Unlike IL-7, which is known to be critical for T cell thymic development, the role of IL-21 in this process is still controversial. IL-21 has been shown to accelerate thymic recovery in mice treated with glucocorticoids and revives the peripheral T cell pool in aged animals. However, mice with a defect in IL-21 signaling exhibit normal thymic cellularity, challenging the importance of this cytokine in the thymic developmental process. Using mixed bone marrow chimeric mice, our studies describe a multilayered role for IL-21 in thymopoiesis. In this system, IL-21R–deficient cells are unable to compete with wild-type populations at different stages of the thymic development. Using a mixed bone marrow chimeric animal model, IL-21 seems to be involved as early as the double-negative 1 stage, and the cells from the knockout compartment have problems transitioning to subsequent double-negative stages. Also, similar to IL-7, IL-21 seems to be involved in the positive selection of double-positive lymphocytes and appears to play a role in the migration of single-positive T cells to the periphery. Although not as critical as IL-7, based on our studies, IL-21 plays an important complementary role in thymic T cell development, which, to date, has been underrecognized.

The thymus provides a unique environment for the development and maturation of T cells. T cell lymphopoiesis is responsible for maintaining a pool of naive peripheral T cells with a broad spectrum of TCR specificities. On the basis of CD4 and CD8 T cell expression, thymopoiesis can be broadly divided into three major stages, namely, double-negative (DN), double-positive (DP), and single-positive (SP) cells. The key events during this process include the entry of lymphoid progenitor cells originating from the bone marrow, the formation of a functional TCR through TCR β-chain and α-chain rearrangement, and positive and negative selection to ensure MHC restriction as well as clearance of autoreactive cells (1).

The role of several common γ-chain cytokines in the thymopoiesis process is well appreciated. Among them, IL-7 is essential for lymphocyte development and survival. Mice deficient in IL-7 and IL-7Rα exhibit significant reductions in T and B cells (2, 3). Specifically, IL-7 is critical for early lymphocyte development by supporting proliferation, survival, and differentiation of the DN subset (4). Additionally, differentiation of positively selected CD8 T cells in the thymus is contingent on IL-7 signaling (5, 6). Similarly, two other common γ-chain cytokines, IL-15 and IL-2, have been reported to be involved in regulatory T cell thymic development (7). In a study conducted a few years ago, it was also demonstrated that IL-18, in synergy with IL-7, can promote bone marrow lymphopoiesis and T cell development (8).

IL-21 is one of the most recently characterized members of the common γ-chain cytokine family (9). It is produced primarily by activated CD4 T cells in the periphery and epithelial cells in the thymus (10, 11). The cytokine is involved in a number of functions, which includes promoting CD4 differentiation, costimulation of activated NK cells, and IgG production by B cells (1113). IL-21R, which is expressed by all lymphocytes, forms a heterodimer with the shared common γ-chain subunit (9). IL-21, unlike IL-7, is not considered to be essential for thymopoiesis as IL-21R knockout (KO) animals exhibit normal thymic cellularity (14). However, it has been reported that IL-21 treatment of mice with glucocorticoids-induced thymic atrophy significantly accelerates the recovery of thymic functions (15). Moreover, in a very recent study, it was demonstrated that the peripheral T cell pool of aged animals was rejuvenated by the administration of IL-21 (16). This could be explained by the ability of IL-21 to induce the expansion of bone marrow–derived hematopoietic progenitor cells (17, 18). Furthermore, a recent in vitro study conducted by Rafei et al. (10) demonstrated that IL-21 has the unique ability to upregulate BCL-6, expand DP thymocytes undergoing positive selection, and increase the production of mature T cells. Additionally, this study showed that, in contrast to IL-7 (5), CD8 T cell differentiation was IL-21 independent. These observations reveal the complex role of IL-21 in enhancing the thymic T cell output in age- or disease-related thymic atrophy.

In this study, we observed that, although IL-21 expression in the thymus was significantly lower than IL-7 and IL-15, every single thymic subset expressed the IL-21R. Considering that normal thymic cellularity in IL-21R KO mice may be attributed to a redundant mechanism(s), we decided to investigate the role of IL-21 in thymic T cell development using wild-type (WT)/IL-21R KO mixed bone marrow chimeric (BMC) mice. In this model, the lack of IL-21 signaling led to various defects, starting as early as the DN1 stage and involved all the subsequent DN stages. Effectively, in vitro coculture of DN1 cells with IL-7 and IL-21 showed greater differentiation than those treated with IL-7 alone. Additionally, the frequency of the more mature DP population was reduced in the KO compartment of the mixed BMC mice. Emigration of SP CD4 and CD8 T cells may also be affected by the lack of IL-21 signaling as these cells expressed lower expression of S1P1R than WT counterparts and exhibited reduced migration to S1P in a transwell migration assay. These findings suggest a complex supplementary role for IL-21 in thymic development that stretches beyond survival and expansion of the different thymic subsets.

C57BL/6, Thy1.1, and CD45.1 congenic mice were obtained from The Jackson Laboratory. IL-21R KO mice (B6N.129-Il21r < tm1Kopf > /J), originally purchased from The Jackson Laboratory, were bred in house under specific pathogen-free conditions at the Animal Research Facility at The George Washington University (Washington, DC). Mixed BMC animals (WT/IL-21R KO at a 1:1 ratio) were generated as previously described (19). Briefly, lethally irradiated Thy1.1 recipients (8 Gy/20 g of body weight) were injected i.v. with 5 × 106 magnetically purified hematopoietic progenitor cells from WT (CD45.1) and IL-21R KO (CD45.2) animals (STEMCELL Technologies). Transferred animals received water supplemented with sulfamethoxazole and trimethoprim (Hi-Tech Pharmacal) for 4–5 wk after injection, and experiments were conducted 4–8 wk post-reconstitution. All animal experiments were approved by The George Washington University School of Medicine and Health Sciences Institutional Animal Care and Use Committee.

IL-2, IL-7, IL-15, and IL-21 were detected by real-time PCR using previously described primers and conditions (19). RNA was isolated from the spleen and thymus with TRIzol reagent (Thermo Fisher Scientific), and cDNA was generated using Moloney murine leukemia virus reverse transcriptase. Amplification was performed with a myIQ Thermocycler (Bio-Rad Laboratories) for 40 cycles comprised of 95°C for 45 s, 61.4°C for 50 s, and 72°C for 45 s. Efficiency for each set of primers was verified using a 2-fold dilution series (20). Gene expression of common γ-chain cytokines was normalized using β-actin as an endogenous control.

Abs used for flow cytometry analysis were purchased from BioLegend (CD45.1 [A20], TCRγδ [GL3], B220 [RA3-6B2], Notch1 [HMN1-12], CCR7 [4B12], and CD62L [Mel.14]) and Thermo Fisher Scientific (CD45.2 [104], Thy1.1/CD90.1 [HIS51], CD4 [GK1.5], CD8 [53.6.7], CD3 [145-2C11], Gr1 [RB6-8C5], CD44 [IM7], CD25 [PC61.5], C-Kit [2B8eBio], CD27 [LG.7F9], IL-21R [eBio4A9], CD5 [53-7.3], CD24 [M1/69], and CD69 [[1H].3F3]). S1P1 (713412) was purchased from R&D Systems.

An Ab mixture (containing anti-CD4, -CD8, -CD19, -B220, -NK1.1, -CD11b, -CD11c, -TCRβ, -TCRγδ, and -Ter119) was used for exclusion of lineage-negative (lin) thymocytes to facilitate the detailed analysis of the various DN subsets.

Cell suspensions were prepared and labeled as previously described (19). Staining for CCR7 was performed at 37°C according to manufacturer’s instructions (BioLegend). Data were acquired using a FACSCalibur Flow Cytometer with a Cytek Biosciences upgrade (Becton Dickinson, Cytek Development). Analysis was carried out with FlowJo software (FlowJo).

Annexin V detection was performed as followed. Briefly, cells were isolated from the thymus and spleen of mixed BMC as described above and rested 4 h at 37°C prior to Annexin V staining in accordance with manufacturer’s recommended protocol (BioLegend).

Migration of thymocytes in response to S1P was assayed as previously described (21). Thymocytes from WT or IL-21R KO mice were serum starved for 2 h and then treated with migration media (IMDM supplemented with 1100 μg/ml delipidated BSA [Sigma-Aldrich], 2 mM l-glutamine, and 25 μg/ml penicillin/streptomycin) containing FTY720 or not (100 nM; Sigma-Aldrich) for 1 h at 37°C and 5% CO2. Briefly, 2.5 × 106 thymocytes were added in the upper chamber of a transwell plate (5-μm pore size; Corning Costar), and a different concentration of S1P (10, 100, and 1000 nM; Sigma-Aldrich) or migration media control were added to the lower chamber. Cells were allowed to migrate for 3 h at 37°C and 5% CO2. Cells from the bottom chamber were then harvested and stained before acquisition on BD FACSCelesta. CountBright Absolute Counting Beads (Thermo Fisher Scientific) were used to determine the exact number of cells migrated for each condition. Migration index was calculated as follows: the number of cells recovered from the bottom chamber/number of cells seeded in the top chamber × 100.

Differentiation of DN subsets in vitro was performed as previously described (22). Briefly, lin population was enriched by negative magnetic selection before DN1 cells (CD44hiCD25lo) were sorted by flow cytometry (purity ≥ 90%). DN1 cells (5 × 103 cells/well) were incubated with the OP9 expressing the DL1 receptor for Notch (OP9-DL1) thymic stromal cell line with either murine rIL-7 (5 ng/ml) (PeproTech), murine rIL-21 (100, 50, or 10 ng/ml) (PeproTech), IL-7 plus IL-21 (5 and 50 ng/ml, respectively), or control with media alone. Cells were passed onto a fresh OP9-DL1 monolayer complemented with the appropriate cytokine treatment every 3 d. Cells were maintained in culture for 10–12 d before harvesting and staining for flow cytometry analysis.

In vivo proliferation of lymphocytes from the spleen or thymus was assessed by bromodeoxyuridine (BrdU) incorporation assay. One group of mixed BMC mice was orally infected with 2 × 107Encephalitozoon cuniculi spores [genotype III maintained as previously described (23)]. All mice were injected i.p. every other day with 1 mg BrdU/mouse as previously described (19), starting 7 d prior to sacrifice. Detection of intranuclear BrdU incorporation was performed according to manufacturer’s instructions (BD Pharmingen).

Results are presented as mean ± SD. Comparison between groups was performed by Student t test throughout the study.

The role of IL-7 in thymopoiesis has been extensively studied, and both IL-7 KO and IL-7R KO animals display severe T cells defect (2, 3). In contrast, the involvement of IL-21, another member of the common γ-chain cytokine family that mediates its functions through a heterodimeric receptor composed of IL-21R and the common γ-chain receptor (9), seems to be more controversial. Early Northern blot analysis determined that IL-21R expression is restricted to lymphoid tissues, namely, the spleen and thymus (9, 24). However, IL-21R KO mice display normal cellularity in both of these tissues (14). Therefore, mRNA levels of different common γ-chain cytokines in the thymus and spleen from C57BL/6 mice were analyzed. As expected, IL-7 and IL-15 are the most predominant members in both thymus and spleen, and as compared with IL-7, IL-2 and IL-21 expressions were significantly lower in both tissues (Fig. 1A). Although levels of IL-7 and IL-15 mRNA in the tissues (thymus and spleen) of IL-21R KO mice were decreased compared with the WT animals, the expression for both cytokines was still substantial (Fig. 1A). Also, IL-21 mRNA expression was significantly reduced in the spleen of the KO mice. Next, the expression of IL-21R by different thymic populations was assessed by flow cytometry. As shown in Fig. 1B, similar to previous findings (15, 25), all subsets in the thymus (DN, DP, SP CD4, and SP CD8) expressed IL-21R, even though the expression was lower in SP CD4 thymocytes as compared with SP CD8 cells. Low expression of IL-21 in the thymus and broad expression of IL-21R by thymic subsets suggest an ambivalent role for IL-21 in thymopoiesis.

FIGURE 1.

Low IL-21 expression in the thymus. (A) Thymus (left) and spleen (right) from WT and IL-21R KO mice were assessed for expression of IL-2, IL-7, IL-15, and IL-21 by real-time PCR. Relative expression is presented as Δ cycle thresholdusing β-actin as a control. Each symbol represents one animal, and graphs represent at least three pooled experiments. (B) IL-21R expression by thymocytes from WT animals was measured by flow cytometry (n = 3–4 mice/experiment). Graph (right) shows the mean fluorescence intensity (MFI) for IL-21R by DN, DP, SP CD4, and CD8 in the thymus of WT mice. Assay was performed at least twice, and data are representative of one experiment.

FIGURE 1.

Low IL-21 expression in the thymus. (A) Thymus (left) and spleen (right) from WT and IL-21R KO mice were assessed for expression of IL-2, IL-7, IL-15, and IL-21 by real-time PCR. Relative expression is presented as Δ cycle thresholdusing β-actin as a control. Each symbol represents one animal, and graphs represent at least three pooled experiments. (B) IL-21R expression by thymocytes from WT animals was measured by flow cytometry (n = 3–4 mice/experiment). Graph (right) shows the mean fluorescence intensity (MFI) for IL-21R by DN, DP, SP CD4, and CD8 in the thymus of WT mice. Assay was performed at least twice, and data are representative of one experiment.

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Several recent reports showed that exogenous administration of IL-21 leads to an increase in thymus size and cellularity (16, 17), suggesting that this cytokine is involved in the development of thymocytes. However, these results were undermined by the fact that IL-21R KO mice exhibit normal T cell and B cell development (14). To better decipher the intrinsic role of IL-21 in the development of different thymic populations, a mixed BMC strategy was used (WT/IL-21R KO at a 1:1 ratio). At 2 mo post-reconstitution, the frequency of IL-21R KO cells in the thymus was significantly lower than the WT counterpart (Fig. 2A). The difference was also detected in the spleen of these animals, although the frequency of IL-21R KO cells decreased earlier in the spleen than in the thymus. The absence of IL-21 signaling also affected the distribution of the different populations in both thymus and spleen. As shown in Fig. 2B, compared with WT cells, frequencies and total numbers of IL-21R KO DN, CD4, and CD8 subsets were reduced in the BMC mice. Because of the reduction in the total number of IL-21R KO cells in BMC mice, we also observed a significant decrease in the total number of DP in these animals. As compared with WT cells, splenic IL-21R KO CD4 and CD8 populations were also significantly decreased in these animals. The frequency and total number of γδ T cells, which arise from immature DN thymocytes that have rearranged the TCRγ and TCRδ loci (26), were also significantly lower in the spleen and thymus from the IL-21R compartment compared with WT counterparts (Fig. 2C). However, the frequency of B cells, eosinophils, macrophages, and monocytes (which includes dendritic cells in our gating strategy) in the spleen were not affected by a lack of IL-21 signaling (Fig. 2D).

FIGURE 2.

Lack of IL-21 signaling affects the thymic T cell development. Role of IL-21 signaling in thymic T cell development was evaluated using mixed BMC (WT; IL-21R KO, 1:1). (A) Mixed BMC mice (WT/IL-21R KO: 1/1) were assessed for frequency of WT (CD45.1) or IL-21R KO (CD45.2) cells in thymus (left) and spleen (right) 1 and 2 mo after reconstitution. (B) Frequency and total number of WT or IL-21R KO DN, DP, CD4, and CD8 T cells in the thymus (left) or CD4 and CD8 T cells in spleen (right) in BMC animals (n = 4–5 mice/experiment). (C) WT and IL-21R KO TCRγδ cells were analyzed in the spleen (left) and lymph nodes (right) of mixed BMC mice. (D) Frequency of B cells (B220+CD3) in WT and IL-21R KO compartments from BMC mice is presented in top panels. In bottom panels, cells gated on B220CD3 are separated between neutrophils (SSChiGr1hi), macrophages (SSCloGr1hi), eosinophils (SSChiGr1int), and monocytes (also including dendritic cells) (SSCloGr1low) (n = 3–4 mice/experiment). Graphs represent pooled data from at least three experiments with three to four mice per experiments (B–D).

FIGURE 2.

Lack of IL-21 signaling affects the thymic T cell development. Role of IL-21 signaling in thymic T cell development was evaluated using mixed BMC (WT; IL-21R KO, 1:1). (A) Mixed BMC mice (WT/IL-21R KO: 1/1) were assessed for frequency of WT (CD45.1) or IL-21R KO (CD45.2) cells in thymus (left) and spleen (right) 1 and 2 mo after reconstitution. (B) Frequency and total number of WT or IL-21R KO DN, DP, CD4, and CD8 T cells in the thymus (left) or CD4 and CD8 T cells in spleen (right) in BMC animals (n = 4–5 mice/experiment). (C) WT and IL-21R KO TCRγδ cells were analyzed in the spleen (left) and lymph nodes (right) of mixed BMC mice. (D) Frequency of B cells (B220+CD3) in WT and IL-21R KO compartments from BMC mice is presented in top panels. In bottom panels, cells gated on B220CD3 are separated between neutrophils (SSChiGr1hi), macrophages (SSCloGr1hi), eosinophils (SSChiGr1int), and monocytes (also including dendritic cells) (SSCloGr1low) (n = 3–4 mice/experiment). Graphs represent pooled data from at least three experiments with three to four mice per experiments (B–D).

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Because the DN population was reduced in the IL-21R KO compartment from BMC mice (Fig. 2B), next, we investigated the earlier stages of the thymic development in these animals. Based on CD44 and CD25 expression by a lin population (lin as defined in 2Materials and Methods section), DN can be subdivided into DN1, DN2, DN3, and DN4 stages (27). However, the distribution of the WT and IL-21R KO cells between these subsets is somewhat different in a chimeric environment and is similar to previously published reports (28, 29). Interestingly, the percentage as well as the total number of DN1 cells (CD44hiCD25lo) were significantly higher in IL-21R KO lin populations as compared with the WT compartment of the BMC, whereas subsequent development stages (DN2–4) were lower among this population (Fig. 3A). However, the identification of the DN1 stage with CD44 and CD25 may not be sufficient because the CD44hiCD25lo subset is comprised of pluripotent early thymic precursor (ETP) along with other cell types (30). ETP and DN2 subsets are defined by a high expression of the stem cell factor receptor, c-Kit (31). A substantial increase in thymic precursors (CD44hic-Kithi) within the lin population was detected in the absence of IL-21 signaling (Fig. 3B). As shown in Fig. 3C, the frequency of ETP was 2–3-fold higher in the IL-21R KO CD44hic-Kithi population as compared with the WT counterpart, whereas the DN2a and DN2b subsets were significantly reduced in the KO compartment (32).

FIGURE 3.

Optimal DN development is dependent on IL-21 signaling. (A) DN1 (CD44hiCD25lo), DN2 (CD44hiCD25hi), DN3 (CD44loCD25hi), and DN4 (CD44loCD25lo) were gated on WT or IL-21R KO lin population from mixed BMC animals at week 8 posttransplant. (B) Frequency and number of thymic precursors (CD44hic-Kithi) were determined within lin population from WT and IL-21R KO compartments from BMC mice. (C). Frequencies and total numbers of WT and IL-21R KO ETP (C-kithiCD25lo), DN2a (C-kithiCD25hi), and DN2b (C-kitloCD25hi) subsets were determined after gating on CD44hiC-kithi lin cells. (D) DN3 subset (CD44loCD25hi) was subdivided into DN3a (FSCloCD27int) and DN3b (FSChiCD27hi). (E) WT and IL-21R KO DN populations were analyzed for Notch1 expression by flow cytometry. (F) Analysis of DN1 cells cocultured with OP9-DL1 cells for 12 d in the presence of IL-7 (5 ng/ml), IL-21 (100 ng/ml), or IL-7 + IL-21 (5 and 50 ng/ml, respectively). Plots are gated on live singlet cells. Lin thymocytes were used as a positive control. Bar graphs represent the frequencies of DN1, DN2, DN3, and DN4 after 12 d of coculture in the presence of IL-7 alone or IL-7 + IL-21 and mean fluorescence intensity (MFI) for CD25 (top) on cells recovered from culture treated with different concentrations of IL-21 (100, 50, 10 and 0 ng/ml). Data are representative of at least two experiments (n = 3–4 mice/experiment).

FIGURE 3.

Optimal DN development is dependent on IL-21 signaling. (A) DN1 (CD44hiCD25lo), DN2 (CD44hiCD25hi), DN3 (CD44loCD25hi), and DN4 (CD44loCD25lo) were gated on WT or IL-21R KO lin population from mixed BMC animals at week 8 posttransplant. (B) Frequency and number of thymic precursors (CD44hic-Kithi) were determined within lin population from WT and IL-21R KO compartments from BMC mice. (C). Frequencies and total numbers of WT and IL-21R KO ETP (C-kithiCD25lo), DN2a (C-kithiCD25hi), and DN2b (C-kitloCD25hi) subsets were determined after gating on CD44hiC-kithi lin cells. (D) DN3 subset (CD44loCD25hi) was subdivided into DN3a (FSCloCD27int) and DN3b (FSChiCD27hi). (E) WT and IL-21R KO DN populations were analyzed for Notch1 expression by flow cytometry. (F) Analysis of DN1 cells cocultured with OP9-DL1 cells for 12 d in the presence of IL-7 (5 ng/ml), IL-21 (100 ng/ml), or IL-7 + IL-21 (5 and 50 ng/ml, respectively). Plots are gated on live singlet cells. Lin thymocytes were used as a positive control. Bar graphs represent the frequencies of DN1, DN2, DN3, and DN4 after 12 d of coculture in the presence of IL-7 alone or IL-7 + IL-21 and mean fluorescence intensity (MFI) for CD25 (top) on cells recovered from culture treated with different concentrations of IL-21 (100, 50, 10 and 0 ng/ml). Data are representative of at least two experiments (n = 3–4 mice/experiment).

Close modal

Subsequently, cell size and CD27 were used to evaluate the subdivision of DN3 into DN3a (pre-β selection cells) and DN3b (post-β selection cells) (33). Our data revealed that both frequency and total number of DN3b (FSChiCD27hi) were significantly lower in IL-21R KO lin populations of BMC as compared with WT counterparts (Fig. 3D). Subsequently, we determined if the defect observed in the IL-21R KO compartment during early thymic stages could be attributed to a dysfunctional Notch signaling pathway because this transcription factor has been reported to play a critical role in the early T cell lineage commitment (34). Interestingly, the expression of Notch 1, which has been shown to be both necessary and sufficient for T cell development (34, 35), was higher in the DN subset from the IL-21R KO compartment as compared with the WT counterpart (Fig. 3E). However, Notch 2 expression was similar between DN from the WT and IL-21R KO compartments (data not shown). The role of IL-21 in DN1 differentiation was further assessed in vitro using the stromal cell line OP9-DL1 (22). For this purpose, sorted DN1 cells were cocultured with OP9-DL1 in the presence of IL-21, IL-7, or IL-7 + IL-21. As shown in Fig. 3F, after 10–12 d of coculture, sorted DN1 cells differentiated into DN2 and DN3 after treatment with IL-7 and IL-7 plus IL-21. However, IL-21 seemed to have an additive effect as a percentage of DN1 remaining in culture were lower when the cells were treated with both IL-7 and IL-21 as compared with those treated with IL-7 alone. Interestingly, IL-21 alone did not promote differentiation to the DN2 or DN3 subsets within the time frame of this experiment, even though CD25 expression was significantly increased with higher concentrations of IL-21 (Fig. 3F). From these observations, it can be postulated that, in the absence of IL-21 signaling, DN1 development seems to be impaired, and processing to the subsequent stages (DN2 and beyond) seems to be significantly compromised.

It has been reported by Haks et al. (36) that intrinsic properties of the TCR–CD3 complex regulate the selection process at the DP checkpoint. Effectively, signal strength through the TCR complex on DP thymocytes regulates the positive and negative selection signals, which lead to the upregulation of surface proteins CD69 and CD5 (37). Therefore, expression of these receptors can be used to monitor the activation and maturation at the DP stage. Our data show that IL-21 signaling is important for the maturation of the DP population as cells from the IL-21R KO compartment exhibit a less mature phenotype with a frequency of postselection DP thymocytes (CD3hiCD5hi) significantly lower than WT cells (Fig. 4A). Also, CD5 expression by DP from the IL-21R KO compartment was significantly lower than the WT counterparts (Fig. 4A). These observations were further emphasized by the analysis of CD3 and CD69, which demonstrated that both the early postpositive selection (CD3hiCD69hi) and the more mature (CD3hiCD69lo) populations were reduced in the IL-21R KO compartment (Fig. 4B). The role of the chemokine receptor CCR7 in thymic maturation is well appreciated and is important for the movement of thymocytes from the cortex to the medulla (38). Interestingly, no difference in the expression of CCR7 by WT and IL-21R KO DP populations from BMC animals was noted (Fig. 4C).

FIGURE 4.

Thymocyte maturation is reduced in the absence of IL-21 signaling. CD3, CD5, and CD69 expression was used to assess the role of IL-21 signaling in the maturation process of total thymic population. (A) Mature thymic cells (CD3hiCD5hi) were evaluated in WT or IL-21R KO compartments from mixed BMC mice at week 8 posttransplant. Histogram shows CD5 expression by thymocytes from WT and IL-21R KO compartments of BMC animals. (B) CD3 and CD69 were used to segregate early postselection (CD3hiCD69hi) and more mature (CD3hiCD69lo) populations in WT or IL-21R KO compartments. (C) WT and IL-21R KO thymocytes were analyzed for CCR7 expression. Flow cytometry plots are representative of at least two experiments (n = 3–4 mice/experiment). Graphs represent pooled data from at least three experiments (A and B, right panels).

FIGURE 4.

Thymocyte maturation is reduced in the absence of IL-21 signaling. CD3, CD5, and CD69 expression was used to assess the role of IL-21 signaling in the maturation process of total thymic population. (A) Mature thymic cells (CD3hiCD5hi) were evaluated in WT or IL-21R KO compartments from mixed BMC mice at week 8 posttransplant. Histogram shows CD5 expression by thymocytes from WT and IL-21R KO compartments of BMC animals. (B) CD3 and CD69 were used to segregate early postselection (CD3hiCD69hi) and more mature (CD3hiCD69lo) populations in WT or IL-21R KO compartments. (C) WT and IL-21R KO thymocytes were analyzed for CCR7 expression. Flow cytometry plots are representative of at least two experiments (n = 3–4 mice/experiment). Graphs represent pooled data from at least three experiments (A and B, right panels).

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Once in the medulla, newly generated SP CD4 and CD8 T cells are functionally immature and express high levels of CD24, a heat-stable Ag (39). During the final maturation steps, SP CD4 and CD8 T cells downregulate CD24 and CD69 before gaining access to the periphery (40). Flow cytometry analysis indicates that the maturation of SP CD4 and CD8 T cells isolated from the thymus of mixed BMC mice is not affected by the lack of IL-21 signaling because no significant differences were noted between the WT and KO compartment (Fig. 5A). SP CD4 and CD8 T cells exhibit a high expression of CD62L and S1P1R, which is facilitated by transcription factor KLF2 expression (41). Similar to the observations made with CD69, levels of CD62L and CD24 expression did not appear to be significantly different between the cells in IL-21R KO and WT compartments from the BMC mice. As shown in Fig. 5B, the frequency of SP mature CD4 and CD8 T cells (CD24loCD62Lhi) were similar between the two compartments (Fig. 5B). Also, the frequency of CD3+ cells among SP CD4 and CD8 T cells are comparable between WT and IL-21R KO compartments (data not shown). After mature SP thymocytes (CD4 and CD8) downregulate CD69 expression, the S1P1 receptor is retained on the surface of the cell, initiating thymic egress when S1P is detected in the vicinity of blood vessels (41). Importantly, both CD4 and CD8 T cells from the IL-21R KO population are unable to express optimal S1P1R levels, which likely compromises their ability to emigrate from the thymus to the peripheral circulation (Fig. 5C). To determine the efficiency of SP CD4 and CD8 T cells from WT and IL-21R KO mice to respond to S1P stimulus, a transwell migration assay was performed (21). As shown in Fig. 5D, both CD4 and CD8 T cells from IL-21R KO exhibit reduced migration to the lower chamber of the transwell at all the concentrations of S1P tested, whereas cells from the WT animals respond to the stimulus. The impaired ability to migrate in response to S1P could contribute to the reduced frequency of IL-21R KO CD4 and CD8 T cells in the spleen of BMC animals. These studies demonstrate that although IL-21 signaling does not seem to play a role in the maturation of SP CD4 or CD8 populations, it most likely is important for their emigration to the periphery.

FIGURE 5.

SP T cells emigration from the thymus is IL-21 dependent. The role of IL-21 signaling in maturation and emigration of thymic SP CD4 and CD8 subsets was analyzed in BMC animals. (A and B) Mature CD4 and CD8 T cells in thymus from BMC were defined as CD24loCD69lo (A) and CD24loCD62Lhi (B). (C) WT and IL-21R KO thymic SP CD4 and CD8 T cells were evaluated for S1P1 expression. Flow cytometry plots are representative of at least two experiments (n = 3–4 mice/experiment). (D) Migration of mature CD4 and CD8 (CD62LhiCD24lo) from WT and IL-21R KO mice in response to different concentrations of S1P (1000, 100, 10, and 0 nM). FTY720 was also used in conjunction with 100 nM of S1P. Statistical significance between WT and IL-21R KO CD4 (*p = 0.0016) and CD8 (*p = 0.0011) T cells was calculated using a Student t test. Graphs represent pooled data from at least three experiments (A and B, right panels).

FIGURE 5.

SP T cells emigration from the thymus is IL-21 dependent. The role of IL-21 signaling in maturation and emigration of thymic SP CD4 and CD8 subsets was analyzed in BMC animals. (A and B) Mature CD4 and CD8 T cells in thymus from BMC were defined as CD24loCD69lo (A) and CD24loCD62Lhi (B). (C) WT and IL-21R KO thymic SP CD4 and CD8 T cells were evaluated for S1P1 expression. Flow cytometry plots are representative of at least two experiments (n = 3–4 mice/experiment). (D) Migration of mature CD4 and CD8 (CD62LhiCD24lo) from WT and IL-21R KO mice in response to different concentrations of S1P (1000, 100, 10, and 0 nM). FTY720 was also used in conjunction with 100 nM of S1P. Statistical significance between WT and IL-21R KO CD4 (*p = 0.0016) and CD8 (*p = 0.0011) T cells was calculated using a Student t test. Graphs represent pooled data from at least three experiments (A and B, right panels).

Close modal

The role of IL-21 in promoting the survival and expansion of T cells in Ag-dependent as well as Ag-independent manner has been previously described (42, 43). Both CD4 and CD8 T cells arise from a very small number of early thymic progenitors, which expand greatly during the process of thymic development. As these cells also undergo apoptosis during the negative selection process, next, we determined the role of IL-21 in the expansion and survival of the different thymic subsets. At first, the survival of thymic subsets was measured by evaluating the expression of proapoptotic protein Annexin V. As shown in Fig. 6A, the cell death pattern was different between WT and IL-21R KO populations in mixed BMC, as the DN population of the IL-21R KO compartment was more apoptotic than WT cells. Annexin V expression in the DP population was not different between the two compartments. Interestingly, we observed an increased expression of Annexin V in the IL-21 KO CD8 T cells in the thymus (Fig. 6A) and the spleen (Fig. 6B) from the mixed BMC animals. Importantly, there was no difference in the expression of Annexin V in CD4 T cells between the IL-21R KO and WT compartments in both thymus and spleen (Fig. 6A, 6B).

FIGURE 6.

IL-21 plays a role in both survival and expansion of stage-specific thymocytes. Annexin V expression was assessed in the different cell populations from the thymus (DN, DP, SP CD4, and SP CD8) (A) and the spleen (CD4 and CD8 T cells) (B) from mixed BMC mice at week 8 post-reconstitution. BrdU incorporation assay was carried out to measure the proliferation of thymic (C) and splenic (D) cell subsets from BMC animals. In vivo BrdU treatment was performed for 7 d before animals were sacrificed, and intranuclear staining for BrdU was analyzed by flow cytometry. (E) BMC animals were infected with E. cuniculi and treated with BrdU 7 d later. Expansion of splenic CD4 and CD8 T cells was measured by BrdU intranuclear staining at day 14 postinfection. Histograms show an overlay of WT and IL-21R KO CD4 (left graph) or CD8 (right graph) from infected BMC mice. Assays were performed at least twice, and data are representative of one experiment (n = 3–4 mice/experiment).

FIGURE 6.

IL-21 plays a role in both survival and expansion of stage-specific thymocytes. Annexin V expression was assessed in the different cell populations from the thymus (DN, DP, SP CD4, and SP CD8) (A) and the spleen (CD4 and CD8 T cells) (B) from mixed BMC mice at week 8 post-reconstitution. BrdU incorporation assay was carried out to measure the proliferation of thymic (C) and splenic (D) cell subsets from BMC animals. In vivo BrdU treatment was performed for 7 d before animals were sacrificed, and intranuclear staining for BrdU was analyzed by flow cytometry. (E) BMC animals were infected with E. cuniculi and treated with BrdU 7 d later. Expansion of splenic CD4 and CD8 T cells was measured by BrdU intranuclear staining at day 14 postinfection. Histograms show an overlay of WT and IL-21R KO CD4 (left graph) or CD8 (right graph) from infected BMC mice. Assays were performed at least twice, and data are representative of one experiment (n = 3–4 mice/experiment).

Close modal

To determine the role of IL-21 in the proliferative ability of the different subsets from the thymus and spleen of BMC animals, we performed a BrdU incorporation assay. As shown in Fig. 6C, impaired IL-21 signaling triggered a slightly lower frequency of proliferative CD8 T cells in the thymus. Conversely, the frequency of thymic DN, DP, and CD4 T cells from the IL-21R KO compartment with newly incorporated BrdU was similar to those in the WT compartment. Interestingly, as compared with the WT compartment, the proliferative ability of both CD4 and CD8 splenic T cells was impaired in the absence of functional IL-21 signaling (Fig. 6D).

To assess the role of IL-21 in the expansion of T cells in an infectious environment, mixed BMC animals were orally infected with E. cuniculi, a pathogen that induces a potent cytotoxic T cell response dependent on IL-21 (19). Although no difference in BrdU incorporation between the WT and IL-21R KO compartments of the infected BMC animals was observed for any of the thymic subsets (DN, DP, CD4, and CD8) (data not shown), proliferative ability of both splenic CD4 and CD8 T cells lacking functional IL-21 signaling was significantly impaired in these animals (Fig. 6E). These studies demonstrate that IL-21 signaling is important for the survival of some thymic subsets (DN and CD8 SP) and the CD8 T cell population in the spleen. Also, this cytokine plays an important role in the expansion of the CD8 T cell population in both the thymus and the periphery.

The observations presented in this manuscript reveal a multilayered role of IL-21 in the thymic development of T lymphocytes and demonstrate the involvement of IL-21 not only in the differentiation of the DN subsets but also in the maturation and emigration process of subsequent thymic populations. The fact that all thymic subsets (DN, DP, CD4, and CD8) express IL-21R suggests a potential role for this cytokine in thymopoiesis. Using BMC mice, we observe that the IL-21R KO population is underrepresented in both the thymus and spleen of chimeric animals. Moreover, this defect is detected as early as the DN1 stage and is followed by a subsequent decrease in the generation of DN2–DN4 populations in the KO compartment. Although the expression of CCR7 by the WT and IL-21R KO populations is not different, IL-21 seems to be involved in the positive selection process. Also, based on our observations, IL-21 signaling does not seem to be important in the maturation of SP lymphocytes (CD4 and CD8); however, the lower S1P1 receptor expression and the diminished ability to migrate to S1P in vitro displayed by IL-21R KO cells can compromise their migration to the periphery.

The thymic T cell development is known to be critically dependent on IL-7 (44). This cytokine, which is produced by thymic epithelial cells, plays a major role from the early stage of DN to the differentiation of CD8 and TCR γδ intraepithelial lymphocytes (44). In the absence of IL-7 signaling, the differentiation of immature thymocytes is compromised, and IL-7R–deficient cells are arrested at the DN3 stage (2, 3). However, the fact that mice with a genetic deficiency in common γ-chain cytokines, not including IL-7, display normal cellularity in both the spleen and thymus has complicated the evaluation of their role in thymopoiesis (44). Therefore, based on this seemingly normal T cell development in IL-21R KO mice and the contradicting fact that IL-21 can improve thymic recovery during aging or after glucocorticoid treatment, a mixed BMC approach (WT and IL-21R KO) was used to determine the intrinsic role of this cytokine in the thymic development. We observed that the frequency of the DN subset and SP T cells (CD4 and CD8) are reduced in the absence of IL-21 signaling. In addition, an increased frequency of the DN1 population is detected in the IL-21R KO compartment; however, these cells do not transition efficiently to subsequent stages (DN2–DN4). As shown by OP9-DL1 and DN1 coculture, IL-21 contributes to the differentiation of DN1 cells into subsequent DN subsets, although IL-7 plays a more predominant role in this process. Interestingly, our data reveal that IL-21 is involved as early as the DN1 stage, which is earlier than IL-7 as this begins only at the DN3 stage and coincides with TCRβ selection of DN thymocytes (4). Although the role of IL-21 in the proliferation and survival of T cells in the periphery has been documented (42, 43), the reduced frequency of the DN population in the IL-21R KO compartment can also be attributed to an increase in apoptosis rather than a defect in their proliferative ability. Also, similar to IL-7, IL-21 does not seem to be involved in the commitment of lymphoid progenitors to a T cell lineage as Notch expression was not decreased by a lack of IL-21 signaling.

Although our studies highlight an important role for IL-21 in the development of DN stages, a lack of IL-21 signaling also seems to affect the transition from postpositive selection (CD3hiCD69hi) into a mature population (CD3hiCD69lo) as the frequency of mature thymocytes is significantly lower in the IL-21R KO compartment. These data are consistent with in vitro studies performed by Rafei et al. (10), showing a strong induction of IL-21R by DP thymocytes undergoing positive selection (10). However, conflicting with these studies (10), our report shows that IL-21R KO DP in mixed BMC exhibit a normal proliferative ability because BrdU incorporation levels are similar to the cells from the WT compartment. The differences can be explained by the fact that we are performing ex vivo studies using BMC mice that address the role of IL-21 signaling in a competitive environment, whereas the data from the studies of Rafei et al. were generated by treating WT thymocytes with IL-21 in vitro.

In addition to the role of IL-21 at the DN and DP stages, studies presented in the manuscript demonstrate that IL-21 is important for the survival and proliferation of SP CD8 cells. A significant increase in Annexin V expression as well as reduced BrdU incorporation were observed in the SP CD8 T cell population from the KO compartment of BMC mice, which is consistent with the previously reported role of this cytokine (42, 43). Conversely, the lack of IL-21 signaling does not seem to affect the survival or expansion of the thymic SP CD4 T cell population. Although the maturation of IL-21R KO SP CD4 or CD8 T cells appears to be normal, the lower expression of S1P1R on both CD4 and CD8 SP cells from the IL-21R KO compartment as well as their lower ability to migrate to S1P stimulation in vitro suggests that these cells have a defect in the ability to migrate to the periphery. As previously reported (12, 42, 43), the role of IL-21 is not limited to the thymic T cell development as our data demonstrate that the lack of IL-21 signaling reduced both CD4 and CD8 T cell expansion in the spleen of mixed BMC mice. Furthermore, the role of IL-21 in the abundance of memory CD8 T cells has been reported (45).These observations emphasize the role of IL-21 in homeostatic proliferation and the survival of both CD4 and CD8 T cells in the periphery, which is very similar to IL-7 (46).

The data presented in this manuscript define a complex role for IL-21 in thymic T cell development. Even though IL-21 levels in the thymus from the WT animals were significantly lower than IL-7 and IL-15, cytokines, which are known to play a role in the thymic development (2, 7), our data demonstrate that a lack of IL-21 signaling leads to a suboptimal thymopoiesis. Although we do not dispute that IL-7 plays a critical role in thymopoiesis (2, 3), our findings are very important in light of the fact that IL-21R KO mice display normal thymic and splenic cellularity (14) and underline the complex role of IL-21 in thymopoiesis. Normal cellularity observed in IL-21R KO mice could be attributed to the overwhelming predominance of IL-7 in the thymus as compared with IL-21. Furthermore, a potentially unidentified redundant mechanism(s) that may be masking the role of IL-21 cannot be ruled out. The data presented in this manuscript demonstrate a multidimensional role for IL-21 in the development of thymic T cells that may be dependent on IL-7. Based on a published report that showed that IL-7 was significantly increased in the thymus of mice treated with IL-21 (16) and our observation that IL-7 production was decreased in IL-21R KO mice, IL-7 and IL-21 may have a synergistic role in T cell thymic development (47). Our studies raise some important issues related to the thymic T cell development, and interactions between IL-7 and IL-21 in this process need to be further studied.

We thank J. C. Zúñiga-Pflücker for providing the OP9-DL1 cell line.

This work was supported by National Institutes of Health Grant AI033325 (to I.A.K.).

Abbreviations used in this article:

BMC

bone marrow chimeric

DN

double-negative

DP

double-positive

ETP

early thymic precursor

lin

lineage-negative

OP9-DL1

OP9 expressing the DL1 receptor for Notch

SP

single-positive.

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The authors have no financial conflicts of interest.