Figure 1.
Expression of IL-21Rα and response to IL-21.
A) Representative flow-cytometry analysis of IL-21Rα-chain on PBS- (upper panels) versus DEX-treated (lower panels) thymocytes. Isotype control are depicted in black whereas test conditions are displayed in red. B) Numbers indicate mean fluorescent intensity of IL-21Rα-chain expression on the surface of DP thymocytes. We tested 4 mice per group, *P<0.00002. C) Representative flow-cytometry analysis of IL-21Rα-chain on fractionated DN thymocytes from PBS- or DEX-treated mice. The isotype control is shown in black. D) Left panel: distribution of DP1 (TCRloCD5lo), DP2 (TCRintCD5hi) and DP3 (TCRhiCD5int) subsets among DP thymocytes from DEX-treated mice. Right panel: representative flow-cytometry analysis of IL-21Rα-chain on DP1 thymocytes derived from DEX-treated animals. E–F) Number of cells recovered following in vitro culture of the following cell subsets with graded concentrations of IL-21: DN thymocytes derived from PBS- or DEX-treated mice, CD69− and CD69+ DP thymocytes from PBS-treated mice, and DP thymocytes derived from DEX-treated mice. We tested 4 mice per group, *P<0.05. All experiments were repeated at least 3 times.
Figure 2.
The effect of rIL-21 administration on thymic recovery following DEX treatment.
A) A representative photograph of thymi derived from treated mice. A group of C57BL/6 mice received equivalent volume of PBS. B) Flow-cytometry/histological analysis of thymi derived from treated mice. C) Total thymic cellularity for all tested groups. D) Absolute numbers of DN, DP, CD4 and CD8 SP T cells from thymi derived from treated mice. PBS only (black), DEX/PBS (red) and DEX/rIL-21 (green). We tested 3 mice per group. Data are representative of 3 separate experiments. E) Representative flow-cytometry analysis of TECs derived from PBS-, DEX/PBS- versus DEX/rIL-21-treated mice. Following TECs gating, IL-21Rα-chain was assessed by flow-cytometry. Isotype controls are displayed by black histogram whereas test condition is represented by red histograms. F) Absolute numbers of TECs from thymi derived from treated mice. We tested 7 mice per group. Data are representative of 3 separate experiments.
Figure 3.
Transcript analysis of molecules implicated in thymocyte survival and expansion.
A–B) qPCR analysis of Bcl2, Bcl2l1 and Mcl1 or (C–D) Bcl6 expression in freshly harvested DN (A–C) or DP (B–D) thymocytes derived from PBS-, DEX/PBS- or DEX/rIL-21-treated WT C57BL/6 mice. Data depict transcript abundance relative to DN (A–C) or DP (B–D) thymocytes. We tested 3 mice per group, *P<0.05. All experiments have been repeated 3 times.
Figure 4.
Administration of rIL-21 to DEX-injected animals induces Bcl-6 expression in DP thymocytes.
A–B) Representative flow-cytometry analysis of Bcl2 expression in DN and DP thymocytes derived from PBS-, DEX/PBS- or DEX/rIL-21-treated mice. C) Compiled mean fluorescent intensity for Bcl2 analysis. D–E) Representative flow-cytometry analysis for Bcl2l1 in DN and DP thymocytes from the same experimental groups. F) Compiled mean fluorescent intensity for Bcl2l1 analysis. G–H) Representative flow-cytometry analysis for Bcl6 in DN and DP thymocytes using same experimental groups. I) Compiled mean fluorescent intensity for Bcl6 analysis. For all experiments performed, we tested 3 mice per group, *P<0.05. Data shown are representative of 3 separate experiments.
Figure 5.
STAT phosphorylation in thymocytes.
A–B) Representative flow-cytometry analysis of pSTATs in fractionated thymocytes derived from PBS- or DEX-treated animals supplemented with 10ng/ml rIL-21 (red histograms) or no cytokines (black histograms). We tested 3–6 mice per group in separate experiments.
Figure 6.
A–B) Flow-cytometry analysis of 15 TCRVβ-chains using intrathymic CD4 (A) or CD8 (B) SP T cells. C–D) Similar flow-cytometry analyses were performed using spleen CD4 (A) or CD8 (B) SP T cells. For both analyses, T cells were derived from PBS- (dark blue), DEX/PBS- (blue) or DEX/rIL-21-treated mice (light blue). We tested 3 mice per group. Data shown are representative of 3 separate experiments.
Figure 7.
Proposed model for rIL-21- accelerated recovery following induction of acute thymic atrophy.
Based on data presented herein, we propose the following model: under normal circumstances, only a small fraction of DP thymocytes (CD69+ post-selection DP3s which represent 6% of DPs) express the IL-21R (step 1). Upon DEX administration, 90–95% of DP thymocytes are depleted by apoptosis within 48 hrs (step 2). At that stage, the thymic cortico-medullary demarcation is blurred. The surviving DPs (CD69− DP1s) upregulate IL-21R on their cell surface and become responsive to rIL-21. Upon rIL-21 administration, pSTAT1, pSTAT3 and pSTAT5 are activated while post-transcriptional mechanisms lead to accumulation of Bcl6. These signaling events accelerate thymic recovery from DEX-induced atrophy.