Cell types required for H-2-restricted cytotoxic responses generated by trinitrobenzene sulfonate-modified syngeneic cells or trinitrophenyl-conjugated proteins.

Murine spleen cells were fractionated over nylon wool or Sephadex G-10 columns, and the cell types involved in the generation of trinitrophenyl (TNP)-specific, H-2 restricted (TNP-self) cytotoxic effector cells were studied from cultures stimulated with trinitrobenzene sulfonate (TNBS)-modified syngeneic cells, TNP-conjugated soluble proteins such as bovine gamma-globulin (TNP-BGG), or bovine serum albumin (TNP-BSA). Unfractionated or nylon nonadherent responding cells generated such effectors, irrespective of whether the cultures were stimulated with TNBS-modified cells or TNP-conjugated proteins. TNP-modified T lymphocytes, B lymphocytes, and phagocyte-enriched spleen cells were all capable of stimulating TNP-self effectors. TNP-self effectors. TNP-self as well as allogeneic cytotoxic responses were dependent on the presence of a radioresistant non-T cell that was removed by Sephadex G-10 fractionation and was replaced by irradiated, Thy 1.2-negative, glass adherent spleen cells, enriched in phagocytic cells. Results obtained by using glass adherent cells that were allogeneic or semi-syngeneic to the responding cells indicated that H-2 homology was not required for efficient glass adherent cell function, and that the H-2 restriction of TNP-self effectors is not determined by these glass adherent cells.     

The role of the macrophage as the stimulator cell in contact sensitivity.

In this study we examined the requirement for the type of stimulator cell for thymus-derived (T) lymphocyte activation to simple chemical haptens. T cells from picryl chloride-immune guinea pigs were challenged in vitro with various trinitrophenyl (TNP)-conjugated syngeneic stimulator cells and the extent of activation was determined by an increase in DNA synthesis. Hapten-specific T cell activation occurred with TNP-conjugated peritoneal exudate cells (PEC) and purified macrophages but not with TNP-conjugated erythrocytes, thymocytes, or nonadherent lymph node cells or PEC. In addition, T cell activation also occurred with TNP-conjugated guinea pig leukemia cells, but only in the presence of macrophages. Furthermore, it was shown that macrophages were required to process and/or present TNP-conjugated leukemia cell antigens rather than simply providing a growth-promoting function. These results suggest that a macrophage-like stimulator cell is required for hapten-specific T cell activation and that this particular stimulator cell may be important in contact sensitivity.     

Induction of I region-restricted hapten-specific cytotoxic T lymphocytes.

Murine cytotoxic T lymphocytes (CTL) reactive to TNP-conjugated syngeneic target cells do lyse to a moderate but significant extent TNP-conjugated, I region compatible but H-2K or H-2D region incompatible target cells. Antibody inhibition experiments and "cold inhibition" experiments indicate that some CTL clones recognize TNP-conjugated targets in association with syngeneic I region determinants independently of H-2K or H-2D gene products. The data suggest that besides TNP-conjugated H-2K or H-2D gene products, in principle, also TNP-conjugated I region determinants do stimulate TNP-specific CTL precursor cells and act as target antigens of TNP-specific CTL.     

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.     

Suppressive effect of ultraviolet-B-irradiation of epidermal cells on the induction of contact sensitivity

Contact sensitivity to trinitrophenyl (TNP) hapten was induced by subcutaneous (s.c.) administration of TNP-modified syngeneic spleen cells or epidermal cells (EC) (TNP-EC). Intraperitoneal (i.p.) inoculation of TNP-EC resulted in a comparable response, whereas i.p. administration of TNP-spleen cells or TNP-modified-ultraviolet (UV)-preirradiated EC (TNP-UV-EC) failed to induce TNP-contact sensitivity responses. The present study investigates the effect of UV-irradiation on the potential of EC for inducing the contact sensitivity response. Exposure of BALB/c mouse EC in vitro to 1600 J/m2 of UV-B before they were modified with TNP had no discernible effect on the Ia-positivity and viability of EC. Coexistence of TNP-UV-EC had no inhibitory effect upon the contact sensitivity response induced by TNP-EC via the i.p. route. The absence of suppressor cell generation was substantiated by the adoptive transfer of spleen cells from mice administered TNP-UV-EC i.p. to normal syngeneic mice. The effect of interleukin 1 (IL-1) or epidermal cell-derived thymocyte-activating factor (ETAF) in restoring the ability of TNP-UV-EC to induce contact sensitivity was examined. IL-1 or ETAF administered along with TNP-spleen cells i.p. induced a potent contact sensitivity response, whereas the same preparations of IL-1 or ETAF were unable to restore the contact sensitivity induction by TNP-UV-EC. The results are discussed in the context of UV-induced cell surface changes of the Langerhans cell population.     

Plant lectin,ATF1011, on the tumor cell surface augments tumor-specific immunity through activation of T cells specific for the lectin

Summary The possibility that a plant lectin as a carrier protein would specifically activate T cells, resulting in the augmentation of antitumor immunity was investigated. ATF1011, a nonmitogenic lectin for T cells purified from Aloe arborescens Mill, bound equally to normal and tumor cells. ATF1011 binding on the MM102 tumor cell surfaces augmented anti-trinitrophenyl (TNP) antibody production of murine splenocytes when the mice were primarily immunized with TNP-conjugated MM102 tumor cells. The alloreactive cytotoxic T cell response was also augmented by allostimulator cells binding ATF1011 on the cell surfaces. These augmented responses may be assumed to be mediated by the activation of helper T cells recognizing ATF1011 as a carrier protein. Killer T cells were induced against ATF1011 antigen in the H-2 restricted manner using syngeneic stimulator cells bearing ATF1011 on the cell surfaces. When this lectin was administered intralesionally into the tumors, induction of cytotoxic effector cells was demonstrated. These results suggest that intralesionally administered ATF1011 binds to the tumor cell membrane and activates T cells specific for this carrier lectin in situ, which results in the augmented induction of systemic antitumor immunity.     

Enhanced TNP-reactive helper T cell activity and its utilization in the induction of amplified tumor immunity that results in tumor regression

The present study was designed to investigate the generation of trinitrophenyl (TNP)-reactive helper T cell activity potent enough to induce the regression of a syngeneic tumor; this occurs by augmenting antitumor-specific immunity through T-T cell interaction. Mice whose skin was painted with trinitrochlorobenzene (TNCB) exhibited a variety of anti-TNP T cell responses, including delayed-type hypersensitivity (DTH) and cytotoxic T cell responses, as well as helper T cell activity. Pretreatment of C3H/He mice with TNP-conjugated copolymer of D-glutamic acid and lysine (TNP-D-GL) or cyclophosphamide, which have been shown, respectively, to inactivate TNP-specific suppressor T cells or suppressor T cells in general, exhibited a slight or marginal augmentation of DTH and cytotoxic potentials when tested 5 wk after TNCB painting. In contrast, the same pretreatment regimens induced an appreciably amplified generation of anti-TNP helper T cell activity. This amplified TNP-helper T cell activity was demonstrated to enhance cytotoxic responses to antigens other than TNP in an antigen-nonspecific way. In fact, such helper T cells enhanced antitumor CTL responses when co-cultured with spleen cells from syngeneic X5563 plasmacytoma-bearing mice in the presence of TNBS-modified X5563 tumor cells. This amplified TNP-helper cell system was utilized for its immunotherapeutic potential. When TNCB was injected into X5563 tumor mass of syngeneic C3H/He mice in which the amplified TNP-helper T cell activity had been generated, an appreciable number of growing tumors was observed to regress. This contrasted with the low incidence of tumor regression observed in mice in which TNP-helper activity had been induced by TNCB painting without inactivation of suppressors. Thus, the present model provides an effective immunotherapeutic manipulation for eliciting enhanced in vivo tumor regression, and emphasizes a role of helper T cells in augmentation of syngeneic tumor immunity.     

Studies on the specificity of in vitro induced lymphocytotoxicity to SV40-transformed fibroblasts.

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from AKR-mice (H-2k) and from BALB/c-mice (H-2d) to syngeneic SV40-transformed fibroblasts. The T cell-dependent cytotoxicity was specific for target cells expressing the same H2-specificity as the immunizing cells. Nontransformed fibroblasts as stimulator cells did not induce efficient cytotoxicity to transformed or nontransformed target cells. Incubation with phytohemagglutinin during the sensitization period modified the specificity of the T cell-mediated lysis of syngeneic SV40-transformed fibroblasts: allogeneic as well as syngeneic target cells were destroyed by these effector cells. However, the polyclonal stimulant activates preferentially cytotoxicity to H2-matched target cells. The in vitro generation of cytotoxic effector cells was restricted to living SV40-transformed fibroblasts as immunizing cells; it was not possible to immunize lymphocytes in the presence of membrane proteins prepared from the SV40-transformed cells. The cytotoxicity of the in vitro immunized lymphocytes was inhibited by incubation with membrane protein preparations from syngeneic or allogeneic SV40-transformed fibroblasts.     

Broadly reactive murine cytotoxic cells induced in vitro under syngeneic conditions

Mouse spleen cells became cytotoxic in short-term 51Cr-release assays for a wide variety of target cells after 5 days of culture in vitro with polyinosinic acid in a system that was otherwise entirely syngeneic. This study characterizes these effector cells with respect to target specificity, effect of donor age, time course of their appearance, mouse strain differences, and expression of differentiation antigens Thy-1, Lyt-1, Lyt-2, NK-1, and asialo GM1. The combination of properties of this cytotoxic cell response that make it unique are that a) the broadly reactive cytotoxic activity developed from unprimed spleen cells in the absence of either foreign cells or foreign serum; b) the response did not peak until 4 to 5 days of culture in vitro; c) the broad reactivity pattern included freshly dispersed primary syngeneic sarcoma cells and cultured syngeneic fibroblasts but did not include syngeneic lymphoblast target cells; d) the response was largely monoclonal as defined by target cell binding; and e) cytotoxic cell activity was sensitive in complement-mediated treatments to both anti-NK and anti-theta but not to anti-Lyt-2, anti-Lyt-1, or anti-asialo GM1. Both high- and low-responding mouse strains have been identified.     

Murine T cell suppression demonstrable in the absence of cytotoxicity and the effect of Cyclosporin A on this system

In vitro culture of murine spleen cells in FCS without prior immunization or allogeneic stimulation leads to the development of spontaneous cytotoxicity. This cytotoxicity is not H-2 restricted and can affect any subsequent in vitro assays using syngeneic cells, especially if those assays include prolonged culture in FCS. Studies on murine spleen cells cultured in NMS, however, led to the detection of a suppressor system that did not display cytotoxic effects. Furthermore, it was found that this suppression, in contrast to the cytotoxicity and suppression generated during culture in FCS, was not sensitive to CYA. The suppressor cell may be an effector or an inducer of suppression and is sensitive to treatment with anti-Thy-1.2 and complement. It is suggested that some in vitro suppression is really due to cytotoxicity that may be directed toward FCS determinants adsorbed onto syngeneic targets.     

Immune response to a syngeneic mammary adenocarcinoma. III. Development of memory and suppressor functions modulating cellular cytotoxicity.

Measurement of the development of cytolytic activity by mammary tumor primed or unprimed syngeneic spleen cells on in vitro monolayers of the 13762 rat mammary tumor operationally defined several subpopulations of lymphoid cells involved in the cytotoxic response. In vitro sensitization of cells from Fischer 344 animals injected 2 to 10 days earlier with 2 x 10(7) viable tumor cells always resulted in a higher and earlier lytic response than cells from non-inoculated animals. Adoptive transfer of the same in vivo primed cells for 5 days in irradiated syngeneic hosts removed any cytotoxic cells originally present but subsequent in vitro sensitization still resulted in a higher and earlier cytolytic response. We defined such cells as "memory" cells for cytotoxicity. Memory cells were radiosensitive and specific for the immunizing target cell. In contrast to cells from animals inoculated for 3 to 10 days, cells obtained 11 and 12 days after immunization had a lower response than unprimed cells on vitro sensitization. The anamnestic response could be restored either by culturing 12-day primed cells in vitro for 2 days without antigen or by adoptive transfer for 5 days into irradiated syngeneic rats. This suggests that another population of cells is present in spleen and suppresses the conversion of memory to cytotoxic cells. A more direct measurement of suppressor cell function was obtained by coincubating tumor-primed and unprimed cells on monolayers during in vitro sensitization. Cells from animals bearing tumors for 5 to 10 days always caused an increase in the response of the mixed lymphocyte groups, whereas 11- to 13-day tumor primed cells always caused a marked decrease in the cytolytic response. These results suggest the following interpretation of the kinetics of cell-mediated cytotoxicity to syngeneic tumor inoculation. Cytotoxic cells appear about 6 days after immunization, reach peak levels 2 days later, and then decrease rapidly. Memory cells are generated at a faster rate, reach peak levels before maximum cytolytic activity, but are then functionally inhibited from converting into differentiated cytotoxic cells by a new population of suppressor cells which reach peak activity about 12 days after immunization.     

Generation of cytotoxic T lymphocytes during coxsackievirus tb-3 infection. II. Characterization of effector cells and demonstration cytotoxicity against viral-infected myofibers1.

This report describes studies characterizing the virus-specific cytotoxic effector cells which are present in the spleens of mice 7 days after infection with Coxsackievirus B-3. An in vitro 51Cr assay employing eyngeneic virus-infected neonatal fibroblasts was used to measure cytotoxic activity. Treatment of immune cells with (anti-thy-1.2) and complement abolished dtheir cytotoxic activity, but no reduction occurred when B cells were removed by incubation with anti-Ig and complement or macrophages eliminated by adherence depletion. The findings therefore imply that the cytotoxic reaction was mediated by sensitized T cells and that B cells and macrophages did not play an important role. Reciprocal assays performed with BALB/c and CBA/J cells showed that Coxsackievirus-immune spleen cells lysed infected syngeneic targets but not allogeneic targets, providing further evidence that cytotoxicity was mediated by effector T cells. In addition and in vitro assay system employing neonatal myocardial cells was developed and used to demonstrate that Coxsackievirus-infected myofibers were susceptible to destruction by immune spleen cells. The evidence suggests that mice infected with Coxsackie B viruses are able to mount a cell-mediated immune response with production of cytotoxic T cells which have the capacity to damage tissues infected with these agents.     

Generation of cytotoxic T lymphocytes from thymocyte precursors to trinitrophenyl-modified self antigens. II. Establishment of a T cell hybrid clone constitutively producing killer-helper factor(s) (KHF) and functional analysis of released KHF

T cell hybridoma lines were constructed by fusion of Mycobacterium tuberculosis-primed and boosted BALB/c T cells with the AKR-derived T lymphoma cell line BW5147. Certain of the hybridomas prepared in this manner secreted constitutively into their culture supernatants biologically active molecules that displayed precursors of cytotoxic T cell activating properties characteristic of killer-helper factor (KHF). Cell surface analysis revealed that the hybridomas were indeed somatic cell hybrids between the two respective partner cells used for fusion. KHF properties of these hybridoma supernatants were verified by their capacity to stimulate peanut agglutinin-binding (PNA+) C3H/He thymocytes to respond in vitro to 2,4,6-trinitrophenyl(TNP)-modified syngeneic stimulator cells in conjunction with suboptimal doses (10 U/ml) of interleukin 2 (IL 2) for the generation of H-2-restricted, TNP-reactive cytotoxic T cells. The biologically active molecules secreted by a T cell hybrid clone (2Y4) were, like conventional KHF, distinct from IL 1, IL 2, or immune interferon (IFN-gamma). The partially purified KHF derived from 2Y4 cells shows activity at apparent m.w. range of 34,000 to 60,000 on gel permeation, and is relatively homogeneous with respect to isoelectric point, which was approximately 4.5 to 4.7. The partially purified 2Y4-KHF is able to augment proliferation of as well as the expression of IL 2 receptors on PNA+ thymocytes in conjunction with IL 2. Finally, addition of 2Y4-KHF on day 0, followed by the addition of IL 2 on day 2 for 7 days of culture was effective in generating potent CTL responses, whereas addition of IL 2 on day 0, followed by the addition of 2Y4-KHF on day 2 to the culture was ineffective.     

Differential effects of BCNU on T cell,macrophage, natural killer and lymphokine-activated killer cell activities in mice bearing a syngeneic tumor

Summary Chloroethylnitrosoureas have been used widely to treat human and experimental animal tumors. We have earlier observed that >90% of the mice transplanted with syngeneic tumors survive following treatment with nitrosoureas such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and furthermore, they resist subsequent challenge with the same tumor. The present investigation was initiated to determine the mechanism by which BCNU brings about this effect. Treatment of tumor cell targets in vivo or in vitro with BCNU, increased their susceptibility to macrophage (MØ)-mediated cytotoxicity as measured in a direct cytotoxicity assay or in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. In contrast, the antitumor cytotoxicity caused by cytotoxic T lymphocytes (CTL), natural killer (NK) cells, or lymphokine-activated killer (LAK) cells, was not altered following BCNU treatment of tumor targets. Studies were also conducted to investigate the direct effect of BCNU in vivo on various cytotoxic effector cells. For this purpose, MØ, NK, LAK, and CTL activities from BCNU-treated-tumor-bearing mice were screened for cytotoxicity against untreated tumor targets in vitro. It was observed that tumor-specific CTL and LAK cell activity increased in BCNU-treated tumor-bearing mice when compared to untreated controls while the cytotoxic potential of NK cells and MØs was not altered. The present study suggests that antitumor drugs such as BCNU are not only tumoricidal but also selectively act in a variety of ways at both the effector and target cell level, leading to overall enhanced antitumor immunity and high rate of cures from the syngeneic tumor challenge.The work at Virginia Polytechnic Institute and State University was supported by NIH grants CA45009 and CA45010 and by a Biomedical Research Support Grant. The work at University of Kentucky was supported by NIH grants CA34052 and CA33629 and by a grant from the Tobacco and Health Institute     

Generation of suppressor lymphocytes during sensitization in culture against a syngeneic tumor: affinity chromatography on insolubilized histamine

We investigated the effect of depletion of histamine-binding lymphoid cells on immunological properties of lymphocytes sensitized in culture against tumor cells. C57BL/6 spleen cells that were sensitized in vitro on monolayers of the syngeneic Lewis lung carcinoma (3LL) became cytotoxic to the tumor cells in vitro after 3 to 5 days of sensitization. Sensitized cells harvested after 4 days of sensitization occasionally enhanced tumor growth in vivo. Fractionation of the sensitized lymphocytes over insolubilized histamine-rabbit serum albumin-Sepharose (HRS) columns decreased or abolished the enhancing activity in vivo and specifically increased the in vitro cytotoxic activity of the depleted lymphocytes. A similar increase in the cytotoxic activity of HRS-fractionated cells was observed in an allogeneic combination of C57BL spleen cells sensitized against C3H fibroblasts. The effect of HRS chromatography on the in vitro cytotoxic activity increased with prolonged incubation of the depleted effector cells with the target cells.     

Effects of sodium periodate modification of lymphocytes on the sensitization and lytic phases of T cell-mediated lympholysis.

Sensitization of mouse splenic lymphocytes in vitro with sodium borohydride, suggesting that the biologic effects of sodium periodate are-treated autologous spleen cells stimulated a one-way mixed lymphocyte reaction and led to the generation of thymus-derived cytotoxic effector cells. These effectors were capable of lysing in 4 hr periodate-treated syngeneic and, to a lesser extent, periodate-treated allogeneic target cells. These results suggest that sensitization by periodate-treated autologous cells could result either from a specific reaction to modified self components or from a nonspecific mitogenic stimulation. Effector cells generated by allogeneic sensitization were detected on periodate-modified targets, irrespective of the H-2 antigens expressed by the targets. The effects of periodate modification on both stimulator and target cells were reversible by sodium periodate are dependent on the formation of a free aldehyde group on cell surface glycoproteins. Pretreatment of stimulator cells with neuroaminidase prevented the effect of periodate treatment, suggesting that the sensitization involves oxidized sialic acid residues. During the 4-hour 51Cr-release assay periodate-treated targets could be used to detect cytotoxic effector cells of any specificity. Fresh spleen cells and lymphocytes cultured for 5 days without antigen or in the presence of lipopolysaccharide did not lyse periodate-treated targets. An increasing level of cytotoxicity was detected on periodate-treated targets when the effector cells were generated, respectively, by stimulation with concanavalin A, by sensitization with periodate-modified autologous cells. Although the lysis of periodate-treated targets is itself nonspecific, effector cell specificity could be determined by selective blocking of the lytic phase with cells syngeneic to the stimulators. These results indicate that a nonspecific interaction can occur between lymphocytes and periodate-treated target cells, but that this interaction leads to lysis only when the lymphocytes were activated to become cytotoxic effectors.     

Cytotoxic lymphocytes induced by soluble factors derived from the medium of leukocyte cultures.

Studies were undertaken to evaluate the cytotoxic capacity of human peripheral blood lymphocytes activated by either supernatants (CFM) derived from lymphocyte cultures or lymphocytes treated for 60 min at 45 degrees C. The effect of the addition of heat-treated cells on the cytotoxic activity of CFM-induced effector cells was also studied. CFM from either unmixed or mixed cultures of lymphocytes was capable of activating cytotoxic effector cells. These effector cells could kill any allogeneic target cells but failed to effect cytotoxicity on the target cells autologous to the responding cells. Both the heat-treated cells and CFM from cultures of these cells also activated lymphocytes to cytotoxic effector cells having specific receptors for nonself antigens. The question of whether heat-treated cells activate cytotoxic cells by themselves or through secreted soluble factor cannot yet be clearly answered. The findings of the present investigation suggest that expression of cytotoxicity induced in MLC is not necessarily restricted to the target cells syngeneic to the stimulator cells, but can be extended to any allogeneic target cells by the indirect effect of soluble factor secreted from stimulated cells that causes a polyclonal activation of cytotoxic precursors in the responding cell populations. The present findings also emphasize the need for caution in the use of heat-treated lymphocytes as innocent-bystander cells in MLC to provide additional cytotoxic specificities in the responder cells, since heat-treated cells alone can activate lymphocytes to cytotoxic effector cells that kill any allogeneic target cells.     

Chicken T lymphocyte clones with specificity for Eimeria tenella. I. Generation and functional characterization

T lymphocyte clones reacting specifically with the antigenic components of Eimeria tenella were generated from splenic lymphocytes of immunized chickens and were maintained for 12 to 14 wk in vitro. These T cell growth factor-dependent T lymphocyte clones from bursectomized and normal chickens proliferated in vitro when stimulated with antigens from different developmental stages of homologous but not heterologous species of the parasite. Specific proliferative responses of the cloned T cells showed an absolute requirement for antigen presentation by histocompatible antigen-presenting cells. Some of the T cell clones exhibited functionally discrete interactions with syngeneic primed B cells; 25% of the T cell clones from immunized normal chickens and 7% of those obtained from immunized bursectomized chickens showed antigen-dependent helper activity and induced specific antibody production by syngeneic primed B cells. Of the T cell clones from immunized normal chickens, 19% showed suppression of in vitro antibody production in comparison to 7% of those isolated from immunized bursectomized chickens. The frequency of cloned T cells with ability to induce cytotoxic activity in macrophages against the sporozoites of E. tenella was much higher in those isolated from bursectomized chickens (80%) than in those isolated from normal chickens. Because both bursectomized and normal chickens can be immunized by repeated infections, differences in the distribution among cloned T cells suggest different effector mechanisms of immunity against coccidiosis in these chickens. Lack of B cells seem to affect the development of T cell immunity as reflected by slower development of immunity and enhanced activation of cytotoxic T cell function.     

Specificities of killing by cytotoxic lymphocytes generated in vivo and in vitro to syngeneic SV40 transformed cells.

Cell-mediated immunity to SV40-transformed C3H and C3H-SW cell lines was measured by using both 51Cr and 125IUdR release assays. Killing by cytotoxic cells generated on in vitro sensitization of immune spleen cells with syngeneic SV40 cells by either assay is specific for syngeneic SV40 transformants. Cytolysis mediated by in vitro sensitized cells is ablated by treatment of the effector cells with anti-theta serum and complement. Intraperitoneal immunization with syngeneic SV40 cells yields two distinct killer-cell populations in the peritoneal exudate when assayed by 125IUdR release. The first, nylon wool nonadherent and sensitive to anti-theta and complement, is indistinguishable from the killers generated in vitro. The second population, present in larger numbers and more efficient on a per-cell basis in killing of SV40 targets than the first, is nylon adherent and is not removed by treatment with anti-theta and complement. This second population will kill any SV40 transformed target, whether syngeneic or allogeneic. The possible roles of T cell and non-T cell effectors in rejection of syngeneic SV40 tumors are discussed.     

Demonstration in vitro of cytotoxic T cells with apparent specificity toward tumor-specific transplantation antigens on chemically induced tumors.

Chemically induced tumors of mice exhibit apparently unique antigenicity upon syngeneic transplantation into appropriately immunized hosts. An in vitro counterpart of this pattern in terms of specificity has not been reported. Data are presented that demonstrate that immune peritoneal exudates contain cells cytotoxic for the specific immunogen tumor but with rare exceptions, not toward other syngeneic methylcholanthrene-induced fibrosarcomas. Only tumors highly immunogenic by transplantation criteria induce cytotoxic PEC regularly; nonimmunogenic tumors consistently fail to do so. The effector cell responsible is eliminated by pretreatment with anti-Thy.1 but not anti-Ig plus complement. In concomitant experiments, PEC populations cytotoxic in vitro also conveyed adoptive protection against the specific tumor in syngeneic hosts. This in vitro assay appears to provide a tool for studying T cell-mediated cytotoxicity toward a set of unique surface antigens present on chemically induced tumors.