PMC

TCR-induced activation of Rap1 and adhesion to ICAM-1 are dependent on ZAP-70 adapter function. (a) Rap1 pulldown assay from Zap70^+/− and Zap70(YYAA) thymocytes. Cells were stimulated with the indicated doses of anti-CD3ε for 2 minutes in the absence of 3-MB-PP1. (b) TCR-induced ICAM-1 adhesion by Zap70^+/− and Zap70(YYAA) T cells. Peripheral CD4⁺ T cells were stimulated as in with the indicated doses of anti-CD3ε. Data are representative of 3 independent experiments.

CD8 memory responses are dependent on ZAP-70 catalytic activity. (a,b) Flow cytometric analysis of LCMV (a) GP^33–41-specific and (b) NP^396–404-specific IFNγ and TNFα production by Zap70^+/− and Zap70(AS) memory CD8⁺ cells. Contour plots are gated on CD8⁺ CD44^high memory cells. The concentrations of 3-MB-PP1 present are indicated above the plots. (c, d) Graphs display the percentage of IFNγ⁺ TNFα⁺ cells at each concentration of 3-MB-PP1 tested. Percentages are of CD8+ CD44^high memory Zap70^+/− (black line) and Zap70(AS) (gray line) cells. Error bars indicate the standard deviation from the mean; n=3. Data are representative of 3 experiments.

Proliferation by CD4⁺ T cells requires ZAP-70 catalytic activity. (a) Purified polyclonal CD4⁺ T cells from Zap70^+/− (gray bars) and Zap70(AS) (black bars) were stimulated with plate-bound anti-CD3ε and anti-CD28 mAbs for 72 hours in the presence of vehicle or 3-MB-PP1; concentrations indicated below the x-axis. Data are presented as the mean ³H thymidine uptake from triplicate cultures. Error bars indicate the standard deviation of the mean. (b) CFSE loaded Zap70^+/− (filled gray histogram) and Zap70(AS) (black line) OT-II T cells were stimulated with irradiated antigen presenting cells and 1 µM OVA^323–339 peptide for 72 hours. The concentration of 3-MB-PP1 present in each culture is indicated above each plot.

Execution of effector T cell functions requires ZAP-70 catalytic activity. (a) Flow cytometric analysis of intracellular cytokine staining for IFNγ and IL-4 by effector T_H1 and T_H2 Zap70^+/− and Zap70(AS) OT-II cells, respectively. Numbers within the dot plots indicate the percentage of cells within each quadrant. The concentrations of 3-MB-PP1 present in each culture are indicated above the dot plots. (b) Lysis of allogeneic ⁵¹Cr labeled P815 target cells by Zap70^+/− and Zap70(AS) CTLs. Cell assay cultures containing effector and target cells were supplemented with vehicle alone (gray line), 10 µM 3-MB-PP1 (black), or CsA (dashed black line). (c) Alloreactive Zap70^+/− (gray bars) and Zap70(AS) (black bars) CTLs, generated as in (b) were stimulated by P815 cells in the presence of 3-MB-PP1 (top) or CsA (bottom). Numbers on the x-axis indicate the final concentration of 3-MB-PP1 (µM) or CsA (µg/ml). Data are representative of 3 experiments.

ZAP-70 catalytic activity is dispensable for T_REG cell suppressive activity in vitro. (a) In vitro suppression assays used CD4⁺CD25⁻ Tconv cells from both Zap70^+/− and Zap70(AS) mice, together with irradiated antigen-presenting cells, anti-CD3ε mAbs, and titrated numbers of Zap70^+/− or Zap70(AS) CD4⁺CD25⁺ T_REG cells. Cultures were set up in the presence of vehicle alone, 5 µM, or 10 µM 3-MB-PP1. Data are presented as the average ³H thymidine uptake in triplicate cultures. Error bars indicate the standard deviation of the mean. (b) Bar graphs show the ³H thymidine uptake of Tconv or T_REG cells alone in the presence of vehicle alone or 3-MB-PP1. (c) Immunoblots of whole cell lysates from purified Zap70^+/− and Zap70(AS) Tconv and T_REG cells were probed for expression of Syk, ZAP-70, and ERK1-ERK2. Data in all panels are representative of at least 3 experiments.

TCR-induced activation of Rap1 and adhesion to ICAM-1 are ZAP-70 kinase-independent. (a) CD4⁺CD25⁻ Tconv and CD4⁺CD25⁺ T_REG cells were loaded with Fluo-3 and Fura Red for analysis of intracellular calcium levels by flow cytometry. Cells were stimulated in the presence of vehicle alone (red) or 3-MB-PP1 (blue). Arrow “1” indicates the addition of biotinylated anti-CD3ε and anti-CD4, arrow “2” indicates the addition of streptavidin, and arrow “3” indicates the addition of ionomycin. Tconv were >99% Foxp3⁻ and T_REG cells were >98% Foxp3⁺. (b) Splenocytes were stimulated ex vivo by crosslinking anti-CD3 mAbs and stained for phospho-ERK. Histograms are gated on Foxp3-negative or Foxp3-positive CD4⁺ cells. Cells were either unstimulated (filled gray histograms) or stimulated in the presence of vehicle alone (red), 5 µM 3-MB-PP1 (green), 10 µM 3-MB-PP1 (blue), or 10 µM 3-MB-PP1 plus PMA (gray line). (c) CrkII was immunoprecipitated (IP) from Zap70(AS) thymocyte lysates. Cells were stimulated by crosslinking anti-CD3 mAbs for 2 minutes. Immunoprecipitated proteins were immunoblotted (IB) for ZAP-70 and CrkII (top). Whole cell lysates were also immunoblotted for the presence of ZAP-70 and CrkII (bottom). (d) Pull-down assay for Rap1-GTP in Zap70^+/− and Zap70(AS) thymocytes. Cells were left unstimulated, or stimulated with crosslinked anti-CD3ε mAb for 2 minutes. The concentrations of 3-MB-PP1 are indicated. Total Rap1 levels in whole cell lysates are shown below. (e) CD4⁺ T cells from Zap70^+/− and Zap70(AS) were stimulated with anti-CD3ε for 10 minutes in wells coated with recombinant ICAM-1. Cells were then washed and counted by flow cytometry. Data in all panels are representative of 3 independent experiments.

T cell activation is dependent on ZAP-70 catalytic activity. (a) Flow cytometric analysis of Fluo-3 loaded Zap70^+/− and Zap70(AS) CD4⁺ cells. For T cells, the arrows indicate 1) the simultaneous addition of 3-MB-PP1 and anti-CD3ε mAb and 2) the addition of crosslinking antibodies. For B cells, the arrow indicates the addition of anti-IgM plus the indicated doses of 3-MB-PP1. (b) Flow cytometric analysis of phospho-ERK staining in Zap70^+/− (top) and Zap70(AS) (bottom) CD4⁺ T cells. Cells were stimulated with biotinylated anti-CD3ε and anti-CD4 plus streptavidin, in the presence of vehicle only (blue line), 5 µM (orange line), 10 µM (green line) and 10 µM 3MB-PP1 plus PMA (thin black line). Unstimulated cells are indicated by the filled gray histogram. (c) % of Zap70^+/− (gray bars) and Zap70(AS) (black bars) T cells positive for CD69 after stimulation with plate-bound anti-CD3ε and anti-CD28 for 16 hours in the presence of 3-MB-PP1. Concentration of 3-MB-PP1 indicated on the x-axis. Percentages are normalized to vehicle-treated cells. (d) Immunoblot analysis of whole cell lysates from purified Zap70^+/− and Zap70(AS) CD4⁺ T cells. Cells were left unstimulated, or stimulated with anti-CD3ε and crosslinking antibodies in the presence of vehicle, 5 µM, or 10 µM 3-MB-PP1 for 2 minutes. Blots probed with antibodies specific for the indicated epitopes. Data are representative of at least 3 independent experiments.