LNs contain heterogeneous subsets of Lin^– DN cells. (A) To estimate the proportion of cells with DN1-DN4 phenotype, lymphoid cells from the thymus, wt LNs, and OM⁺ LNs were stained for CD25, CD44, and lineage markers (CD3ϵ, CD8α, CD8β, CD11b, CD45R/B220, Ly6C, Ly6G, NK1.1, TER-119, TCRβ, and TCRγ). Numbers in the various quadrants correspond to percentage of Lin^– cells stained with CD25 and CD44. (B) Number of cells with DN1-DN4 phenotype in the 3 lymphoid organs (mean ± SD; n = 3). (C) Number of c-Kit^–, c-Kit^lo, and c-Kit^hi DN1 phenotype cells per 10⁶ lymphoid cells. Gated Lin^–CD44⁺CD25^– cells were stained for c-Kit (mean ± SD; n = 3). No DN1 phenotype c-Kit^hi cells were detected in the wt and OM⁺ LNs (*). (D) Expression of c-Kit on DN1 phenotype cells (Lin^–CD44⁺CD25^–) and of Sca-1 on both c-Kit^lo and c-Kit^hi subsets are shown for each organ. Numbers correspond to percentages of cells in each quadrant. Data in panels A and D are representative of 1 experiment out of 3.