Figure 3. SLP-76 T is required in mature T cells for up-regulation of activation markers and for proliferation.

Splenocytes isolated from SLP-76^cHET and SLP-76^cKO mice were left unstimulated (shaded histograms) or were stimulated overnight with soluble anti-CD3 (open histograms). Cells were surface stained with CD4, CD8, CD69 and CD25. Expression of CD69 (A) and CD25 (B) on CD4⁺YFP⁺ and CD8⁺YFP⁺ are shown. Representative of greater than five experiments. (C) Splenocytes were labeled with CFSE and stimulated in vitro for seventy-two hours then stained with CD4 and CD8. Proliferation measured by dilution of CFSE in CD4 gated (left panel) and CD8 gated (right panel) are shown. SLP-76^cKO cells are shown with a dotted line and SLP-76^cHET by solid line. Representative of two experiments (D) CFSE-labeled Thy1.2+ T cells from SLP-76^cHET and SLP-76^cKO mice were adoptively transferred into RAG2^−/−CD45^SJL mice. Suspensions taken from lymph nodes seven days after transfer gated on CD45^B6 donor, B220⁻ then CD4 (left) or CD8 (right) are shown. Representative of two experiments. (E) Splenocytes from SLP-76^cHet (dashed line) and SLP-76^cKO (solid line) were adoptively transferred into CD45^SJL and RAG2^−/− CD45^SJL recipients. BRDU was administered from during days 5 through 7. Lymph node suspensions gated on CD45^B6 donor, YFP⁺ then CD4 (left) or CD8 (right) are shown. Shaded histograms represent BRDU incorporation into cells transferred into non-lymphopenic CD45^SJL mice, where no LIP is expected. Representative of 2 experiments with a total of 9 recipient mice in each.