Nck recruitment to the TCR required for ZAP70 activation during thymic development

Abstract

The adaptor protein Nck is inducibly recruited through its SH3.1 domain to a proline-rich sequence (PRS) in CD3ε after TCR engagement. However, experiments with a knockin mutant bearing an 8-aa replacement of the PRS have indicated that Nck binding to the TCR is constitutive, and that it promotes the degradation of the TCR in preselection double-positive (DP) CD4(+)CD8(+) thymocytes. To clarify these discrepancies, we have generated a new knockin mouse line (KI-PRS) bearing a conservative mutation in the PRS resulting from the replacement of the two central prolines. Thymocytes of KI-PRS mice are partly arrested at each step at which pre-TCR or TCR signaling is required. The mutation prevents the trigger-dependent inducible recruitment of endogenous Nck to the TCR but does not impair TCR degradation. However, KI-PRS preselection DP thymocytes show impaired tyrosine phosphorylation of CD3ζ, as well as impaired recruitment of ZAP70 to the TCR and impaired ZAP70 activation. Our results indicate that Nck is recruited to the TCR in an inducible manner in DP thymocytes, and that this recruitment is required for the activation of early TCR-dependent events. Differences in the extent of PRS mutation could explain the phenotypic differences in both knockin mice.

Figures

FIGURE 1.

Mutation of the two central prolines in CD3ε abolishes Nck recruitment to the TCR. (A) Cartoon of the cytoplasmic tail of CD3ε illustrating different sequence motifs. A cartoon indicating the mutations in the PRS previously described (in boldface) is shown (right panel). (B) Pull-down (pd) of lysates of COS cells transfected with either Flag-tagged WT or CD3ε double proline mutant (fεwt or fεmut) with GST-SH3.1 analyzed in immunoblots (IB) probed with anti-Flag. Relative densitometric values of three samples run in parallel are shown (right panel). Data are representative of two experiments. (C) Thymocytes and splenocytes isolated from either WT or KI-PRS mice were left unstimulated or stimulated with anti-CD3 for 5 min before lysis and GST-SH3.1 pd. Relative densitometric values of three samples run in parallel are shown (right panels). Data are representative of three experiments. (D) Preselection DP thymocytes from WT mice were enriched by panning on anti-TCRβ–coated plates at 0°C. The double-color plot shows that 94% of the nonattached thymocytes were DP for CD4 and CD8, whereas the single-color histogram demonstrates that all TCRbright and TCRint were removed. The purified preselection DP WT thymocytes were stimulated with anti-CD3 for the times indicated and lysed. Subsequently, a GST-SH3.1 pd and anti-CD3ζ IB were performed. Relative densitometric values of three samples run in parallel are shown (right panel). Data are representative of two experiments. (E) Nontransgenic WT and KI-PRS total thymocytes were stimulated with anti-CD3, and the lysates precipitated with anti-CD3 (Ip) were analyzed in IB probed with anti-Nck (top). Anti-CD3ζ served as a loading control. Likewise, total cell lysates were immunoblotted with anti-Nck to reveal equal levels of expression. Relative densitometric values of three samples run in parallel are shown (right panel). (F) A similar experiment was carried out with thymocytes from OT-I TCR transgenic mice that were stimulated with T2-Kb APCs loaded with OVAp for the times indicated. Relative densitometric values of three samples run in parallel are shown (right panel). Data are representative of three experiments. BRS, basic amino acid–rich sequence; EC, extracellular domain; ER-RS, endoplasmic reticulum retention sequence; IC, intracellular domain; PTB, phosphotyrosine-binding motif; PxxP, consensus SH3 domain-binding motif; TM, transmembrane domain.

FIGURE 2.

High TCR expression in KI-PRS DP thymocytes is not the result of lower degradation. (A) Distribution in the four major thymic populations of nontransgenic WT and KI-PRS mice according to CD4 and CD8 expression. TCR expression for WT (shadowed) and KI-PRS (solid line) mice are shown in the histograms. Mean fluorescence intensity (MFI) values for TCRβ expression are indicated in gray (WT) and black (KI-PRS) type. Data are representative of six experiments. (B) Total thymocytes were incubated with or without anti-CD3 for the times indicated, and anti-CD3ζ immunoprecipitates were probed in nonreducing immunoblots with anti-ubiquitin. Based on their mobility, the lower band appears to be the monoubiquitinated and the upper band the bis-ubiquitinated CD3ζ homodimer. The membranes were reprobed with anti-CD3ζ (bottom panels), and relative densitometric values of three samples run in parallel are shown (right panels). Data are representative of three experiments.

FIGURE 3.

Mutation of the PRS arrests thymocyte development at intermediate stages of differentiation. (A) Flow cytometry of OT-I TCR transgenic WT and KI-PRS thymocytes showing the percentage of DP and postselection transitional (CD4+CD8low and CD4low CD8low) thymocyte populations. Absolute cell number in each subpopulation counted in three mice per group is shown below. Data are representative of five experiments. (B) CD5 and transgenic TCR (Vα2) analysis of DP subpopulations (top histograms). Lower histograms show the expression of positive selection markers in the total DP population and in the DP3 subpopulation. Absolute cell number in each DP subpopulation counted in three mice per group is shown to the right. Data are representative of five experiments. (C) Flow cytometry of nontransgenic WT and KI-PRS thymocytes showing the percentage of DP, transitional (CD4+CD8low), CD4SP, and CD8SP thymocyte populations. DP thymocytes were analyzed for the expression of CD5 and TCR (CD3) to assess their distribution in the DP1, DP2, and DP3 subpopulations. Absolute cell number in each thymic subpopulation was counted in four mice per group. Data are representative of three experiments. (D) Flow cytometry analysis of CD24 and TCR expression in nontransgenic WT and KI-PRS mice allowed the thymocytes to be divided into four subpopulations (a–d). These subpopulations were reanalyzed according to CD4 and CD8 expression (bottom panels). Absolute cell number in the a–d subpopulation, counted in four mice per group, is shown (right panel). Data are representative of three experiments.

FIGURE 4.

Partial arrest of thymocyte differentiation at the pre-TCR stage in nontransgenic and OT-I TCR transgenic KI-PRS mice. (A) Flow cytometry of thymocytes from nontransgenic WT and KI-PRS mice showing thymocyte distribution in the four major DN subpopulations. Thymocytes were gated in the DN quadrant according to CD4 and CD8 expression, and the selected cells were reanalyzed for the expression of CD25 and CD44 markers. The DN3 and DN4 subpopulations correspond to CD44−CD25+ and CD44−CD25− DN thymocytes, respectively. Absolute cell number in each thymic subpopulation was counted in four mice per group (right panel). Data are representative of six experiments. (B) The analysis described in (A) was also performed on thymocytes from OT-I TCR transgenic WT and KI-PRS mice. Absolute cell number in each thymic subpopulation was counted in three mice per group (right panel). Data are representative of six experiments.

FIGURE 5.

PRS mutation impairs early activation events in thymocytes. (A) Intracellular flow cytometry analysis of ZAP70 expression in total DP and DP1-DP3 subpopulations. Histograms corresponding to WT are shadowed, and those corresponding to KI-PRS are indicated by solid lines. Mean fluorescence intensity (MFI) values for WT and KI-PRS mice are shown in gray and black type, respectively. Data are representative of three experiments. (B) Intracellular flow cytometry analysis of phospho-Y319 ZAP70 in total DP, in DP1-DP3 subpopulations, and in transitional (CD4+CD8low and CD4lowCD8low) and mature (CD8SP) thymocytes. Data are representative of three experiments. (C) Intracellular flow cytometry analysis of phospho-Y292 ZAP70 of thymocyte populations as in (B). Data are representative of three experiments. (D) Phospho-CD3 was analyzed after stimulation of OT-I thymocytes from WT and KI-PRS mice with T2-Kb APCs loaded with OVAp, probing anti-CD3 precipitates in immunoblots with anti-phosphotyrosine. The membrane was reprobed with an Ab specific for the phosphorylated form of the N-terminal tyrosine of the first ITAM of CD3ζ (EM-26) and with an Ab directed against total ZAP70. Data are representative of three experiments.