Anti-tumor cytostatic mechanism and delayed-type hypersensitivity against a syngeneic murine tumor. Comparison between neonatally thymectomized mice and congenitally athymic nude mice

We studied the anti-tumor mechanism against a syngeneic tumor using a BALB/c-MA tumor system by cytolysis and cytostasis assays in vitro comparing mice neonatally thymectomized at 1 day or 7 days after birth (NTx-1, NTx-7), sham-operated (sham) mice, and congenitally athymic nude BALB/c mice. NTx-1 mice showed more rapid tumor growth and a slightly lower degree of strong cytostatic activity in peritoneal exudate cells (PEC) than NTx-7 or sham mice. Nude mice showed more rapid MA growth than NTx-1 mice and no cytostatic activity in PEC. After immunization with mitomycin C-treated MA (MMC-MA), NTx-1 mice acquired an immunoprophylactic capacity against MA and showed cytostatic activity and delayed footpad reaction (DFR) to MA, however, nude mice showed no acquisition of such an immunity, or cytostatic activity, or DFR to MA. These differences between NTx-1 and nude mice could be well-explained by less capacity of nude mice to produce a macrophage-activating factor, which activates macrophages to exert cytostasis and DFR. However, NTx-1 mice could not reject MA by immunization with MMC-MA in CFA (MMC-MA/CFA), although such immunized sham mice could eliminate MA completely. Both PEC and spleen cells from Sham mice immunized with MMC-MA/CFA showed cytostatic activity, whereas NTx-1 mice showed cytostatic activity of the same level in PEC and less in spleen cells compared to Sham mice. Cytolytic activity was never detected throughout this study in a BALB/c-MA system. These data suggest that cytostasis plays an important role in antitumor immunity against a syngeneic MA tumor and that two types of cytostasis is included from the standpoint of thymus-dependency of ontogenic development, relatively low and high.     

The immunological role of the thymus in the pathogenesis of virus-induced lymphomas. Suppression of cytolytic T-cell function

Thymectomy of young adult mice has been found to prevent virus-induced lymphomas which develop as the animals age. Thymectomy protects mice by removing a source of suppressor T cells which inhibit the generation of cytolytic T cells against autochthonous tumors. Furthermore, suppression is specific since T cells are regulated in their capacity to respond to syngeneic but not allogeneic tumor cells. To determine if suppression could be adoptively transferred, lethally irradiated, bone-marrow-reconstituted mice were inoculated with T cells from either normal or thymectomized mice. Only T cells from thymectomized animals transferred enhanced T-cell reactivity to syngeneic tumor cells. More importantly, T cells from thymectomized mice injected with virus protected recipients challenged with lethal doses of syngeneic tumor cells. We conclude that thymectomy protects mice from developing virus-induced T-cell lymphomas by removing a source of suppressor T cells which regulates the activity of specific cytolytic T cells directed against autochthonous tumor cells.     

The immune response to a chemically induced fibrosarcoma

Summary A clone of C3H10T 1/2 fibroblasts transformed in vitro with the carcinogen 3-methylcholanthrene readily produced tumors when as few as 10³ cells were injected into immunocompetent adult syngeneic mice. A non-transformed clone of the same parentage did not produce tumors. Because the cell-mediated immune response has an important role in inhibiting the growth of tumors, we have compared the ability of both these transformed and non-transformed fibroblasts to stimulate and to act as targets in cell-mediated cytotoxicity (CMC) assays. This model is unique in that studies of the immune response to tumors rarely have or utilize appropriate normal controls. When both types of irradiated fibroblasts were used as stimulators in vitro, neither syngeneic nor allogeneic effector spleen cells capable of efficiently lysing the tumor fibroblasts were generated. In contrast, the normal fibroblasts could both stimulate and be lysed by allogeneic cytolytic T cells (CTL). However, the tumor fibroblasts could be lysed by allogeneic effector spleen cells that had been sensitized to C3H/He spleen cells. These results suggest that the expression of alloantigenic determinants necessary for stimulating a CMC response may vary substantially among normal cell types. They further indicate that the tumor cells are not resistant to lysis by appropriately stimulated effector cells. Thus, they must express antigenic determinants necessary for immune lysis and they do not inhibit the functional expression of cytolytic cells once generated. Consequently, tumor growth in vivo may be dependent, in part, upon a failure of the syngeneic host's immunocompetent cells to respond appropriately to the tumor cells. Additional data are provided which suggest that this failure is attributable in large part to immunosuppressive properties of the tumor cells.     

Studies of thymopoietin pentapeptide (TP5) on experimental tumors: I. TP5 relieves immunosuppression in tumor-bearing mice

The allogeneic and syngeneic immune responses of tumor-bearing mice (C57BL/6 mice bearing 3LL and DBA mice bearing P815) were evaluated by the cytotoxic lymphocyte precursor unit (CLP-U) and MLC. In general, tumor-bearing mice showed slightly enhanced immune responses 4 days after tumor inoculation. This enhanced immune response rapidly declined and about 7–10 days after tumor inoculation, both allogeneic and syngeneic responses were markedly lower than normal. Mice treated with TP5, starting 2 weeks before tumor inoculation, retained normal or enhanced allogeneic and syngeneic responses up to 3 weeks after tumor inoculation. When this tumor-induced suppressive effect was studied in cell transfer experiments, spleen cells from tumor-bearing mice enhanced the growth of tumors in syngeneic recipients whereas spleen cells from TP5-treated mice inhibited the growth of tumors in syngeneic recipients. Moreover, the spleen cells from TP5-treated mice also showed enhanced cytotoxic activity against tumor cells in vitro. These findings suggest that the tumors, after a transient stimulatory phase, induced immune suppressive mechanisms in the hosts' immune defenses. Treatment with TP5 prevented the development of these immune suppressive effects and spleen cells from TP5-treated tumor-bearing mice inhibited tumor growth in freshly tumor-inoculated recipients.     

Generation of cytotoxic lymphocytes to syngeneic tumor by using co-stimulator (Interleukin 2)

Cytotoxic lymphocytes (CL) highly active against the syngeneic mastocytoma, P815, were generated from spleen cells of DBA mice cultured with co-stimulator (Interleukin 2) and P815. More CL activity was generated from spleen cells of P815 tumor-bearing mice than from spleen cells of normal mice. Thymus cells from tumor-bearing mice, however, did not produce increased CL activity. Most of the CL were Thy 1 and Ly 1 positive. The optimal culture conditions and kinetics were similar to those for the generation of allogeneic cytotoxic T lymphocytes. The cytotoxic activity against syngeneic P815 was similar in magnitude to the response of DBA spleen cells to allogeneic tumor lines and to the response of allogeneic CBA spleen cells to P815. Although CL generated from tumor-bearing mice did not lyse normal DBA cells, they did lyse, to a much lesser degree, a number of tumor cell lines other than the sensitizing P815. This nonspecific lysis was not H2 restricted nor was it restricted to tumors of lymphoid origin. Generation of nonspecific cytolytic activity was antigen independent, occurring in the presence of co-stimulator alone.     

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.     

Graft versus neuroblastoma reaction is efficiently elicited by allogeneic bone marrow transplantation through cytolytic activity in the absence of GVHD

Continuous efforts are dedicated to develop immunotherapeutic approaches to neuroblastoma (NB), a tumor that relapses at high rates following high-dose conventional cytotoxic therapy and autologous bone marrow cell (BMC) reconstitution. This study presents a series of transplant experiments aiming to evaluate the efficacy of allogeneic BMC transplantation. Neuro-2a cells were found to express low levels of class I major histocompatibility complex (MHC) antigens. While radiation and syngeneic bone marrow transplantation (BMT) reduced tumor growth (P < 0.001), allogeneic BMT further impaired subcutaneous development of Neuro-2a cells (P < 0.001). Allogeneic donor-derived T cells displayed direct cytotoxic activity against Neuro-2a in vitro, a mechanism of immune-mediated suppression of tumor growth. The proliferation of lymphocytes from congenic mice bearing subcutaneous tumors was inhibited by tumor lysate, suggesting that a soluble factor suppresses cytotoxic activity of syngeneic lymphocytes. However, the growth of Neuro-2a cells was impaired when implanted into chimeric mice at various times after syngeneic and allogeneic BMT. F1 (donor-host) splenocytes were infused attempting to foster immune reconstitution, however they engrafted transiently and had no effect on tumor growth. Taken together, these data indicate: (1) Neuro-2a cells express MHC antigens and immunogenic tumor associated antigens. (2) Allogeneic BMT is a significantly better platform to develop graft versus tumor (GVT) immunotherapy to NB as compared to syngeneic (autologous) immuno-hematopoietic reconstitution. (3) An effective GVT reaction in tumor bearing mice is primed by MHC disparity and targets tumor associated antigens.     

Helper factor(s) augment in vitro induction of tumor-specific immunity

Helper factor supernatants derived from alloantigen-activated murine lymphocytes augment the generation of cytolytic effector cells to syngeneic tumor cells. The effects are dose dependent and vary with the syngeneic tumor cell system studied. The effector cells are specific for the tumor-associated antigen(s) utilized for their induction, and are sensitive to lysis with anti-T-cell serum (Thy 1.2), but are insensitive to lysis with an allogeneic anti-NK-cell serum. The helper factor supernatants also augment the production of a “tissue-culture-induced” cytolytic cell (cultured NK cell), which is resistant to treatment with both anti-Thy 1.2 and anti-NK serum.     

Immune response to a syngeneic mammary adenocarcinoma. II. In vitro generation of cytotoxic lymphocytes.

A method is described for the consistent in vitro generation cytotoxic cells by incubating Fischer 344 rat spleen cells on monolayers of a syngeneic mammary adenocarcinoma. Significant cytotoxicity by in vitro culture is generated as early as 3 days after initiation and effector cells are cytolytic only toward target cells of the sensitizing monolayer. Reciprocal sensitization with allogeneic fibroblasts as the immunizing monolayer yielded effector cells cytolytic for the fibroblasts but without effect on the mammary tumor. The consistency in the generation of cytotoxic cells by in vitro culture should permit its standardized use in following other related immune phenomena such as blocking by serologic factors and suppression, recritment of memory for cytotoxic function.     

Studies of H-2 restriction in cell-mediated cytotoxicity and transplantation immunity to Leukemia-associated antigens.

H-2 dependency of T cell-mediated cytotoxicity and transplantation immunity to leukemia-associated antigens has been investigated. Through the use of a 20-hr 125IUdR release assay, it was found that the induction of T cell-mediated cytotoxicity against Friend virus-induced leukemias of different H-2 haplotype orgins could be produced by immunization with both syngeneic and allogeneic tumor cells; the effector cells that were generated by syngeneic immunization could also provide effective killing of allogeneic tumor cells, although the killing of allogeneic targets might require a longer incubation time (20 to 40 hr). Furthermore, in vivo transplantation immunity against Friend virus-induced leukemias also was induced by immunization with both syngeneic and allogeneic tumors and syngeneic immunization could induce specific protection against the challenge with a-logeneic tumor in x-irradiated hosts. These findings clearly indicate that, both at the sensitizing phase and effector phase of the immune response, there is no strict H-2 dependency for T cell-mediated cytotoxicity or in in vivo transplantation imunity to leukemia-associated antigens.     

Effect of allogeneic tumor cells,interleukin-2 and interleukin-6, on the growth of subcutaneous syngeneic tumors

In the present study we demonstrate the ability of allogeneic M3 tumor cells to induce an antitumor response against the syngeneic tumor, when injected locally together with syngeneic B16 melanoma cells. The replacement of the allogeneic tumor cells with either syngeneic or allogeneic splenocytes had no effect on the growth of the syngeneic tumor. Systemic administration of both interleukin-2 (IL-2) and IL-6 did not affect the antitumor response induced by allogeneic tumor cells. When mice, previously injected with B16 and M3 cells, were rechallenged subcutaneously with B16 tumor cells at a different anatomical site, an inhibitory effect in some, but not all, experiments was observed. Systemic injections of either IL-2 or IL-6 did not alter the antitumor effects of the allogeneic and syngeneic tumor-cell mixtures. The significance of our results in developing immunotherapy modalities based on active immunization with allogeneic tumor cells and selected cytokines is discussed.This study was supported by a grant from the Israeli Cancer Association     

B7-H3 enhances tumor immunity in vivo by costimulating rapid clonal expansion of antigen-specific CD8+ cytolytic T cells

B7-H3 is a B7 family molecule with T cell costimulatory function in vitro. The in vivo role of B7-H3 in the stimulation of tumor immunity is unclear. We report here that expression of B7-H3 by transfection of the mouse P815 tumor line enhances its immunogenicity, leading to the regression of tumors and amplification of a tumor-specific CD8+ CTL response in syngeneic mice. Tumor cells engineered to express B7-H3 elicit a rapid clonal expansion of P1A tumor Ag-specific CD8+ CTL in lymphoid organs in vivo and acquire the ability to directly stimulate T cell growth, division, and development of cytolytic activity in vitro. Our results thus establish a role for B7-H3 in the costimulation of T cell immune responses in vivo.     

Suppression of cell-mediated immunity to challenge with P 815 mastocytoma in concanavalin A-treated mice

C57Bl/6 (B6) mice allogeneic to the P 815 mastocytoma tumor cell line when treated with concanavalin A prior to and at frequent intervals following challenge intraperitoneally with 10⁷ tumor cells showed a significant suppression of their cell-mediated immune response at 9–10 days when compared with untreated animals. Suppression of the immune response of mice syngeneic (DBA/2) or hybrid (BDF₁) to the tumor was also evidenced by increased mortality rates in concanavalin A-treated animals. The suppression of cell-mediated cytotoxicity observed in B6 mice treated with concanavalin A could be reversed by pretreatment with 20 mg silica injected intraperitoneally 7 days prior to challenge. These results suggest that macrophages play a significant role in the concanavalin A-induced immune suppression observed in this in vivo tumor-host system.     

Graft-versus-host reaction-like phenomenon induced by BCG in mice lethally irradiated and transferred with syngeneic bone marrow cells.

Heat-killed BCG in paraffin exerted a lethal effect on CS7BL/6 mice irradiated lethally and transferred with syngeneic bone marrow cells. Such an effect was not detectable when mice were subjected to adult thymectomy and used as the hosts. Lymphoid cells from such nonthymectomized mice exhibited cytotoxicity to syngeneic tumor cells but not to allogeneic tumor cells in an in vivo cytotoxicity test and induced splenomegaly in sublethally irradiated syngeneic recipients after systemic transfer. The cytotoxicity of such lymphoid cells was abolished by a treatment with anti-θ serum and complement. In the bone marrow of mice irradiated and transferred with bone marrow cells, the number of nucleated cells, the ratio of myeloid to erythroid cell series, and the percentage of lymphocytes were increasd by BCG injection. These results suggest the possibility that self-tolerance may be broken by BCG stimulation in the process of reconstitution of lymphoid cells in the irradiated mice.     

Prolongation of H--Y incompatible skin grafts in mice by neonatal spleen cells

Prolongation of H--Y incompatible skin graft survival can be achieved by the injection of adult female recipients with neonatal spleen cells. The suppressive effect depends mainly on the sex of neonatal syngeneic and allogeneic donors.     

Immunologic response in vitro after thymechtomy in patients with myasthenia gravis.

Thymectomy in adult animals impairs immune functions such as lymphocyte response to phytohemagglutinin (PHA) and to allogeneic cells. The responses of lymphocytes from 18 myasthenia gravis patients, 12 of whom had undergone thymectomy, were studied; the interval between thymectomy and investigation ranged from 1 month to 26 years (mean, 9.5 years). Peripheral blood lymphocytes were cultured in autochthonous plasma or homologous AB serum. In vitro responses to stimulation with PHA, concanavalin A and allogeneic monomuclear cells were within the 95% range of normal responses in all patients. Because our findings contrast with the definite immune defects resulting from thymectomy found in adult animals, longer follow-up is needed.     

Specific inhibition of cytotoxic memory cells produced against UV-induced tumors in UV-irradiated mice.

Cytotoxic responses of UV-irradiated mice against syngeneic UV-induced tumors were measured by using a 51Cr-release assay to determine if UV treatment induced a specific reduction of cytotoxic activity. The in vivo and in vitro primary responses against syngeneic tumors and allogeneic cells were unaffected, as was the "memory" response (in vivo stimulation, in vitro restimulation) against alloantigens. In contrast, the memory response of UV-treated mice against syngeneic, UV-induced tumors was consistently and significantly depressed. The cytotoxicity generated by tumor cell stimulation in vivo or in vitro was tumor-specific and T cell-dependent. Since the primary response against syngeneic UV-induced tumors produces apparently normal amounts of tumor-specific cytotoxic activity, UV-treated mice may not reject transplanted syngeneic tumors because of too few T effector memory cells. These results imply that, at least in this system, tumor rejection depends mostly on the secondary responses against tumor antigens and that at least one carcinogen can, indirectly, specifically regulate immune responses.     

Neonatal induction of tolerance to skeletal tissue allografts without immunosuppression

Vascularized allogeneic skeletal tissue transplantation without the need for host immunosuppression would increase reconstructive options for treating congenital and acquired defects. Because the immune system of a fetus or neonate is immature, it may be possible to induce tolerance to allogeneic skeletal tissues by alloantigen injection during this permissive period. Within 12 hours after birth, 17 neonatal Lewis rats were injected through the superficial temporal vein with 3.5 to 5 million Brown Norway bone marrow cells in 0.1 ml normal saline. Ten weeks after the injection, peripheral blood from the Lewis rats was analyzed for the presence of Brown Norway cells to determine hemopoietic chimerism. The Lewis rats then received a heterotopic, vascularized limb tissue transplant (consisting of the knee, the distal femur, the proximal tibia, and the surrounding muscle on a femoral vascular pedicle) from Brown Norway rat donors to determine their tolerance to the allogeneic tissue. A positive control group (n = 6) consisted of syngeneic transplants from Lewis rats into naive Lewis rats to demonstrate survival of transplants. A negative control group (n = 6) consisted of Brown Norway transplants into naive Lewis rats not receiving bone marrow or other immunosuppressive treatment. The animals were assessed for transplant viability 30 days after transplantation using histologic and bone fluorochrome analysis. All the syngeneic controls (Lewis to Lewis) remained viable throughout the experiment, whereas all the Brown Norway to Lewis controls had rejected. Ten of the 17 allografts transplanted into bone marrow recipients were viable at 30 days, with profuse bleeding from the ends of the bone graft and the surrounding graft muscle. The percent of chimerism correlated with survival, with 3.31 percent (SD = 1.9) of peripheral blood, Brown Norway chimerism present in the prolonged survival groups and 0.75 percent (SD = 0.5) of Brown Norway chimerism in the rejected graft group. This study demonstrated prolonged survival of allogeneic skeletal tissue without immunosuppression after early neonatal injection of allogeneic bone marrow in a rat model.     

Thy and Ly markers on lymphocytes initiating tumor rejection

Nonadherent spleen cells from mice specifically immune to the syngeneic methylcholanthrene-induced fibrosarcoma H-7 caused suppression of tumor growth in footpads of nonimmune mice. The cells were Thy-1 positive, largely Ly-1 positive, but Ly-2 and Ly-3 negative, and resembled T cells causing delayed-type hypersensitivity reactions rather than cytolytic T cells. It is suggested that some tumors fail to elicit the production of rapidly cytolytic T cells but can be rejected by a mechanism akin to that of delayed-type hypersensitivity, mediated by a different class of T cells working in collaboration with macrophages.     

Augmentation of in vivo cytotoxic T lymphocyte activity and reduction of tumor growth by large multivalent immunogen.

Class I alloantigen incorporated into cell-size supported membranes provides an effective stimulus for in vitro stimulation of CTL responses. When alloantigen-bearing cell-size (5 microns) microspheres, termed large multivalent immunogen (LMI), were administered in vivo, no primary cytotoxic response to the Ag could be detected. However, coadministration of LMI and allogeneic tumor stimulator cells resulted in substantial augmentation of the resulting CTL response, compared with that obtained from mice that received just stimulator cells. Responses were augmented only when the same alloantigen was present on the LMI and on the stimulator cells, and the effector cells remained specific for the cognate alloantigen-bearing targets. The physical form of the alloantigen was critical for augmentation; alloantigen in liposomes had no effect on response levels. Tumor cell Ag in the form of purified plasma membrane vesicles can also be incorporated onto the surface of cell-size microspheres. As with allogeneic responses, tumor Ag on LMI specifically augmented the in vivo CTL activity generated in response to irradiated tumor cells in syngeneic mice. Administration of Ag-bearing LMI to mice inoculated i.p. with live P815, EL4, or RDM4 tumor cells resulted in a significant reduction in growth of the tumors in their syngeneic hosts. Similarly, LMI treatment significantly reduced growth of P815 as a solid s.c. tumor. LMI-mediated growth reduction occurred only when plasma membrane Ag from the cognate tumor was used to prepare the LMI, and Ag in the form of free plasma membrane vesicles was not effective. Although Ag has been used to manipulate in vivo humoral and Th responses, this has proven to be much more difficult for CTL responses. The ability of Ag-bearing LMI to affect significantly the in vivo levels of cytolytic response and to reduce syngeneic tumor growth has potential for application to tumor immunotherapy and, possibly, treatment of other diseases in which CTL can provide a protective effect.