The Syk and ZAP-70 SH2-containing tyrosine kinases are implicated in pre-T cell receptor signaling

Abstract

An early stage in thymocyte development, after rearrangement of the beta chain genes of the T cell receptor (TCR), involves expression of the pre-TCR complex and accompanying differentiation of CD4(-)CD8(-) double negative (DN) cells to CD4(+)CD8(+) double positive (DP) cells. The ZAP-70 and Syk tyrosine kinases each contain two N-terminal SH2 domains that bind phosphorylated motifs in antigen receptor subunits and are implicated in pre-T receptor signaling. However, mice deficient in either ZAP-70 or Syk have no defect in the formation of DP thymocytes. Here we show that, in mice lacking both Syk and ZAP-70, DN thymocytes undergo beta chain gene rearrangement but fail to initiate clonal expansion and are incapable of differentiating into DP cells after expression of the pre-TCR. These data suggest that the ZAP-70 and Syk tyrosine kinases have crucial but overlapping functions in signaling from the pre-TCR and hence in early thymocyte development.

Figure 1

Thymocyte numbers in neonatal mice. The averaged numbers of thymocytes from 1-day-old wild-type (wt) and mutant neonates are plotted in the bar graph as shown. Black dots indicate the actual thymocyte count of each sample.

Figure 2

Arrested thymocyte development in syk^−/−; zap-70^−/− double mutants. (A) FACScan analysis of thymocytes from 1-day-old neonates with anti-CD4 (fluorescein isothiocyanate) and anti-CD8 (phycoerythrin) antibodies. The percentage of cells in the four quadrants are indicated in the top right-hand corner. (B) FACScan analysis of thymocytes with anti-CD25 (fluorescein isothiocyanate) and anti-CD44 (phycoerythrin) antibodies. Only histograms of CD25 expression are depicted, and the percentage of the CD25⁺ population in each group is shown under the bar denoted M1. (C) FACScan analysis of thymocytes with anti-CD25 (fluorescein isothiocyanate) and anti-CD44 (phycoerythrin) antibodies. The percentage of cells in the three regions, R3, R4, and R5, are shown in the boxes beneath.

Figure 3

Arrested development of DN thymocytes in syk^−/−; zap-70^−/− mutant mice. (A) Comparison of the gene rearrangement activity of the TCR β chain locus in wild-type (wt), syk^−/−; zap-70^−/− mutant, and rag-2 mice. The products of rearrangement, as detected by PCR, between the V_β or the D_β gene segments and the J_β2.1–J_β2.6 gene segments are indicated. GL marks the PCR products of germ line sequence between D_β and J_β2.6 segments. (B) Cell size distribution of CD25⁺ thymocytes. Total thymocytes stained with CD25 and CD44 antibodies (as described in Fig. 2B) were gated for CD25 expression and analyzed for cell-size distribution base on the forward scatter values. The results from a wild-type (solid line) mouse and a syk^−/−; zap-70^−/− mutant mouse (shaded area) are overlaid in the histogram as shown. The smaller “E” and the larger “L” subsets are marked. (C) FACScan analysis of thymocytes with anti-CD3 (fluorescein isothiocyanate) and anti-TCR β antibodies. The percentages of thymocytes in the four quadrants are shown in the top right-hand corner.

Figure 4

The roles of Syk and ZAP-70 in B and T cell development. The roles of Syk and ZAP-70 at different points in the development of the αβ T lineage (A), the γδ T lineage (B), and the B cell lineage (C) are depicted. The development of the γδ lineage is derived from the common intermediate T cell precursors as shown in A at a point before pre-TCR signaling (CD44⁻CD25⁺ “E”). The cell types whose development is most obviously affected by mutations in syk or zap-70 are shaded. This summary is based on the present study and others reported (, –12, 29).