Figure 2. CAR stimulation promotes less intense phosphorylation of CD3 chains and proximal TCR signaling adaptors.

(A) Schematic of experimental design stimulating bi-specific T cells with beads coated with TCR (SCT/CD28) or CAR (ROR1) antigens. (B to D) Graphs show mean log₂FC of the indicated phosphorylation (PO₄) sites as depicted in (A) from two or three LC-MS/MS experiments with T cells from two unique donors. (E) Comparison of the mean log₂FC of phosphorylation sites after TCR or CAR stimulation with SCT/CD28 or ROR1 beads. Red dots specify sites that possessed mean log₂FC values differing by ≥ 1 between TCR and CAR stimulated samples at 10, 45, and 90 min relative to control bead stimulation. (F) Graph shows mean log₂FC of phosphorylation of CD3δ, CD3ε, and CD3γ ITAMs. The log₂FC in phosphorylation of both ITAM tyrosines was averaged. Data are means from two or three LC-MS/MS experiments with T cells from two unique donors. (G) Heat map shows mean log₂FC of select phosphorylation sites at the 10-min time point. (H) Graph shows mean log₂FC of select phosphorylation sites in LAT. (I) Western blot analysis for CD3ζ, CD3ζ pTyr¹⁴², LAT and LAT pTyr²²⁰, ZAP-70, ZAP-70 pTyr³¹⁹, ZAP-70 pTyr⁴⁹³, PLC-γ1, and PLC-γ1 pTyr⁷⁸³, and PLC-γ1 pSer¹²⁴⁸ (p = phosphorylation) in the bi-specific T cell lysates utilized for LC-MS/MS experiments. Blots are representative of three experiments using T cells prepared from two unique donors; compiled data is quantified in fig. S4A. (J) Western blot analysis for LAT, LAT pTyr²²⁰, ZAP-70, ZAP-70 pTyr³¹⁹, PLC-γ1, and PLC-γ1 pTyr⁷⁸³ in lysates from bi-specific T cells expressing a BB/ζ CAR after 10 min of stimulation with control, SCT/CD28, or ROR1 beads. Blots are representative of three experiments using T cells prepared from two unique donors; compiled data is quantified in fig. S4B.