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):4656-4664 May 17, 2012 ©2012 by American Society of Hematology

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 108 558 ± 19 694 and, with the addition of IL-21, was 109 311 ± 25 925. Data are representative of at least 5 independent experiments. *P < .05; **P < .01; ***P < .001. Kesley Attridge et al. Blood 2012;119:4656-4664 ©2012 by American Society of Hematology

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 78 190 ± 2095. (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 149 504 ± 9868. Data are representative of at least 3 independent experiments. *P < .05. **P < .01. ns indicates not significant. Kesley Attridge et al. Blood 2012;119:4656-4664 ©2012 by American Society of Hematology

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;119:4656-4664 ©2012 by American Society of Hematology

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 91 011 ± 5998. Data are representative of at least 3 independent experiments. **P < .01; ***P < .001. Kesley Attridge et al. Blood 2012;119:4656-4664 ©2012 by American Society of Hematology

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 94 497 ± 8704. (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 < .001. ns indicates not significant. Kesley Attridge et al. Blood 2012;119:4656-4664 ©2012 by American Society of Hematology

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 111 995 ± 22 306. Data are representative of at least 3 independent experiments. *P < .05; **P < .01. ***P < .001. ns indicates not significant. Kesley Attridge et al. Blood 2012;119:4656-4664 ©2012 by American Society of Hematology

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+DO11.10+ 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;119:4656-4664 ©2012 by American Society of Hematology