Hyperresponse to T-cell receptor signaling and apoptosis of Id1 transgenic thymocytes

Abstract

The basic helix-loop-helix transcription factors, E2A and HEB, play important roles in T-cell development at multiple checkpoints. Expression of their inhibitor, Id1, abolishes the function of both transcription factors in a dose-dependent manner. The Id1 transgenic thymus is characterized by an accumulation of CD4- CD8- CD44+ CD25- thymocytes, a dramatic reduction of CD4+ CD8+ thymocytes, and an abundance of apoptotic cells. Here we show that these apoptotic cells carry functional T-cell receptors (TCRs), suggesting that apoptosis occurs during T-cell maturation. In contrast, viable Id1 transgenic CD4 single positive T cells exhibit costimulation-independent proliferation upon treatment with anti-CD3 antibody, probably due to a hyperresponse to TCR signaling. Furthermore, Id1 expression causes apoptosis of CD4 and CD8 double- or single-positive thymocytes in HY- or AND-TCR transgenic mice under conditions that normally support positive selection. Collectively, these results suggest that E2A and HEB proteins are crucial for controlling the threshold for TCR signaling, and Id1 expression lowers the threshold, resulting in apoptosis of developing thymocytes.

FIG. 1.

Enhanced apoptosis of DP thymocytes in Id1 transgenic mice. (A) EMSA with nuclear extracts prepared with total thymocytes from wild-type and heterozygous Id1 transgenic mice. The E-box and Oct-1 probes were as described previously (24). As a control, EMSA with a nuclear extract from the WEHI231 B-cell line was performed with or without anti-E47 antibodies to supershift the E47 containing complexes as marked with arrows. The amount of Oct-1 binding complex serves as a control for the amount of nuclear extract in each sample. (B). Thymocytes from wild-type and Id1 transgenic mice on the FVB/N background were stained with the indicated antibodies. The number in each quadrant is the percentage of the subset of thymocytes. (C). Sorted viable DP thymocytes were stained with Annexin V-FITC with or without 8 h of culture at 37°C. The percentage of Annexin-positive cells is shown on top of the gate.

FIG. 2.

Isolation of genomic DNA from Id1 transgenic mice for TCR gene rearrangement assays. The scheme for isolation of genomic DNA from viable and apoptotic cells is diagrammed. Aliquots of DNA samples prepared were analyzed by agarose gel electrophoresis.

FIG. 3.

Normal TCRβ chain usage in Id1 transgenic thymocytes. Thymocytes from wild-type and Id1 transgenic mice on FVB/N (A) and C57BL/6 (B) backgrounds were stained with FITC-conjugated antibodies specific for each of the indicated Vβ chains. The percentage of each Vβ specific population out of total thymocytes is shown.

FIG. 4.

Costimulation-independent proliferation of CD4⁺ thymocytes from Id1 transgenic mice. (A) Sorted CD4⁺ thymocytes were plated in triplicates (2 × 10⁵ cells per well) in a 96-well plate coated with indicated concentrations of anti-CD3ɛ antibody with or without soluble anti-CD28 MAb (2 μg/ml) and incubated for 48 h. Cells were pulsed with 1 μCi of [³H]thymidine per well for the last 18 h of incubation. The amount of [³H]thymidine incorporated by proliferating thymocytes was measured by scintillation counting. (B) Sorted CD4⁺ thymocytes from wild-type and Id1 transgenic mice were stimulated with plate-bound anti-CD3ɛ antibody (10 μg/ml) in a 96-well plate with or without PMA (2.5 ng/ml) for 48 or 72 h. [³H]thymidine incorporation was measured as described for panel A.

FIG. 5.

CD4⁺ thymocytes from Id1 transgenic mice do not display the phenotype of memory T cells. (A) Surface marker expression in CD4⁺ thymocytes. Thymocytes from wild-type and Id1 transgenic mice were stained with anti-CD4 and anti-CD8 plus one of the antibodies specific for the indicated antigens. The levels of the indicated surface antigens on gated CD4 SP thymocytes are shown in histograms. Solid lines represent wild-type thymocytes, and shaded areas designate Id1 transgenic thymocytes. (B) Cytokine secretion by activated CD4⁺ thymocytes. CD4⁺ thymocytes (2 × 10⁵ cells per well) were stimulated for 48 h with plate-bound anti-CD3ɛ (10 μg/ml) with or without soluble anti-CD28 monoclonal antibody (2 μg/ml). Culture media were collected and analyzed for the indicated cytokines by enzyme-linked immunosorbent assay. Bars: 1, wild-type CD4⁺ thymocytes stimulated with anti-CD3ɛ and anti-CD28; 2, Id1 transgenic CD4⁺ thymocytes stimulated with anti-CD3ɛ alone; 3, Id1 transgenic CD4⁺ thymocytes stimulated with anti-CD3ɛ and anti-CD28.

FIG. 6.

NF-κB is necessary but not sufficient for costimulation-independent proliferation. (A) EMSA for NF-κB DNA-binding activities. CD4⁺ thymocytes (2 × 10⁵ cells per well) were stimulated with indicated concentrations of plate-bound anti-CD3ɛ with or without soluble anti-CD28 for 24 h. Nuclear extracts were prepared from the stimulated and unstimulated thymocytes and used in EMSAs. The indicated subunits present in each of the specific NF-κB binding complexes were identified by supershift assays (data not shown). A ubiquitously expressed nonspecific binding complex (NS) served as a loading control. (B) Inhibition of CD4⁺ thymocyte proliferation by an inhibitor of NF-κB activation. Proliferation cultures were set up as described for Fig. 5B, except the inhibitor peptide (NBD) or control peptide (NBA) were included at the concentration of 200 μM. [³H]thymidine incorporation was measured as described for Fig. 4A.

FIG. 7.

Effects of Id1 on thymocyte positive selection in H-Y TCR transgenic mice. (A) Total thymocytes from littermates with indicated genotypes were analyzed by fluorescence-activated cell sorting. The numbers shown indicate the percentages of the subsets. Total thymic cellularity is shown in parenthesis. Male (M) and female (F) mice are as labeled. Eleven litters of mice were analyzed and identical results were obtained. The data shown are representative of analyses of one such litter. Histograms of anti-VαT3.70 staining of total thymocytes (B) or the CD3ɛ^hi population (C) represent HY-TCR expression. The percentage of HY-TCR-positive cells is shown on top of the gate.

FIG. 8.

Effect of Id1 on thymocyte positive selection in AND transgenic mice. (A) Thymocytes from littermates with the indicated genotypes were stained with anti-CD4 and anti-CD8 antibodies. The numbers shown indicate the percentages of the subsets. The total thymic cellularity is shown in parentheses. Eight litters of mice were analyzed, and identical results were obtained each time. The data shown are representative of analyses for one such litter. (B) Detection of apoptosis. Total thymocytes from mice with the indicated genotypes were labeled with FITC-conjugated dUTP and analyzed by flow cytometry. The percentage of apoptotic cells is shown on top of the gate.