Figure 2.

All early T-cell developmental stages are found in the spleen of Tg8 mice. A) Spleen, bone marrow (BM) and liver tissues from 7-week-old Tg8 animals were homogenized and stained. DN cells were negatively gated on lineage (Lin = CD3/4/8/19/11c/F4/80/Gr1/FcεRI/TCRγδ), positively gated on Thy1.2⁺, and classified as DN1-DN4 with anti-CD25 and -CD44 antibodies. Data are representative of n = 4 mice for each genotype. B) DN1/DN2/DN3/DN4 cells from spleen of Tg8 mice identified as in (A) were stained with antibodies against pre-TCRα (pTα). Representative data from n = 4 mice. C) Spleen and bone marrow cells from 3-week-old Tg8 and WT animals were homogenized and stained. LSK cells were negatively gated on lineage (Lin = CD3/4/8/19/11c/F4/80/Gr1/FcεRI/TCRγδ), positively gated on cKit expression, and then analyzed for Sca1 expression. Data are representative of n=4 mice for each genotype. CD4⁺CD8⁺ cells D) and progenitor LSK cells E) are found at the bridging channels (BC) of the marginal sinus endothelia surrounding the peri-arteriolar lymphoid sheaths. Frozen spleen sections from 14-day-old Tg8 and WT mice were fixed and stained with anti-CD4, anti-CD8, anti-MADCAM, anti-c-Kit, anti-FcεRI and secondary antibodies conjugated with fluorescent dyes. Arrows indicate BCs, which are the discontinuities of the marginal sinus endothelia stained with MADCAM in both (D) and (E). Arrowheads indicate CD4⁺CD8⁺ (D) and LSK cells (E). Data are representative of n = 5 mice per genotype.