Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Mar 1;182(5):2859-67.
doi: 10.4049/jimmunol.0802978.

IL-21 mediates suppressive effects via its induction of IL-10

Rosanne Spolski  1 , Hyoung-Pyo Kim, Wei Zhu, David E Levy, Warren J Leonard
Affiliations

IL-21 mediates suppressive effects via its induction of IL-10

Rosanne Spolski et al. J Immunol. .

Abstract

IL-21 is a pleiotropic cytokine that is required for normal Ig production. We previously showed that IL-21 was elevated in BXSB-Yaa mice with systemic lupus erythematosus. These mice also had elevated IL-10 levels, and we now show that IL-21 induces IL-10 mRNA and protein, suggesting unexpected immunosuppressive activities for IL-21. Indeed, Th1 priming with IL-21 leads to accumulation of cells with immunosuppressive activity, and IL-21 overexpression decreases specific Ab production after immunization in an IL-10-dependent fashion. Moreover, we show that IL-21 signaling is required for maximal induction of IL-10 by IL-6 or IL-27. Overall, our data indicate that IL-21 regulates immune responses at least in part by inducing IL-10 and reveal unanticipated immunosuppressive actions for this cytokine.

PubMed Disclaimer

Figures

Figure 1
Figure 1
IL-21 induces IL-10 expression. (A and B) Splenic T cells from either IL-21R KO mice and their littermate controls (A) or from IL-21 TG mice and their littermate controls (B) were purified by negative selection and stimulated in vitro with anti-CD3 (2 ug/ml) + anti-CD28 (1 ug/ml) for 48 h. Secreted IL-10, IL-4, and IFNγ were measured by ELISA using specific kits (BD PharMingen). Shown is an experiment representative of three individual experiments. *P < 0.01; **P < 0.001. The genetic backgrounds for the IL-21R KO mice and IL-21 transgenic mice differ (see Methods).
Figure 2
Figure 2
IL-21 induces IL-10 mRNA in CD4+ and CD8+ T cells. (A and B) Splenic CD4+ or CD8+ T cells from Balb/c mice were stimulated with anti-CD3 (2 ug/ml) + anti-CD28 (1 ug/ml) without or with IL-21 for the indicated times and IL-10 mRNA levels measured by real-time PCR. The control refers to cells not exposed to either anti-CD3+ anti-CD28 or to IL-21. IL-21 alone did not induce IL-10 mRNA above control levels in either CD4+ or CD8+ T cells (not shown). (C) Naive CD4+ or CD8+ T cells were stimulated with anti-CD3 + anti-CD28 in the absence or presence of IL-21 for 48 h, at which point culture supernatants were assayed for IL-10 by ELISA (BD Pharmingen). Data are representative of three individual experiments. *P < 0.05; **P < 0.01.
Figure 3
Figure 3
IL-21-induced expression of IL-10 is mediated by STAT3. (A) IL-21-mediated induction of IL-10 is diminished in CD4+ and CD8+ splenic T cells from Stat3 KO mice (upper panels), whereas IFN-γ is not diminished (lower panels). CD4+ and CD8+ splenic T cells were isolated from Stat3 KO mice or WT littermates and stimulated with anti-CD3 (2 ug/ml) + anti-CD28 (1 ug/ml) in the absence or presence of IL-21 for 6 h. Relative IL-10 and IFN-γ mRNA levels were quantified by real-time PCR. *P < 0.05; **P<0.01 (B) Constitutively activated STAT3 expression augments basal and IL-21-induced IL-10 protein expression. CD4+ and CD8+ T cells from Balb/c mice were transduced with a retrovirus encoding a constitutively activated form of STAT3. Cells were stimulated overnight with anti-CD3 (2 ug/ml) + anti-CD28 (1 ug/ml) without or with IL-21 and stained intracellularly with antibodies to IL-10. FACS analysis of gated GFP-positive CD4+ or CD8+ T cells is shown. Data shown are representative of three individual experiments.
Figure 4
Figure 4
IL-21 induces IL-10 mRNA and protein expression in Th1 and Tc1 cells. (A and B) Naive CD4+ or CD8+ T cells were polarized under either Th1/Tc1 or Th2/Tc2 conditions in the absence or presence of IL-21 and then expanded in IL-2. In (A), polarized cells were then stimulated with PMA + ionomycin for 6 h and relative levels of IFN-γ, IL-4, and IL-10 mRNA were quantified by real-time PCR. In (B), Th1/Tc1 and Th2/Tc2 polarized cells were re-stimulated with anti-CD3 + anti-CD28 for 48 h in the absence or presence of IL-21, and levels of IL-10 secreted in culture supernatants were measured by ELISA. Statistically significant differences, when present, are indicated by asterisks: *P < 0.05; **P < 0.01; ***P < 0.001. (C) Th1/Tc1 and Th2/Tc2 cells that had been polarized either in the absence or presence of IL-21 were re-stimulated for 6 h with PMA + ionomycin in the presence of Golgi stop for the last 4 h. Intracellular levels of IL-10 and IFN-γ were assessed by flow cytometry. The quadrants were determined based on staining with isotype control antibodies. Data shown are representative of three individual experiments.
Figure 5
Figure 5
IL-21 controls expression of IL-10 in Th17/Tc17 primary cells and is required for normal induction of IL-10 by IL-6 or IL-27. (A) Naive CD4+ or CD8+ T cells from WT or IL-21R KO mice were polarized in vitro in the presence of TGFβ + IL-6 for 4 days and re-stimulated for 6 h with PMA + ionomycin in the presence of Golgi stop for the last 4 h. Intracellular levels of IL-10 and IL-17 were assessed by flow cytometry. The quadrants were determined based on staining with isotype control antibodies. (B) WT CD4+ T cells were stimulated under neutral conditions with anti-CD3 + anti-CD28 in the presence or absence of TGFβ and IL-6 or IL-27 for 4 days, at which time culture supernatants were assayed by ELISA for IL-21. (C) CD4+ T cells from either WT or IL-21R KO mice were stimulated with anti-CD3 + anti-CD28 + anti-IFNγ + anti-IL-4 in the presence of TGFβ with either IL-6, IL-21, or IL-27 for 4 days and then re-stimulated for 6 h with PMA + ionomycin in the presence of Golgi stop for the last 4 h. Intracellular levels of IL-10 and IL-17 were assessed by flow cytometry. (D) The percentage of IL-10 positive cells in (c) are represented in a bar graph. (E) CD4+ and CD8+ T cells were pre-activated with anti-CD3 + anti-CD28 for 48 h, washed and rested overnight, and were re-stimulated without or with IL-6, IL-21 or IL-27 for 6 h. RNA was then isolated and relative IL-10 mRNA levels quantified by real-time PCR. (F) Parallel culture supernatants were assayed at 48 h for IL-10 by ELISA. *P<0.01; **P<0.05.
Figure 6
Figure 6
IL-21 augments IL-10 expression even in committed Tc1 or Tc17 primary cells and in Th1 committed AE7 cells. (A) CD8+ T cells were subjected to three rounds of polarization under either Tc1 or Tc17 conditions in either the absence or presence of IL-21 and were then stimulated with or without anti-CD3/CD28 in either the absence or presence of IL-21 for 24 h, at which point IL-10 levels were measured in culture supernatants. (B) AE7 cells were TCR stimulated and were then expanded in IL-2, rested overnight and were then re-stimulated with PMA + ionomycin in either the presence or absence of IL-21 and IL-2. Culture supernatants were assayed by ELISA at 24 h for IL-10 and IFN-γ. Data shown are representative of three separate experiments. *P < 0.05.
Figure 7
Figure 7
Th1 cells polarized in the presence of IL-21 inhibit the activation of naive CD8+ T cells, and this inhibition is IL-10-dependent. Naive OT-1 cells were activated with peptide/APC in the absence or presence of Th1 cells that were primed in the presence or absence of IL-21. (A) CD25 surface expression on gated Vα2+ OT-1 cells was measured by flow cytometry at 48 h in cells treated either with a control Ab (upper panels) or anti-IL-10R (lower panels). MFIs are indicated in each profile. (B) Proliferation of OT-1 cells was assessed by CFSE dilution at 48 h after peptide/APC stimulation. Shown is the effect of the addition of Th1 cells primed in the absence vs. presence of IL-21. The percentage of cells in the indicated region is shown. MFIs were 96, 237, and 351 for OT-1, OT-1 + Th1, and OT-1 + Th1/IL-21, respectively. These proliferation assays were performed in the absence of exogenous IL-2. (C) CD25 surface expression on OT-1 cells from WT mice (upper two panels) or IL-10 KO mice (lower two panels) was measured at 48 h without co-culture (left panel) or after co-culture with Th1 cells (middle panels) or Th1 cells polarized with IL-21 (right panels). MFIs are indicated in each profile. Shown is an experiment representative of three individual experiments.
Figure 8
Figure 8
IL-21 transgenic mice exhibit diminished production of specific antibody in response to primary immunization with ovalbumin, and this inhibition is partially dependent on the presence of IL-10. IL-21 transgenic mice and non-transgenic littermates on IL-10 KO and WT backgrounds were immunized i.p. with 100 ug ovalbumin in alum. At day 7 after primary immunization, serum levels of OVA-specific immunoglobulin isotypes were assessed by ELISA. Data are mean ± SEM from three experiments (2−4 animals per experiment). Statistical significance was determined using Student's t test. *The statistical difference between TG/WT and TG/IL-10 KO was P < 0.001 for IgM, P < 0.002 for IgG1, P < 0.02 for IgG2a, and ) < 0.009 for IgG2b.
See this image and copyright information in PMC

References

    1. Mosmann TR, Coffman RL. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol. 1989;7:145–173. - PubMed
    1. Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol. 2007;25:821–852. - PubMed
    1. O'Garra A, Vieira P. Regulatory T cells and mechanisms of immune system control. Nat Med. 2004;10:801–805. - PubMed
    1. Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol. 2001;19:683–765. - PubMed
    1. McGeachy MJ, Bak-Jensen KS, Chen Y, Tato CM, Blumenschein W, McClanahan T, Cua DJ. TGF-beta and IL-6 drive the production of IL-17 and IL-10 by T cells and restrain T(H)-17 cell-mediated pathology. Nat Immunol. 2007;8:1390–1397. - PubMed

Publication types

MeSH terms