Inflammatory cells in solid murine neoplasms. IV. Cytolytic T lymphocytes isolated from regressing or progressing Moloney sarcomas.

Highly purified suspensions of intratumoral T lymphocytes, recovered 11 and 13 days after induction of regressing or progressing Moloney sarcomas, were compared in their ability to lyse specifically the MSC cells used for tumor induction. Cytolytic activity, expressed in terms of lytic units/10(6) T cells, was similar for intratumoral T cell suspensions obtained 11 days after induction of either regressing (3.1 +/- 1.3 LU/10(6) T cells) or progressing (4.3 +/- 1.8) neoplasms. By 13 days post-induction, regressing tumors contained T lymphocytes with an increased cytolytic activity (11.1 +/- 4.5) whereas those from progressing tumors were strikingly less able to kill MSC cells (less than or equal to 0.2). This dramatic loss in cytotoxicity could not be attributed to errors associated with the enzymatic disaggregation method, inhibition by copurified endogenous tumor cells, or immunosuppression induced by viral infection. The changes in functional activity of intratumoral T lymphocytes from the two types of sarcoma appeared to be correlated with the stage of neoplasia. In this model system, cytolytic activity of T lymphocytes increased during spontaneous tumor regression whereas losses in cytotoxicity occurred coincident with the onset of inexorable progression.     

In vitro reactivity of splenic lymphocytes from normal and UV-irradiated mice against syngeneic UV-induced tumors.

Skin tumors induced in mice by chronic ultraviolet (UV) irradiation are highly antigenic and are frequently immunologically rejected upon transplantation to normal syngeneic recipients. In this study we characterized this immune response with an in vitro microcytotoxicity test. Cytotoxic activity was present in the spleen cells of mice given a single injection of syngeneic UV-induced fibrosarcoma cells. After removal of adherent spleen cells, the remaining splenic lymphocytes were specifically cytotoxic for the immunizing tumor and showed no cross-reactivity with other syngeneic UV-induced or methylcholanthrene-induced tumors of similar histologic type. The level of cell-mediated reactivity against UV-induced tumors was quite high compared to that obtained with syngeneic tumors induced by methylcholanthrene, and the cytotoxicity was attributable to a population of theta antigen-bearing lymphocytes. With this in vitro test, we compared the response of normal mice, which reject a syngeneic tumor challenge, with that of UV-irradiated mice, in which the syngeneic UV-induced tumors grow progressively. After tumor cell inoculation, lymphocytes form the unirradiated (regressor) mice showed a high degree of cytotoxicity that reached a maximum level 8 days after injection. In contrast, no reactivity could be detected in the spleens of tumor-challenged UV-irradiated (progressor) mice.     

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.     

Differential activation of cytotoxic and suppressor T cells against syngeneic tumors in the mouse.

The cytotoxic T cell against a methylcholanthrene-induced sarcoma, S1509a, was induced in syngeneic mice by deliberate immunization with mitomycin C (MMC)-treated live tumor cells. The soluble tumor antigen (STA) extracted from the same tumor by 3 M KCl was, however, unable to induce the cytotoxic T cell upon immunization, although it was able to activate predominantly the suppressor T cell that then specifically suppressed the effect of the cytotoxic T cell against the homologous tumor. The suppressor T cell generated by STA had the same characteristics as those found in tumor-bearing animals: 1) The suppressor T cell has a very strict specificity against individual tumors; 2) The cell expresses cell surface determinants controlled by genes in the I-J subregion of the mouse H-2 complex. The activity of the cytotoxic T cell was completely inhibited by live tumor cells but not by STA, whereas that of the suppressor T cell was neutralized by STA. The results that cytotoxic and suppressor T cells are activated under different conditions, and that the antigenic determinants recognizable by these two cell types are not the same. The soluble extract contains only the determinants recognizable by the suppressor T cell, and the cytotoxic T cell can be activated only by the determinants associated with self antigen present on the surface of live tumor cells.     

Cellular interaction between cytotoxic and suppressor T cells against syngeneic tumors in the mouse.

Splenic T cells from animals bearing growing syngeneic tumors specifically inhibited the effector process of tumor cell lysis by the cytotoxic T cell which had been activated in vitro by mitomycin C treated homologous tumor. The suppression was strictly specific for the individual tumor by which suppressor cells were generated, whereas in some cases cytotoxic T cells generated by two closely related sarcomas showed a certain degree of crossreactivity. This suggests that suppressor and cytotoxic T cells recognize different antigenic moieties on tumor cells; one unique to the individual tumor and the other shared by related tumor cell lines.The suppressor T cell from tumor bearing animals possessed Ia antigen controlled by a gene in I-J subregion of H-2 major histocompatibility complex. Cytotoxic T cells generated by some but not all syngeneic tumors were also killed by anti-Ia and complement; however, the Ia antigen on such cytotoxic T cells was found to be controlled by a locus in I-A subregion. In general, the cytotoxic T cells generated by newly established tumor cell lines had Ia antigen, whereas some old cell lines, which were capable of growing across the H-2 barrier, activated the Ia negative cytotoxic T cell. These results collectively indicate that the immunological resistance against tumors is dependent on the balance of activations of the cytotoxic and suppressor T cells with different specificities and phenotypic expressions.     

Antigenic reactivation in vitro of the cytotoxic activity of lymphoid cells from mice bearing syngeneic tumors]

The cytotoxic activity of lymphoid cells from mice bearing syngeneic sarcomas is increased when these cells are preincubated on heavily irradiated cells of the same tumours. This secondary stimulation is highest at the time when the cytotoxicity measured directly is relatively low. In consequence, whatever the age of the tumour, the level of cytotoxicity reached (35-45 %) cannot be increased further in normal conditions.     

Number of macrophages and distribution of mitotic activity in regressing and progressing Moloney sarcomas.

The concentration of macrophages per gram tumor was as much as five times greater in regressing compared to progressing Moloney sarcomas. Infiltrating monocular cells, including macrophages, were closely associated with a cessation of mitotic activity in tumors.     

Immune reactivity of frozen-thawed murine spleen cells.

Spleen cells from mice immunized with SRBC were subjected to controlled rate freezing to ?100 °C. Complete recovery of PFC was obtained with DMSO used as the cryopreservative. Simple dilution of spleen cells in DMSO, or a single cycle of freezing and thawing in DMSO prior to short-term culture, resulted in early loss of recoverable cells. A single cycle of freezing and thawing inhibited the in vitro immune response to SRBC while having little effect on the response to TNP-T4. The in vitro blastogenic responses to LPS and PHA-P were severely reduced in cultures of frozen and thawed cells.     

Cell-mediated immune response to syngeneic ultraviolet-induced tumors. II. The properties and antigenic specificities of cytotoxic T lymphocytes generated in vitro following removal from syngeneic tumor-immunized mice.

The immune responses of C3Hf mice to syngeneic fibrosarcomas induced with either ultraviolet light or methychlolanthrene (MCA) were measured in vitro by the ability of cytotoxic lymphocytes (CTL) from immunized animals to kill ⁵¹Cr-labeled tumor targets in a 6-hr assay. The CTL were generated by the in vitro culturing of draining popliteal lymph node (DLN) cells derived from animals that were footpad immunized 8 days previously. It was determined that CTL activity could be generated using DLN from both normal (uv tumorresistant) and uv-exposed (uv tumor-susceptible) C3H mice. The kinetics of CTL generation between these two groups, however, was different in that the lymphocytes from normal animals appeared to differentiate into CTL faster than the lymphocytes from the uv-irradiated mice. The in vitro generation of CTL activity was found to be extremely radiosensitive and was also inhibited by the presence of viable tumor cells within the cell culture. Once generated, it was observed that the CTL were extremely insensitive to the effects of gamma irradiation. It was also established that the CTL is a T lymphocyte that appears to be Ia^?. The CTL derived from mice immunized to syngeneic uv- or MCA-induced tumors were capable of expressing cross-reactive non-MHC-restricted killing of multiple tumor targets. Cold cell inhibition experiments confirmed the presence of cross-reactive determinants on various tumors and also established the presence within a single CTL preparation of effector cells with specificity for both the unique tumor specific transplantation antigens as well as the common (cross-reactive) tumor-associated antigens.     

Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors.

FBL-3 is a highly immunogenic murine leukemia of C57BL/6 origin induced by Friend murine leukemia virus (MuLV). Immunization of C57BL/6 mice with FBL-3 readily elicits CD8+ cytotoxic T lymphocytes (CTL) capable of lysing FBL-3 as well as syngeneic leukemias induced by Moloney and Rauscher MuLV. The aim of this current study was to identify the immunogenic epitope(s) recognized by the FBL-3-specific CD8+ CTL. A series of FBL-3-specific CD8+ CTL clones were generated from C57BL/6 mice immunized to FBL-3. The majority of CTL clones (32 of 38) were specific for F-MuLV gag-encoded antigen. By using a series of recombinant vaccinia viruses expressing full-length and truncated F-MuLV gag genes, the antigenic epitope recognized by the FBL-3 gag-specific CTL clones, as well as by bulk-cultured CTL from spleens of mice immune to FBL-3, was localized to the leader sequence of gPr80gag protein. The precise amino acid sequence of the CTL epitope in the leader sequence was identified as CCLCLTVFL (positions 85-93) by examining lysis of targets incubated with a series of synthetic leader sequence peptides. No evidence of other CTL epitopes in the gPr80gag or Pr65gag core virion structural polyproteins was found. The identity of CCLCLTVFL as the target peptide was validated by showing that immunization with the peptide elicited CTL that lysed FBL-3. The CTL elicited by the Gag peptide also specifically lysed syngeneic leukemia cells induced by Moloney and Rauscher MuLV (MBL-2 and RBL-5). The transmembrane peptide was shown to be the major gag-encoded antigenic epitope recognized by bulk-cultured CTL derived from C57BL/6 mice immunized to MBL-2 or RBL-5. Thus, the CTL epitope of FBL-3 is localized to the transmembrane anchor domain of the nonstructural Gag polyprotein and is shared by leukemia/lymphoma cell lines induced by Friend, Moloney, and Rauscher MuLV.     

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.     

Effector phenotypes and mechanisms of antitumor immune reactivity of tumor-immunized and tumor-bearing mice in two syngeneic tumors.

By using two different syngeneic tumors, Meth A sarcoma and RL male 1 lymphoma of BALB/c origin, the present study was designed to investigate the subset(s) of T cells mediating in vivo antitumor immune responses and some of the effector mechanisms of in vivo protective immunity in BALB/c mice immunized against tumor or bearing tumor. Spleen cells from the mice immunized against Meth A tumor or bearing Meth A tumor inhibited the growth of Meth A tumor in the Winn assay. In the Meth A-immunized mice, L3T4+ (CD4+) cells played a major role in mediating the inhibitory activity against Meth A tumor growth, whereas in the Meth A-bearing mice, the antitumor protective immunity was mediated by both L3T4+ and Lyt-2+ (CD8+) cells. Spleen cells from the Meth A-immunized or Meth A-bearing mice were not able to generate cytotoxic T lymphocytes (CTL) directed against Meth A tumor after the in vitro restimulation of spleen cells with mitomycin C (MMC)-treated Meth A cells, while fresh spleen cells from the Meth A-immunized or Meth A-bearing mice were able to induce the strong delayed-type hypersensitivity (DTH) responses to Meth A tumor. The DTH response to Meth A tumor was mediated by L3T4+ cells in the Meth A-immunized mice and by both L3T4+ and Lyt-2+ cells in the Meth A-bearing mice. In the similar experiments performed in the RL male 1 lymphoma, the antitumor activity in spleen cells from the RL male 1-immunized or RL male 1-bearing mice depended on Lyt-2+ but not L3T4+ cells in the Winn assay. When spleen cells from the RL male 1-immunized or RL male 1-bearing mice were cultured with MMC-treated RL male 1 cells for 5 days, an appreciable CTL response to RL male 1 tumor was induced. These results suggest that the nature of tumor and/or tumor antigens determines which T cell subset is required to exhibit the protective immunity against tumor and thus the different effector mechanisms could be induced in the different tumor models. Furthermore, these data support the conclusion that antitumor T cell responses are affected by the immune state of host to tumor.     

Stimulation of lymphoid cells from normal and immune mice by syngeneic BALB/c plasma cell tumors.

Lymphoid cells from normal and immunized BALB/c mice could be stimulated in vitro by syngeneic PCT contrasted with an absence of response to a number of other tumors. Maximal responses of normal cells to PCT were found to occur 5 days after the initiation of the cultures at an optimal responding:stimulation cell ratio of 1:2. MLTI activity of normal cells could not be blocked or enhanced by PCT myeloma protein products indicating that MLTI reactivity was directed against non-idiotypec cell surface determinants. Lymphoid cells from immunized mice demonstrated increased MLTI responses to cells of the immunizing tumor but not to other PCT, indicating that the post-immunization MLTI responses were primarily to individual rather than shared tumor cell surface antigens. Activity of both normal and immunized spleen cells was found to involve thymus-derived lymphocytes. The persistence of residual MLTI activity after treatment with anti-theta serum and complement, however, implicated participation of non-theta antigen-bearing cells in MLTI reactivity. From these data, we conclude that lymphoid cells from un-immunized mice are capable of T cell-dependent reactivity to syngeneic PCT-associated antigens and that elevations in these reactivities after immunization may reflect specific cellular immune responses.