Fig 2: AMG 330-induced downstream signaling in T cells is strongly enhanced by CD86 expression on target cells. Kinetics of phosphorylation (% positive) of A Akt, B ERK1/2, and C ZAP70 in T cells after 1, 3, 5, 10, and 20 min of AMG 330-mediated engagement with different Ba/F3 cell constructs. Percentage of phosphorylated D Akt, E ERK1/2, and F ZAP70 in T cells after 10 min of engagement. Overlays of representative flow cytometry histograms after 10 min of engagement are shown. AMG 330 concentration = 5 ng/ml; E:T ratio = 1:1; n = 7–8; Error bars represent mean ± SEM; Statistical analysis: One-way ANOVA or Mixed-effects analysis with Dunnett's multiple comparisons test; ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Fig 3: AMG 330 induces TCR triggering characterized by CD45 exclusion from and CD33 clustering within the synapse. A Representative spinning disc confocal microscope images of AMG 330 (BiTE® molecule) and c BiTE molecule-mediated conjugates formed of a CD33-transduced Raji B cell and a reconstituted HEK-T cell. B Line profiles of CD45 (green), CD33 (blue), and AMG 330 (red) intensities across a conjugate interface equivalent to that shown in a representative image in panel A. C Total number of AMG 330-induced T-cell–CD33+ CD86± PD-L1± Ba/F3 cell conjugates after 20 min, assessed by flow cytometry. D Representative imaging flow cytometric analysis of AMG 330-induced T-cell–CD33+ CD86± PD-L1± Ba/F3 cell conjugation: brightfield (BF, gray), Hoechst staining (purple), Ba/F3 cell (GFP+; green), T cell (CD45+; magenta), LFA-1 (yellow), and overlay of Ba/F3, T-cell and LFA-1 channels. E Median intensity of LFA-1 accumulation at the interface of AMG 330-and c BiTE molecule-induced T-cell–CD33+ CD86± PD-L1± Ba/F3 cell conjugates. Statistical analysis: One-way ANOVA with Dunnett's multiple comparisons test; ns p > 0.05, *p ≤ 0.05

Fig 4: Lenalidomide reverses negative impact of IFNγ and TNFα on AMG 330-mediated T-cell function and synapse formation in co-cultures with primary AML samples. A Expression of CD33 (MFI), CD86 (% CD33+), and PD-L1 (% CD33+) on primary AML samples after pretreatment ± IFNγ and TNFα (PD-L1ind) for 48 h. B AMG 330-mediated cytolytic capacity, granzyme B expression, IFNγ and IL-2 secretion of HD T cells against non-pretreated and PD-L1ind primary AML samples. C Fold change of AMG 330-mediated cytolytic capacity, granzyme B expression, IFNγ and IL-2 secretion of HD T cells against PD-L1ind primary AML samples ± lenalidomide or/and nivolumab after 72 h of co-culture. D Fold change of median intensity of LFA-1 accumulation at the interface of AMG 330-induced T-cell–primary AML cell conjugates. Primary AML samples were pretreated ± IFNγ and TNFα for 48 h and HD T cells were pretreated ± lenalidomide for 24 h; AMG 330 concentration = 5 ng/ml; Lenalidomide = 10 µM; Nivolumab = 10 µg/ml; E:T ratio = 1:4 (panels B and C) or 1:1 (panel D); n = 4–14; Error bars represent mean ± SEM; Statistical analysis: Paired t-test (panel A and B) and One-way ANOVA with Dunnett's multiple comparisons test (Panels C and D); ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

Fig 5: T-cell co-signaling receptors are expressed on leukemic cells and transduced Ba/F3 sublines. A MFI ratio of cell surface expression of CD80 (green), CD86 (blue) and PD-L1 (orange) in CD45dimSSClow primary AML samples (n = 107–377). MFI ratios ≥ 1.5 (dashed lines) indicate positivity. B Percentage of CD80-, CD86- and PD-L1-positive cells and C distribution of CD86 and PD-L1 expression intensities within primary AML samples (n = 432–521). D Antigen count of CD33 (red), CD86 (blue) and PD-L1 (orange) on transduced Ba/F3 sublines (n = 3) with E representative histograms