CX₃CR1 is required for T cell retention in chronically inflamed skin. (A) Equal numbers of LACK-specific CX₃CR1^gfp/gfp and CX₃CR1^+/gfp Th2 or Th1 cells were coinjected into WT recipient mice at day 0. Recipients were sensitized with LACK at days 1 and 4 and analyzed at days 3 and 7. Donor cells were analyzed by flow cytometry 3 d later in inguinal draining LNs (dLN) after staining with antibodies against CD45, CD4, Thy1.1, Thy1.2, annexin-V, and 7-AAD. Data show mean frequencies of annexin-V⁺ cells among donor cells ± SEM. (B) CX₃CR1^+/gfp and CX₃CR1^gfp/gfp Th2 cells were infected with a Bcl2 or an empty retroviral vector. Equal numbers of transduced cells were coinjected into recipients that were further sensitized with one round of LACK sensitization. Data show the mean of donor cell frequency in skin (bottom) and inguinal draining LN (top) of individual mice in one representative experiment out of two (n = 5 mice per group). *, P < 0.05. (C) AD was induced in WT mice as described in the legend of Fig. 1, and equal numbers of LACK-specific CX₃CR1^gfp/gfp and CX₃CR1^+/gfp Th1 (top) or Th2 (bottom) cells were coinjected at day 41. Donor cells were analyzed in skin, draining LN, and spleen by flow cytometry. Data show representative flow cytometry profiles. Numbers indicate frequencies ± SEM of donor Th1 (top) or Th2 (bottom) cells among the CD4⁺ T cell population. One representative experiment out of two is shown (n = 12 mice per group). (right) 18 h before sacrifice, recipient mice were injected with BrdU and donor cells were analyzed by flow cytometry after staining with anti-BrdU, anti-Thy1.1, anti-Thy1.2, anti-CD4, or anti-CD45 antibodies. Data show representative cytometry profiles, and numbers indicate the mean (n = 12 mice). One representative experiment out of two is shown. (D) Equal numbers of LACK-specific CX₃CR1^gfp/gfp and CX₃CR1^+/gfp effector cells were coinjected into WT recipient mice at day 0. Recipients were sensitized with LACK at days 1 and 4. At day 4, some recipient mice topically received LACK together with CX₃-AT. 3 d later, the frequencies of donor cells were analyzed in skin and draining LNs by flow cytometry. Data show mean of donor cell frequency of individual mice in one representative experiment out of two (n = 5 mice per group). *, P < 0.05. (E–G) AD was induced as described in the legend of Fig. 1. During the last round of sensitization, some mice also received CX₃-AT (50 µg/mouse) through the patch together with LACK as indicated in the figure. (E) May-Grünwald Giemsa staining of skin sections. (F) Epidermal thickness. (G) Mast cell and eosinophil numbers. Data for E–G show mean of cell numbers in one representative experiment out of two (n = 6 mice per group). *, P < 0.05. (H–J) CX₃CR1^gfp/gfp mice were injected with polyclonal naive WT or CX₃CR1^gfp/gfp (KO) CD4⁺ T cells 24 h before AD induction. During the last round of sensitization, mice also received CX₃-AT (50 µg/mouse) through the patch together with LACK as indicated in the figure. (H) Epidermal thickness. (I and J) Mast cell and eosinophil numbers. Data for H–J show mean of cell numbers in one representative experiment (n = 5 mice per group). *, P < 0.05. Error bars indicate SEM.