T-Cell Proliferation, Memory, and Effector Phenotypes

Purified T-cells were subjected to CFSE-based proliferation assay, as previously reported (7, 10). Briefly, purified T-cells from immunized and re-challenged mice were labeled with CFSE (5 µM) by incubating for 20 min at 37°C in a 5% CO₂ humidified atmosphere. CFSE labeled T-cells (0.5×10⁶) were co-cultured with APCs (0.5×10⁶) and stimulated with rMOMP (5 μg/mL) in round-bottom polypropylene tissue culture tubes and incubated for 120 h at 37°C. After incubation, the cells were harvested and stained using CD3-APC-Cy7, CD4-PerCP-Cy5.5, CD62L-APC, and CD44-PE to evaluate T-cell proliferation and memory (CD44^high CD62L^high) and effector (CD44^high CD62L^low) phenotypes. Stained cells were washed, fixed, and data were acquired on a BD LSR II flow cytometer and analyzed using FCS Express 6 FLOW (De Novo Software, Pasadena, CA). Gating on CFSE⁺ T-cells was used for the selection of CD3⁺CD4⁺ T-cell populations. Histogram fluorescence intensities were used to quantify the proliferating and resting T-cells amongst the total CFSE⁺CD3⁺CD4⁺ T-cells.