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Figure S6.Antigen-specific T cells up-regulated Tim-3 expression over a 5-d stimulation. PBMCs from seven chronically HIV-1–infected individuals were stained with CFSE and stimulated with pooled HIV-1–Gag/Nef peptides or pooled CEF peptides for 5 d with the addition of either 2 mg/ml sTim-3 or an expression control. At the end of this period, cells were stained with monoclonal antibodies to CD3, CD8, CD4, and Tim-3. Shown are flow cytometry plots of CFSE by Tim-3 after gating on CD3+CD8+ or CD3+CD4+ viable cells in a representative HIV-1–infected individual. In response to antigenic stimuli, a population of Tim-3–expressing cells emerges. All cells that have proliferated (diluted CFSE) in response to CEF express high levels of Tim-3. The expression of Tim-3 in response to prolonged antigenic stimuli provides an opportunity for limiting immune responses by the engagement of Tim-3 ligands. In response to HIV-1–Gag/Nef, a population of Tim-3–expressing cells that fail to divide is observed, potentially because of preexisting Tim-3 expression on HIV-1–specific T cells as demonstrated in Fig. 2. Addition of sTim-3 restores the proliferation of a subset of these Gag/Nef-specific T cells. An additional observation from this experiment is that in both the CD8+ and CD4+ populations, incubation with sTim-3 results in higher levels of Tim-3 expression on Tim-3+ cells. This is consistent with an effective block of the Tim-3–Tim-3L interactions, resulting in less cycling of Tim-3.