Figure 5. TNFR2 deficient naïve CD4 cells are less proliferative to TCR stimulation in vitro.

Naïve CD4 cells (CD4⁺CD25⁻CD45RB^hi) were flow-sorted from WT or TNFR2^−/− mice. The cells were seeded into a 96-well plate at 5 × 10⁴ cells per well. Aliquot of cells were stained with CFSE. The cells were stimulated with plate-bound anti-CD3 antibody (A,C) or with soluble anti-CD28 antibody (B,D). The proliferation of cells was evaluated either with a ³H thymidine incorporation assay after 72 hours incubation (A,B), or analyzed by FACS after 120 hours incubation (C,D). Grey histogram represent TNFR2^−/− cells, open solid line histogram represent WT cells. Typical FACS data are shown on the left and summary (N = 3, MFI of CFSE expression) was shown on the right. (E,F) WT or TNFR2^−/− naïve CD4 cells were stimulated with APCs either from WT mice (E) or from TNFR2^−/− mice (F) in the presence of 1 μg/ml of anti-CD3. The proliferation was evaluated 72 hours after incubation with a ³H thymidine incorporation assay. Data shown are representative of three separate experiments with similar results. Comparison of the difference between two groups, *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed Student t Test).