Figure 4. ACAID suppresses CTL function, and this suppression does not appear to be directly mediated by CD4⁺CD25⁺FoxP3⁺ Treg. Splenocytes were harvested on days 4, 8, and 12 (n=3 per time point) post immunization from positive control mice (A) or ACAID expressing mice (B). Following their alloantigen stimulation in vitro, responder cells were plated with allospecific eGFP⁺ target cells at ratios of 2.5, 5, and 10 to 1 (responder to target) in triplicate wells. Baseline activity was obtained with in vitro allostimulated naïve splenocytes. Flow cytometry was used to enumerate target cell survival, values that are inversely proportional to CTL function. An asterisk indicates statistical significance as calculated by ANOVA. C: ACAID-induced Treg do not directly suppress CTL function. Baseline CTL cultures were incubated at a 2.5:1 ratio and splenic Treg magnetically sorted from ACAID-induced mice were added to CTL assays. No statistical (NS) difference was observed as calculated via Student’s t-test.