Split tolerance to the MHC class I molecule H-2Dd in animals transgenic for its soluble analog

To determine whether the function of MHC molecules in tolerance and education is related to cell surface expression, we have produced two strains of transgenic mice in the C57Bl/6 background that express soluble analogs of the H-2D(d) class I protein. The transgenes were stably integrated and genetically transmitted in a Mendelian fashion. Messenger RNA for the hybrid genes was detected in all tissues analyzed in a class I-like pattern of expression, with the highest levels in lymphoid tissues. All mice bearing the transgenes expressed relatively high levels (0.1 mg/ml) of the encoded protein in their serum as assessed by Western blotting and enzyme-linked immunosorbent assay (ELISA). Gel filtration chromatography showed that the soluble H-2D(d) protein exists as a heterodimer with beta2-microglobulin and as higher order multimers in serum. Lymphoid cells from the transgenic mice showed no cell surface expression of the soluble class I protein in indirect immunofluorescence assays. Splenocytes from two independently derived transgenic lines generated primary cytotoxic and proliferative responses directed against membrane H-2D(d) antigens. Mice of both strains rejected tail skin from donors that differed from the B6 background at the H-2D(d) locus only, but with delayed kinetics compared to nontransgenic littermate controls. Mice expressing the transgenic protein on immunization did not produce antibodies that recognized soluble H-2D(d) in ELISA, whereas B6 mice generated strong antibody responses to challenge with splenocytes bearing cell surface H-2D(d). Thus, transgenic mice expressing soluble H-2D(d) were partially tolerant to stimulation by membrane-bound H-2D(d). As with the activation of T-cells, the induction and maintenance of immunologic tolerance apparently displayed different requirements depending upon the T-cell subpopulation involved.