Extended Data Fig. 8: iCAR design, expression and impact on primary human NK cell signaling and function.
From: KIR-based inhibitory CARs overcome CAR-NK cell trogocytosis-mediated fratricide and tumor escape
(a) Schematic diagram of the viral vector design for the different anti-CD19 iCARs; TM: transmembrane; SE: signaling endodomain. (b) Cell surface expression of aCAR19, 19scFv, or iCAR19 constructs by flow cytometry in transduced primary human NK cells. Dot plots are representative of three different donors. Inset numbers indicate the percentage of CAR-expressing cells within the indicated gated regions. (c and d) Flow cytometric expression of (c) phos-SHP1 (pSHP1) and (d) phos-Syk/Zap70 in NK cells expressing CAR19, 19scFv or the different iCAR19 constructs (n = 5 donors) in response to stimulation with CD19+ Raji cells; the following bar graphs show their expression, determined by gMFI normalized to the unstimulated NK cell population. (e-h) CD107a (left), TNF-α (middle), and IFN-γ (right) production by NK cells transduced with CAR19, 19scFv, or the different iCAR19 constructs in response to 6-hr stimulation with (e) CD19+ K562 cells (K562gCD19+), (f) K562, (g) RajiCD19+, or (h) CD19− RajiCD19-KO target cells (n = 5 donors). (i and j) Incucyte analyses of percentage (%) caspase 3/7 events in (i) RajiCD19+ and (j) autoNKgCD19+ target cells after co-culture with NK cells expressing CAR19, 19scFv or the different iCAR19 constructs, (representative for three donors). (k and l) Cumulative population doubling (PD) of NK cells expressing CAR19, 19scFv or the different iCAR19 constructs over 70 days of culture with (k) IL-2 only or with (l) IL-2 plus weekly uAPC stimulation (n = 3 donors). (m) tCD19 expression on NK cells transduced with CAR19, 19scFv or the different iCAR19 constructs, presented as ratio of TROG+/TROG− cells at different time points during co-culture with RajiCD19+ cells (n = 3 donors). P values were determined by two-tailed one-way ANOVA in panels c-h, or two-tailed two-way ANOVA in panels i-m. Data were assessed in flow cytometry in panels b,c,d,e,f,g,h,m, and shown by mean + s.e.m. Each symbol represents an individual donor.
