Recombinant galectin-9 induces apoptosis in EBV-specific CD4⁺ T cells. (A) Intense membrane expression of Tim-3 is detected on 4 EBV-specific CD4⁺ T cell clones of Th1 subtype but not on polyclonal CD4⁺ T cells sorted directly from PBMCs (CD4⁺ PBMC; gray curve, control staining). EBV-specific clones are directed against the EBV-proteins EBNA3C (GB3C), gp350 or BLLF1 (JM1H2), EBNA1, and BZLF1. (B) A recombinant modified form of galectin-9 with increased stability (Gal-9 NC) induces apoptosis in JM1H2 CD4⁺ T cells with an ID₅₀ of approximately 100 pg/mL (apoptosis was assessed according to the percentage of annexin V–positive cells). (C) Recombinant wild-type galectin-9 (gal-9 WT) at a concentration of 100 pg/mL induces apoptosis in GB3C T cells. Induction of apoptosis is suppressed by preincubation of the T cells with 10 μg/mL blocking anti–Tim-3 monoclonal antibody, but not a control IgG.