The Ets-1 transcription factor is required for complete pre-T cell receptor function and allelic exclusion at the T cell receptor beta locus

Abstract

The pre-T cell receptor (TCR) functions as a critical checkpoint during alphabeta T cell development. Signaling through the pre-TCR controls the differentiation of immature CD4(-)CD8(-)CD25(+)CD44(-) [double-negative (DN)3] thymocytes into CD4(+)CD8(+) double-positive (DP) cells through the CD4(-)CD8(-)CD25(-)CD44(-)(DN4) stage. In addition, pre-TCR activity triggers expansion and survival of thymocytes and inhibits TCRbeta gene rearrangement through a process referred to as allelic exclusion. Whereas many proteins involved in the pre-TCR transduction cascade have been identified, little is known about the nuclear factors associated with receptor function. Here, we use gene targeting to inactivate the Ets-1 transcription factor in mice and analyze pre-TCR function in developing Ets-1-deficient (Ets-1(-/-)) thymocytes. We find that inactivation of Ets-1 impairs the development of DN3 into DP thymocytes and induces an elevated rate of cell death in the DN4 subset. This defect appears specific to the alphabeta lineage because gammadelta T cells maturate efficiently. Finally, the percentage of thymocytes coexpressing two different TCRbeta chains is increased in the Ets-1(-/-) background and, in contrast with wild type, forced activation of pre-TCR signaling does not block endogenous TCRbeta gene rearrangement. These data identify Ets-1 as a critical transcription factor for pre-TCR functioning and for allelic exclusion at the TCRbeta locus.

Fig. 1.

Ets-1 is required for efficient differentiation of αβ thymocytes. (A and B) Flow cytometric analysis of thymocytes from wild-type/RAG-2^-/- (Wt → RAG) and Ets-1^-/-/RAG-2^-/- (Ets-1^-/- → RAG) chimera mice. Numbers represent the percentage of cells found in the indicated quadrant or region. Histograms show the level of CD5 expression on CD4⁺CD8⁺ DP-gated thymocytes. Wild-type thymocytes are indicated by fine lines, and Ets-1^-/- thymocytes are indicated by thick lines. (C) Analysis of thymus cell numbers. Thymus cell suspensions were counted, stained with anti-CD3-APC, anti-CD8-FITC, anti-CD4-bi, and anti-γδ-PE, and the percentages of triple-negative (CD4^-CD8^-CD3^-), DP (CD4⁺CD8⁺), SP4 (CD4⁺), SP8 (CD8⁺), and γδ⁺ cells determined by FACS. Cell numbers were calculated by multiplying the total number of thymocytes by the percentage of each subset. Wild-type and Ets-1^-/- subsets are indicated by open and shaded bars, respectively. These results are representative of six independent chimeras generated from different fetal liver cell injections. For each staining, potential RAG2^-/--derived cells were gated out by using the Ly5.2 cell-surface marker. Heterozygous chimeras were identical to wild-type chimeras and are not shown.

Fig. 2.

Impaired development of Ets-1^-/--immature thymocytes. (A) Flow cytometric analysis of immature thymocytes from wild-type/RAG-2^-/-/γc^-/- (Wt → RAG/γc) and Ets-1^-/-/RAG-2^-/-/γc^-/- (Ets-1^-/- → RAG/γc) chimera mice. Thymus cell suspensions were stained with anti-CD8-bi, anti-CD4-bi, anti-CD3-bi, anti-Ly5.1-bi, anti-CD25-PE, anti-CD44-FITC, and propidium iodide. Dot plots represent CD25 and CD44 expression on live (propidium iodide^-) CD3^-CD4^-CD8^-Ly5.1^- thymocytes. Numbers indicate the percentage of cells in each quadrant. (B) Flow cytometric analysis of day 18.5 p.c. embryonic thymocytes from wild-type (Wt) and Ets-1^-/-. Thymus cell suspensions were stained with anti-CD8-FITC and anti-CD4-PE and analyzed by FACS. For analysis of early thymocytes (Lower), cells were stained with anti-CD8-APC, anti-CD4-APC, anti-CD3-APC, anti-CD25-PE, and anti-CD44-FITC. Dot plots represent the expression of CD25 and CD44 on CD3^-CD4^-CD8^- triple-negative thymocytes. Percentages of cells are shown in the dot plots. (C) Cell-cycle analysis of sorted CD25⁺CD3^-CD4^-CD8^- (DN2/3), CD25^-CD44^-CD3^-CD4^-CD8^- (DN4), and CD4⁺CD8⁺ (DP) thymocytes of wild-type (Wt) and Ets-1^-/- day 18.5 p.c. embryos. The percentages of BrdUrd⁺ cycling cells and of BrdUrd^- quiescent cells are shown in each dot plot. (D) Cell death analysis. Thymocytes of adult wild-type (Wt) and Ets-1^-/- mice were stained with CD25-, CD44-, CD3-, CD4-, and CD8-specific antibodies, further labeled with annexin V, and analyzed by FACS. The percentage of annexin V⁺ cells among CD25^-CD44^-CD3^-CD4^-CD8^- (DN4) is shown in each histogram. These results are representative of three independent experiments.

Fig. 3.

Allelic exclusion at the TCRβ locus is inefficient in Ets-1^-/- thymocytes. (A) Thymocytes from wild-type/RAG-2^-/- (Wt → RAG), Ets-1^-/-/RAG-2^-/- (Ets-1^-/- → RAG), TCRβ-transgenic wild-type/RAG-2^-/- (WtTgb → RAG), and TCRβ-transgenic Ets-1^-/-/RAG-2^-/- (Ets-1^-/-Tgb → RAG) chimera mice were stained with anti-CD3-FITC, anti-Vβ8-PE, anti-Vβ5-bi, anti-Vβ7-bi, and anti-Vβ9-bi antibodies and analyzed by FACS. CD3⁺, Vβ8⁺ cells (Left) were analyzed for Vβ 5,7,9 expression (Right). The percentage of Vβ8⁺ cells that also express Vβ 5,7,9 is shown in the dot plot. (B) Thymocytes from wild type/RAG-2^-/- (Wt → RAG), Ets-1^-/-/RAG-2^-/- (Ets-1^-/- → RAG) chimera mice carrying a TCRβ transgene (Tgβ) or not transgenic (Non Tg), were stained with antibodies directed against CD4, CD8, and Ly5.2 and analyzed by FACS. Dot plots represent CD4 and CD8 expression on Ly5.2⁺ cells. The percentage of DN and DP cells is shown in the dot plots. (C) PCR analysis of TCRβ gene rearrangements. Genomic DNA isolated from thymocytes of nontransgenic or TCRβ-transgenic (Tβ), wild-type (+/+), or Ets-1^-/- (-/-) chimera mice was amplified with primers that detect rearrangements among Dβ2 and Jβ2(Upper)or among Vβ11 or Vβ5 and Jβ2(Lower). PCR products were analyzed by Southern blot. Lanes Kd and 0 correspond to RAG2^-/- kidney and to no DNA, respectively. For Dβ2 and each Vβ, there are six bands that correspond to rearrangements to the Jβ2.1 through Jβ2.6 gene segments. (Upper) Triangles represent serial dilutions at a 1:5 ratio of wild type (+/+) thymocyte DNA into RAG2^-/- kidney (Kd) DNA. Products generated by the germ-line Dβ2-Jβ2 (GL) region are indicated. (Lower) Two quantities of DNA (100 and 20 ng) were used in the assay. Equivalent loading was confirmed by amplification of a fragment of the Hprt gene.

Fig. 4.

Analysis of thymocyte development in Ets-1^-/- chimeras mice expressing a Lck^F505 transgene. (A) Thymocytes from wild-type/RAG-2^-/- (Wt → RAG), Ets-1^-/-/RAG-2^-/- (Ets-1^-/- → RAG) chimera mice expressing an activated Lck transgene (Lck^*Tg) or none (Non Tg) were analyzed for CD4 and CD8 expression. The percentage of cells falling in each quadrant is indicated. (B) Histogram showing CD25 expression on CD3^-CD4^-CD8^-Ly5.2⁺ thymocytes from wild-type/RAG-2^-/- (Wt → RAG), Ets-1^-/-/RAG-2^-/- (Ets-1^-/- → RAG) chimera mice expressing Lck^F505 (thick lines) or nontransgenic (dotted lines). The percentages of cells falling in the gate are shown in the histograms. (C) Thymocytes from the indicated chimeras (referenced as in A) were analyzed for cell-surface expression of CD3 and TCRαβ. The percentages of CD3⁺ and TCRαβ⁺ cells are shown in the dot plots. For each staining, potential RAG2^-/--derived cells were gated out by using the Ly5.2 cell-surface marker. (D) Histogram showing the total number of cells in the thymus of RAG chimera mice injected with wild-type (+/+), Ets-1 (-/-), wild-type/Lck^* transgenic (+/+ Lck^*), and Ets-1^-/-/Lck^* transgenic (-/- Lck^*) fetal liver cells. These results are representative of three independent chimeras of each genotype.