The fine specificity of alloimmune cytotoxic T lymphocytes (CTL) was investigated in CTL responses across the smallest known H-2 differences, those based on mutation at a single H-2 locus. CTL were generated in all possible mixed lymphocyte culture (MLC) combinations among seven H-2Kb mutants and the mouse strain of origin, C57BL/6 (B6-H-2b). CTL were also generated in all F1 hybrid responder/homozygous stimulator-cell combinations among four Kb mutants and B6-H-2b. CTL activity was measured in cell-mediated lympholysis (CML) against target cells from all Kb mutants and B6-H-2b. Cross-reactivity against targets other than the MLC stimulator was extensive and led to the distinction of 64 CML target determinants. Two Kb mutants (B6-H-2bm6 and B6.C-H-2bm9) showed identical typing for all 64 CML determinants. CML reactions after MLC between these two haplotypes were mutually negative. The mutants B6-H-2bm3 and B6.C-H-2bm11 showed identical typing for 47 of the 64 determinants. Their close relationship is in agreement with the finding that H-2bm3 anti-H-2bm11 CTL were the only ones that exclusively lysed target cells of stimulator-cell genotype. On the basis of CML typing, the sequence of relatedness of the mutants with H-2b is as follows: bm6/bm9-bm10-bm3-bm1-bm11, bm6/bm9 being the closest to, and bm11 the most distant from H-2b. The extensive cross-reactivity of alloimmune CTL appears to reflect immunogenetic complexity rather than lack of specificity. Comparison with other reports supports the notion that the system of Kb CML determinants, recognized by alloimmune CTL, is at least partially overlapping with the H-2Kb specificity repertoire involved in the associative T cell recognition of virus-infected cells.

The B6.C-H-2bm12 mutant is described and evidence is presented for the mutational site occurring in the IA subregion. The mutant is of the gain and loss type as bm12 in equilibrium or formed from C57BL/6 grafts are rejected in 14-16 d. Mapping studies by the gene-complementation method using H-2 recombinant strains place the mutation in the K or IA regions of the H-2 complex and furthermore, the use of this test and the use of other H-2 mutants indicate that H-2Kb is not the site of the mutation, making the IA region the most likely site. Serological analysis with a battery of H-2b, Iab, and other Ia sera, both by cytotoxicity, rosetting, and also by absorption analysis, indicated no alteration in H-2 specificities, particularly in H-2.K33. By contrast, all of the Iab specificities coded for by the IA subregion (Ia.3, 8, 9, 15, and possibly 20) are extensively altered and are either absent or greatly reduced in amount indicating an extensive alteration in the Ia-bearing molecule. The bm12 mutant strongly stimulates the parental C57BL/6 strain in an mixed lymphocyte reaction (MLR), and the reciprocal also occurs, the degree of stimulation being similar to that obtained with K + IA differences originating in another H-2 haplotype and points to the mutation effecting the Lad-1 locus. The presence of an extensive histocompatibility change, a marked alteration in the serologically detected Ia specificities, and a strong MLR, all produced by the one mutation, provides strong evidence for the identity of the Ia-1, Lad-1, and H-2(IA) loci in the IA subregion. The bm12 mutant should be of value in determining the relationship of Ia specificities, Ir genes, and other phenomena effected by the I region.