FIG. 3.

Inhibition of LFA-1-ICAM-1-dependent T-cell-APC interaction and IL-2 production by Rap1N17 or SPA-1. (A) Inhibition of cell aggregation by Rap1N17 and SPA-1. 3A9 T cells were transfected with empty vector, or genes encoding Rap1N17, Spa-1, or H-rasN17, and more than five stable clones for each construct were isolated for the experiment. The result is representative of five independent clones, which gave similar results. They were cultured with CH27 B cells in the presence of antigen, as described in Fig. 2A. Original magnification, ×100. (B) Conjugate formation of 3A9 T cells (green) and CH27 B cells as APC (red) loaded with (+HEL) or without (No antigen) antigen. T cells were mixed with an equal number of APC and incubated for 30 min at 37°C. Nonspecific aggregates were disrupted by vortexing, and the samples were analyzed by flow cytometry. A representative set of two-dimensional plots is shown. The number in each plot is the percentage of conjugates. (C) Inhibition of IL-2 production by Rap1N17 and SPA-1. 3A9 T cells transfected with the vector alone, Rap1N17, or Spa-1 were incubated with CH27 B cells as described in Fig. 2A, and the supernatants were harvested for IL-2 measurement. An optical density at 437 nm of 1 was equal to 0.25 ng of recombinant mouse IL-2/ml. Bars represent the average and standard error of two representative experiments performed in triplicate with each of five clones.