(A) Surface staining of CD4 and CD8 on Ctrp4^–/– or WT thymocytes. Numbers in quadrants indicate the percentages of different stage cells, including CD4^–CD8^– DN, CD4⁺CD8⁺ DP, CD8^–CD4⁺ single-positive, and CD4^–CD8⁺ single-positive T cells (n = 10 animals per group from 1 representative experiment of 3). (B) Flow cytometry analysis of the transition between the different populations of DN T cell precursors in the thymus, which were marked by the differential expression of CD44 and CD25. DN1:CD44⁺CD25^–, DN2:CD44⁺CD25⁺; DN3:CD44^–CD25⁺; DN4: CD44^–CD25^–. (C and D) Representative plots showed the percentages of naive (CD44^loCD62L^hi) and memory/effector (CD44^hiCD62L^lo) CD4⁺ T cells (C) and CD8⁺ T cells (D) in the spleens of Ctrp4^–/– or WT mice. (E) Flow cytometry analyses of Th1 (IFN-γ⁺) and Th17 (IL-17A⁺) effector T cells in the spleens of Ctrp4^–/– and WT mice (n = 5/group). Data are represented as the frequency of CD4⁺CD44⁺ cells. (F) Gene expression of Il17a, Il17f, Rorc, or Ctrp4 mRNA in CD4⁺ T cells from Ctrp4^–/– or WT mice (n = 5/group) were analyzed by quantitative real-time PCR. Values were normalized against Gapdh. Data are represented as mean ± SEM and are from 1 of 3 independent experiments with similar results. Statistical significance was determined using 2-tailed, unpaired Student’s t test or Mann-Whitney U test as appropriate after assessing for distribution. *P < 0.05; **P < 0.001.