β2TTT/AAA integrin knock-in CD4 T-cell activation in vitro and in vivo is normal. (A) Purified naïve CD4 T cells were activated in vitro using soluble anti-CD3 (2.5 μg/mL) plus IL-2 (20 ng/mL), plate-bound anti-CD3 (2.5 μg/mL at 37°C for 4 hours), or phorbol ester (PdBu, 50 nM). WT (solid line) and KI (dashed line) T-cell activation was measured 24 hours later by expression of the typical activation markers: CD69, CD44, CD25, and CD62L. Histograms are representative of N = 4. (B) IL-2 production by WT and KI T cells after 24-hour stimulation with plate-bound anti-CD3 or PdBu. Data are pooled from N = 4, with mean ± SEM displayed. (C) WT and KI CD4 T cells were labeled with CFSE before activation with soluble anti-CD3 plus IL-2. Proliferation was measured by flow cytometry. Data are pooled from N = 2 and are representative of N = 4. (D) WT and KI mice received an intravenous adoptive transfer of LPS-matured, peptide-loaded WT DCs, and the antigen-specific CD4 T-cell response in the spleen was measured using MHC class II tetramers 7 days later. Data are pooled from 2 independent experiments, with 5 mice per group per experiment. Each data point represents an individual mouse, with mean indicated. The control group received no adoptive transfer. Isotype shows staining of the WT immunization group with an irrelevant peptide-tetramer. Student t test was used to calculate P values. NS, not significant.
