1. IL-21 inhibits T cell IL-2 production and impairs Treg homeostasis
by Kesley Attridge, Chun Jing Wang, Lukasz Wardzinski, Rupert Kenefeck, Jayne L. Chamberlain, Claire Manzotti, Manfred Kopf, and Lucy S. K. Walker
Blood
Volume 119(20):
May 17, 2012
©2012 by American Society of Hematology

2. IL-21 counteracts the ability of Tregs to inhibit T-cell proliferation and activation marker expression.
IL-21 counteracts the ability of Tregs to inhibit T-cell proliferation and activation marker expression. CFSE-labeled BALB/c.Thy1.1+ CD4+CD25− Tconv (2.5 × 104) were cultured with anti-CD3 and CD19+ B cells (5 × 104) with the indicated ratios of Thy1.2+CD4+CD25+ Tregs alone or in the presence of 100 ng/mL IL-21. At day 3, cells were analyzed by flow cytometry. The relative cell number of conventional T cells (A) and their expression profiles for CD25 and CD69 (B), and CFSE (C) are shown. The average Tconv cell number in the absence of cytokine or Tregs was ± and, with the addition of IL-21, was ± Data are representative of at least 5 independent experiments. *P < .05; **P < .01; ***P < .001.
Kesley Attridge et al. Blood 2012;119:
©2012 by American Society of Hematology

3. Conventional T cells are the major target for IL-21 during release from Treg-mediated suppression.
Conventional T cells are the major target for IL-21 during release from Treg-mediated suppression. (A) BALB/c lymph node Tconv (CD4+Foxp3−), Tregs (CD4+Foxp3+), and B cells (CD19+) were stained by flow cytometry for expression of IL-21R. (B) CD4+CD25− Tconv (2.5 × 104) were cultured with anti-CD3 and CD19+ B cells (5 × 104) with the indicated ratios of CD4+CD25+ Tregs in the presence of IL-21. Cell populations were deficient for the IL-21R as indicated. Tconv cell count at day 3 is shown. The average Tconv cell number in the absence of Tregs was ± (C) IL-21R−/−CD4+CD25− Tconv (2.5 × 104) were cultured with anti-CD3 and IL-21R−/−CD19+ B cells (5 × 104) with the indicated ratios of IL-21R+/+CD4+CD25+ Tregs alone or in the presence of IL-21. Relative Tconv cell count at day 3 is shown. The average Tconv cell number in the absence of cytokine or Treg was ± Data are representative of at least 3 independent experiments. *P < .05. **P < .01. ns indicates not significant.
Kesley Attridge et al. Blood 2012;119:
©2012 by American Society of Hematology

4. Effect of IL-21 on Tconv surface marker expression and Foxp3 induction.
Effect of IL-21 on Tconv surface marker expression and Foxp3 induction. (A) BALB/c CD4+CD25− Tconv (2.5 × 104) and CD19+ B cells (5 × 104) were cultured alone, with anti-CD3, with IL-21, or with both for 15 to 18 hours. Surface marker expression on gated Tconv is shown. (B) BALB/c Thy1.1+CD4+CD25− T conv (2.5 × 104) were cultured with anti-CD3, CD19+ B cells (5 × 104), and Thy1.2+CD4+CD25+ Tregs (2.5 × 104) alone or in the presence of IL-21. Three days later, Foxp3 expression by Thy1.1+CD4+ cells and Thy1.2+CD4+ cells was determined. There was negligible Foxp3 induction in Tconv (Foxp3 expression in gated Tregs is shown as a positive control). Data are representative of at least 3 independent experiments.
Kesley Attridge et al. Blood 2012;119:
©2012 by American Society of Hematology

5. IL-21 suppresses IL-2 and IFN-γ production by conventional T cells.
IL-21 suppresses IL-2 and IFN-γ production by conventional T cells. BALB/c CD4+CD25− Tconv (2.5 × 104) were cultured with anti-CD3 and CD19+ B cells (5 × 104) alone or in the presence of IL-21. Intracellular cytokine staining was performed at day 3. The proportion of T cells expressing the indicated cytokine (A), and representative contour plots for IL-2 and IFN-γ expression (B) are shown. (C) BALB/c CD4+CD25− Tconv (2.5 × 104) were cultured with anti-CD3 and CD19+ B cells (5 × 104) with the indicated ratios of CD4+CD25+ Tregs alone or in the presence of either anti–IFN-γ antibody or IFN-γ. IL-21 was added where indicated. Relative Tconv cell counts at day 3 are shown. The average Tconv cell number in the absence of cytokine, blocking antibody, or Tregs was ± Data are representative of at least 3 independent experiments. **P < .01; ***P < .001.
Kesley Attridge et al. Blood 2012;119:
©2012 by American Society of Hematology

6. IL-21 can substitute for IL-2 in conventional, but not regulatory, T cells.
IL-21 can substitute for IL-2 in conventional, but not regulatory, T cells. (A) BALB/c CD4+CD25− Tconv (2.5 × 104) were cultured with anti-CD3 and 5 × 104 CD19+ B cells alone, with anti–IL-2 antibody, or with both anti–IL-2 Ab and 200 ng/mL IL-21. Relative Tconv cell counts are shown at day 3. The average Tconv cell number in the absence of cytokine or Tregs was ± (B) BALB/c CD4+CD25+ Tregs (2.5 × 104) were cultured alone or in the presence of either 20 ng/mL IL-2 or 200 ng/mL IL-21. Representative staining for Foxp3 and CD25 and relative cell counts are shown at day 3. Data are representative of at least 3 independent experiments. **P < .01; ***P < ns indicates not significant.
Kesley Attridge et al. Blood 2012;119:
©2012 by American Society of Hematology

7. IL-21 indirectly affects Treg homeostasis.
IL-21 indirectly affects Treg homeostasis. (A) BALB/c CD4+CD25− Tconv (2.5 × 104) were cultured with anti-CD3 and CD19+ B cells (5 × 104) alone or in the presence of IL-21. Plots show secreted IL-2 levels for gated CD4+Foxp3− cells at day 3. (B) IL-21R–deficient CD4+CD25− Tconv were cultured with anti-CD3 and CD19+ B cells. Plots show intracellular IL-2 staining at day 3 for gated CD4+Foxp3− cells. (C) BALB/c CD4+CD25− Tconv (2.5 × 104) were cultured with anti-CD3, CD19+ B cells (5 × 104), and CD4+CD25+ Tregs (1.25 × 104) alone or in the presence of IL-21. The percentage of CD4+Foxp3− cells staining for intracellular IL-2 at day 3, and representative contour plots, are shown. (D) BALB/c CD4+CD25− Tconv (2.5 × 104) were cultured with anti-CD3, CD19+ B cells (5 × 104), and CD4+CD25+ Tregs (1.25 × 104) alone or in the presence of either IL-21 or anti–IL-2 Ab. The relative number of CD4+Foxp3+ Tregs is shown at day 3. (E) BALB/c IL-21R−/−CD4+CD25− Tconv (2.5 × 104) were cultured with anti-CD3, CD19+ B cells (5 × 104), and IL-21R+/+CD4+CD25+ Tregs (1.25 × 104) in the presence or absence of IL-21. The relative number of CD4+Foxp3+ Tregs is shown at day 3. (F) CD4+CD25− Tconv (2.5 × 104) were cultured with anti-CD3, CD19+ B cells (5 × 104) and CD4+CD25+ Tregs (1.25 × 104) alone or in the presence of IL-21. Histograms represent pSTAT5 staining for gated CD4+Foxp3+ cells at day 3 in the presence or absence of IL-21 compared with levels in unstimulated control T cells. (G) CD4+CD25− Tconv (2.5 × 104) were cultured with anti-CD3 and CD19+ B cells (5 × 104) alone or with CD4+CD25+ Tregs (2.5 × 104) in the presence of IL-21 where indicated. Tregs were preincubated alone or with 20 ng/mL IL-2. At day 3, cells were analyzed by flow cytometry, and the relative Tconv cell count is shown. The average Tconv cell number in the absence of cytokine or Treg was ± Data are representative of at least 3 independent experiments. *P < .05; **P < .01. ***P < ns indicates not significant.
Kesley Attridge et al. Blood 2012;119:
©2012 by American Society of Hematology

8. IL-21 down-regulates IL-2 and counteracts Treg suppression in vivo.
IL-21 down-regulates IL-2 and counteracts Treg suppression in vivo. (A) Thy1.1+ CD4+ DO11.10 T cells (1 × 106) were adoptively transferred into Thy1.2+ BALB/c recipients. Mice were immunized with OVA/alum intraperitoneally on day 1 and received daily intraperitoneal injections of IL-21 (1 μg) or vehicle control (PBS). Plots represent secreted IL-2, and graph represents collated IL-2 mean fluorescence intensity for Thy1.1+CD4+ T cells harvested from the spleen at day 5. (B) Thy1.1+ CD4+ DO11.10 T cells (1 × 106) were adoptively transferred into Thy1.2+ BALB/c recipients alone or with Thy1.2+CD4+CD25+ DO11.10 Tregs (2.5 × 105). Treg populations were preincubated alone or with 20 ng/mL IL-2 as indicated. Mice were immunized with OVA/alum intraperitoneally on day 1 and received daily intraperitoneal injections of IL-21 (1 μg) or vehicle control. At day 5, absolute numbers of Thy1.1+CD4+DO cells in the spleen were determined by flow cytometry. Data are representative of at least 2 independent experiments. *P < .05. ns indicates not significant.
Kesley Attridge et al. Blood 2012;119:
©2012 by American Society of Hematology