Figure 1.

A greater portion of LAG-3, compared with CD4, is stored intracellularly. (A) Freshly isolated CD4⁺ T cells from C57BL/6 mice were activated with plate-bound anti-CD3 and anti-CD28 for 72 h. Activated T cells were harvested and treated, or not, with pronase to remove surface CD4 and LAG-3. Cells were fixed and permeabilized before staining for intracellular expression analysis following pronase treatment. Treated cells were stained with anti-CD4 or anti-LAG-3 and analyzed by flow cytometry. (B) Normal naïve CD4⁺ T cells isolated from spleens and lymph nodes of C57BL/6 mice were activated with plate-bound anti-CD3 and anti-CD28 for 72 h. T cells were collected, stained for CD4 and LAG-3 and analyzed by flow cytometry. For surface plus intracellular staining, cells were fixed and permeabilized before staining. Percentage of intracellular CD4 or LAG-3 was calculated by dividing total (surface plus intracellular) CD4 or LAG-3 from the amount of intracellular CD4 or LAG-3. Results are expressed as mean +SEM of three independent experiments. (C and D) LAG-3⁺ CD4⁺ 3A9 T-cell hybridomas and OTII TCR transgenic T cells (previously stimulated with APC plus OVA326–339 peptide) were surface-biotinylated and then treated with pronase, or not. Whole cell lysates were immunoprecipitated with (C) anti-CD4 mAb or (D) anti-LAG-3 mAb and probed either with streptavidin, anti-CD4.D1/2 antisera (upper panels) or anti-LAG-3.D1 antisera (lower panels).