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beta 2-Microglobulin knockout mice (beta 2-m-/-) with MHC class I expression deficiency are able to develop functional TCR(+)-alpha beta, CD8+ CTLs in response to tumor cell injection. The i.p. injection of beta 2-m-/- mice with tumor results in the massive accumulation of highly lytic CD8+ CTLs in the peritoneum and causes the local recruitment of CD8+ T cells into lymph nodes and spleens of immune animals. The accumulation of CD8+ CTLs in peritoneum is accompanied by the rejection of tumor cells and the survival of animals. The deficiency in MHC class I expression in beta 2-m/- mice is reflected in the delayed tumor rejection and CD8+ cell accumulation during the primary anti-tumor response in comparison with normal mice. The secondary response, however, is identical in normal and MHC class I-deficient mice. The rejection of tumor cells appears to be MHC class I directed because no rejection of tumors, no accumulation of CD8+ CTLs, and no survival of animals were observed when syngeneic tumor cells were used for injection with the notable exception of anti-minor Ag response. The Ag specificity of CD8+ CTLs in beta 2-m-/- mice is demonstrated using a panel of tumor target cells and class I transfectants. Although no substantial differences were found in the number and specificity of peritoneal CD8+ CTLs in beta 2-m-/- and normal mice using tumor rejection studies, the analysis of TCR-V beta phenotype using the panel of mAbs revealed the reduction in proportion of TCR-V beta 5 and TCR-V beta 6 used by CD8+ cell population from beta 2-m-/- mice. Development of lytic and H-2-directed CD8+ cells in regional lymph nodes was also observed after footpad immunization of beta 2-m-/- mice with TNP-labeled C57BL/6 splenocytes, suggesting anti-minor Ag reaction.

Anti-wild-type (B6) H-2Kbm mutant (bm) CTL were induced in the regional lymph nodes by 2 injections (with 2 week interval) of bm mice into foot-pads with B6 irradiated splenocytes. CTL were tested 7 days after the boost, including 3 days precultivation in monoculture (required for high CTL activity in bm). Active bm4 CTL inducible in vivo but not in the mixed lymphocyte culture (MLC), while bm1, bm3 and their F1 hybrids with BALB/c were equally active in both models. In vivo induced bm3 CTL were cloned with B6 irradiated splenocytes stimulators in the presence of rat interleukine-2. Of 9 Thy1.2 positive narrow-specific CTL clones 2 displayed cross-reactivity to allogeneic target cells (TC): the 1st lysed H-2Kk [TC B10.A(2R)] and the 2nd H-2Kd [TC B10.D2(R101)]. The results witness for non-identity of the in vivo and in vitro induced CTL. The variable cross-reactivity of the narrow-specific CTL clones possibly occur because of receptors' affinity difference.

Differences in the generation of anti-H-2Kb wild-type specific cytotoxic T lymphocytes (CTL) and specific suppressor T cells (SSTC) were investigated in H-2Kbm mutant mouse strains. To this end, optimal conditions for in vivo induction of highly active CTL in this mutant system were found and the T-cell origin of CTL and SSTC was confirmed using the anti-Thy-1.2 monoclonal antibody G4. Unlike the CTL, which were generated in vivo by any of the mutant strains tested (bm1, bm3, and bm4), the SSTC were only produced by bm3, whose H-2Kbm3 antigen, in contrast to the other H-2Kbm molecules, differs from wild type by serologically defined determinants. The high activity of anti-wild type bm4 CTL induced in vivo contrasted with a low activity of such CTL induced in mixed lymphocyte culture (MLC). This appeared to be the property of bm4 only, but not of bm1 or bm3, and it was reproduced in the reciprocal system B10 anti-bm4. CTL generation could be restored in the MLC by the addition of concanavalin A supernate or a mixture of bm4 and bm12 stimulator cells. Three of the six in vivo induced and in vitro propagated bm3 anti-B6 CTL clones demonstrated selective cross-reactivity to only one of the third-party H-2K molecules used, either Kk, Kd, or Kbm4. The present results indicate that (a) the SSTC and antibody recognize similar H-2Kb epitopes; (b) the H-2Kb epitopes recognized by the CTL and SSTC are not identical; (c) the genetic control of CTL generation in vivo is distinct from that in MLC, and (d) the affinities of antigen-specific receptors on T-cell clones of the same specificity may be different, leading to their individual cross-reactivity patterns.