During the past year, we have succeeded in growing several lines of alloreactive normal T cells. BALB/c spleen cells obtained from mice immunized against C57B1/6 cells have been grown in IL-2-supplemented media for 3 months and four "clones" have been isolated by limit dilution cloning. We are now typing the clones for a variety of surface markers. Preliminary results indicated them to be Thy-positive, H-2KD-positive, TL-negative, and Ly-1.1-positive. These clones have a reactivity pattern that is unusual. Although made in BALB/c (H-2D) against B1/6 (H-2B), three of them are far more reactive against H-2K. The basis of this is being explored. The murine T-cell alloantigen, Thy-1.2, has been transferred into a Thy-1.1-bearing, mouse T-cell lymphoma, BW5147, using DNA extracted from BALB/c thymocytes (Thy-1.2-expressing cells) and the calcium phosphate precipitation method for DNA-mediated gene transfer. Two weeks later, they were reanalyzed for the number of "stable" transformants expressing Thy-1.2. Eight percent (0.1% of the original population) of the cells of the planned population and 5% of the sorted population were stained, as compared to 2% of the control. In one experiment, the 8% positive population was resorted and was subsequently found to be 28% positive 1 month later. In the absence of repeated selection, the majority of the cells lose the transformed gene but some long-term lines can be obtained. Southern blotting analysis reveals that these cells do in fact carry two different Thy-1.1 genes. We have further refined this methodology and have succeeded in transferring several human macrophage surface markers into murine cells. (LB)