Composition and phenotype of CD45–thymic stromal cells changes with age. At various time points, CD45– stromal cells were stained for (A) MHC II versus EpCAM, (B) MHC II versus MTS-15, and (C) MHC II versus Ly51. Regions indicate CD45– stroma composed of TECs (MHC IIhi and MHC IIlo at adult time points), fibroblasts, cTECs (MHC II+/Ly51+), and mTECs (MHC II+/Ly51–). All dot plots are gated on CD45– thymic stromal cells isolated from at least 3 thymi pooled at the ages indicated and are representative of 2 to 5 experiments. Bar graphs of the ratios of (D) TEC/fibroblasts, (E) mTEC/cTEC, and (F) MHC IIhi/MHC IIlo TECs in 4-week-old and middle-aged (12-month-old) thymi. Means and standard errors are plotted, and groups were compared using a Mann-Whitney rank sum U test. (*P < .05, **P < .01, ***P < .001). (G) Cytospins of purified cTECs, mTEChi subset, and mTEClo subset from 4- to 6-week-old mice stained with anti-K5 (green) and anti-K8 (red). (H) The mean proportion of TEC subsets that expressed K5, K8, or both (double positive, DP) with standard error from 3 experiments.