Reduction of the DN3 precursor population in CCX-CKR^−/− mice. (A) Representative dot plots of DN subsets showing expression of CD25 and CD44 expression within the lineage negative compartment (defined as CD3⁻CD4⁻CD8⁻CD11c⁻CD45R⁻Gr1⁻). DN populations were further classified by CD117 expression, DN1 CD117⁺, DN2 CD117⁺, DN3 CD117^−/lo, and DN4 CD117⁻. Numbers adjacent to gates indicate percent positive in each. (B) Quantitation of proportions (top) and total number (bottom) of DN subsets. Data are pooled from 3 independent experiments: WT, n = 8; and CCX-CKR^−/−, n = 8. Two-tailed unpaired t test: *P = .011, **P = .0022. (C) Representative histogram of CD117 expression on DN3 cells (left) and quantitation of CD117 expression (right) from mice used in panel B. Two-tailed unpaired t test: **P = .0095. (D) Analysis of DN cell localization in thymus sections stained with anti-CD25 (red) and anti-CD44 cells (green) of WT (top left), CCX-CKR^−/− (top middle), and CCR7^−/− (top right). CD25⁺CD44⁺ cells appear yellow. Images below show colocalization of CD25⁺CD44⁺ cells (white) with CMJ indicated by dotted line. Representative images from 2 independent experiments: WT, n = 9; CCX-CKR^−/−, n = 10; and CCR7^−/−, n = 9. Scale bar represents 250 μm. (E) Quantitation of CD25⁺CD44⁺ cell distribution in thymus sections. (F) Enumeration of CD25⁺ cells in the cortex of thymic sections ± SD. One-way ANOVA with Bonferroni posttest: ***P < .0001. Two to 4 fields of view per thymus from those used in panel D were analyzed in panels E and F. (B-C) Bars represents mean values.