Isolation of rat/rat T cell hybrids using W/Fu C58(NT)D as fusion partner

Functional rat/rat T cell hybrids were isolated for the first time by the fusion of spleen cells from rats tolerized to the hapten TNP to a HAT-sensitive rat thymoma (C58(NT)D). 11 fusions using different protocols were attempted to assess the optimal conditions for high hybridization frequency of the desired specificity. Interestingly, the cell density requirement of the non-transformed fusion partner took precedence over that of the C58 cell line, i.e., rat cells needed to be at high density after fusion, but others have reported that mouse cells prefer a much lower density even when the same line (C58) is used. Six fusions yielded hybridomas with between 3% and 70% of wells containing hybrids after three weeks of culture, depending on the protocol used. Phenotypically, all of the hybrids and clones tested expressed the W3/25 (rat CD4) antigen, but no OX-8 (rat CD8) or immunoglobulin molecules. A minority of hybrids were found to secrete factors able to suppress (a) proliferation, (b) antibody production, and (c) cells bearing IL-2 receptors, but none appeared to suppress the production of IL-2 itself. In contrast to non-transformed rat T cell lines, the T hybrids isolated were easy to grow to high densities, clone and freeze without the need to add exogenous antigen or lymphokines to the cultures at any stage.