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

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.

Figure 1. Greater dependence on catalytic activity of Syk versus Zap-70 for β selection

(a) FTOC of e15.5 Zap70(AS) thymic lobes was performed for 4 days with vehicle alone (DMSO), 5 µM 3-MB-PP1, 1 µM BAY61-3606, or both inhibitors. Overlayed histograms show CD27 expression on gated CD25⁺ CD44⁻ DN3 cells from fetal thymic lobes cultured with the indicated inhibitors. (b) Flow cytometry plots are gated on total CD4⁻CD8⁻ DN and TCR γδ negative cells. The numbers indicate the percentage of cells within each quadrant. (c) Total cell numbers for a single fetal thymic lobe cultured under the indicated inhibitor conditions on day 3. Bar graphs display the mean total cell numbers (± s.e.m.) from three independent experiments. Data in panels (a,b) are from one representative experiment out of 3 independent experiments. *P <0.05, **P <0.005, ***P <0.0005, NS not significant (Student’s t-test).

Figure 2. Positive selection requires continuous Zap-70 catalytic activity

(a) Zap70(AS) FTOC samples after 5 days of culture in the presence of the indicated concentrations of 3-MB-PP1. Flow cytometry plots are gated on total viable cells (top), DP cells (second row), CD4⁺SP cells (third row), and CD8⁺SP cells (bottom row). The numbers indicate the percentage of cells within each gate. (b) Fetal thymic lobes were cultured in the absence of inhibitor for 4 days (left column, “before pulse”). On day 4, lobes were cultured with DMSO only (middle column, “Day 6 DMSO”) or 5 µM 3-MB-PP1 (right column, “Day 6 3-MB-PP1”) for an additional 48 hand analyzed. Data in (a) and (b) are representative of 2 and 3 independent experiments, respectively.

Figure 3. Positive selection in thymic slices requires Zap-70 catalytic activity

Pre-selection DP cells were generated by transferring bone marrow from Zap-70(AS) OT-I donor mice into irradiated B2m^−/− recipients. (a) Pre-selection CD45.2⁺Zap70(AS) OT-I DP cells were introduced onto CD45.1⁺B2m^−/− or WT thymic slices in the presence of DMSO alone or 3-MB-PP1 and analyzed at 24, 48, 60, or 72 hs for the presence of CD8⁺SP cells. The percentages of CD8⁺SP cells were determined among the CD45.2⁺ cells, and normalized relative to the average of the 72hr DMSO samples. The graph displays the average percentage of CD8⁺SP (± s.e.m.) from technical triplicate samples. Data are from one representative experiment out of three independent experiments. (b) Histograms show the expression of CD5 (top) and CD69 (bottom) on DP cells from panel (a) after 24 h of culture. (c) Graphs show the corrected calcium ratio and interval speed of two (each panel) representative individual Zap70(AS) OT-I cells detected by two-photon imaging in WT thymic slices. (d) Each horizontal line represents an individual Zap70(AS) OT-I cell track within a WT thymic slice. The arrows represent the time point at which DMSO or 10 µM 3-MB-PP1 was added to the thymic slices. The black segments represent the time points during which elevated [Ca²⁺]_i was detected. (e) Imaging data was converted to dot plot form to display the calcium ratio versus time. Each dot represents a single time point for each cell. The horizontal red line delineates signaling from non-signaling cells. The numbers represent the percentage of events with low or high [Ca²⁺]_i levels within each gate. Imaging data are representative of three movies from two independent experiments. *P <0.05, **P <0.005, NS not significant (Student’s t-test).

Figure 4. Temporal and dose-dependent requirements for Zap-70 catalytic activity during positive selection

(a,b) Pre-selection Zap-70(AS) OT-I DP cells were cultured on WT thymic slices for a total of 72 h. Schematics on the left indicate the time intervals during which 2.5 µM 3-MB-PP1 was added (closed bars), or cultured without inhibitor (open bars). Graphs display the mean percentage (± s.e.m.) of CD8⁺SP cells from technical triplicate samples normalized relative to the DMSO treated control sample. Data in panel (a) are from one representative experiment out of three independent experiments. Panel (b) displays data compiled from 5 independent experiments. (c) CD5 expression on DP cells from panel (b), after 72 h of culture on thymic slices. Data are from one representative experiment out of three independent experiments. (d) Pre-selection Zap70^+/− or Zap-70(AS) OT-I DP cells were introduced onto B2m^−/− or WT thymic slices in the constant presence of the indicated concentrations of 3-MB-PP1 or DMSO alone for 72 h. Graph shows technical triplicate samples for each condition, and the horizontal line represents the mean and error bars show (± s.e.m.). (e) CD5 expression on DP cells after 48 h of culture. Data shown in (d) and (e) are from one representative experiment out of three independent experiments. *P <0.05, **P <0.005, ***P <0.0005, NS not significant (Student’s t-test).

Figure 5. Invariant Zap-70 dependent signal threshold for positive selection

(a) Pre-selection OT-I Zap-70(AS)-Nur77-GFP DP cells were cultured on WT thymic slices for 72 hours in the presence of the indicated concentrations of 3-MB-PP1. Graph displays the average percentage of CD8⁺SP (± s.e.m.) from technical triplicate samples detected at 72 hours, normalized to the average of the DMSO only control samples.(b) Histograms show GFP expression by the CD8⁺SP populations from indicated inhibitor treatment conditions. Closed gray histogram shows the GFP expression on DP cells cultured on non-selecting B2m^−/− thymic slices. (c) Zap-70(AS)-Nur77-GFP thymic lobes were cultured for 7 days in the presence of the indicated concentrations of 3-MB-PP1. Flow cytometry plots are gated on DP cells and the numbers indicate the percentage of cells within the TCRβ^hi CD69⁺ population (top). Histograms show GFP expression of TCRβ⁻ CD69⁻ DP cells (black) versus TCRβ^hi CD69⁺ cells (red) (bottom). (d) GFP expression of the TCRβ^hi CD69⁺ cells from Zap-70(AS)-Nur77-GFP FTOC. Data from (a,b) are representative of 2 independent experiments. Data from (c,d) are representative of 3 independent experiments. *P <0.05, **P <0.0005, NS not significant (Student’s t-test).

Figure 6. Negative selection requires Zap-70 catalytic activity in a dose and time-dependent manner

Pre-selection CD45.2⁺Zap70^+/− or Zap70(AS) OT-I DP cells were co-transferred with dye-labeled, CD45.2⁺ F5 TCR DP cells at a 1:1 ratio, onto CD45.1⁺ WT thymic slices. Slices were then cultured with or without 0.1 nM OVA peptide to induce negative selection. All graphs in this figure display the technical triplicate samples for each condition, with the horizontal line representing the mean OT-I: F5 ratio (± s.e.m.), normalized to the average ratio from the control samples, specified below. (a) After 24 h of culture with constant exposure to the indicated inhibitors, slices were analyzed for the ratio of viable OT-I cells to F5 cells. Ratios were normalized to the control sample (DMSO alone, 0 nM OVA). Inhibitor concentrations: 3-MB-PP1 (5 µM), HXJ42 (1 µM), BAY61-3606 (1 µM). (b) Histograms show CD69 expression on viable DP cells from slices cultured with the indicated inhibitors. (c) Zap-70(AS) OT-I DP thymocytes were cultured on B2m^−/− or WT thymic slices with OVA and analyzed at the indicated time points. Ratios were normalized to the 0 hr samples. (d) Histogram shows CD69 expression on viable DP cells at each indicated time point. (e) HXJ42 (1 µM) was added to thymic slices at the indicated times after co-introduction of pre-selection OT-I thymocytes and OVA peptide. Ratios were normalized to the average of the (DMSO, 0 nM OVA) control samples. Horizontal bars represent the average of technical triplicate samples (± s.e.m.). (f) Histogram shows CD69 expression on viable DP cells at 24 h. (g) Zap-70(AS) OT-I DP thymocytes were cultured on WT thymic slices for 24 h with OVA in the presence of the indicated concentrations of HXJ42. Horizontal bars represent the average of technical triplicate samples (± s.e.m.). Ratios were normalized to the average of the (DMSO, 0 nM OVA) control samples. All panels show data from one representative experiment out of 3 independent experiments. *P <0.05, **P <0.005, ***P <0.0005, NS not significant (Student’s t-test).