Fig 1: Biochemical effects of aLag-3 and aPD-1 Ab treatment. (A) 2D12 T-cell hybridomas expressing Lag-3 and PD-1 were stimulated with MCC peptide and DC-1 cells with or without addition of 20 nM of control Ab (ctrl), aLag-3 (28G10), aPD-1 (DX400), the cocktail of aLag-3 and aPD-1 Abs, or PMA plus ionophore (Iono+PMA). IL-2 production in the culture supernatant after 24 hours was measured. (B) 2D12 T-cell hybridoma expressing Lag-3 and PD-1 was stimulated with MCC peptide, and DC-1 cells with or without blocking Abs were lysed at the indicated time after stimulation. Tyrosine phosphorylation levels were analyzed by Western blotting using indicated anti-phospho-tyrosine Abs. The quantitation of the blots is shown on the right. * and ** mean p < 0.1 and 0.01, respectively. MCC, moth cytochrome c; Ab, antibody; PMA, phorbol myristate acetate.
Fig 2: Effects of bispecific Lag-3/PD-1 Abs on Lag-3 and PD-1 cluster formation and function. (A) Naïve AND-tg T cells were stimulated with MCC and DC-1 cells with or without control IgG, the cocktail of aLag-3 and aPD-1 Abs, bsAb, or bsVHH. IL-2 production in the culture supernatant after 24 hours was measured. * and ** mean p < 0.1 and 0.01, respectively. (B) Representative images of AND-tg T cells co-expressing Lag-3-GFP, PD-1-CFP, and TCR upon Ag stimulation on planar bilayer in the presence of control Ab, the cocktail of aLag-3 and aPD-1 Abs, bsAb, or bsVHH are shown. Abs, antibodies; MCC, moth cytochrome c; TCR, T-cell receptor.
Fig 3: Effects of aLag-3 blocking Abs on Lag-3 cluster formation. (A) The Lag-3-GFP expressing AND-tg/Lag-3 KO T cells were pre-incubated with 20 nM or 200 nM aLag-3 Ab (28G10) and examined on planer bilayers. The representative images of Lag-3-GFP (green) and TCR (red), merged, and background subtracted merged with co-localized (with TCR-MC) image (white) at 60 sec after activation started are shown. (B) The peak value of total cluster intensity in T cells from panel (A) is plotted. (C) IL-2 production by naïve AND-tg T cells stimulated by graded concentrations (0.01 μM to 10 μM) of MCC and APC in the presence or absence of aLag-3 Ab at 48 hours are plotted. (D–F) Lag-3-GFP expressing AND-tg T cells were treated with a panel of aLag-3 Abs (28G10, C9B7W, 1F3, 9B7, and 17D8). (D) Representative images of Lag-3 cluster and TCR-MC formation after Abs were added at time 0, and the images were taken 60 sec after activation. (E) The peak value of total cluster intensities of panel (D) is plotted. (F) IL-2 production by T cells at 48 hours in the presence of aLag-3 Abs is shown. The leftmost columns represent unstimulated conditions. Experiments in panels (C, F) were performed in triplicate, and the average ± SD is shown. * and ** mean p < 0.1 and 0.01. aLag-3, anti-Lag-3; Abs, antibodies; TCR, T-cell receptor; TCR-MC, T-cell receptor microcluster; MCC, moth cytochrome c; APC, antigen-presenting cell.
Fig 4: Simultaneous analysis of Lag-3, PD-1, and TCR cluster formation. (A) Representative images of Lag-3-GFP, PD-1-CFP, TCR, co-localization of Lag-3-GFP and TCR, co-localization of PD-1-CFP and TCR, and co-localization of PD-1-CFP and Lag-3-GFP following treatment with control IgG (ctrl), aLag-3 Ab (28G10), aPD-1 Ab (DX400), or the cocktail of aLag-3 Ab and aPD-1 Ab (20 nM) are shown. (B) The peak values of total cluster intensities of the images in panel (A) are plotted. (C) IL-2 production by naïve AND-tg T cells upon stimulation with MCC (0 μM, 0.1 μM, 1 μM, or 10 μM) and APC in the presence of 20 nM of control IgG (ctrl IgG), aLag-3, aPD-1, or the cocktail of aLag-3 and aPD-1 Abs (20 nM each) for 48 hours are shown. Experiments were performed in triplicate, and the average ± SD is shown. * and ** mean p < 0.1 and 0.01, respectively, using Student’s t-test. The cocktail showed combination-enhancing effects. TCR, T-cell receptor; MCC, moth cytochrome c; APC, antigen-presenting cell.
Fig 5: Lag-3 forms clusters co-localized with TCR-MCs. (A) T cells from AND-tg/Lag-3 KO mice expressing Lag-3-GFP were incubated with MCC(K99A) peptide-loaded APC for 10 min and fixed. The images from side of conjugated cells were taken by confocal microscopy. Representative images of Lag-3-GFP (green), TCR stained by aCD3ε Ab (red), and merged, and the bright field (BF) revealed Lag-3 accumulation at immune synapse. (B) Lag-3-GFP-expressing AND-tg T cells were analyzed on planer bilayer, and the TIRF images every 10 sec (s) are shown. Lag-3-GFP formed clusters and co-localized with TCR-MCs (TCRβ). Single-colored clusters in panels (B, D, F) are expressed in gray for better resolution. (C) The total cluster intensity, a metric derived from cluster number and each cluster’s intensity, is plotted as a function of time for the images in panel (B) Lag-3-GFP is shown in green and TCRβ in red, and yellow denotes overlapping co-association. (D) 2D12 T-cell hybridoma cells expressing Lag3(ΔCP)-GFP were stained with H57 for TCR. Left: the images of Lag-3(ΔCP)-GFP (green) and TCR (red), merged on planer bilayer, are shown. Lag-3(ΔCP)-GFP showed intact cluster formation and co-localization with TCR-MCs. Right: statistical analysis of the peak total cluster intensity values, Lag-3-GFP, TCR, and co-localized clusters for Lag-3-GFP (gray) and Lag3(ΔCP)-GFP (black) are plotted. (E) IL-2 production from Lag-3-GFP or Lag-3(ΔCP)-GFP expressing 2D12 hybridoma cells. The cells were stimulated with 10 μM MCC peptide-pulsed DC-1 cells or PMA plus ionophore for 24 hours. Experiments were performed in triplicate, and the average ± SD is shown. **p < 0.01. Single-colored clusters in panels (B, D) as well as Figures 2A, D , 3A , 4A , 6B . Supplementary Figure 4A is expressed in gray for better resolution. TCR-MCs, T-cell receptor microclusters; APC, antigen-presenting cell; TIRF, total internal reflection fluorescence microscopy; MCC, moth cytochrome c; PMA, phorbol myristate acetate; Ab, antibody.
Supplier Page from Enzo Life Sciences, Inc. for LAG-3 (mouse):Fc (mouse), (recombinant)