CCR6^–/– mice have a markedly diminished DTH response to allogeneic BALB/c splenocytes. (a) CCR6^–/– (n = 10; circles) and CCR6^+/+ (n = 11; squares) animals were sensitized by intravenous injection of 10⁶ BALB/c splenocytes. Five days later, mice were challenged by injecting 13 × 10⁶ BALB/c splenocytes into their right footpads, and swelling was measured 24 hours later. Individual data and the mean value for each group are presented. The two-tailed t-test value for these data was P = 0.000016. (b) In vitro MLR to allogeneic BALB/c splenocytes. Lymphocytes were prepared from IAB LNs from CCR6^+/+ (filled squares) and CCR6^–/– mice (filled circles) and cultured in 96-well plates (2 × 10⁵ cells/well) with increasing amounts of stimulator allogeneic BALB/c splenocytes. After 72 hours, cultures were pulsed with ³H-thymidine for 24 hours and cell proliferation was estimated. Background proliferation is also shown (open symbols). Each point represents the average value of two separate groups, each consisting of pooled LNs from two mice. Assays were performed in triplicate. (c) Adoptive transfer of sensitized CCR6^+/+ CD4⁺ T cells to CCR6^–/– mice restores their ability to produce a DTH response. Unsensitized CCR6^+/+ and CCR6^–/– mice were adoptively transferred with 2 × 10⁷ CD4⁺ T cell–enriched preparations purified from sensitized CCR6^+/+ and CCR6^–/– donors, as indicated. T cells from CCR6^–/– IAB LNs were also allowed to proliferate in an in vitro MLR assay with BALB/c splenocytes before being injected to CCR6^–/– hosts (++). After 16 hours, animals were challenged with 13 × 10⁶ BALB/c splenocytes in footpads, and swelling was measured 24 hours later.