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IL-7 coordinates proliferation, differentiation and Tcra recombination during thymocyte β-selection.

Boudil A, Matei IR, Shih HY, Bogdanoski G, Yuan JS, Chang SG, Montpellier B, Kowalski PE, Voisin V, Bashir S, Bader GD, Krangel MS, Guidos CJ - Nat. Immunol. (2015)

Bottom Line: Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate but rearranged Tcra prematurely and differentiated rapidly.Deletion of Bcl6 partially restored the self-renewal of DN4 cells in the absence of IL-7, but overexpression of BCL2 did not.Thus, IL-7 critically acts cooperatively with signaling via the pre-TCR and Notch1 to coordinate proliferation, differentiation and Tcra recombination during β-selection.

View Article: PubMed Central - PubMed

Affiliation: 1] Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, Toronto, Canada. [2] Department of Immunology, University of Toronto, Toronto, Canada.

ABSTRACT
Signaling via the pre-T cell antigen receptor (pre-TCR) and the receptor Notch1 induces transient self-renewal (β-selection) of TCRβ(+) CD4(-)CD8(-) double-negative stage 3 (DN3) and DN4 progenitor cells that differentiate into CD4(+)CD8(+) double-positive (DP) thymocytes, which then rearrange the locus encoding the TCR α-chain (Tcra). Interleukin 7 (IL-7) promotes the survival of TCRβ(-) DN thymocytes by inducing expression of the pro-survival molecule Bcl-2, but the functions of IL-7 during β-selection have remained unclear. Here we found that IL-7 signaled TCRβ(+) DN3 and DN4 thymocytes to upregulate genes encoding molecules involved in cell growth and repressed the gene encoding the transcriptional repressor Bcl-6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate but rearranged Tcra prematurely and differentiated rapidly. Deletion of Bcl6 partially restored the self-renewal of DN4 cells in the absence of IL-7, but overexpression of BCL2 did not. Thus, IL-7 critically acts cooperatively with signaling via the pre-TCR and Notch1 to coordinate proliferation, differentiation and Tcra recombination during β-selection.

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IL-7 signaling promotes DN4 growth and proliferation in vivo.(a) Flow cytometric analysis of forward scatter (FSC), an indicator of cellsize and metabolism, of each subset identified as described in Supplementary Fig. 1.:Il7r+/+ (shaded) versusIl7r−/− (open) (Top).Il7r+/+ (shaded) versusIl7r−/− BCL2 (open) (Bottom).(b) Flow cytometric quantification of BrdU incorporation in DN4 cells (Top)from Il7r+/+ andIl7r−/− mice, assessed 2h after the firstBrdU injection and identified as shown in Supplementary Fig. 1. Bar graphs show % BrdU+ cells(mean +/− SD) in each subset fromIl7r+/+Il7r−/−and Il7r−/−BCL2 mice (Bottom) (3 biological replicates/group). (c) Flowcytometric analysis of CD71 (Top) or CD98 (Bottom) vs CD25 expression, shown as 5%probability contour plots gated on DN CD3 Lin icTCRβ+ thymocytes(d) Histograms show flow cytometric quantification of CD71 expression (Top)or CFSE (Bottom) after WT DN4 thymocytes were cultured in Med (shaded) or IL-7 (open) for15 or 40h. (e) WT DN4 thymocytes (5×103/well, indicated bydotted horizontal line) were cultured with OP9-DL4 cells and media containing theindicated IL-7 concentrations for 48h. Bar graphs show the mean (+/− SD) number ofDN cells recovered (3 technical replicates/group). The significance of differences betweengroups was assessed in (b) and (e) using one-way ANOVA withNewman-Keuls post hoc t-test. Similar results were obtained in 3(a, b, c, e) or 2 independent experiments (d).*P<0.001, **P<0.01,***P<0.05.
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Figure 4: IL-7 signaling promotes DN4 growth and proliferation in vivo.(a) Flow cytometric analysis of forward scatter (FSC), an indicator of cellsize and metabolism, of each subset identified as described in Supplementary Fig. 1.:Il7r+/+ (shaded) versusIl7r−/− (open) (Top).Il7r+/+ (shaded) versusIl7r−/− BCL2 (open) (Bottom).(b) Flow cytometric quantification of BrdU incorporation in DN4 cells (Top)from Il7r+/+ andIl7r−/− mice, assessed 2h after the firstBrdU injection and identified as shown in Supplementary Fig. 1. Bar graphs show % BrdU+ cells(mean +/− SD) in each subset fromIl7r+/+Il7r−/−and Il7r−/−BCL2 mice (Bottom) (3 biological replicates/group). (c) Flowcytometric analysis of CD71 (Top) or CD98 (Bottom) vs CD25 expression, shown as 5%probability contour plots gated on DN CD3 Lin icTCRβ+ thymocytes(d) Histograms show flow cytometric quantification of CD71 expression (Top)or CFSE (Bottom) after WT DN4 thymocytes were cultured in Med (shaded) or IL-7 (open) for15 or 40h. (e) WT DN4 thymocytes (5×103/well, indicated bydotted horizontal line) were cultured with OP9-DL4 cells and media containing theindicated IL-7 concentrations for 48h. Bar graphs show the mean (+/− SD) number ofDN cells recovered (3 technical replicates/group). The significance of differences betweengroups was assessed in (b) and (e) using one-way ANOVA withNewman-Keuls post hoc t-test. Similar results were obtained in 3(a, b, c, e) or 2 independent experiments (d).*P<0.001, **P<0.01,***P<0.05.

Mentions: Since IL-7 significantly increased expression of many genes that regulatemetabolism, signaling and growth, we evaluated the impact of IL-7 deficiency on cell size,a reflection of cellular metabolism and proliferation during β-selection. Althoughthe size of DN3b cells from Il7r+/+ vsIl7r−/− mice was similar,Il7r−/− DN4 cells were much smaller thantheir WT counterparts, suggesting loss of trophic signaling (Fig. 4a). Furthermore, IL-7 deficiency significantly impaired BrDU uptake byIl7r−/− DN4 cells, but not DN3b cells (Fig. 4b). BCL2 over-expression did notprevent atrophy or restore proliferation ofIl7r−/− DN4 cells, strongly suggesting thatIL-7 signaling is required to maintain DN4 thymocyte trophic responses and proliferationin vivo, rather than simply to maintain Bcl2-dependent survival.


IL-7 coordinates proliferation, differentiation and Tcra recombination during thymocyte β-selection.

Boudil A, Matei IR, Shih HY, Bogdanoski G, Yuan JS, Chang SG, Montpellier B, Kowalski PE, Voisin V, Bashir S, Bader GD, Krangel MS, Guidos CJ - Nat. Immunol. (2015)

IL-7 signaling promotes DN4 growth and proliferation in vivo.(a) Flow cytometric analysis of forward scatter (FSC), an indicator of cellsize and metabolism, of each subset identified as described in Supplementary Fig. 1.:Il7r+/+ (shaded) versusIl7r−/− (open) (Top).Il7r+/+ (shaded) versusIl7r−/− BCL2 (open) (Bottom).(b) Flow cytometric quantification of BrdU incorporation in DN4 cells (Top)from Il7r+/+ andIl7r−/− mice, assessed 2h after the firstBrdU injection and identified as shown in Supplementary Fig. 1. Bar graphs show % BrdU+ cells(mean +/− SD) in each subset fromIl7r+/+Il7r−/−and Il7r−/−BCL2 mice (Bottom) (3 biological replicates/group). (c) Flowcytometric analysis of CD71 (Top) or CD98 (Bottom) vs CD25 expression, shown as 5%probability contour plots gated on DN CD3 Lin icTCRβ+ thymocytes(d) Histograms show flow cytometric quantification of CD71 expression (Top)or CFSE (Bottom) after WT DN4 thymocytes were cultured in Med (shaded) or IL-7 (open) for15 or 40h. (e) WT DN4 thymocytes (5×103/well, indicated bydotted horizontal line) were cultured with OP9-DL4 cells and media containing theindicated IL-7 concentrations for 48h. Bar graphs show the mean (+/− SD) number ofDN cells recovered (3 technical replicates/group). The significance of differences betweengroups was assessed in (b) and (e) using one-way ANOVA withNewman-Keuls post hoc t-test. Similar results were obtained in 3(a, b, c, e) or 2 independent experiments (d).*P<0.001, **P<0.01,***P<0.05.
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Figure 4: IL-7 signaling promotes DN4 growth and proliferation in vivo.(a) Flow cytometric analysis of forward scatter (FSC), an indicator of cellsize and metabolism, of each subset identified as described in Supplementary Fig. 1.:Il7r+/+ (shaded) versusIl7r−/− (open) (Top).Il7r+/+ (shaded) versusIl7r−/− BCL2 (open) (Bottom).(b) Flow cytometric quantification of BrdU incorporation in DN4 cells (Top)from Il7r+/+ andIl7r−/− mice, assessed 2h after the firstBrdU injection and identified as shown in Supplementary Fig. 1. Bar graphs show % BrdU+ cells(mean +/− SD) in each subset fromIl7r+/+Il7r−/−and Il7r−/−BCL2 mice (Bottom) (3 biological replicates/group). (c) Flowcytometric analysis of CD71 (Top) or CD98 (Bottom) vs CD25 expression, shown as 5%probability contour plots gated on DN CD3 Lin icTCRβ+ thymocytes(d) Histograms show flow cytometric quantification of CD71 expression (Top)or CFSE (Bottom) after WT DN4 thymocytes were cultured in Med (shaded) or IL-7 (open) for15 or 40h. (e) WT DN4 thymocytes (5×103/well, indicated bydotted horizontal line) were cultured with OP9-DL4 cells and media containing theindicated IL-7 concentrations for 48h. Bar graphs show the mean (+/− SD) number ofDN cells recovered (3 technical replicates/group). The significance of differences betweengroups was assessed in (b) and (e) using one-way ANOVA withNewman-Keuls post hoc t-test. Similar results were obtained in 3(a, b, c, e) or 2 independent experiments (d).*P<0.001, **P<0.01,***P<0.05.
Mentions: Since IL-7 significantly increased expression of many genes that regulatemetabolism, signaling and growth, we evaluated the impact of IL-7 deficiency on cell size,a reflection of cellular metabolism and proliferation during β-selection. Althoughthe size of DN3b cells from Il7r+/+ vsIl7r−/− mice was similar,Il7r−/− DN4 cells were much smaller thantheir WT counterparts, suggesting loss of trophic signaling (Fig. 4a). Furthermore, IL-7 deficiency significantly impaired BrDU uptake byIl7r−/− DN4 cells, but not DN3b cells (Fig. 4b). BCL2 over-expression did notprevent atrophy or restore proliferation ofIl7r−/− DN4 cells, strongly suggesting thatIL-7 signaling is required to maintain DN4 thymocyte trophic responses and proliferationin vivo, rather than simply to maintain Bcl2-dependent survival.

Bottom Line: Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate but rearranged Tcra prematurely and differentiated rapidly.Deletion of Bcl6 partially restored the self-renewal of DN4 cells in the absence of IL-7, but overexpression of BCL2 did not.Thus, IL-7 critically acts cooperatively with signaling via the pre-TCR and Notch1 to coordinate proliferation, differentiation and Tcra recombination during β-selection.

View Article: PubMed Central - PubMed

Affiliation: 1] Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, Toronto, Canada. [2] Department of Immunology, University of Toronto, Toronto, Canada.

ABSTRACT
Signaling via the pre-T cell antigen receptor (pre-TCR) and the receptor Notch1 induces transient self-renewal (β-selection) of TCRβ(+) CD4(-)CD8(-) double-negative stage 3 (DN3) and DN4 progenitor cells that differentiate into CD4(+)CD8(+) double-positive (DP) thymocytes, which then rearrange the locus encoding the TCR α-chain (Tcra). Interleukin 7 (IL-7) promotes the survival of TCRβ(-) DN thymocytes by inducing expression of the pro-survival molecule Bcl-2, but the functions of IL-7 during β-selection have remained unclear. Here we found that IL-7 signaled TCRβ(+) DN3 and DN4 thymocytes to upregulate genes encoding molecules involved in cell growth and repressed the gene encoding the transcriptional repressor Bcl-6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate but rearranged Tcra prematurely and differentiated rapidly. Deletion of Bcl6 partially restored the self-renewal of DN4 cells in the absence of IL-7, but overexpression of BCL2 did not. Thus, IL-7 critically acts cooperatively with signaling via the pre-TCR and Notch1 to coordinate proliferation, differentiation and Tcra recombination during β-selection.

Show MeSH
Related in: MedlinePlus