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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 prevents premature recombination of Tcra locusin DN4 subset. (a,b) Genomic DNA from DN4, ISP, eDP (CD71hiFSChi) and lDP (CD71lo FSClo) thymocytes was analyzedby qPCR to measure Vα-to-Jαrecombination using primers for Trav17 (Vα17) and proximalTraj61 (Jα61) and Traj56 (Jα56)segments (a), or primers for Trav12 (which detect multiple,widely distributed Trav12 (Vα12) family members) and centralTraj42 (Jα42) and Traj30 (Jα30)segments vs distal Traj17 (Jα17) segment (b). Bargraphs show relative Jα usage (mean ± SD) in each subset,normalized to levels detected in unfractionated WT thymocytes (n=4, 2biological replicates/subset/genotype and 2 technical replicate/sort). ND: not detected.(c) Bar graphs depict normalized expression values (Log2 scale)for Rag1, Rag2 and Cish from the Illumina mRNAexpression profiling shown in Fig. 3. FDR-adjustedq-values for each comparison are indicated: *FDRq<0.0001. (d) qRT–PCR quantification ofRag1 and Rag2 mRNA in sortedIl7+/+ andIl7−/− DN4 thymocytes (normalized toCD45, 3 technical replicates/group). The significance of differencesbetween groups was assessed using Student’s t-test as describedfor Fig. 1: *P<0.0001,**P<0.01. (e) Western blotting for Rag2 (top) andβ-actin (bottom) protein in the indicated subsets fromIl7+/+ andIl7−/− thymi. Protein extracts from totalRag2+/+ andRag2−/− thymocytes were used as positive andnegative controls, respectively. Similar results for each subset were obtained in 2 ormore (a, b), 1 (d) and 3 (e) independentexperiments.
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Figure 8: IL-7 signaling prevents premature recombination of Tcra locusin DN4 subset. (a,b) Genomic DNA from DN4, ISP, eDP (CD71hiFSChi) and lDP (CD71lo FSClo) thymocytes was analyzedby qPCR to measure Vα-to-Jαrecombination using primers for Trav17 (Vα17) and proximalTraj61 (Jα61) and Traj56 (Jα56)segments (a), or primers for Trav12 (which detect multiple,widely distributed Trav12 (Vα12) family members) and centralTraj42 (Jα42) and Traj30 (Jα30)segments vs distal Traj17 (Jα17) segment (b). Bargraphs show relative Jα usage (mean ± SD) in each subset,normalized to levels detected in unfractionated WT thymocytes (n=4, 2biological replicates/subset/genotype and 2 technical replicate/sort). ND: not detected.(c) Bar graphs depict normalized expression values (Log2 scale)for Rag1, Rag2 and Cish from the Illumina mRNAexpression profiling shown in Fig. 3. FDR-adjustedq-values for each comparison are indicated: *FDRq<0.0001. (d) qRT–PCR quantification ofRag1 and Rag2 mRNA in sortedIl7+/+ andIl7−/− DN4 thymocytes (normalized toCD45, 3 technical replicates/group). The significance of differencesbetween groups was assessed using Student’s t-test as describedfor Fig. 1: *P<0.0001,**P<0.01. (e) Western blotting for Rag2 (top) andβ-actin (bottom) protein in the indicated subsets fromIl7+/+ andIl7−/− thymi. Protein extracts from totalRag2+/+ andRag2−/− thymocytes were used as positive andnegative controls, respectively. Similar results for each subset were obtained in 2 ormore (a, b), 1 (d) and 3 (e) independentexperiments.

Mentions: As expected, we detected very low amounts of primary and secondaryTcra rearrangements in cycling WT DN4, ISP and CD71+CD98+ eDP cells, whereas we detected high levels of both primary andsecondary rearrangements in post-mitotic CD71− CD98−lDP cells (Fig. 8a,b). In striking contrast,Il7−/− DN4 cells had abundant primary andsecondary rearrangements that were comparable in frequency to those found in WT lDP cells.Thus, abnormally quiescent DN4 cells from IL-7-deficient mice undergo premature andextensive Tcra recombination. Nonetheless,Il7−/− ISP and eDP cells had very low levelsof both primary and secondary Tcra rearrangements, similar to levels seenin WT ISP and eDP cells. Furthermore, we did not detect Tcrarearrangements in Il7−/− DN3b cells (data notshown). The sharp contrast in Tcra rearrangement status between quiescentDN4 relative to cycling ISP and eDP cells fromIl7−/− mice provides further evidence thatDN4 cells are not precursors of ISP and eDP cells in these mutant mice.


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 prevents premature recombination of Tcra locusin DN4 subset. (a,b) Genomic DNA from DN4, ISP, eDP (CD71hiFSChi) and lDP (CD71lo FSClo) thymocytes was analyzedby qPCR to measure Vα-to-Jαrecombination using primers for Trav17 (Vα17) and proximalTraj61 (Jα61) and Traj56 (Jα56)segments (a), or primers for Trav12 (which detect multiple,widely distributed Trav12 (Vα12) family members) and centralTraj42 (Jα42) and Traj30 (Jα30)segments vs distal Traj17 (Jα17) segment (b). Bargraphs show relative Jα usage (mean ± SD) in each subset,normalized to levels detected in unfractionated WT thymocytes (n=4, 2biological replicates/subset/genotype and 2 technical replicate/sort). ND: not detected.(c) Bar graphs depict normalized expression values (Log2 scale)for Rag1, Rag2 and Cish from the Illumina mRNAexpression profiling shown in Fig. 3. FDR-adjustedq-values for each comparison are indicated: *FDRq<0.0001. (d) qRT–PCR quantification ofRag1 and Rag2 mRNA in sortedIl7+/+ andIl7−/− DN4 thymocytes (normalized toCD45, 3 technical replicates/group). The significance of differencesbetween groups was assessed using Student’s t-test as describedfor Fig. 1: *P<0.0001,**P<0.01. (e) Western blotting for Rag2 (top) andβ-actin (bottom) protein in the indicated subsets fromIl7+/+ andIl7−/− thymi. Protein extracts from totalRag2+/+ andRag2−/− thymocytes were used as positive andnegative controls, respectively. Similar results for each subset were obtained in 2 ormore (a, b), 1 (d) and 3 (e) independentexperiments.
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Figure 8: IL-7 signaling prevents premature recombination of Tcra locusin DN4 subset. (a,b) Genomic DNA from DN4, ISP, eDP (CD71hiFSChi) and lDP (CD71lo FSClo) thymocytes was analyzedby qPCR to measure Vα-to-Jαrecombination using primers for Trav17 (Vα17) and proximalTraj61 (Jα61) and Traj56 (Jα56)segments (a), or primers for Trav12 (which detect multiple,widely distributed Trav12 (Vα12) family members) and centralTraj42 (Jα42) and Traj30 (Jα30)segments vs distal Traj17 (Jα17) segment (b). Bargraphs show relative Jα usage (mean ± SD) in each subset,normalized to levels detected in unfractionated WT thymocytes (n=4, 2biological replicates/subset/genotype and 2 technical replicate/sort). ND: not detected.(c) Bar graphs depict normalized expression values (Log2 scale)for Rag1, Rag2 and Cish from the Illumina mRNAexpression profiling shown in Fig. 3. FDR-adjustedq-values for each comparison are indicated: *FDRq<0.0001. (d) qRT–PCR quantification ofRag1 and Rag2 mRNA in sortedIl7+/+ andIl7−/− DN4 thymocytes (normalized toCD45, 3 technical replicates/group). The significance of differencesbetween groups was assessed using Student’s t-test as describedfor Fig. 1: *P<0.0001,**P<0.01. (e) Western blotting for Rag2 (top) andβ-actin (bottom) protein in the indicated subsets fromIl7+/+ andIl7−/− thymi. Protein extracts from totalRag2+/+ andRag2−/− thymocytes were used as positive andnegative controls, respectively. Similar results for each subset were obtained in 2 ormore (a, b), 1 (d) and 3 (e) independentexperiments.
Mentions: As expected, we detected very low amounts of primary and secondaryTcra rearrangements in cycling WT DN4, ISP and CD71+CD98+ eDP cells, whereas we detected high levels of both primary andsecondary rearrangements in post-mitotic CD71− CD98−lDP cells (Fig. 8a,b). In striking contrast,Il7−/− DN4 cells had abundant primary andsecondary rearrangements that were comparable in frequency to those found in WT lDP cells.Thus, abnormally quiescent DN4 cells from IL-7-deficient mice undergo premature andextensive Tcra recombination. Nonetheless,Il7−/− ISP and eDP cells had very low levelsof both primary and secondary Tcra rearrangements, similar to levels seenin WT ISP and eDP cells. Furthermore, we did not detect Tcrarearrangements in Il7−/− DN3b cells (data notshown). The sharp contrast in Tcra rearrangement status between quiescentDN4 relative to cycling ISP and eDP cells fromIl7−/− mice provides further evidence thatDN4 cells are not precursors of ISP and eDP cells in these mutant mice.

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