In total, six types of IS were quantified, including typical IS (ET), ETE, ETT, ETET, ETEE, and ETTT. (a) The fraction of typical IS and variants under different conditions; (b) The composition of IS variants (ETE, ETT, ETET, ETEE, ETTT) under different conditions; (c) The positive correlation between the fraction of IS variants (% of total IS) and total IS formation (effector cell % engaged). The formula and R2 of linear regressions are shown. (d) The E:T ratios involved in total IS and IS variants. Experimental setup: Condition 1, 2 X, E:T(1), CD3(L), CD19(M), 100 ng/mL, 60 min; Condition 2, 4 X, E:T(1), CD3(L), CD19(M), 100 ng/mL, 60 min; Condition 3, 2 X, E:T(1), CD3(H), CD19(M), 100 ng/mL, 60 min; Condition 4, 2 X, E:T(1), CD3(L), CD19(L), 100 ng/mL, 60 min; Condition 5, 4 X, E:T(6), CD3(L), CD19(M), 100 ng/mL, 60 min; Condition 6, 4 X, E:T(0.17), CD3(L), CD19(M), 100 ng/mL, 60 min; Conditions 7–12 are the same as Conditions 1–6, except with lower bispecific T cell engager (BiTE) concentrations (20 ng/mL).