Extended Data Fig. 1.

(a) Thymidine incorporation proliferation assay with purified CD4⁺ T cells from spleen and lymph nodes of LAG3^+/+ and LAG3^−/− mice stimulated with plate bound CD3ε and CD28 Abs for 72h in the absence (a) or presence of isotype control or anti-LAG3 blocking antibodies (b). (c) LAG3 expression of LAG3^+/+ CD4⁺ T cells, isolated as above following activation with CD3ε and CD28 Abs at 48hr. Cells were gated on a live lymphocyte gate, and CD4. (d) Diagram depicting TIRF microscopy analysis on stimulating lipid bilayer. (e) Western blot analysis and quantification of TCR–proximal signaling events in Lag3^+/+ or Lag3^−/− CD4⁺ T cells stimulated for 48 hr with CD3ε and CD28 Abs to induce LAG3 expression, allowed to rest in the presence of IL-2 and then re-stimulated for 5, 15 and 30 min with anti-CD3. Quantitation determined by percent density of Lag3^−/− compared to Lag3^+/+. Data in (a) represents the mean of n=16 (LAG3^+/+) and n= 16 (LAG3^−/−) individual mice. Data in (b) represents the mean of n=6 (LAG3^+/+) and n= 6 (LAG3^−/−) individual mice. Data in (e) represents the mean of 4–8 individual experiments. Statistical analysis performed using Student’s unpaired two-sided t test with P values noted in figures.