Preparation of Naïve CD4 T cell suspension

To further investigate whether LFA-1 contributes to T cell differentiation and their functional responses, CD4 T cells were cultured with or without BIRT377 (500 ng/mL). A total of 20 mice (wildtype, FFID: IMSR_JAX:000664; 10 females and 10 males, 8–10 week-old) were compared in this study. In each experiment, 5 male and 5 female mice were used, with two repeat experiments (total of 10 male and 10 female mice). No handling occurred with these mice. Mice were sacrificed with CO₂ asphyxiation, followed by cervical dislocation. Under sterile conditions, spleens and lymph nodes (cervical, inguinal and brachial) were collected in tubes containing ice-cold PBS with 2% fetal bovine serum (FBS; Gibco, Thermofisher Scientific, MA, USA). Spleens and lymph nodes were disrupted using a micro-plunger to press the tissues through a 70 μm nylon mesh. Cells were centrifuged at 300 × g for 10 min at 4 °C and resuspended at 1 × 10⁸ nucleated cells/mL in PBS with 2% FBS and 1mM EDTA (ethylene diaminetetraacetic acid). Naïve CD4 T cells (CD4⁺CD44^lowCD62L^high) were isolated using EasySep^™ Naïve CD4 T Cell Isolation Kit, per manufacturer’s instructions (Stemcell Technologies, BC, Canada). In this technique, non-naïve T cells were labeled with biotinylated antibodies and magnetic particles, allowing for the collection of desired naïve T cells using an EasySep^™ magnet (Stemcell Technologies, BC, Canada). Live cells were counted on a hemocytometer.