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Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development

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An Erratum to this article was published on 16 January 2015

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Abstract

The catalytic activity of Zap70 is crucial for T cell antigen receptor (TCR) signaling, but the quantitative and temporal requirements for its function in thymocyte development are not known. Using a chemical-genetic system to selectively and reversibly inhibit Zap70 catalytic activity in a model of synchronized thymic selection, we showed that CD4+CD8+ thymocytes integrate multiple, transient, Zap70-dependent signals over more than 36 h to reach a cumulative threshold for positive selection, whereas 1 h of signaling was sufficient for negative selection. Titration of Zap70 activity resulted in graded reductions in positive and negative selection but did not decrease the cumulative TCR signals integrated by positively selected OT-I cells, which revealed heterogeneity, even among CD4+CD8+ thymocytes expressing identical TCRs undergoing positive selection.

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Figure 1: Greater dependence on catalytic activity of Syk versus Zap70 for β selection.
Figure 2: Positive selection requires continuous Zap70 catalytic activity.
Figure 3: Positive selection in thymic slices requires Zap70 catalytic activity.
Figure 4: Temporal and dose-dependent requirements for Zap70 catalytic activity during positive selection.
Figure 5: Invariant Zap70-dependent signal threshold for positive selection.
Figure 6: Negative selection requires Zap70 catalytic activity in a dose-dependent and time-dependent manner.

Change history

  • 23 June 2014

    In the version of this article initially published online, the second sentence of the legend to Figure 6e was incorrect. It should read "HXJ42 (1 μM) was added...". The error has been corrected in the HTML version of this article.

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Acknowledgements

We thank A. Roque for animal husbandry and C. Zhang for synthesis of 3-MB-PP1 and HXJ42. This work was supported by the Arthritis Foundation postdoctoral fellowship 5476 (to B.B.A.-Y.), the California Institute of Regenerative Medicine postdoctoral training grant T1-00007 (to H.J.M.), graduate student training grant TG2-01164 (to J.O.R.), the Rosalind Russell Medical Research Foundation Bechtel Award (to J.Z.), Arthritis National Research Foundation grant (to J.Z.), and US National Institutes of Health grants K08 AR059723 (to J.Z.), AI064227 (to E.A.R.), AI091580 (to A.W.) and RC2AR058947 (to A.W.).

Author information

Authors and Affiliations

Authors

Contributions

B.B.A.-Y., H.J.M., E.A.R. and A.W. designed the experiments. B.B.A.-Y., H.J.M., J.O.R. and D.A.C. performed the experiments. J.Z. and K.M.S. provided advice and reagents. B.B.A.-Y., H.J.M., E.A.R. and A.W. wrote the manuscript.

Corresponding authors

Correspondence to Ellen A Robey or Arthur Weiss.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Specificity of Zap70(AS) and Syk inhibitors.

(a) Flow cytometric analysis of calcium ratio in Zap70+/– and Zap70(AS) thymocytes stimulated with soluble anti-CD3 (arrow “1”) and crosslinking goat anti-hamster secondary antibodies (arrow “2”) in the presence of the indicated concentrations of 3-MB-PP1. Histograms show the calcium ratio in the DP population. One of three independent experiments is shown. (b) Immunoblot analysis of whole cell lysates from Zap70+/– and Zap70(AS) thymocytes stimulated by crosslinking anti-CD3 antibodies for 2 min. Blots show phosphorylated Erk (top) or total Erk1 and Erk2 (bottom). One of three independent experiments is shown. (c) Flow cytometric analysis of calcium ratio in Zap70(AS) splenocytes stimulated with soluble anti-CD3 in the presence of either 10 μM 3-MB-PP1 or 1 μM BAY61-3606 (arrow “1”), and crosslinking goat anti-hamster secondary antibodies plus anti-IgM (arrow “2”). Histograms are gated on CD19+ B cells (left) or CD90.2+ T cells (right). Arrow “3” indicates the addition of ionomycin. The black line represents cells stimulated in the presence of DMSO alone, and the blue and red lines represent stimulation the presence of BAY61-3606 and 3-MB-PP1, respectively. (d) Flow cytometry plots show CD4 and CD8 expression of total viable cells in Day 0-3 FTOC samples. Numbers indicate the percentage of cells within each quadrant. Plots show one representative fetal thymic lobe out of n = 3 independent experiments.

Supplementary Figure 2 Titration of Zap70 inhibition in FTOC does not change the ratio of CD4+SP to CD8+SP cells.

(a) Dot plots show CD4 and CD8 expression by TCRβhi cells from the Zap70(AS) FTOC samples treated with each indicated concentration of 3-MB-PP1, as in Figure 2a. Numbers indicate the percentage of cells within each gate. (b) Flow cytometry plots are gated on TCRβhi CD4+SP cells (top) or TCRβhi CD8+SP cells (bottom) cells and show expression of TCRβ and CD24. (c) Graphs display the percentage of TCRβhi CD5hi DP cells (left) and TCRβhi CD24lo CD4+SP and CD8+SP cells (middle, left) in fetal thymic lobes cultured in the presence of each indicated concentration of 3-MB-PP1. Shown are two independent experiments. (d) Graphs display the average percentage of TCRβhi CD5hi DP cells (± s.e.m.) (left) and total numbers of TCRβhi CD24lo CD4+SP and CD8+SP cells (middle, right) as in Figure 2b (n = 3 samples). Lines connect data points from the same experiment. Data are from one of two independent experiments (a,b), cumulative of two (c) or three (d) independent experiments. *P <0.05 (unpaired two tailed Student's t-test).

Supplementary Figure 3 Recovery of calcium signaling events after washout of Zap70 inhibitor from thymic slices.

(a) Graphs show individual Zap70(AS) OT-I cell tracks in WT thymic slices. Each horizontal line represents an individual cell track within a WT thymic slice, beginning 7 min (left, n = 60 cell tracks) or 135 min (right, n = 94 cell tracks) after 3-MB-PP1 was washed away from the thymic slice. The red segments represent the time points during which elevated [Ca2+]i was detected. Data are one representative movie of three movies from two independent experiments. (b) Graphs show the average percentage of Zap70(AS) OT-I DP cells (± s.e.m.) exhibiting elevated [Ca2+]i within a WT thymic slice 2 h after 3-MB-PP1 washout, compared to a sample that was exposed to DMSO only. Data are cumulative of two movies from independent experiments (Washout, n = 145 cell tracks; DMSO, n = 120 cell tracks) NS, not significant (unpaired two tailed Student's t-test).

Supplementary Figure 4 Effect of Zap70 inhibition on nur77-GFP reporter transgene expression.

(a) Histograms on the left show flow cytometric analysis of GFP expression by pre-selection OT-I Zap70(AS)-nur77-GFP DP thymocytes stimulated by each indicated concentration of plate-bound anti-CD3 (top), or a single concentration of anti-CD3 (10 μg/ml) and each indicated concentration of 3-MB-PP1 (bottom) for 6 h. Populations shown are gated on viable, TCR Va2hi CD69+, DP cells. Histograms on the right compare GFP expression by the gated GFP+ TCR Va2hi CD69+, DP cells from each condition (n = 3 samples). (b) WT and Bim–/– nur77-GFP DP thymocytes were analyzed by flow cytometry for expression of TCRβ and CD69. Pre-selection (TCRβlo CD69-, in red) and post-selection (TCRβhi CD69+, in blue) populations are color-coded. Histograms (bottom) compare GFP expression by pre-selection and post-selection cells. (c) Overlaid histograms compare GFP expression by WT (closed) and Bim–/– (open) pre-selection and post-selection DP thymocytes. (d) Flow cytometry data shown in Figure 5c, reanalyzed to compare GFP expression among DP cells subdivided into TCRβlo CD5lo (DP1), TCRβint CD5hi (DP2) and TCRβhi CD5hi (DP3) populations. Numbers above the histograms indicate the concentration of 3-MB-PP1. (e) Histogram shows GFP expression of DP3 cells from FTOC cultured in the presence of each indicated concentration of 3-MB-PP1. Data are representative of 3 independent experiments (a), three biological replicates (b,c), and 3 fetal thymic lobes from 3 independent experiments.

Supplementary Figure 5 Negative selection assay and specific and potent inhibition of Zap70(AS) by HXJ42.

(a) Flow cytometry plots show GFP and eFluor670 expression by total viable cells in thymic slices 24 h after addition of OVA, or no peptide, and exposed to each indicated inhibitor, as in Figure 6a. Zap70+/– and Zap70(AS) OT-I cells were labeled with eFluor670, and control F5 TCR transgenic cells expressed GFP. Numbers indicate the percentage of cells within each gate, which were used to calculate the OT-I to F5 ratio in Figure 6a. One of three technical replicates is shown, from one of three independent experiments. (b) Small molecule inhibitor HXJ42. (c) Immunoblot analysis of lysates from Zap70+/– and Zap70(AS) thymocytes stimulated with soluble anti-CD3 and crosslinking goat anti-hamster secondary antibodies in the presence of each indicated concentration of 3-MB-PP1 or HXJ42 for a total of 2 min. Immunoblots were probed for phosphorylated Lat (Tyrosine 132), Erk (Threonine 202 and Tyrosine 204), and total actin. Immunoblots are from one of three independent experiments. (d) Graphs display the mean 3H counts per minute normalized to the DMSO-treated controls (proliferation) ± s.e.m. (n = 3 samples) of Zap70+/– and Zap70(AS) CD4+ cells stimulated for 72 h in the presence of each indicated concentration of 3-MB-PP1 or HXJ42. One of two independent experiments is shown. (e) Graphs show the corrected calcium ratio and interval speed of one representative OT-I cell on a positively selecting WT slice (left, n = 102 signaling cells), or two representative OT-I cells on a negatively selecting WT slice in the presence of 1 nM OVA (middle and right, n = 188 signaling cells) from three movies from two independent experiments for each condition.

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Supplementary Data 1

Supplementary software. (ZIP 17 kb)

Two-photon imaging of preselection Zap70(AS) OT-I DP cell migration and intracellular calcium concentration in thymic slices.

Left, OT-I thymocytes in nonselecting (B2m–/–) slices in the absence of inhibitor. Middle, OT-I thymocytes under positive selecting (WT slice) conditions with DMSO addition after 10 min of a 40-min movie. Right, positive selecting (WT slice) conditions with the addition of 3-MB-PP1 after 10 min of a 40-min movie. Top, z-projection of fluorescence emission of Indo-1 dye in unbound (green) and calcium bound (red) forms. Bottom, intracellular calcium ratio of calcium bound/unbound Indo-1 dye represented as a heat map. Frames were collected every 20 s for 40 min, 2-4 h after cells were added to the slice. Imaging data are representative of three movies from two independent experiments. (MOV 2219 kb)

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Au-Yeung, B., Melichar, H., Ross, J. et al. Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development. Nat Immunol 15, 687–694 (2014). https://doi.org/10.1038/ni.2918

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