Rat anti cd4 rm4 5

Spleen, popliteal lymph node (pLN) and joint footpad of CHIKV-infected mice were harvested at 6 dpi and 7 dpi. Isolation of splenocytes, pLN and joint footpad cells were processed as described4 (link)21 (link)26 (link). Isolated cells from spleen, joint footpad and pLN were first stained with Live/dead Fixable Aqua Dead Cell Stain Kit (1:400) (Life Technologies) for 20 min. Cell were then washed and resuspended in staining buffer (2% fetal bovine serum in PBS). Staining was performed using rat anti-CD45 (30-F11, BD Pharmingen), rat anti-CD4 (RM4–5, BD Pharmingen), rat anti-CD3 (17A2, Biolegend), rat anti-CD25 (PC61.5, eBioscience), hamster anti-CTLA-4 (UC10-4B9, eBioscience) and rat anti-CD44 (IM7, eBioscience) antibodies for 15 min. Stained cells were then washed with PBS and fixed using IC Fixation Buffer (eBioscience). Intracellular staining of rat anti-Foxp3 (FJK-16s, eBioscience) was done according to manufacturer’s instructions. Data acquisition and analyzes were done as described above.

Murine splenocytes or pLN were isolated as described above. Isolated leukocytes were resuspended in complete RPMI medium (10% fetal bovine serum (FBS) in RPMI medium) containing 0, 1, 10 or 100 ng/ml murine IL-2 (Peprotech). Stimulation was performed at 37 °C for 30 mins. Stimulation was terminated by the addition of 1X BD Phosflow^TM Lyse/Fix Buffer (BD Bioscience) at 37 °C for 10 mins. Permeabilization was performed using Perm Buffer III (BD Bioscience) on ice for 30 mins. Cells were then washed 3 times with staining buffer to completely remove traces of Perm Buffer III before proceeding to stain with rat anti-CD4 (RM4–5, BD Bioscience), rat anti-CD25 (7D4, eBioscience), rat anti-Foxp3 (FJK-16s, eBioscience) and anti-pSTAT5 (pY694) (BD Bioscience) antibodies for 1 hr. Human peripheral blood mononuclear cells (PBMCs) were obtained from blood of healthy donors using Ficoll-Plaque (GE Healthcare)67 (link). Stimulation of PBMCs was done as above except that human IL-2 (Peprotech) was used and stimulation was done at 37 °C for 15 mins instead. Staining was done using mouse anti-human CD3 (UCHT1, BD Bioscience), mouse anti-human CD4 (SK3, BD Bioscience), mouse anti-human CD45RA (HI100, eBioscience), rat anti-human Foxp3 (PCH101, BD Bioscience) and mouse anti-pSTAT5 (pY694, BD Bioscience) antibodies for 1 hr.