Activation of autoreactive T-cell clones from NZB.H-2bm12 mice

We have previously established H-2bm12-restricted autoreactive T-cell clones from NZB.H-2bm12 mice which induce in-vitro production of IgG anti-dsDNA antibodies by syngeneic B cells. However, the mechanism underlying the activation of autoreactive T cells is not clear. We have taken advantage of the existence of L cells which were co-transfected with the A alpha b gene and independent A beta genes comprising all the permutations of amino acid residues distinguishing A beta b from A beta bm12. Using this panel of L cells expressing recombinant I-A molecules, 6/6 autoreactive T-cell lines responded significantly to the FT7.2 L cell transfectant expressing wild-type I-Abm12 as well as control APC from NZB.H-2bm12 or B6.C-H-2bm12 mice, but not to the other recombinant L-cell transfectants or to allogeneic APC. The fixation of APC with paraformaldehyde prior to co-culture led to dramatically diminished reactivity of all the autoreactive cloned T-cell lines tested. Interestingly, the inability of FT7.2 L cells to induce T-cell activation after paraformaldehyde fixation could be reconstituted by the addition of B/monocyte cells, but not T cells, from NZB.H-2bm12, NZB.H-2b or NZB(H-2d) mice and to a significantly lesser extent, from C57BL/6 (H-2b) and BALB/c (H-2d) mice. Conversely, when treated with either mitomycin C or cycloheximide, before incubation with autoreactive T cells and fixed FT7.2 cells, the ability of spleen cells from NZB.H-2b mice to reconstitute reactivity was reduced. Controls for these observations included KLH-specific T-cell clones from NZB.H-2bm12 mice, the T-cell hybridoma H66.3.6.54 specific for beef insulin and restricted to I-Ab, the IBM026 hybridoma specific for OVA and restricted to I-Abm12, and the TH2.2 hybridoma, and I-Ab antigen-presenting cell line.