Interleukin-4 impairs granzyme-mediated cytotoxicity of Simian virus 40 large tumor antigen-specific CTL in BALB/c mice

In this report we analyzed the impact of interleukin-4 (IL-4) on tumor-associated simian virus 40 (SV40) large T-antigen (TAg)-specific CD8⁺ cytotoxic T cells during rejection of syngeneic SV40 transformed mKSA tumor cells in BALB/c mice. Strikingly, challenge of naïve mice with low doses of mKSA tumor cells revealed a CD8⁺ T cell-dependent prolonged survival time of naïve IL-4^?/? mice. In mice immunized with SV40 TAg we observed in IL-4^?/? mice, or in wild type mice treated with neutralizing anti-IL-4 monoclonal antibody, a strongly enhanced TAg-specific cytotoxicity of tumor associated CD8⁺ T cells. The enhanced cytotoxicity in IL-4^?/? mice was accompanied by a significant increase in the fraction of CD8⁺ tumor associated T-cells expressing the cytotoxic effector molecules granzyme A and B and in granzyme B-specific enzymatic activity. The data suggest that endogenous IL-4 can suppress the generation of CD8⁺ CTL expressing cytotoxic effector molecules especially when the antigen induces only a very weak CTL response.     

Delayed-type hypersensitivity responses to H-Y: Characterization and mapping ofIr genes

This paper examines the delayed-type hypersensitivity (DTH) response to male (H-Y) antigen(s). Female mice of theH?2 ^b haplotype developed delayed footpad reaction to syngeneic or allogenic male thymus and spleen cells after priming with syngeneic male thymus and spleen cells. The reaction peaks at 24 h, has classical DTH histology and is specific to H-Y antigen as it is not elicited with female cells. Cell transfer studies show that donor/recipient matching at theI?B ^b subregion is necessary for sucessful transfer of DTH and that the effective primed population is Thy-1⁺, Lyt-1⁺, 2^?. DTH response to H-Y antigen appears to be confined to mice of theH?2 ^b haplotype. There appears to be a lack of associative recognition between H-Y antigen and MHC-coded determinants in the effector phase of DTH, and macrophage processing of H-Y seems likely, since nonresponder haplotypes can elicit the DTH response. Studies withH?2 ^b recombinant mouse strains indicate that the dominantIr gene is located in theI?B region. Female F₁ hybrid mice derived from matings of strains not involvingH?2 ^b haplotype failed to develop DTH to H-Y. In summary, these data imply that a complete correlation exists between DTH to H-Y and the ability to reject male skin graft, suggesting that the effector mechanisms of skin-graft rejection may closely involve DTH cells.     

Suppressor cell activity in mice bearing a progressively growing Simian virus 40-induced sarcoma

Summary General and cell-mediated immunity (CMI) were investigated in BALB/c mice bearing progressively growing Simian virus 40-induced (mKSA) sarcoma by means of the Winn tumor cell neutralization (WN), ¹²⁵I isotopic footpad (IFP), lymphoproliferative (LP) and plaque-forming cell (PFC) assays. Correlation between depressed antitumor immunity and the IFP responses was observed in tumor-bearing (TB) mice. Depressed LP responses to both T- and B-cell mitogens were observed in both early and late stages of tumor growth. Results obtained with the PFC assay similarly demonstrated depressed humoral immunity to sheep red blood cells (SRBC). Suppressor cell activity was demonstrated in cocultivation experiments in which spleen cells of TB mice were mixed with normal spleen cells. Treatment of TB spleen cells by passage through Sephadex G-10 columns or incubation on plastic surfaces to deplete the adherent cells restored LP responses. Cocultivation of Sephadex G-10- or plastic-adherent cells from TB mice with normal spleen cells significantly reduced mitogen-induced LP responses of normal cells. Examination of cell surface markers indicated an increase in the proportion of spleen cells bearing Fc receptors, which correlated with progressive mKSA tumor growth. There was also a correlation between Fc receptor-bearing spleen cells and macrophages, as shown by nonspecific esterase staining. These results indicate that depressed LP and PFC responses and the appearance of suppressor cells in mKSA tumor-bearing mice parallel an impaired ability to recognize (IFP responses) and neutralize (WN responses) tumor cells.     

Mechanisms of genetic control of immune responses

The mechanisms underlying Ir gene control of CMI were addressed by examining the DTH and Tprlf responses specific for the synthetic polymers GT, GAT, and GA. We show that BALB/c mice (GAT/GA responders, GT nonresponders) primed with GT fail to develop DTH and Tprlf responses specific for GT, GAT, or GA. GAT immunization resulted in DTH responses that could be elicited not only with GAT and GA but also with GT, demonstrating that GT-specific T_DH are present in nonresponder mice. GT-specific DTH was transferred with Thy-1⁺ Lyt-1⁺2^–, H-2 Irestricted, nylon wool nonadherent cells. GA-primed BALB/c mice developed GAT- and GA-, but not GT-apecific DTH responses, indicating that GA and GT do not cross-react at the T-cell level. The ability of GAT [but not a mixture of GA plus GT, or GT electrostatically complexed to the immunogenic carrier MBSA (GT-MBSA)] to induce GT-specific DTH suggested a requirement for covalent linkage of stimulatory GA and nonstimulatory GT determinants present on the GAT molecule. Similarly, GT-specific in vitro Tprlf responses could be demonstrated in GAT-primed mice exhibiting significant levels of GT-specific DTH but not in GT- or GT-MBSA-primed mice. Tolerization experiments also suggested that GT-specific Th were involved in the development of GT-specific DTH in GAT-primed mice. The GT nonresponsiveness of BALB/c mice for DTH and Tprlf responses could not be reversed by treatments designed to abrogate Ts activity (priming with GT-MBSA and CY injection), nor could GT-primed cells be shown to inhibit the development or elicitation of GT-specific CMI in GAT-primed mice during the afferent and/or efferent stages of DTH. Our results suggest that GT nonresponsiveness does not result from the absence of GT-specific T cells or preferential induction of Ts. The results are discussed in the context of hole-in-the-repertoire and antigen presentation (determinant selection) models of Ir gene control.Abbreviations used in this paper APC antigen-presenting cells - BSA bovine serum albumin - BSS Mishell-Dutton balanced salt solution - CFA complete Freund's adjuvant - CMI cell-mediated immunity - CY cyclophosphamide - DTH delayed-type hypersensitivity - GA poly(Glu⁶⁰Ala⁴⁰) - GAT poly (Glu⁶⁰Ala³⁰Tyr¹⁰) - GT poly(Glu⁵⁰Tyr⁵⁰) - GT-MBSA GT complexed to methylated bovine serum albumin - It immune response - LN lymph node - PPD purified protein derivative of tuberculin - TDH DTH T cells - Th helper T cells - Tprlf T-cell proliferation - Ts suppressor T cells - TsF T-cell suppressor factor(s)     

Feasibility of UV-inactivated vaccinia virus in the modification of tumor cells for augmentation of their immunogenicity

Summary We compared the immunity induced by tumor cells modified with UV-inactivated purified vaccinia virus (UV-VV) and with live purified vaccinia virus (L-VV). C3H/HeN mice were inoculated i.p. with UV-VV or L-VV after whole-body irradiation with 150 rads of X-rays (priming). After 3 weeks the mice were immunized i.p. 3 times at weekly intervals with syngeneic X5563 or MH134 cells that had been adsorbed in vitro with UV-VV or infected with L-VV and subsequently irradiated with 10⁴ rads of X-rays. Then 1 week after the last immunization, the mice were challenged s.c. with X5563 viable tumor cells or challenged i.p. with MH134 viable tumor cells. The 50% lethal dose (TLD₅₀) of X5563 in mice primed and immunized with UV-VV (UV-VV group) on s.c. challenge (10^6.06) was the same as for mice treated with L-VV (L-VV group), whereas the TLD₅₀ of unprimed or nonimmunized mice (control group) was 10^2.61. The TLD₅₀ of MH134 in the UV-VV treated group on i.p. challenge (10^6.48) was similar to that of the L-VV treated group (10^6.54), while the TLD₅₀ of the control group was 10^1.00. The difference between the TLD₅₀ values of X5563 on s.c. challenge of mice primed and immunized with UV-VV or L-VV and control mice was 10^3.4. The difference between the TLD₅₀ values of MH134 on i.p. challenge of primed and immunized mice and control mice was 10^5.5. These results indicate that the in vivo helper function of UV-VV is similar to that of L-VV and that the augmenting effect of this protocol depends on the kind of tumor.     

Recombinant human tumor necrosis factor mediates regression of a murine sarcoma in vivo via Lyt-2+ cells

Summary The role of an immune response in recombinant-human-tumor-necrosis-factor(rHTNF)-mediated regression of a weakly immunogenic, MCA-106 sarcoma in vivo was examined. C57BL/6 mice bearing established 10-day s.c. tumor were treated with single i.v. doses (8 g) of rHTNF. rHTNF administration resulted in marked hemorrhagic necrosis and subsequent regression of tumor in treated mice. Mice cured of MCA-106 sarcoma by rHTNF specifically rejected a subsequent challenge (5×10⁵ cells) of the same tumor (P<0.01) but not of the antigenically distinct, syngeneic MCA-105 sarcoma. Tumor bearers were depleted in vivo of selective T-cell subsets by the systemic administration of specific monoclonal antibodies before rHTNF therapy. rHTNF-induced regression, but not hemorrhagic necrosis of the MCA-106 sarcoma was blocked in mice depleted of Lyt-2⁺ cells, but not of L3T4⁺ cells. The in vivo role of T-cell subsets in rHTNF-mediated tumor regression is discussed.Howard Hughes Medical Institute Research Scholar     

Growth inhibition of murine mammary carcinoma by monoclonal IgE antibodies specific for the mammary tumor virus

Summary Two IgE-producing hybridomas were established from spleen cells of Balb/c mice, which had been immunized with mouse mammary tumor virus (MMTV). These IgE monoclonal antibodies (mAbs) reacted specifically with the major envelope glycoprotein (gp36) of MMTV, as established by the immunoblot assay and by passive cutaneous anaphylaxis. The effect of the IgE mAbs (produced by clone A8) on the growth of the MMTV-secreting mammary adenocarcinoma H2712 was investigated in syngeneic C3H/HeJ mice. The mice were inoculated s.c. with either 10⁵ (100 × LD₅₀) or 10⁶ (1000 × LD₅₀) tumor cells and received repeated i.p. injections of 25 µg anti-gp36 IgE mAbs at 4-day intervals for 8 weeks. This treatment prevented the development of subcutaneous tumors in 50% of the animals. Similar protection was observed when the tumor cells (10⁵/animal) were injected i.p. 4 days prior to the beginning of the i.p. treatment consisting of injections of 25 µg mAbs at 4-day intervals for 6 weeks. However, these mAbs did not protect C3H/HeJ mice against the MMTV-negative MA16/c carcinoma cells. Hence, these results support the view that IgE-mediated cytotoxic mechanisms may play an immunologically specific antitumor surveillance role and that laboratory-induced antitumor IgE mAbs have the potential of specific therapeutic agents for in vivo destruction of tumor cells.     

Treating tumor-bearing mice with vitamin D3 diminishes tumor-induced myelopoiesis and associated immunosuppression,and reduces tumor metastasis and recurrence

Metastatic Lewis lung carcinoma (LLC-LN7) tumors that secrete granulocyte/macrophage-colonystimulating factor (GM-CSF) stimulate myelopoiesis and induce bone marrow-derived immunosuppressor cells that are homologous to granulocyte/macrophage progenitor cells. In vitro treatment of the LLC-LN7 cells with 1,25-dihydroxyvitamin D₃ reduced tumor cell production of suppressor-inducing activity, although suppressor-inducing activity could be restored by reconstituting the tumor supernatants with recombinant GM-CSF. Treatment of mice having LLC-LN7 tumors with vitamin D₃ reduced tumor production of GM-CSF and the frequency of myeloid progenitor cells. This was associated with a reduction in immunosuppressor activity and an increase in T cell function. Vitamin D₃ treatment of mice having palpable tumors transiently retarded tumor growth, but caused a prominent reduction in tumor metastasis. Treating mice with vitamin D₃ after tumor excision resulted in a reduction in the tumor-induced myelopoietic stimulation and associated immunosuppressive activity, and enhanced T cell function. These mice had a markedly reduced incidence of tumor recurrence. The results of this study suggest that vitamin D₃ treatment of mice with GM-CSF-secreting tumors can interrupt the myelopoiesis-associated immunosuppressor cascade and, in turn, reduce tumor metastasis and recurrence.This study was supported in part by grants from the Medical Research Service of the Department of Veterans Affairs and by grants CA-45080 and CA-48080 from the National Institutes of Health     

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.     

Vaccination with metastasis-related tumor associated antigen TPD52 and CpG/ODN induces protective tumor immunity

Tumor protein D52 (TPD52) is involved in transformation and metastasis and has been shown to be over-expressed in tumor cells compared to normal cells and tissues. Murine TPD52 (mD52) shares 86% protein identity with the human TPD52 orthologue (hD52). To study TPD52 protein as a target for active vaccination recombinant, mD52 was administered as a protein-based vaccine. Naïve mice were immunized with either mD52 protein and CpG/ODN as a molecular adjuvant or CpG/ODN alone. Two weeks following the final immunization, mice were challenged s.c. with syngeneic tumor cells that over-express mD52. Two distinct murine tumor cell lines were used for challenge in this model, mKSA and 3T3.mD52. Half of the mice immunized with mD52 and CpG/ODN rejected or delayed onset of mKSA s.c. tumor cell growth, and 40% of mice challenged with 3T3.mD52 rejected s.c. tumor growth, as well as the formation of spontaneous lethal lung metastases. Mice immunized with mD52 and CpG/ODN generated detectable mD52-specific IgG antibody responses indicating that mD52 protein vaccination induced an adaptive immune response. In addition, mice that rejected tumor challenge generated tumor-specific cytotoxic T lymphocytes’ responses. Importantly, microscopic and gross evaluation of organs from mD52 immunized mice revealed no evidence of autoimmunity as assessed by absence of T cell infiltration and absence of microscopic pathology. Together, these data demonstrate that mD52 vaccination induces an immune response that is capable of rejecting tumors that over-express mD52 without the induction of harmful autoimmunity.     

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.     

Secondary delayed-type hypersensitivity to sheep red blood cells in mice: a long-lived memory phenomenon

Immunization of mice with sheep red blood cells (SRBC) can induce the capacity to react with a secondary delayed-type hypersensitivity (DTH) immune response upon a booster injection of the antigen. In this paper the kinetics of secondary DTH after intravenous (iv) immunization with various doses of SRBC was studied by means of the foot swelling test. Dose-response studies showed that maximal secondary DTH responsiveness was obtained by iv administration of a priming dose of 3 × 10⁴ SRBC and a booster dose of 3 × 10⁵ SRBC 2 months later. Secondary DTH in such treated mice was characterized by an earlier appearance of the state of DTH, an earlier peak reactivity, and an increased intensity of the DTH response as compared to the primary DTH response. Up to 1 year after priming, a secondary DTH could be elicited, indicating the long-lived character of this memory phenomenon. With increasing intervals between the priming and booster injection, a gradual shift to a later time, of the peak secondary DTH reactivity was found. The capacity of primed mice to react with an increased intensity upon a booster injection could be adoptively transferred into lethally irradiated recipients by means of spleen cells obtained from primed mice. This phenomenon appeared to be highly dependent on Thy 1.2⁺ cells and on the booster dose of SRBC. The DTH reaction, evoked in such recipients, showed a prolonged time course.     

Immunization of syngeneic mice against malignant melanoma with subcellular membrane fractions of a bromodeoxyuridine-grown variant

Summary Syngeneic C57BL/6 mice immunized with non-tumorigenic B16 melanoma cells and crude membrane fractions are able to reject challenge with the tumorigenic B₅59 parent clone. The immunogenic C₃471 variant was derived from the malignant B₅59 clone by continuous growth in the presence of 1 g 5-bromodeoxyuridine (BrdUrd)/ml. Fifty-one percent (35 of 69) of mice immunized by three inoculations of 10⁶ cell equivalents of C₃471 crude membranes (CM) isolated by nitrogen cavitation remained tumor-free for at least 50 days after challenge with a tumorigenic dose of B₅59 cells. This compares favorably with the virtually 100% protection evoked by 10⁶ viable C₃471 cells. For those CM-immunized mice failing to reject B₅59 challenge, the mean latent period for tumor formation was significantly increased (P0.001) over controls. In addition, mice immunized with cultured C₃471 cells were able to reject, with equal efficiency, challenge with either cultured or tumor-derived B₅59 cells, indicating that the immunogen(s) present on C₃471 cells and CMs was (were) not a tissue culture artifact. Freshly prepared syngeneic fascia cells and membranes as well as CM prepared from cultured malignant B₅59 cells had no tumor rejection activity. In vivo tumor rejection activity in plasma membrane vesicles prepared from C₃471 cells by formaldehyde treatment also demonstrated tumor rejection activity. The host response to CMs, as to C₃471 cells, could be transferred by lymphoid cells from mice immunized with C₃471 CMs or cells. Co-injection of leucocytes from CM-immunized mice together with a tumorigenic dose of B₅59 cells into immunocompetent syngeneic mice resulted in abrogation of B₅59 tumorigenicity. The tumor rejection antigen(s) induced or increased by growth in BrdUrd has not yet been characterized biochemically, but is likely to involve a cell surface component common to both cell types and is retained by crude membrane fractions. The use of subcellular fractions from a spontaneous melanoma grown with BrdUrd, which elicits immunity against the malignant tumor, represents a model with immunoprophylactic potential for human neoplasia. Abbreviations used in this paper are: B16, melanoma of C57BL/6 mouse of spontaneous origin; B₅59, tumorigenic B16 clonal derivative; BrdUrd, 5-bromodeoxyuridine; C₃471, nontumorigenic BrdUrd-grown B16 clonal derivative; ceq, cell equivalents; CM, crude membrane; FBS, fetal bovine serum; MEM, minimal essential medium; MEMF, MEM containing 25 mM formaldehyde; MLP, mean latent period for tumor formation; MTV, mean tumor volume; pc, post challenge; PC, peritoneal cells; PMV, plasma membrane vesicles; TRA, tumor rejection antigen     

Simian virus 40 large-T-antigen-specific rejection of mKSA tumor cells in BALB/c mice is critically dependent on both strictly tumor-associated, tumor-specific CD8(+) cytotoxic T lymphocytes and CD4(+) T helper cells

Protective immunity of BALB/c mice immunized with simian virus 40 (SV40) large T antigen (TAg) against SV40-transformed, TAg-expressing mKSA tumor cells is critically dependent on both CD8(+) and CD4(+) T lymphocytes. By depleting mice of T-cell subsets at different times before and after tumor challenge, we found that at all times, CD4(+) and CD8(+) cells both were equally important in establishing and maintaining a protective immune response. CD4(+) cells do not contribute to tumor eradication by directly lysing mKSA cells. However, CD4(+) lymphocytes provide help to CD8(+) cells to proliferate and to mature into fully active cytotoxic T lymphocytes (CTL). Depletion of CD4(+) cells by a single injection of CD4-specific monoclonal antibody at any time from directly before injection of the vaccinating antigen to up to 7 days after tumor challenge inhibited the generation of cytolytic CD8(+) lymphocytes. T helper cells in this system secrete the typical Th-1 cytokines interleukin 2 (IL-2) and gamma interferon. Because in this system TAg-specific CD8(+) cells secrete only minute amounts of IL-2, it appears that T helper cells provide these cytokines for CD8(+) T cells. Moreover, this helper effect of CD4(+) T cells in mKSA tumor rejection in BALB/c mice does not simply improve the activity of TAg-specific CD8(+) CTL but actually enables them to mature into cytolytic effector cells. Beyond this activity, the presence of T helper cells is necessary even in the late phase of tumor cell rejection in order to maintain protective immunity. However, despite the support of CD4(+) T helper cells, the tumor-specific CTL response is so weak that only at the site of tumor cell inoculation and not in the spleen or in the regional lymph nodes can TAg-specific CTL be detected.     

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.     

Role of the Innate Immune Response and Tumor Immunity Associated with Simian Virus 40 Large Tumor Antigen

We examined properties of the innate immune response against the tumor-specific antigen simian virus 40 (SV40) large tumor antigen (Tag) following experimental pulmonary metastasis in naive mice. Approximately 14 days after mKSA tumor cell challenge, expression of inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and RANTES was upregulated in splenocytes harvested from mice, as assessed by flow cytometry and antibody array assays. This response was hypothesized to activate and induce tumor-directed NK cell lysis since IL-2-stimulated NK cells mediated tumor cell destruction in vitro. The necessary function of NK cells was further validated in vivo through selected antibody depletion of NK cells, which resulted in an overwhelming lung tumor burden relative to that in animals receiving a control rabbit IgG depletion regimen. Interestingly, mice achieved increased protection from experimental pulmonary metastasis when NK cells were further activated indirectly through in vivo administration of poly(I:C), a Toll-like receptor 3 (TLR3) agonist. In a separate study, mice receiving treatments of poly(I:C) and recombinant SV40 Tag protein immunization mounted effective tumor immunity in an established experimental pulmonary metastasis setting. Initiating broad-based immunity with poly(I:C) was observed to induce a Th1 bias in the SV40 Tag antibody response that led to successful antitumor responses not observed in animals treated only with poly(I:C) or SV40 Tag. These data have direct implications for immunotherapeutic strategies incorporating methods to elicit inflammatory reactions, particularly NK cell-driven lysis, against malignant cell types that express a tumor-specific antigen such as SV40 Tag.Considerable interest has been directed toward the role innate immunity plays in reducing malignant growth and progression. Although the innate system by broad definition is not endowed with the antigen specificity and memory recall of adaptive immunity, natural killer (NK) cells are an innate effector population that shares most properties with the adaptive arm of the immune system, excluding receptor rearrangement (28). Interestingly, NK cells can be employed to directly target and destroy malignant cell types through diverse pathways that include tumor major histocompatibility complex class I (MHC-I) loss and upregulation of stress-inducible protein ligands for the NK cell activating receptor NKG2D (24, 29). Much effort is under way in human clinical trials to manipulate NK cell properties for directed therapies against cancer (13, 29).One strategy in eliciting innate immunity in general involves activating the Toll-like receptor (TLR) family, which are preferentially expressed by innate effectors such as NK cells, macrophages, and dendritic cells (DCs) (26). TLR ligands include a variety of pathogen-associated molecular patterns with differing downstream responses based on the cell type involved and specific TLR activated. In TLR-expressing cells, signal transduction pathways follow a MyD88-independent course to produce type I interferons (IFNs) (e.g., TLR3) or a MyD88-dependent pathway that results in the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and IL-6 and expression of costimulatory molecules such as CD40, CD80, and CD86 (e.g., TLR4 and TLR9) (2, 12, 23, 26). In the case of TLR3, activation by poly(I:C) causes DCs and additional accessory cells to secrete type I interferons and IL-12, activating NK cells and prompting NK cell secretion of IFN-γ among other effects (14, 20). Ultimately, modulation of TLR activation results in the generation of a range of cytokines that promote inflammation, Th1 bias, and NK cell-directed killing that can be utilized in a beneficial manner for tumor treatment strategies.TLR agonist incorporation alongside vaccine strategies has resulted in promising results in mouse models of cancer (12). Indeed, the TLR7 agonist imiquimod is an effective FDA-approved topical compound used to treat superficial basal-cell carcinoma and external genital warts (9). However, to our knowledge, modulating TLR activity while also incorporating recombinant simian virus 40 (SV40) large tumor antigen (Tag) protein immunizations in a therapeutic tumor setting has not been previously reported. SV40 Tag is a clinically relevant tumor-specific antigen that has been shown to be expressed by a number of human malignancies, including malignant pleural mesothelioma (MPM), and represents a potential target for immunotherapeutic strategies.Our laboratory has previously defined a unique role for antibody-dependent cell-mediated cytotoxicity (ADCC) reactions—specific against SV40 Tag—promoting cytotoxic T-lymphocyte (CTL) activity in response to neoantigens through cross-presentation of tumor cell debris in a model of experimental pulmonary metastasis (16, 17). In this report, we analyze the role of innate immunity in mediating tumor cell lysis during the early course of tumorigenesis in the absence of vaccination. Overall, we find that activated NK cells are necessary effector cells in achieving antitumor reactions and providing partial tumor immunity during the onset of tumorigenesis and that these functioning NK cells are likely activated in vivo due to inflammation as a result of tumor growth and progression. The burden of tumor challenge could be further reduced in naive animals with the indirect activation of NK cells using poly(I:C) as a TLR3 agonist prior to and during malignant dissemination. Interestingly, in an established pulmonary tumor setting, therapeutic treatment of mice with poly(I:C) and recombinant SV40 Tag resulted in enhanced protection that was not observed using poly(I:C) or SV40 Tag alone. One effect of instituting poly(I:C) treatment alongside SV40 Tag immunizations was a Th1 skewing of the SV40 Tag IgG antibody response that correlated with therapeutic tumor protection.Our results have direct implications for the prevention and treatment of malignancies, such as MPM, that express the SV40 Tag oncoprotein. Combining specific aspects of innate and adaptive immunity by targeting both NK cells and humoral activity against SV40 Tag, respectively, represents a novel and clinically significant immunotherapeutic strategy for potential use in patients.     

Development of hyporesponsiveness of natural killer cells to augmentation of activity after multiple treatments with biological response modifiers

Summary Four biological response modifiers (BRMs), MVE-2 (maleic anhydride divinyl ether), Corynebacterium parvum (C. Parvum), PolyICLC (polyinosinic:polycytidylic acid stabilized with poly-l-lysine), and mouse -interferon (-IFN), were tested to assess whether repeated treatments would repeatedly induce or sustain augmented levels of natural killer (NK) cell activity and/or macrophage (M0)-mediated inhibition of tumor cell growth. In contrast to a significant increase in splenic NK activity obtained with a single treatment with each of the agents, multiple treatments with these BRMs led to a progressive decrease in the degree of augmentation of NK activity. In contrast, multiple injections with these agents resulted in sustained augmentation of M0-mediated reactivity. Separation of the spleen cells by Percoll discontinuous density gradient centrifugation indicated that with mice treated once with each BRM high levels of NK activity were detected in the lower density fractions and that these fractions contained a higher percentage of large granular lymphocytes (LGLs) than that found in comparable fractions from normal mice. In contrast, cells in the lower density fractions from mice that received multiple treatments had decreased NK activity and an appreciably lower proportion of LGLs. These results indicate that the development of hyporesponsiveness to augmentation of splenic NK-cell activity following multiple treatments with BRMs may be attributable to a decreased percentage of LGLs, the effector cell population responsible for NK cell-mediated cytotoxicity. Abbreviations used in this paper: BRMs, biological response modifiers; MVE-2, maleic anhydride divinyl ether of molecular weight 15,500; C. parvum, Corynebacterium parvum; PolyICLC, polyinosinic-polycytidylic acid stabilized with poly-l-lysine in carboxymethylcellulose; IFN, interferon; NK cells, natural killer cells; M0, macrophage; LGLs, large granular lymphocytes; PGE, prostaglandin E; FBS, fetal bovine serum; PBS, phosphate-buffer saline composed of 4.86 g NaCl, 0.306 g KH₂PO₄, and 2,417 g NaHPO₄ in 100 ml H₂O adjusted to pH 7.2; LPS, lipopolysaccharide     

Active treatment of murine tumors with a highly attenuated vaccinia virus expressing the tumor associated antigen 5T4 (TroVax) is CD4+ T cell dependent and antibody mediated

5T4 is a tumor associated antigen that is expressed on the surface of a wide spectrum of human adenocarcinomas. The highly attenuated virus, modified vaccinia Ankara, has been engineered to express human 5T4 (h5T4). In a pre-clinical murine model, the recombinant virus (TroVax) induces protection against challenge with CT26–h5T4 (a syngeneic tumor line expressing h5T4). Anti-tumor activity is long lived, with protection still evident 6 months after the final vaccination. In a therapeutic setting, injection of mice with TroVax results in a reduction in tumor burden of >90%. Depletion of CD8⁺ T cells has no effect upon therapy in the active treatment model, whereas depletion of CD4⁺ T cells completely abrogates anti-tumor activity. In a prophylactic setting, depletion of CD4⁺ and CD8⁺ T cells after the induction of a h5T4 immune response has no deleterious effect on protection following challenge with CT26–h5T4. In light of these studies, the role of antibodies in protection against tumor challenge was investigated. 5T4 specific polyclonal serum decreased tumor burden by approximately 70%. Thus, we conclude that CD4⁺ T cells are essential for the induction of a protective immune response and that antibodies are the likely effector moiety in this xenogeneic murine tumor model.     

Selective induction and inhibition of direct and lectin-dependent cell-mediated cytotoxic reactivity

The immune reactivity of mice (C57BL/6, H-2^b) which had been challenged with various numbers (10²–10⁸) of allogeneic tumor cells (P815, H-2^d) was assessed at various times after challenge. Challenge with a high dose (10⁸) of tumor cells resulted in the development of direct cytotoxicity (DCMC), lectin-dependent cytotoxicity (LDCC), delayed-type hypersensitivity (DTH), and antibody production, whereas challenge with lower doses (< 10⁶) of tumor cells favored development of DTH and LDCC with marginal or no DCMC or antibody production. Spleen cells from low-dose alloimmune animals failed to produce DCMC when cultured with P815 cells in vitro and were capable of nonspecifically suppressing the DCMC response of normal spleen cells in MLC. Treatment with cyclophosphamide (100 mg/kg) prior to alloimmunization did not alter the pattern of DTH and cytotoxic reactivity, although treatment after alloimmunization was immunosuppressive for all forms of reactivity. When low-dose challenge was followed by cyclophosphamide treatment and a subsequent high-dose challenge, selective inhibition of DTH, LDCC, and suppressor activity, but not DCMC, was observed. The data suggest that (a) the initial challenge dose plays a significant role in determining which effector and regulatory populations will be activated and what direction the expression of immune reactivity will take; (b) the activated responding populations of DTH, DCMC, and LDCC effector cells are sensitive to cyclophosphamide treatment, whereas the precursors of each are resistant to the effects of the drug; (c) low-dose alloimmunization may be used in combination with cyclophosphamide treatment to modulate DTH, DCMC, and LDCC reactivity in a selective manner; (d) the cytotoxic effector cells responding to highdose challenge and mediating DCMC and those responding to low-dose challenge and mediating LDCC appear to arise from distinct precursor populations.     

Paradoxical Effect of Pertussis Toxin on the Delayed Hypersensitivity Response to Autoantigens in Mice

Background

Pertussis toxin (PTX), an exotoxin of Bordetella pertussis, enhances the development of experimental autoimmune diseases such as experimental autoimmune uveitis (EAU) and experimental autoimmune encephalomyelitis (EAE) in rodent models. The mechanisms of the promotion of experimental autoimmune diseases by PTX may be based upon PTX-induced disruption of the blood eye/brain barriers facilitating the infiltration of inflammatory cells, the modulation of inflammatory cell migration and the enhancement of the activation of inflammatory cells. We hypothesized that the facilitation of experimental autoimmunity by PTX suggests that its influence on the in vivo immune response to auto-antigen may differ from its influence on non-self antigens.

Methodology/Principal Findings

We have evaluated the effect of PTX on the simultaneous generation of delayed type hypersensitivity (DTH) responses and autoimmune responses to uveitogenic interphotoreceptor retinoid binding protein peptide (IRBP_161–180), encephalitogenic myelin oligodendrocyte glycoprotein peptide (MOG_35–55) or ovalbumin (OVA). PTX injection of mice immunized to IRBP peptide_161–180 led to (i) the development of EAU as shown by histopathology of the retina, (ii) pro-inflammatory cytokine production by splenocytes in response to IRBP peptide _161–180, and (iii) symptomatic EAE in mice immunized with encephalitogenic MOG peptide_35–55. However, mice that received PTX had a reduced DTH response to IRBP_161–180 peptide or MOG peptide_35–55 when challenged distal to the site affected by autoreactive T cells. Moreover, footpad challenge with MOG_35–55 peptide reduced EAE in mice immunized with MOG peptide. In contrast, the use of PTX when immunizing with OVA protein or an OVA immunogenic peptide did not affect the DTH response to OVA.

Conclusions/Significance

The results suggest that that the reduced DTH response in mice receiving PTX may be specific for autoantigens and autoantigen-reactive T cells are diverted away from ectopic sites that received the autoantigen and towards the tissue site of the autoantigen.