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. 2002 Jun;106(2):212-21.
doi: 10.1046/j.1365-2567.2002.01402.x.

Interleukin-7 inhibits pre-T-cell differentiation induced by the pre-T-cell receptor signal and the effect is mimicked by hGM-CSF in hGM-CSF receptor transgenic mice

Yuko Yasuda  1 , Azusa Kaneko, Ichiko Nishijima, Shoichiro Miyatake, Ken-Ichi Arai
Affiliations

Interleukin-7 inhibits pre-T-cell differentiation induced by the pre-T-cell receptor signal and the effect is mimicked by hGM-CSF in hGM-CSF receptor transgenic mice

Yuko Yasuda et al. Immunology. 2002 Jun.

Abstract

We have previously reported that human granulocyte-macrophage colony-stimulating factor (hGM-CSF) causes a stage-specific inhibition of T-cell receptor (TCR) alphabeta cell development in the thymus of transgenic mice constitutively expressing the hGM-CSF receptor. Since it has been reported that the addition of interleukin-7 (IL-7) to fetal thymic organ culture (FTOC) has similar effects, we compared the effects of IL-7 and hGM-CSF on TCR(alphabeta) cell development in hGM-CSF receptor transgenic mice. We reconstituted fetal lobes with sorted pre-T, or post pre-T CD4(-)CD8(-) precursor cells. The addition of either IL-7 or hGM-CSF to these cultures suppressed further differentiation of pre-T cells but not post pre-T cells. At the same time, the cell number was increased, suggesting that pre-T-cell proliferation is stimulated by these cytokines. Furthermore, the differentiation of recombination-activating gene-1 (RAG-1)-deficient pre-T cells in response to anti-CD3 antibody stimulation was suppressed by either IL-7 or hGM-CSF, suggesting that these cytokines inhibit the pre-T-cell receptor (pre-TCR) signal. This inhibition is unexpected because the pre-TCR signal and the IL-7 signal have previously been considered to be co-operative. Recent analysis of the downstream events of IL-7 receptor and GM-CSF receptor revealed that they share common signal transduction molecules. Our results show that IL-7 is able to promote pre-T cell proliferation and to suppress differentiation induced by the pre-TCR signal. GM-CSF can mimic these biological activities of IL-7 when the pre-T cells express GM-CSF receptors. Our data suggest that both timing and level of activation of the IL-7 signalling pathway must be precisely regulated to facilitate the differentiation of thymocytes.

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Figures

Figure 1
Figure 1
Effects of IL-7 and hGM-CSF on FTOC development. Day 14 fetal thymic lobes were harvested from hGMR transgenic embryos and placed in FTOC. Lobes were cultured in medium alone or in the presence of 500 U/ml of IL-7 or 50 ng/ml of hGM-CSF. FTOC were harvested on day 11 and examined for expression of CD4, CD8, TCRαβ and TCRγδ. Percentages of cells in each CD4/CD8 population are shown in each quadrant. In the histograms, percentages of TCRαβ+ cells (left group of histograms) or TCRγδ+ cells (right group of histograms) are shown. Addition of either IL-7 or hGM-CSF into FTOC resulted in the inhibition of TCRαβ cell development but not TCRγδ cell development. The results are representative of three separate experiments.
Figure 2
Figure 2
Effects of IL-7 and hGM-CSF on the differentiation of triple negative (TN) thymocyte precursor subsets from hGMR transgenic mice. Pre-T and post pre-T cells were sorted from transgenic mice, transferred into 2-deoxyguanosine-depleted wild-type fetal lobes, and then cultured in medium alone or in the presence of 500 U/ml of IL-7 or 50 ng/ml of hGM-CSF. Pre-T-cell cultures were harvested on day 14 and post pre-T cell cultures on day 8. (a) The mean values of the total cell number per lobe after each treatment are presented as percentages of that obtained from FTOC medium alone. The mean values obtained from FTOC medium alone were 43 000/lobe for pre-T FTOC, 85 000/lobe for post pre-T FTOC. Mean and standard deviation (SD) were calculated from results of three separate experiments. Statistics are based on the Student's t-test. *P<0·02. (b) Expression of CD4, CD8, TCRαβ and TCRγδ. Percentages of cells in each CD4/CD8 population are shown in each quadrant. The results are representative of three separate experiments. The addition of either IL-7 or hGM-CSF to the cultures suppressed further differentiation of pre-T cells but not that of post pre-T cells.
Figure 3
Figure 3
Effects of IL-7 and hGM-CSF on pre-T and post pre-T-cell survival in single-cell suspension culture. Pre-T and post pre-T cells were sorted from trangenic mice and 105 cells were cultured in medium alone or in the presence of 500 U/ml of IL-7 or 50 ng/ml of hGM-CSF in 96-well plates. Cells were harvested after 3 days culture and counted. IL-7 supported the survival of pre-T cells but not that of post pre-T cells; hGM-CSF supported the survival of both pre-T and post pre-T cells. Mean and SD were calculated from results of three separate experiments. Statistics are based on the Student's t-test. *P<0·01, **P<0·05.
Figure 4
Figure 4
Effects of IL-7 and hGM-CSF on the pre-TCR signal. hGMR+/−RAG-1−/− thymic lobes were stimulated by anti-CD3 antibody, mimicking the pre-TCR signal. Day 14 fetal thymic lobes were harvested from hGMR−/−RAG-1−/− embryos and placed into FTOC. Lobes were cultured in medium alone or in the presence of 500 U/ml of IL-7 or 50 ng/ml of hGM-CSF for 2 days, then stimulated with 25 µg/ml of anti-CD3 antibody (+αCD3) or control hamster IgG (−αCD3). After 6 days of further culture, FTOC were harvested. (a) The mean values of the total cell number per lobe after each treatment are presented as percentages of that obtained from FTOC medium alone without anti-CD3 antibody. The mean value obtained from FTOC medium alone without anti-CD3 antibody was 51 000/lobe. Mean and standard deviation (SD) were calculated from results of five separate experiments. Statistics are based on the Student's t-test. *P<0·01, **P<0·05. (b) Expression of CD4, CD8 and CD25. Percentages of cells in each CD4/CD8 population are shown in each quadrant. The results are representative of five separate experiments. Stimulation by anti-CD3 antibody did not overcome the suppression of differentiation by either IL-7 or hGM-CSF. To exclude any effects mediated by stromal cells, hGMR+/−RAG-1−/− pre-T cells were sorted, transferred into 2-deoxyguanosine-depleted wild-type fetal lobes, and cultured in the presence of 50 ng/ml of hGM-CSF for 2 days, then stimulated with 20 µg/ml of anti-CD3 antibody. (c) The mean values of the total cell number per lobe after each treatment are presented as percentages of that obtained from FTOC with hGM-CSF and without anti-CD3 antibody. The mean value obtained from FTOC with hGM-CSF and without anti-CD3 antibody was 113 000/lobe. Mean and standard deviation (SD) were calculated from results of three separate experiments. Statistics are based on the Student's t-test. *P<0·05. (d) Expression of CD25 (solid lines). Down-regulation of CD25 expression was observed with anti-CD3 antibody. The results are representative of three separate experiments. Dotted lines, isotype control staining.
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