FIG. 5.

Characterization of αβTCR, γδTCR, and CD69 expression in thymocytes from Prep1^i/i mice. (A and B) Flow cytometric analysis of intracellular TCRβ expression in thymocytes from Prep1^i/i and Prep1^+/+ mice. Purified DN cells were surface stained with PE-conjugated anti-CD44 and APC-conjugated anti-CD25 antibodies. DP thymocytes were directly gated after surface staining with PE-conjugated anti-CD4 and APC-conjugated anti-CD8 antibodies. Viable cells were gated according to their forward scatter (FCS) and side scatter (SCS) characteristics. Surface TCRβ was preblocked with excess unlabeled, purified MAb. Thymocytes were fixed, permeabilized, and stained intracellularly with FITC-conjugated anti-TCRβ antibody (gray histograms) or with control immunoglobulin (thin lines). (A) One example of DN4 gated thymocytes is shown. The percentage of icTCRβ-positive thymocytes is shown above the bar. (B) Average percentages of DN3, DN4, and DP thymocytes positive for icTCRβ from six Prep1^i/i or wild-type control mice. (C and D) Flow cytometric analysis of surface αβTCR and γδTCR expression in total thymocytes from Prep1^i/i and Prep1^+/+ mice. (C) Total thymocytes were stained with FITC-conjugated anti-TCRβ and APC-conjugated anti-TCRγδ antibodies. Viable cells were gated according to their FCS and SCS characteristics. The percentages of αβTCR^high and γδTCR⁺ cells are shown. One representative experiment of eight independent experiments is shown. (D) Average percentage of thymocytes positive for αβTCR (αβ) and γδTCR (γδ) for eight mice each for Prep1^i/i, heterozygous, and wild-type control mice. Values that were significantly different (P < 0.001) are indicated (***). (E) Total thymocytes were stained with FITC-conjugated anti-TCRβ and PE-conjugated anti-CD69 antibodies. Viable cells were gated according to their FCS and SCS characteristics. The percentages of CD69⁺αβTCR^high and CD69⁺αβTCR^low cells are shown in the squares. One representative experiment of eight independent experiments is shown.