Fig. 3: Characterization of TIM-3/Gal-9/PD-1 tri-molecular interaction.

a, b TIM-3 ECD binding to plate-immobilized GST-Gal-9C (a) or GST-Gal-9N (b) in the presence of increasing concentrations of PD-1 ECD. c PD-1 ECD binding to plate-immobilized TIM-3 ECD or Gal-9. d TIM-3 ECD binding to plate-immobilized PD-1 in the presence of increasing concentrations of Gal-9. e Duolink assay of PD-1 and TIM-3 association in Gal-9 KO Jurkat cells co-expressing the two receptors with or without Gal-9. Scale bar: 10 μm. Dashed lines represent mean values; error bars represent SD. Statistical differences were assessed using unpaired two-tailed t-tests. n = 254 cells examined for each group over two independent experiments. ****P < 0.0001. f Jurkat cells expressing PD-1 (myc tagged) and TIM-3 (3xFlag tagged) individually or together were incubated with or without 2 μg/ml exogenous Gal-9 followed by IP/western blotting with indicated antibodies. g, h IP/Western analysis of Jurkat cells expressing TIM-3 and 3xFlag tagged wildtype PD-1 or PD-1(N116Q) mutant, individually or in indicated combinations, in the presence or absence of lactose. i–k Jurkat cells expressing PD-1 (i) or TIM-3 (j) or both (k) were incubated with or without Gal-9, and then lysed in a detergent buffer and centrifuged. Protein levels in the supernatants (S) and pellets (P) were determined by western blotting with the indicated antibodies. l Schematic diagram showing TIM-3/Gal-9/PD-1 tri-molecular interactions. TIM-3 and PD-1 dimerize through their intracellular domains. Gal-9 crosslinks TIM-3/PD-1 dimers with its N-CRD (green) and C-CRD (orange) to form galectin/glycoprotein lattices. Data are representative of two (a–i) or three (j, k) independent experiments. Source data are provided as a Source data file.