Figure 2. Secretory granule release in CTLs.

(A) CTLs were generated in a 3- to 6-day MLR. Release of granzyme A from CTLs was induced by cross-linking of the TCR with anti-CD3ε mAb for 4 hours. Enzymatic activity in supernatants was determined. Secretion is expressed as percentage of maximal activity, measured for Triton X-100 cell lysates. Data are mean ± SD of 7 independent experiments performed in triplicate. *P < 0.02, Student’s t test. (B) Cell surface translocation of CTLA4. CTLs were stimulated for 4 hours with plate-bound mAbs (anti-CD3ε, 5 μg/ml; anti-CD28, 3 μg/ml) (+CD3) or left untreated (-CD3). Cells were stained for CTLA4 and CD8 expression without permeabilization, and viable cells were gated for analysis of CTLA4 fluorescence by flow cytometry. One representative example of 6 independent experiments is shown. Gray histogram shows the isotype control. (C) Release of IFN-γ upon TCR activation. Supernatants from A were analyzed by ELISA. Data are mean ± SD of 6 independent experiments. (D) Cytolytic activity of CTLs (H-2^b haplotype) at different ratios of effector cells to a fixed number of allogeneic P815 target cells (H-2^d haplotype). Data represent mean ± SD of quadruplicate experiments. One representative experiment of 8 is shown.