Alloreactive CCR2^-/- CD8⁺ T cells have intact proliferation, activation, IFN-γ production, and cytolytic activity. (A) MLR with WT and CCR2^-/- CD8⁺ T-cell effectors and irradiated C3FeB6F1 stimulators. Bars represent mean ± SEM for specific proliferation of 12 replicate wells from one representative experiment out of 3; cpm indicates counts per minute. (B,C) Sublethally irradiated (900 cGy) C3FeB6F1 mice received CFSE-labeled WT or CCR2^-/- T cells. Recipient spleens were harvested after 72 hours for FACS analysis. Data shown are from one representative mouse out of 4 mice from 2 experiments. (B) Histogram overlays for CFSE-labeled WT (black line) and CCR2^-/- (gray area) donor CD8⁺ T cells. Markers indicate nonproliferative cells (I), slow-proliferative cells (II), and fast-proliferative T cells (III). (C) Dot plots of CFSE and CD44 expression on WT and CCR2^-/- donor CD8⁺ T cells. (D-G) Lethally irradiated (1300 cGy) C3FeB6F1 received 5 × 10⁶ TCD WT BM cells in combination with 3 × 10⁶ WT (▪) or CCR2^-/- (□) CD8⁺ T cells. (D) Recipient spleens were harvested at day 7 for FACS analysis of CD25, CD44, and CD62L expression. Shown is the mean ± SEM for 4 mice per group. (E) Serum cytokine levels were measured by ELISA at indicated time points. Shown is the mean ± SEM for 8 to 12 mice per group per time point. (F) Recipient spleens were harvested at day 7 and restimulated for 12 hours with irradiated TCD C3FeB6F1 splenocytes. Cells were subsequently stained for intracellular IFN-γ. Shown is the mean ± SEM for 4 mice per group. (G) Mice were killed on day 7, and splenocytes were used as effectors in a ⁵¹Cr cytotoxicity assay. Targets were allogeneic 32Dp210 and third party P815. Shown is the mean specific lysis ± SEM for 4 mice per group.