Acquisition of a functional T cell receptor during T lymphocyte development is enforced by HEB and E2A transcription factors - PubMed (original) (raw)

Acquisition of a functional T cell receptor during T lymphocyte development is enforced by HEB and E2A transcription factors

Mary Elizabeth Jones et al. Immunity. 2007 Dec.

Abstract

The T cell receptor (TCR) is required for positive selection and the subsequent transition from the CD4(+)CD8(+) double-positive (DP) to the CD4(+) or CD8(+) single-positive (SP) stage of alphabeta T cell development. The molecular mechanism that maintains DP fate prior to the acquisition of a functional TCR is not clear. We have shown here that the structurally and functionally related transcription factors HEB and E2A work together to maintain DP fate and to control the DP to SP transition. Simultaneous deletion of HEB and E2A in DP thymocytes was sufficient for DP to SP transition independent of TCR. Loss of HEB and E2A allowed DP cells to bypass the requirement for TCR-mediated positive selection, downregulate DP-associated genes, and upregulate SP-specific genes. These results identify HEB and E2A as the gatekeepers that maintain cells at the DP stage of development until a functional alphabetaTCR is produced.

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Figures

Figure 1

Figure 1. T Cell-Specific Deletion of HEB and E2A Generates Peripheral CD8TCR− Cells

(A) Representative staining of indicated tissues from 2-month-old _Tcf12_f/f_Tcfe2a_f/fCD4Cre− control and _Tcf12_f/f_Tcfe2a_f/fCD4Cre+ mice for CD4, CD8α, and TCRβ. Percentages in each quadrant are displayed. (B) Cell number in the thymus of 2–3-month-old mice (n = 9). (C) Cell numbers per 10,000 events collected from lymph node (LN) of 2–7-month-old mice (_Tcf12_f/f_Tcfe2a_f/fCD4Cre− n = 7, _Tcf12_f/f_Tcfe2a_f/fCD4Cre+ n = 8). ***p < 0.001 and *p = 0.012, Student’s t test, two-tailed. Graphed results in (B) and (C) are means with error bars representing SD.

Figure 2

Figure 2. HEB and E2A Double-Deficient CD8TCR+ and CD8TCR+ Cells Produce IFN-γ and Upregulate Activation Markers upon Stimulation

In vitro culture of LN cells isolated from _Tcf12_f/f_Tcfe2a_f/fCD4Cre− and _Tcf12_f/f_Tcfe2a_f/fCD4Cre+ mice with or without (unstimulated) PMA and ionomycin for 6 hr. Cells were analyzed by FACS analysis for intracellular IFN-γ and surface CD69 and CD44 expression. Plots are gated on CD8+ cells, and percentages in each quadrant are displayed. Data are representative of three independent experiments.

Figure 3

Figure 3. CD8TCR− Cells Are T Cells Developing in the Absence of a Functional TCR

(A) TCRβ V to DJ rearrangement analysis on DNA from _Tcf12_f/f_Tcfe2a_f/fCD4Cre− and _Tcf12_f/f_Tcfe2a_f/fCD4Cre+ sorted thymus (DP) and LN (CD8TCR+, CD8TCR−) populations used Vβ8 5′ consensus and Jβ2.7 3′ primers. Rearrangement products involving Jβ2.1–Jβ2.7 are shown. _Lat_−/− (Zhang et al., 1999) and _Rag2_−/− total thymocyte DNA were used as positive and negative controls, respectively. CD14 was used as a loading control. Molecular weight marker is labeled (M). (B) Intracellular TCRβ expression in specified populations from _Tcf12_f/f_Tcfe2a_f/fCD4Cre+ LN. (C) RT-PCR analysis for TCRα (Cα) expression in sorted populations from _Tcf12_f/f_Tcfe2a_f/fCD4Cre− and _Tcf12_f/f_Tcfe2a_f/fCD4Cre+ mice. _Rag2_−/− total thymocyte cDNA was used as a negative control, and GAPDH was used as a loading control. Three-fold serial dilutions are as shown. (D) Phenotype of thymus and LN cells from EαΔ and _Tcf12_f/f_Tcfe2a_f/fCD4Cre+Eα Δ mice. Percentages in each quadrant are displayed.

Figure 4

Figure 4. _Tcf12f/fTcfe2a_f/fCD4Cre+ DP Thymocytes Survive Poorly, but Differentiate to SP Cells fEfficiently in Culture

(A) Quantitative RT-PCR analysis of RORγt expression in sorted _Tcf12_f/f_Tcfe2a_f/fCD4Cre− and _Tcf12_f/f_Tcfe2a_f/fCD4Cre+ DP cells. Samples were normalized to the expression of GAPDH. Data are from duplicates of two independent experiments (n = 4). ***p < 0.001, Student’s t test, two-tailed. Graphed results are means with error bars representing standard error of the mean (SEM). (B and C) Ex vivo culture analysis of sorted DP thymocytes from _Tcf12_f/f_Tcfe2a_f/fCD4Cre− and _Tcf12_f/f_Tcfe2a_f/fCD4Cre+ mice. (B) DP cells were plated in media alone and analyzed for Annexin V expression by FACS at 0, 6, 18, 25, and 43 hr after plating. Wild-type (B6) and Eα ΔDP cells were used as additional controls. Data are representative of two independent experiments. (C) DP cells were plated on a layer of total thymic stromal cells (day 0) and analyzed by FACS analysis for CD4, CD8, and TCRβ expression on day 1–3. Percentages in each quadrant are displayed. TCRβ expression within the _Tcf12_f/f_Tcfe2a_f/fCD4Cre+ CD8SP gate is shown for day 3. Data are representative of three independent experiments.

Figure 5

Figure 5. Loss of HEB and E2A Initiates CD8 T Cell Maturation and Thymic Egress in the Absence of a TCR-Mediated Positive-Selection Signal

(A) Volcano plot from microarray data comparing gene expression in _Tcf12_f/f_Tcfe2a_f/fCD4Cre+ and _Tcf12_f/f_Tcfe2a_f/fCD4Cre− DP thymocytes. Changes in gene expression are shown as a ratio of _Tcf12_f/f_Tcfe2a_f/fCD4Cre+ to _Tcf12_f/f_Tcfe2a_f/fCD4Cre− cells. Upper plot shows the 15,730 genes remaining after quality filtering, with the 285 genes with greater than 2-fold change and t test p value ≤ 0.05 in red. Lower plot highlights a few genes of interest. (B) Quantitative RT-PCR analysis of Gfi1, Mad1l1, KLF2, and Foxo1 expression in sorted _Tcf12_f/f_Tcfe2a_f/fCD4Cre− and _Tcf12_f/f_Tcfe2a_f/fCD4Cre+ DP cells. Samples were normalized to the expression of GAPDH. Data are from duplicates of two independent experiments (n = 4). ***p < 0.001 and **p = 0.0054, Student’s t test, two-tailed. Graphed results are means with error bars representing SEM. (C and D) FACS analysis of IL-7Rα, CCR7, and CXCR4 expression in DP compared to SP stage in thymus from _Tcf12_f/f_Tcfe2a_f/fCD4Cre− and _Tcf12_f/f _Tcfe2a_f/fCD4Cre+ mice. Cells are pregated on CD4+CD8+ (DP), CD4+CD8− (CD4SP), and CD4−CD8+ (CD8SP) populations. (C) Histograms display IL-7Rα, CCR7, and CXCR4 expression in designated populations from individual stainings. (D) FACS plots demonstrate coordinated expression of CCR7 and IL-7Rα or CXCR4 in designated populations with percentages in each quadrant displayed. Data are representative of two independent experiments.

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