Fig 2: Regulation of TCR signaling by LYP downstream of ZAP70. A Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in Jurkat cells transfected with Vav and stimulated with CD3 plus CD28 antibodies for 6 h, as indicated. The insert shows the IB of the Vav and LYP proteins expressed. *P < 0.05 and **P < 0.01 for comparison of cells transfected with different plasmids and cells transfected with empty vector (pEF). B Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in Jurkat cells transfected with rac-QL, a dominant active mutant of rac, and LYP and and stimulated with CD3 plus CD28 antibodies for 6 h. The insert shows the IB of LYP. *P < 0.05 and **P < 0.01 for comparison of cells transfected with different plasmids and cells transfected with empty vector (pEF) C Activation of a luciferase reporter gene driven by the IL-2 minimal promoter in wild type and 494 Jurkat cells transfected with LYP and SLP76 as indicated. The cells were left untreated or stimulated with CD3/CD28 antibodies for for 6 h. The insert shows the IB of the SLP76 and LYP proteins expressed. *P < 0.05 **P < 0.01 and ***P < 0.001 for comparison of cells transfected with different plasmids and cells transfected with empty vector (pEF). D Wild type and 494 JK cells were stimulated with CD3/CD28 antibodies during the indicated periods of time. Phosphorylation of Y128 of SLP76 in each condition was measured in cell lysates by IB. Similarly, phosphorylation of LCK in Y394 and Y493 are shown. E, Wild type Jurkat cells and cells deficient in LYP (JK 494) transfected with LYP were stimulated with CD3/CD28 antibodies for 15 min and the phosphorylation of Y128 of SLP76, and Y191 of LAT were detected in each condition by IB

Fig 3: E-Syt proteins modulate signaling and functionality of T cells via PM DAG levels.(A) Left: E-Syt dimer located between the ER and PM in the unstimulated T cell. Green arrows indicate the transfer of DAG (green lipid structure) from the PM to the ER. No phosphorylation of TCR signaling proteins (ZAP70, LAT, and MAPK) or IL-2 production. Right: The absence of E-Syt protein dimer at ER-PM junction sites leads to the accumulation of DAG (green lipid structure) at the PM. DAG accumulation at the PM leads to the phosphorylation of TCR signaling proteins (ZAP70, LAT, and MAPK) and eventually IL-2 production. (B) Left: E-Syt dimer located between the ER and PM in the activated T-cell mediate the transfer of DAG (green lipid structure) from the PM to the ER. Normal phosphorylation of TCR signaling proteins (ZAP70, LAT, and MAPK) and IL-2 production expected. Right: The absence of E-Syt protein dimer at ER-PM junction sites in an activated T cell leads to the vast increase of DAG (green lipid structure) at the PM. DAG accumulation leads to the enhanced phosphorylation of TCR signaling proteins (ZAP70, LAT, and MAPK) and IL-2 production. This figure was created with BioRender.com. Source data are available online for this figure.

Fig 4: E-Syt1 and -2 knockdown in primary CD8+ T cells enhances cytotoxic functions through increased TCR signaling.(A) Western blot analysis and quantification of cell lysates from human CD8+ T cells transfected with either non-targeting (NT), E-Syt1, E-Syt2, E-Syt1&2, or Munc13-4 siRNA. The data shown represent the mean ± SEM of at least five independent experiments. Significance was determined by one-way ANOVA with Bonferroni’s multiple comparisons test against the respective E-Syt protein levels in control NT siRNA (****P < 0.0001). (B) CD107a degranulation assay of siRNA-treated human CD8+ T cells incubated in the absence (unstimulated) or the presence of P815 target cells coated with human anti-CD3 antibody (stimulated) for various time points (30–150 min) and analyzed for the appearance of CD107a on the cell surface via flow cytometry. Representative flow plots of unstimulated and stimulated CD8+ NT and E-Syt1 knockdown cells activated for 60 min. Graph of the percentage of CD107a + CD8 + T cells across all activation time points. Plots are a mean ± SEM of at least three independent experiments. Significance was determined by two-way ANOVA with Bonferroni’s multiple comparisons test against control NT siRNA (*P < 0.0332). (C) Measurement of lymphocyte-mediated cytotoxicity of siRNA-treated human CD8+ T cells against P815 target cells using EarlyTox Caspase-3/7 NucView-488 assay. The percentage of P815 target cell death was tested every 5 min for over 5 h. Plots are a mean ± SEM of at least three independent experiments. Significance was determined by two-way ANOVA with Bonferroni’s multiple comparisons test against control NT siRNA (**P < 0.0021, ****P < 0.0001). (D–F) Flow cytometry analysis of CD3 + CD8+ gated T cells from human PBMCs using signaling events measured by phosphorylation levels of early TCR signaling proteins ZAP70 at Y292 (B) LAT at Y226 (C) and PLC-γ1 at Y783 (D) upon stimulation with anti-CD3 and CD28 antibody-coated beads for indicated times. Plots show the change in median fluorescence intensity (MFI) between the indicated time of stimulation and t = 0. Plots are a mean ± SEM of at least three independent experiments. Significance was determined by two-way ANOVA with Bonferroni’s multiple comparisons test against control NT siRNA CD8+ T cells (*P < 0.0332, **P < 0.0021). Source data are available online for this figure.

Fig 5: Expression dynamics of adhesion molecules on CAR T cells and the effect on CARIS avidity.(A) Hypothetical schematic of mechanisms of LFA-1 activation. (B to D) Flow cytometry for CD11a (B) and CD18 (C) expression on HER2-CAR T cells at baseline and for aLFA-1 (D) in response to plate bound recombinant HER2, using an LFA-1 extended-open conformation-specific antibody (m24). MFI, mean fluorescence intensity. (E) WB for membrane fractions from HER2-CAR T cells at baseline (0 min) and in conjugation with LN229 (15 and 60 min) probed for CAR (CD3ζ), LFA-1 (CD18), LAT, and CD71. (F) Percentage LFA-1 (CD18) recruited to the LR fractions from (E). (G) ImageStream flow cytometry for HER2-CAR T cells conjugated with LN229 (30 min). CARs (emerald GFP), actin (phalloidin), nuclei (DAPI), and aLFA-1 (m24). (H to J) Fluorescence intensity of activated aLFA-1 (H) and its correlation with actin intensity (I) or CAR intensity (J) at the CARIS of mature synapses. N = 3; n = 9007. (K and L) Flow cytometry for ICAM-1 expression at baseline on CD4 and CD8 (K) and product (L) of HER2-CAR T cells. (M) Schematic representation of z-Movi acoustic force microscopy for testing CARIS avidity. A force ramp of 0 to 1000 pN was applied to pull HER2-CAR T cells off an LN229 monolayer toward acoustic nodes. (N) z-Movi ultrasound microscopy measuring the percentage of HER2-CAR T cells bound to LN229 at 500 pN after 2, 5, 20, or 40 min of incubation. (O) Detachment forces for individual cells after 5 or 40 min of incubation. Median represents the force needed to detach 50% of CAR T cells (EF50). Mean of three donors + SD [(B) to (D), (F), (K), and (L)], data pooled from three donors [(H) to (J)] or three to five avidity runs [(N) and (O)] compared using one-way [(B), (C), and (O)] or RM two-way Holm-Sidak corrected ANOVA [(D) and (N), ratio paired t test (F), Mann-Whitney U test (H), or Spearman correlation (non-Gaussian distribution) [(I) and (J)]. *P < 0.05, **P < 0.01, ****P < 0.0001. (A) and (M) created using Biorender.com.