T cell clones specific for hybrid I-A molecules. Discrimination with monoclonal anti-I-A (k) antibodies

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Abstract
Alloreactive and soluble antigen-reactive, I-A-restricted T cell clones were examined for their ability to recognize hybrid I-A antigens. Several clones that recognized hybrid I-Ab/I-Ak molecules on (C57BL/6 .times. A/J)F1 [(B6A)F1] [mouse] spleen cells were studied. Clones that recognized hybrid I-A molecules of the .**GRAPHIC**. type were distinguished from those that recognized .**GRAPHIC**. molecules. This conclusion was reached by considering data from the following 3 independent types of experiments. Monoclonal antibodies were used to inhibit T cell stimulation. Antibodies 10.2.16 and H116.32 distinguished 2 mutually exclusive families of T cell clones. One group of clones was inhibited by 10.2.16 and not H116.32, the other group exhibited reciprocal inhibition. T cell proliferation was assayed using antigen-presenting cells from B6.C-H-2bm12 (bm12) and [bm12 .times. B10.A(4R)]F1 mice. Because the bm12 strain has a mutation that results in an altered .**GRAPHIC**. polypeptide chain .**GRAPHIC**. clones that could recognize the [bm12 .times. B10.A(4R)]F1 cells were apparently recognizing .**GRAPHIC**. molecules. Alternatively, clones not recognizing [bm12 .times. B10.A(4R)]F1 cells had specificity for .**GRAPHIC**. molecules. I-A molecules immunoprecipitated from radiolabeled (B6A)F1 splenocyte extracts were analyzed by 2-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These experiments confirmed an earlier report that antibody 10.2.16 recognized determinants on the .**GRAPHIC**. chain. Antibody H116.32 immunoprecipitated products consistent with recognition of .**GRAPHIC**. determinants. These 3 types of results offer conclusive evidence that T cell clones recognizing hybrid I-A molecules use either .**GRAPHIC**. or .**GRAPHIC**. molecules as recognition or restriction sites. Clones whose proliferation was supported by [bm12 .times. B10.A(4R)]F1 cells and blocked by anti-I-Ak antibody 10-2.16 recognized .**GRAPHIC**. molecules. Clones that were blocked by antibody H116.32 and did not recognize [bm 12 .times. B10.A(4R)]F1 cells use a recognition site(s) on .**GRAPHIC**. molecules. It was demonstrated both functionally and biochemically that hybrid F1 I-A molecules of the structure .**GRAPHIC**. and .**GRAPHIC**. both exist on (B6A)F1 splenocytes and that both configurations are used in immune recognition phenomena.