Winn Assays Research Articles

Cell-mediated suppression of tumor immunity has a non-specific component. I. Evidence from transplantation tests.

We have first demonstrated that the growth of a transplanted, 3-methyl-cholanthrene-induced BALB/c sarcoma, MCA-1425, in specifically immune syngeneic mice, was facilitated when the MCA-1425 tumor cells were injected together with either a mixture of irradiated MCA-1425 cells and thymus cells from mice immune to MCA-1425 or a mixture of irradiated cells from an antigenically different BALB/c sarcoma, MCA-1460, and thymus cells from mice immune to MCA-1460. We then performed Winn assays to study the induction of an immune response that could prevent the outgrowth of MCA-1425 cells in irradiated syngeneic recipients. Tumor immunity was found to be suppressed, if the mice immunized by a subcutaneous transplant of MCA-1425 cells and used as lymphocyte donors, were injected intraperitoneally with cells from either a different sarcoma, MCA-1460, or from 12-day BALB/c embryos. We tentatively attribute our finding to antigen-non-specific suppressor effector cells and postulate that an antigen-specific event is often involved in their induction.

Original H-2d and alien H-2k-like antigens of a BALB/c fibrosarcoma as defined by in vitro and in vivo studies of cell-mediated immunity.

AbstractThe present study examines the immunosensitivity and the immunogenicity of both original H‐2d and alien H‐2k‐like antigens of the BALB/c (H‐2d) fibrosarcoma C‐1 as detected by in vitro and in vivo cell‐mediated cytotoxicity (CMC) assays. It was found that 51Cr‐labeled C‐1 cells were lysed in vitro by C 57 BL/6 anti‐H‐2d lymphocytes. The specificity of this reaction was shown by cold inhibition experiments in which the anti‐H‐2d cytotoxic activity on YC8 (H‐2d) targets was inhibited by unlabeled YC8 or C‐1 but not by C3UR11 (H‐2k) tumor cells. Both Dd‐ and Kd‐encoded antigens were recognized by appropriate cytotoxic effectors. The immunogenicity of H‐2d antigens of C‐1 was revealed by the ability of C 57 BL/6 anti‐C‐1 lymphocytes to lyse YC8 targets. The expression of H‐2k‐like alien alloantigens on C‐1 was indicated by the finding that anti‐H‐2k cytotoxic T lymphocytes (CTL), generated by culturing BALB/c spleen cells immune to BALB.K (H‐2k), C3Hf (H‐2k) or A (H‐2a = H‐2k/d) tissues with the cells of the same strain used for immunization, lysed C‐1 targets. The cytotoxicity of these anti‐H‐2k CTL against C 3 UR11 (H‐2k) targets could be specifically inhibited by cold C 3 UR 11 or C‐1 cells but not by two other BALB/c tumors. Using recombinant H‐2‐congenic mice, it was shown that both Dk and Kk antigens were recognized by CTL on C‐1 cells. The immunogenicity of the H‐2k‐like antigens, however, could not be detected in vitro. In fact, effector spleen cells from BALB/c mice immune to C‐1 did not develop any detectable cytotoxicity against C 3 UR 11 targets as assayed either by a direct in vitro test or after in vitro restimulation with C‐1 sarcoma cells. A similar experimental design was adopted in Winn assays carried out by mixing spleen cells of BALB/c immune mice with either C‐1 or C3 UR 11 targets and injecting the mixtures in BALB/c or hybrid recipients. These in vivo tests revealed the presence of both H‐2d (Kd and Dd) and H‐2k‐like (Kk and Dk) antigens on C‐1. At variance with the in vitro CMC assays, however, the Winn assay also detected the immunogenicity of the H‐2k alien antigens, since BALB/c anti‐C‐1 spleen cells were able to significantly reduce the growth of C 3 UR 11 lymphoma cells in (BALB/c × C 3 Hf)F1 hosts.

Activity of Lymphoid Cells Separated From Mammary Tumors in Blastogenesis and Winn Assays<xref ref-type="fn" rid="fn2">2</xref><xref ref-type="fn" rid="fn3">3</xref>

Lymphoid cells isolated from mouse mammary tumors by isokinetic gradients were not stimulated in vitro by either phytohemagglutinin or a soluble, tumor-associated antigen extract even though splenocytes from the tumor-bearing mice were responsive to both. In in vivo Winn assays, lymphoid cells isolated from tumors markedly stimulated tumor growth rate. The effect on growth rate was abrogated by exposure of the isolated lymphoid cells to antilymphocyte serum and complement.

T-T hybridoma product specifically suppresses tumor immunity.

Culture supernatants of spleen or thymus cells from BALB/c mice bearing transplanted, syngeneic, methylcholanthrene-induced sarcomas suppress T lymphocyte-mediated lysis of cells from the tumor borne by the donor of the spleen or thymus cells. On this basis, we hybridized thymus cells from mice bearing sarcoma MCA-1490 with cells from the T lymphoma BW5147. The hybrids (hybridomas) formed were tested for production of factors that could suppress T lymphocyte-mediated lysis of MCA-1490 cells. One hybridoma, and a clone derived from it, produced factors that suppressed the lysis of MCA-1490 cells in vitro. In addition, these factors enhanced the growth of MCA-1490 in immune mice and prevented the destruction of MCA-1490 cells by immune lymphocytes in tumor neutralization (Winn) assays. In vitro lysis of cells from another MCA-induced sarcoma by immune lymphocytes was not suppressed. The suppressor factors did not affect the proliferative response of BALB/c lymphocytes to mitogens or the generation of a cytotoxic response to C57BL/6 alloantigens. Neither did they inhibit the generation of primary or secondary cytotoxic responses to murine leukemia virus-related antigens present on a BALB/c lymphoma line, LSTRA. Although our findings suggest that these suppressor factors are specific for MCA-1490, their specificity for antigens restricted to this tumor needs further definition.

Immunologic Heterogeneity of Tumor Cell Subpopulations From a Single Mouse Mammary Tumor<xref ref-type="fn" rid="FN2">2</xref>

Five subpopulations (66, 67, 68H, 168, and 4.10LM) obtained from a single BALB/cfC3H mammary adenocarcinoma were used to assess intratumor immunologic heterogeneity. BALB/c and BALB/cfC3H mice were immunized with each of the subpopulations, and lymph node cells (LNC) from immunized animals were tested for cell-mediated immunity (CMI) to each subpopulation in vitro by chromium release and microcytotoxicity tests and in vivo by Winn assays. The immunogenic character of the subpopulations differed markedly. The pattern of cross-reactivity indicated that at least two determinants were involved, one of which was probably a viral antigen. The viral antigen was expressed on 4 subpopulations (66, 68H, 168, and 4.10LM). The other determinant was immunogenic in both BALB/c and BALB/cfC3H mice and was expressed on 4 subpopulations (66, 67, 168, and 4.10lm). Thus 1 subpopulation (68H) expressed only the viral antigen, 1 (67) expressed only the other antigen, and 3 (66, 168, and 4.10LM) expressed both. Expression of the determinants showed qualitative and quantitative variations. Quantitative differences were noted by the relative effectiveness of the subpopulations to induce CMI and by the relative sensitivities to LNC-mediated killing. Qualitative differences were indicated by the occurrence of unidirectional cross-reactivities between some pairs of subpopulations; a determinant could be expressed so that the subpopulation could induce cytotoxic cells but not be sensitive to them or vice versa.

Tumour-derived lymphoid cells prevent tumour growth in Winn assays.

Tumour-derived lymphoid cells prevent tumour growth in Winn assays.

Assessment of syngeneic cell-mediated immunity to mouse mammary tumors by three different assays

The purpose of this study was to compare the MCT, CRT, and Winn assays for assessment of CMI to syngeneic tumors. Spontaneous and MMTV-induced mammary tumors in Balb/c and Balb/cfC3H mice were used. LNC were obtained at various times relative to S.C. injection of sensitizing tumor cells and surgical removal of the outgrowth. In direct comparison to Winn assays, CRT often gave false negatives (i.e., LNC could inhibit tumor growth in the Winn assay without being cytolytic), but when LNC were cytolytic in the CRT, they inhibited in Winn assays. Thus, a positive CRT was associated with a positive Winn assay. In contrast, results from MCT tests were generally not associated with those obtained by CRT or Winn assays. This divergence in assays was independent of differences in status of the LNC donor. With all three assays, stimulation of tumor growth or increased survival was occasionally observed. For the CRT this was manifested in the release of smaller amounts of 51Cr in the presence of ‘immune’ LNC than in the presence of normal LNC. For MCT and Winn assays it was seen in better tumor growth after treatment with LNC than after treatment with medium alone. Detection of this phenomenon was independent among the three assays.

Cell-mediated reactivity to antigens shared by Moloney-virus-induced lymphomas (LSTRA) and certain 3-methylcholanthrene-induced mouse sarcomas.

Spleen cells (SC) both from BALB/c mice whose primary Moloney sarcoma virus (MSV)-induced sarcomas had spontaneously regressed and from normal, untreated BALB/c mice, were co-cultivated for 5 days with mitomycin-C-treated LSTRA cells; LSTRA is a BALB/c Moloney lymphoma which shares cell surface antigens with MSV-indiced sarcomas. These SC, referred to as CMR and CU cells, respectively, were shown to be cytotoxic to LSTRA cells in 3 h 51Cr-release assays; CMR cells showed, in most cases, the greatest lytic activity against LSTRA targets. The same SC were also reactive, in 20-h microcytotoxicity and 51Crassays, against target cells from a variety of transplanted sarcomas indiced by 3-methylcholanthrene (MCA) in Balb/c mice. The highest reactivity was seen when CMR or CU cells were tested against target cells from sarcoma lines that expressed an NB-ecotropic MuLV cross-reacting serologically with Moloney virus. Reactivity against isotope-labelled tumor cells expressing MuLV-associated cell surface antigens could be competititively inhibited by adding unlabelled tumor cells expressing such antigens. Finally, Winn assays were performed in which CMR cells strongly inhibited the outgrowth of cells from three sarcoma lines that express the NB-ecotropic MuLV. There was less but significant inhibition of cells from some other MCA sarcomas, either negative for the expression of MuLV-associated antigens or expressing the N-ecotropic endogenous BALB/c MuLV. CU cells enhanced tumor outgrowth in Winn assays at least as often as they inhibited it.

In vitro induction of tumor-specific immunity. IV. Specific adoptive immunotherapy with cytotoxic T cells induced in vitro to plasmacytoma antigens

Specifically activated cytotoxic T cells (CL's) were raised by cocultivation in vitro of normal spleen cells with syngeneic plasmacytoma cells. These CL's were fully active both in vitro in lysing 51Cr-labelled tumor cells and in in vivo Winn assays, in inhibiting the growth of tumor cells in syngeneic mice when inoculated admixed with the tumor cells, even at ratios of 2 CL's to 1 tumor cell. However the same CL populations were only marginally effective in an immunotherapy model in which CL's were injected intravenously and tumor cells subcutaneously or intramuscularly (at a ratio of up to 100 CL/1 tumor cell). It is suggested that the in vitro conditions alter cell surface properties, thereby interfering with normal cell traffic, and that if this problem can be overcome, in vitro educated CL's may constitute a potential source of activated cells for human tumor immunotherapy.

Cellular immune reactivity in vitro and tumor rejection provided by tumor-associated antigens of friend-virus-induced leukemia.

Cell-mediated immunity (CMI) and tumor rejection were studied in the Friend virus leukemia system of C57Bl/6 mice. Mice were immunized with Friend leukemia virus (FLV) or X-irradiated FBL-3 leukemic cells and studied temporally for the development of CMI reactivity by assays of 51Cr release lymphocyte cytotoxicity, lymphocyte transformation, migration inhibition, Winn tumor cell neutralization and transplantation rejection. High levels of specific lymphocyte cytotoxicity were observed by day 7 f0llowing FLV infection; this reactivity reached a peak between 17 and 21 days, and returned to background levels by day 36. Further, positive Winn assays were obtained with spleen cells from mice immunized with FLV at times when the mice resisted live FBL-3 tumor challenge. Positive lymphocyte transformation was obtained with spleen cells from mice immunized with FLV or FBL-3, but not with cells from normal mice or mice immune to a syngeneic methycholanthrene-induced tumor, when cultured with papain-soluble FBL-3 or RBL-5 tumor-cell extracts or mitomycin-C (MMC)-treated FBL-3 or RBL-5 cells. Positive reactivity in the lymphocyte transformation assay occurred after reactivity had peaked in the lymphocyte cytotoxicity test. Similar positive macrophage migration inhibition patterns were also obtained with peritoneal exudate cells (PEC) from FLV-immunized mice using papain-solubilized tumor-associated antigen (TAA) from FBL-3 cells. These data suggest that sequential development and modulation of CMI reactivity occurs as observed in different assays following immunization in this system.

Immunogenicity, antigenicity and mechanisms of tumor rejection of mineral-oil-induced plasmacytomas in syngeneic BALB/c mice.

AbstractTransplantation immunity studies were conducted to determine whether mineral‐oil‐induced plasmacytomas of syngeneic BALB/c mice possess tumor‐associated transplantation antigens (TATAs). In a series of experiments to determine whether TATA could be detected, it was established that the optimum immunization regimen against TATA was obtained by immunizing syngeneic hosts with an intradermal inoculation of viable plasmacytoma cells, followed by therapeutic drug‐induced tumor regression with aniline mustard. This immunization regimen induced transplantation immunity not only to the homologous tumor employed for immunization, but was effective in demonstrating some cross‐protection when mice were rechallenged with other plasmacytomas. Thus, definite cross‐reactivity of antigens was observed between some plasmacytomas, but others shown to possess TATA did not share antigens. These, therefore, may possess distinct TATA (s). Further, studies with Winn assays employing cells from BALB/c mice immune to plasmacytoma cells support the idea that cell‐mediated immune functions are responsible for syngeneic plasmacytoma rejection. Specificity studies in subsequent Winn assays demonstrated that the kill was directed against plasmacytoma cells and not against TATA (s) of an MLV‐induced Moloney leukemia and SV40 virus‐induced tumor cells. The nature of the TATA (s) detected is not known, but they probably represent new “cell‐associated” antigens acquired during malignant transformation. Finally, the selective removal of the thymus‐dependent cell component of the immune lymphocyte population with anti‐theta serum and complement eliminated the tumor‐neutralizing properties of the immune cells in the Winn assay.