Analysis of TCR diversity.

<p>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.</p

Administration of rIL-21 to DEX-injected animals induces Bcl-6 expression in DP thymocytes.

<p>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, *<i>P</i><0.05. Data shown are representative of 3 separate experiments.</p

Proposed model for rIL-21- accelerated recovery following induction of acute thymic atrophy.

<p>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.</p

STAT phosphorylation in thymocytes.

<p>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.</p

Interleukin-21 Accelerates Thymic Recovery from Glucocorticoïd-Induced Atrophy

<div><p>Both physiological and psychological stress cause thymic atrophy via glucocorticoïd (GC)-dependent apoptosis of double-positive (DP) thymocytes. Given the pervasiveness of stress, GC-induced thymic atrophy is arguably the most common type of acquired immunodeficiency. We recently reported that interleukin-21 (IL-21) has a unique ability to expand the small subset of DP thymocytes (CD69⁺) which are ongoing positive selection, and that administration of IL-21 increases thymic output in aged mice. The goal of this study was to evaluate whether IL-21 could mitigate GC-induced thymic atrophy. In contrast to double-negative (DN) and single-positive (SP) thymocytes, most DP thymocytes (CD69⁻) do not constitutively express the IL-21 receptor (IL-21R). Accordingly, CD69⁻ DP thymocytes from PBS-treated mice were unresponsive to IL-21 administration. However, following GC injection, surviving CD69⁻ DP thymocytes up-regulated IL-21R and responded to IL-21 treatment as evidenced by enhancement of Bcl6 expression and phosphorylation of STAT1, STAT3 and STAT5. Consequently, IL-21 administration to GC-treated mice accelerated thymic recovery by expanding considerably DP thymocytes and, to a lesser extent, DN thymocytes. However, IL-21-induced expansion of DN/DP thymocytes did not alter the diversity of the intrathymic or peripheral T-cell receptor (TCR) repertoire. We conclude that IL-21 dramatically accelerates recovery from GC-induced thymic atrophy.</p></div

The effect of rIL-21 administration on thymic recovery following DEX treatment.

<p>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.</p

Transcript analysis of molecules implicated in thymocyte survival and expansion.

<p>A–B) qPCR analysis of <i>Bcl2</i>, <i>Bcl2l1</i> and <i>Mcl1</i> or (C–D) <i>Bcl6</i> 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, *<i>P</i><0.05. All experiments have been repeated 3 times.</p

Additional file 1: Figure S1. of Interleukin-21 promotes thymopoiesis recovery following hematopoietic stem cell transplantation

Gating strategies for LSKs. BM cells collected from WT or IL-21R−/− C57BL/6 mice were treated in vitro for proliferation, then stained with the Lin−Sca1+c-kit+ antibody cocktail prior to Ki-67+ incorporation analysis. All described experiments were conducted at least three times with n = 5/group. Figure S2. Functional characterization of T cells. (A) Representative cell trace dilution analysis on CD4+ or CD8+ T cells derived from ctl (unirradiated), PBS-treated, or IL-21-treated LP/J recipient mice. (B) Cytokine quantification by ELISA from T cells derived from the same groups described in panel (A). (C) Representative flow cytometry analysis of granzyme B expression. (D) Quantification of T cells expressing granzyme B. For all presented studies, T cells were stimulated with CD3-CD28 dynabeads for 48 h prior to analyses. All described experiments were conducted at least three times with an n = 5/group. Figure S3. Molecular characterization of T cells. T cells sorted from ctl, PBS-treated, or IL-21-treated LP/J recipient mice were analyzed for their expression of various transcription factors involved in T cell differentiation. All described experiments were conducted at least three times with n = 5/group. Figure S4. Gating strategies for Breg analysis. For detection of IL-10-producing Bregs, CD19+ B cells were first isolated from spleens of treated mice (isotype shown by the filled gray histogram), then stained after in vitro treatment with CD1d and CD5 antibodies. The B cell subset CD1dhiCD5+ was gated prior to IL-10 assessment by intracellular staining. All described experiments were conducted at least three times with n = 5/group. (PDF 2111 kb