Fig. 3. TIM-3⁺ regulatory T cells suppress better than TIM-3^— regulatory T cells.

Regulatory (CD4⁺CD25^highCD127^low/—; Tregs) T cells were sorted by flow cytometry and activated with anti-CD3/anti-CD28 for 3 days. After 3 days TIM-3⁺ and TIM-3^— regulatory T cells were resorted by flow cytometry and responder (CD4⁺CD25^low/— CD127⁺; Tresp) T cells were sorted in parallel. CFSE-labeled Tresp were cocultured with TIM-3⁺ or TIM-3^— Tregs at different Tregs:Tresp ratios ranging from 1:2 to 1:8. At day 3, cells were collected, stained for viability and fixed. A. Proliferation of unstimulated (upper left panel) or stimulated (upper right panel) Tresp alone and Tresp and TIM-3⁺ or TIM-3^— Treg co-cultures (lower panels left and right respectively), one representative experiment. B. Percentage of suppression as per the following formula: %suppression = [1-(Tresp in coculture / Tresp alone)] x 100. Statistical analyses of 3 independent experiments with two donors per experiment.