechanisms of tumor immunity induced by the virally encoded tumor-specific antigen simian virus 40 (SV40) large tumor antigen (Tag). The mechanistic aspects of SV40 Tag-induced tumor immunity have been examined within an experimental murine model of pulmonary metastasis. To date, CD4⁺ T cells and SV40 Tag-specific antibody have been implicated as required immune components within this murine system in order to achieve complete systemic tumor immunity (18). These studies demonstrated that during the course of immunization with SV40 Tag (i.e., the induction-phase response), CD4⁺ T cells were required to induce an SV40 Tag humoral response. The specific role of the antibody response against an experimental tumor cell challenge was observed to involve ADCC-mediated clearance pathways (4, 23).In the present study, we further characterize the immunologic response to SV40 Tag immunization by observing the necessary immune cell components following experimental challenge (i.e., the effector-phase response) with a tumor cell line expressing SV40 Tag. With the development of an SV40 Tag antibody response following SV40 Tag immunization in vivo, CD8⁺ T-cell depletion during the effector phase resulted in the formation of lung tumor foci and decreased survival not observed with the abrogation of CD4⁺ T cells. SV40 Tag-immunized mice also displayed a heightened Th1 response and CD8⁺ CTL activity after experimental tumor cell challenge, as assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays. In all, these data indicate that CD8⁺ T cells mediate tumor immunity following antibody activation in response to the tumor-specific antigen SV40 Tag. We hypothesize that CD8⁺ T-cell activity is initiated due to cross-presentation mechanisms as a result of ADCC activity against SV40 Tag. We are not aware of another published report that formulates a role for ADCC activity against a viral oncoprotein in this manner in order to engage CD8⁺ T-cell activation.SV40 Tag has been reported to be expressed in a number of human cancers, including malignant pleural mesothelioma and non-Hodgkin''s lymphoma, although a causal link between SV40 infection and tumorigenesis is uncertain (11, 24, 35). The results of this study have direct implications for the construction of an appropriate immunotherapeutic strategy for patients suffering malignancies expressing the SV40 Tag tumor-specific antigen.     

Characterization of in vitro reactivity by BCG-treated guinea pigs to syngeneic line-10 hepatocarcinoma

Summary The purpose of this study was to characterize in vitro the systemic tumor immunity induced by a BCG-intratumoral injection in line-10 hepatocarcinoma established in the skin of inbred guinea pigs (strain 2). Macrophages from BCG-tumor-cured guinea pigs at effector to target cell ratios of 10:1 and 100:1 were cytotoxic in vitro to line-10 tumor cells, and this cytotoxicity was potentiated by autologous serum. Significant cytotoxicity of lymphocytes from BCG-tumor-cured guinea pigs could only be achieved at ratios of 10,000:1, and no effect of autologous serum could be demonstrated. Lymphocytes from both normal and BCG-tumor-cured (line-10 immune) guinea pigs had a significant cytotoxic effect on the highly antigenic line-1 cells at ratios of 1:10,000. Macrophages from both normal and line-10 immune guinea pigs were cytotoxic to line-1 target cells at ratios of 1:100. With respect to specific cytotoxicity (cytotoxicity above and beyond levels achieved with effector cells from normal animals), the only significant difference was demonstrated when line-10 served as target cells and the effector cells were isolated from BCG-tumor-cured (line-10 immune) guinea pigs. Abbreviations used in this paper: BCG, Bacillus Calmette-Guérin; CMEM, complete minimum essential medium; cpm, counts per minute; HBSS, Hanks' balanced salt solution; i.d. intradermally; i.p., intraperitoneally; PEC, peritoneal exudate cells; SDA, superficial distal axillary; ¹²⁵IdUrd, [¹²⁵I]iododeoxyuridine.     

Effects of colony-stimulating factors on cellular cytotoxicity

 Colony-stimulating factors (CSF) are used clinically in the treatment of chemotherapy-induced myelosuppression and in support of bone marrow transplantation. As CSF are known to have pleiotropic functions, their effects on cellular cytotoxicity were analysed in vitro against bladder carcinoma cell lines. By means of an L-[³H]methionine-release assay, the cytotoxicity of peripheral blood mononuclear cells against the natural-killer(NK)-cell-resistant bladder carcinoma cell lines BT-A and SBC-7 was measured using different effector/target-cell ratios. Costimulatory effects of granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage-colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and stem cell factor (SCF) on the generation of lymphokine-activated killer (LAK), bacillus Calmette-Guérin-activated killer (BAK) and natural killer (NK) cell cytotoxicity were investigated in this assay. Furthermore, the effect of CSF on proliferation of urothelial tumor cells in vitro was determined by a [³H]thymidine DNA-labelling technique. GM-CSF, but not G-CSF, IL-3 or SCF, was able to increase NK, BAK and LAK cytotoxicity in a dose-dependent manner. No acceleration of carcinoma cell proliferation was evident under the conditions of our assay. These data indicate the costimulatory effect of GM-CSF on cellular cytotoxicity, which might be used for immunotherapeutic purposes. Received: 30 July 1996 / Accepted: 20 December 1996     

Active specific immunotherapy of residual micrometastasis

Summary A vaccine of Bacillus Calmette-Guérin (BCG) admixed with tumor cells induced systemic immunity and had a therapeutic effect on subclinical, disseminated micrometastasis. Inbred strain-2 guinea-pigs given IV injections of 5×10³ to 10⁶ syngeneic L10 hepatocarcinoma cells were vaccinated after metastatic foci were established in the lung parenchyma. The purpose of this study was to establish the variables that can be manipulated to assure optimal immunotherapy while minimizing deleterious side effects of the BCG. In the present study we examined the variables of source, dose, and ratio of BCG to tumor cells. Four BCG sources (lyophilized Tice and Connaught; fresh-frozen Phipps and Tice) were compared. No significant differences among these BCG preparations could be detected with respect to adjuvant potential when they were admixed with attenuated tumor cells in a vaccine. The dose study clearly demonstrated that a BCG dose dependency exists with relation to induction of effective cell-mediated immunity or survival from disseminated micrometastatic disease. Furthermore, evaluations of dose versus ratio of BCG to tumor cells also supported a BCG dose dependency, with the lowest effective BCG dose being directly influenced by tumor burden of the host. Cutaneous reactivity and hypersensitivity of the primary and secondary immunization sites of tumor-bearing animals treated with effective and ineffective vaccines supported the direct association of reaction to BCG and specific tumor immunity. However, when an in vitro leukocyte migration inhibition assay was used, the degree of reactivity to BCG could not be exploited as a quantitative, diagnostic monitor of effective systemic tumor immunity.     

Signalling pathway of tumor necrosis factor in normal and tumor cells

Summary Several aspects of the activity and effects of tumor necrosis factor (TNF) were investigated to gain further insight into its cytotoxic mechanism. The relation between number of TNF receptors and TNF susceptibility of both tumor cells and normal cells was studied, utilizing a specific binding assay. Among the tumor cells, a fairly close correlation (r=0.855) was observed between receptor number and sensitivity to TNF. No cytotoxic effect by TNF was observed on any of the normal cells tested, even though TNF receptors were shown to be present, and cell proliferation was apparently stimulated by TNF in some cases. TNF internalization and intracellular distribution were studied by pulse-labelling and Percoll density gradient centrifugation. In L-M (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) cells and HEL (human embryonic lung cells, non-sensitive to TNF cytotoxicity) cells, receptor-bound ¹²⁵I-labelled recombinant human TNF was rapidly internalized and delivered to lysosomes within 15–30 min, and this was followed by degradation and release into the culture medium. The presence of either a cytoskeletal disrupting agent or a lysosomotropic agent was observed to inhibit the cytotoxic effect of TNF, thus also indicating that TNF internalization, followed by delivery to lysosomes, is essential in the cytolytic mechanism of TNF.As observed by [³H]uridine incorporation, TNF did not affect RNA synthesis in L-R cells (TNF-resistant cell lines derived from L-M cells) and HEL cells, but markedly stimulated (by 3.5 times) RNA synthesis in L-M cells.