TNF-α production by alloreactive CD8⁺ T cells correlates with in vivo cytotoxic activity in mice treated with costimulation blockade. C57BL/6 mice were (A) left naive, (B) injected with BALB/c DST, or (C-D) treated with anti-CD154 mAb and a BALB/c DST. Six days later peripheral blood samples were stimulated in vitro with BALB/c (H2^d) splenocytes and then stained for cell surface markers and for TNF-α. The values shown in the dot plots represent the percentage of CD8⁺ T cells producing TNF-α. One day after the cytokine assay, CFSE-labeled BALB/c (H2^d) and C57BL/6 (H2^b) splenocytes were transferred at equal ratios into the mice, and the survival of each population was examined 20 hours later. All recipient mice were treated with a mAb specific for NK1.1 to deplete NK cells. Survival of the BALB/c splenocytes is shown in representative histograms, and the values represent the percent specific lysis of BALB/c splenocytes. A summary of TNF-α production and percent specific lysis is shown in panel D (n = 15). The data are representative of 3 separate experiments.