To test whether alloimmunization with H-2 or/and non-H-2 different normal tissues may increase the immunity to syngeneic tumors, groups of BALB/c (H-2d) mice were immunized with a series of allogeneic lymphoid cells and then challenged i.p. with syngeneic lymphoma cells. The outgrowth of otherwise lethal doses of the Moloney virus-induced lymphoma YC8 and of its clones was inhibited in BALB/c mice immune to DBA/2 (H-2d), C3Hf (H-2k), C3H.SW (H-2b), C3H.OH (H-2o2) and to B10 background tissues but not in mice immunized to A/He, BALB.K (H-2k) or BALB.B (H-2b) normal tissues. Anti-YC8 effect was also induced by immunizing BALB/c recipients with a pool of five different allogeneic cell lines which included C3Hf, C57BL/6J (H-2b), N:NIH (H-2q), B10.M (H-2f), and DBA/2 lymphoid cells. No growth inhibition of other BALB/c lymphomas induced by Moloney virus (LSTRA), X-rays (RL male I) or urethane (UR-1) was evident in alloimmune mice. In vivo transfer of growth inhibition of YC8 was obtained with BALB/c anti-B10.D2 peritoneal exudate cells in a Winn assay. The ability of these alloimmune lymphoid cells to delay significantly the survival time of BALB/c mice injected with the mixture of immune cell and YC8 cells was abrogated by anti-Thy 1.2 plus C' treatment. In addition, nu/nu BALB/c mice were unable to develop resistance to YC8 outgrowth after alloimmunization. The results of this study show that: (1) syngeneic growth of a lymphoma can be prevented by alloimmunization with normal cells; (2) this cross-reaction involved non-H-2 antigens; (3) the phenomenon appeared to be mediated by T cells.

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The DNA of 22 fibrosarcomas, newly induced in BALB/c mice by subcutaneous doses of 3-methylcholanthrene (3-MCA), was tested in NIH 3T3 transformation assay. Activation of K-ras and N-ras was found in 7 and 3 cases respectively. No H-ras activation was detected. Polymerase chain reaction and oligonucleotide hybridization performed on the DNA of the 22 sarcomas revealed 5 cases of K-ras mutation at codon 12, 3 at codon 13 and 1 at both codons. One case of K13 mutation was not detectable by transfection. Three cases of mutation at codon 61 of N-ras were also found, one of which was simultaneous with a K12 mutation. Tumor-specific transplantation antigens (TSTA) were assessed in the 22 original tumors. Altogether 16 sarcomas were immunogenic, with the highest frequency of TSTA+ tumors (10/11 and 5/6) in the groups given 1.0 and 0.1 mg of 3-MCA respectively, the lowest (1/5) in that with 0.01 mg of carcinogen; ras mutations occurred in the DNAs of 11 out of the 16 TSTA+ sarcomas, but none of the DNAs of the 6 TSTA- tumors showed ras mutation. The results suggest that 3-MCA-induced transformation of subcutaneous fibroblasts can involve mutations in codons 12, 13 or 61 of K- and N- but not H-ras gene and that such mutation is accompanied by the expression of TSTA.

The 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.

W e have previously shown that the methylcholanthrene-induced BALB/c fibrosarcoma C-1 syngeneically transplanted in vivo expressed, i n addition t o i t s original H-Zd and tumor-associated transplantation antigens, also the foreign H-2Kk 23, 1,5, 11.25 specificities. In the present study we have investigated the expression and the immunogenicity of these antigens on an in vitro line of C-1 tumor. The binding of C57BL/6J o r C3Hf anti-BALB/c sera o r that of monospecific alloantisera t o H-2d specificities on plated C-1 cells (evaluated by the indirect isotopic '251-antiglobulin assay) showed that the expression of H-Zd antigens was very low o r undetectable on the tumor kept in vitro. Absorption by the in vivo and in vitro maintained C-I cells of the complement-dependent cytotoxicity on BALB/c normal lymphoid cells of the C75BL/6J anti-BALB/c serum confirmed the lower expression of H-2d antigens on C-1 kept in vitro as compared t o the in vivo transplanted tumor. The H-Zk antigens found on C-1 were also examined by the above methods using BALB/c anti-C3Hf, anti-AKR, anti-C57BL/6J polyspecific sera and also monospecific alloantisera directed t o H-Zk antigens. The C-1 cultured cells displayed a significant reduction of H-Zk antigens as compared t o the in vivo C-1 tumor, the antigen 5 being undetectable on the in vitro cells.Allogeneic and syngeneic sera raised against in vitro o r in vivo C-1 cells were tested on either BALB/c o r C3Hf normal lymph-node cells by the complement-dependent cytotoxicity assay. A significantly higher titer of cytotoxic antibodies t o H-Zd and t o H-2k was obtained i n the allogeneic and syngeneic sera respectively, after immunization with the in vivo C-1 cells as compared t o sera raised against the in vitro tumor line. Transplantation studies showed that both in vitro and in vivo C-1 lines possess the same tumor-associated transplantation antigen whose expression was nonsignificantly reduced on the cultured cells.