Fig 2: Expression of Zap70C564A increases proximity between Lck and the TCR. An in situ Proximity Ligation Assay (PLA) between the TCR (CD3e) and Lck was performed to study the proximity between Lck and the TCR; a red fluorescent signal indicates that the distance between Lck and the TCR (CD3e) is less than 80 nm. (A) Anti-CD3e (OKT3) was used to simulate Jurkat cells for 5 min at 37 °C. Technical PLA controls were performed using both anti-Lck and anti-CD3e primary antibodies (left panel), the anti-Lck primary antibody alone (middle panel), or the anti-CD3e antibody alone (right panel). In all samples, both secondary antibodies were used. The bar graph shows the quantification of two independent experiments. One-way ANOVA was used to perform statistical analyses. Mean values ± SEM are shown. **** p < 0.0001. (B) As additional PLA controls, we used parental Jurkat T cells (expressing both CD3e and Lck) (left panel) and CRISPR/Cas9 gene edited Lck-deficient (J.Lck KO, middle panel) or CD3e-deficient (J.CD3e KO, right panel) Jurkat T-cell variants. The PLA was performed as indicated in (A). The bar diagram shows the quantification of two independent experiments. Statistical analysis was performed as in (A). (C) Zap70-deficient P116 cells stably expressing no Zap70 (Zap70neg), Zap70wt or Zap70C564A were either left unstimulated, stimulated with anti-CD3e (OKT3), or stimulated with pervanadate (PerV) for 5 min at 37 °C. Nuclei were stained with DAPI. Data of three independent experiments are shown (upper right bar diagram), n = ~700 cells per sample. (D) Values were set to one for ZAP70wt for each experiment and each condition to best visualize the effect of the C564A mutation. Statistical analysis was performed using unpaired Student’s t test. Mean values + SEM are shown, ns = not significant; * p < 0.1.

Fig 3: The R821H mutation in PIK3CD is associated with diminished activation of AKT and increased T cell proliferation and calcium flux. (A) Immunoblots of phosphorylated AKT (P-AKT), p70 S6K (P-p70 S6K), and ZAP70 (P-ZAP70) in T cell blasts from a control (Ctrl.) and the patient (Pat.) stimulated with anti-CD3 antibody for 0, 1, 2, 4, and 8 min. Total AKT and ACTIN were used as loading controls. Molecular weight markers are shown on the right. (B) Densitometry quantifications of immunoblots of P-AKT of T cells stimulated as in A for 0, 5, and 10 min. (C) FACS histograms of intracellular P-AKT in activated T cells not stimulated (no stim.) or stimulated with anti-CD3 in the presence or not of the p110d pharmacological inhibitor IC87114 (IC). (D and E) FACS dot-plots and corresponding histograms of proliferation assays (after gating on CD3+ cells) assessed with the CFSE dye. T cell blasts (D) or PBMCs (E) stimulated or not with immobilized 1 µg ml-1 anti-CD3 (D, left), different doses of anti-CD3 (D, right) or anti-CD3/CD28 coated beads (E). Staining with anti-CD25 antibody as an activation marker. Numbers ahead each peak correspond to the number of divisions. Calculated proliferation indexes from FACS data are shown on the right. (F) Intracellular Ca2+ levels by real time flow cytometry in T cell blasts or PBMCs of Ctrl. (gray) or Pat. (black line) after stimulation with anti-CD3 and a cross-linker. (G) Same as A, with immunoblots of phosphorylated ERK (P-ERK). (H) Normalized densitometry quantifications of P-ERK immunoblots. Stimulation of patient cells at 5 min was used as 100%. *, P < 0.1; ***, P < 0.001; ****, P < 0.0001 with unpaired (B) or paired (D, E, and H) Student’s t test from data of five (B), two (0.1 and 10 µg ml-1; D), or four (0 and 1 µg ml-1; D) or four (E and H) independent experiments with mean and SD. In D, at 10 µg ml-1, P = 0.052 as indicated. Data are representative of three independent experiments for A, C, and F. A.U., arbitrary units.

Fig 4: Flotillin expression is required for conjugate formation and T cell signaling. a Representative flow cytometry dot plots of wildtype and Flotillin1/2 KO Jurkat T cells conjugated to SEE pulsed Raji B cells and stained with cell viability dye and CFDA, respectively. b Percentage of stable T–B cell conjugates determined as violet dye and CFDA double positives, relative to total cell population. c Flow cytometry quantification of WT and flotillin1/2 KO Jurkat T cells activated with soluble CD3e and CD28 and fixed prior or 5, 10, and 20 min after activation. Cells were stained with antibodies against phosphorylated (left) TCR?-pY142, (middle) Zap70-pY493, and (right) ERK-pY202/204. d Representative immunofluorescence microscopy images of WT and flotillin1/2 KO Jurkat T cells stained with antibodies against nuclear factor of activated T-cells (NFAT) and e the integrated NFAT fluorescence intensity over the Hoechst-stained nucleus in WT and flotillin1/2 KO Jurkat T cells activated or not with anti-CD3e and CD28 antibodies. f Representative immunofluorescence microscopy images of WT and flotillin1/2 KO Jurkat T cells stained with antibodies against T-cell-specific adaptor protein (TSAd) and g the integrated TSAd fluorescence intensity over the Hoechst-stained nucleus in WT and flotillin1/2 KO Jurkat T cells activated or not with anti-CD3e and CD28 antibodies. Scale bars, 5 µm. Data obtained from three or more independent experiments. Error bars indicate mean (±SEM). *p < 0.01; **p < 0.001; ****p < 0.00001; Mann–Whitney t-test

Fig 5: Acylation-deficient mutant of ZAP-70 is catalytically active.A, Schematic representation of ZAP-70 showing the position of the S-acylated cysteine residue (C564) in the kinase domain. B, In vitro kinase assay performed using ZAP-70 immunoprecipitated from P116 (ZAP-70 null) Jurkat T cells expressing WT or acylation-deficient C564R ZAP-70. Phosphorylation of SLP-76 was detected using phospho-SLP-76 (Y128) antibody. C, Quantified data showing phosphorylation of SLP-76 by WT or C564R ZAP-70. Data shown are representative of three independent biological repeats and represented as mean ± SEM, normalized to total SLP-76.