Regulatory T cells are not responsible for low alloresponse mediated by memory phenotype T cells. (A) Sorting strategy for CD25⁻ memory T cells. We first gated on CD25⁻ T cells. The rest of the sorting strategies for naive and memory phenotype T cells are the same as those described in Figure 1A. Shown in the third column are cells from region A in the second column. Numbers represent percentages of parent gates. (B) Phenotypes of sorted T-cell subsets. Shown in the third column are cells from region A in the second column. (C) Mixed lymphocyte reaction (C57BL/6–anti-BALB/c). Proliferation was determined by ³H-thymidine incorporation. Results represent mean ± SD of triplicate wells. Data are representative of 2 experiments with similar results. *P < .01, memory versus other groups; #P < .01, naive versus bulk. (D) In vivo GVHD (C57BL/6→BALB/c) assay. Sorted T-cell subsets were transplanted into lethally irradiated recipients along with T-cell–depleted BM cells. Mice were monitored for the development of GVHD. Data were pooled from 2 independent experiments. For survival, P < .05 (T_CM versus naive). For body weight, P was not significant for TCD BM only versus T_CM. (E) Coculture assay. T cells with putative regulatory activity from C57BL/6 mice were added to the 5-day mixed lymphocyte cultures (C57BL/6–anti-BALB/c). Cocultured cells were added to the responder cells at 2 different ratios. Proliferation was determined by ³H-thymidine incorporation. Results represent mean ± SD of triplicate wells. Data are representative of 3 experiments with similar results. *P < .05, bulk versus medium.