Figure 4. CD8.4 enhances proximal signaling in OT-I thymocytes.

(A-G) Thymocytes from CD8WT or CD8.4 OT-I Rag2^−/−β2m^−/− mice were stimulated with 100 nM K^b-OVA, -Q4R7, or -Q4H7 tetramers or left unstimulated (ns). Results were normalized to the average of signal from unstimulated CD8WT or CD8.4 cells in each experiment. (A) Phosphorylation of TCRζ (Y142) was analyzed by flow cytometry on CD4+CD8+ population. Mean ± SEM, n=6. (B-C, G) Phosphorylation of LAT, ZAP70, and Erk was analyzed using phospho-specific Abs. Reprobing the membranes for total ZAP70, LAT, and Erk served as respective loading controls. (D-F) Phosphorylation of LAT, SLP76, and VAV in whole cell lysates was determined using anti-pTyr Ab and anti-actin Ab. as a loading control by Western blotting. Mean ± SEM, n=4. Statistical significance was tested using student's T test (1 tailed, unequal variance): * p ≤ 0.05, ** p <0.01. See also Figure S3. (H) CD8WT or CD8.4 OT-I DP thymocytes were loaded with Indo-1 and stimulated with 200 nM K^b-OVA, -Q4R7, or -Q4H7 tetramers or 1.5 μM ionomycine. Calcium mobilization was analyzed by flow cytometry. Ca²⁺ response index is shown (see Extended Matherial and Methods). A representative experiment from a total of 3 is shown.