A role for IL-27 in limiting T regulatory cell populations

Abstract

IL-27 is a cytokine that regulates Th function during autoimmune and pathogen-induced immune responses. Although previous studies have shown that regulatory T cells (Tregs) express the IL-27R, and that IL-27 inhibits forkhead box P3 upregulation in vitro, little is known about how IL-27 influences Tregs in vivo. The studies presented in this article show that mice that overexpress IL-27 had decreased Treg frequencies and developed spontaneous inflammation. Although IL-27 did not cause mature Tregs to downregulate forkhead box P3, transgenic overexpression in vivo limited the size of a differentiating Treg population in a bone marrow chimera model, which correlated with reduced production of IL-2, a vital cytokine for Treg maintenance. These data identify an indirect role for IL-27 in shaping the Treg pool.

Fig. 1

IL-27 tg mice succumbed to an inflammatory disease. A. IL-27 tg mice died at 8–12 wk of age. Data represent 8 WT or IL-27 tg mice; p<0.0001 by Kaplan and Meier logrank test. B. Ten wk-old IL-27 tg mice had inflammatory infiltrates (→) in the liver, pancreas, kidney, and lung. Images displayed are representative of 6 WT or IL-27 tg mice (bar is 100 μm in liver, lung, and kidney images, and 50 μm for pancreas images).

Fig. 2

IL-27 tg mice developed systemic inflammation by 7 wk of age. A. Compared to WT mice, IL-27 tg mice were lymphopenic in the spleen and LN, but had similar PEC counts. Data represent SEM for 10 WT and 9 IL-27 tg mice from 5 independent experiments; spleen p=0.0003, LN p<0.0001, PEC p=0.0653. B. A greater percentage of CD4⁺ and CD8⁺ T cells in the spleen, LN, and PEC from IL-27 tg mice expressed CD69 than those from WT mice. Plots were gated on CD3⁺CD4⁺ or CD3⁺CD8⁺ cells. Data represent SEM for 10 WT and 9 IL-27 tg mice from 5 independent experiments; spleen CD4⁺ p=0.0076, spleen CD8⁺ p=0.0002, LN CD4⁺ p=0.003, LN CD8⁺ p<0.0001, PEC CD4⁺ p<0.0001, PEC CD8⁺ p<0.0001. C. Following stimulation, a greater percentage of splenic CD8⁺ T cells from IL-27 tg mice produced IFN-γ than those from WT mice. Plots were gated on CD8⁺ cells. Data represent SEM for 11 WT and 10 IL-27 tg mice from 4 independent experiments; p=0.0003. D. IL-27 tg mice had higher amounts of IFN-γ, IL-10, IL-6, and TNF-α in the serum than WT mice, and an equivalent amount of macrophage-derived chemokine (MDC). Data represent SEM for 5 WT and IL-27 tg mice from an assay performed by Rules Based Medicine; IFN-γ p=0.0317, IL-10 p=0.0079, IL-5 p=0.0079, IL-6 p=0.0159, MDC p=1.0, TNF-α p=0.0079. N.D. is not detected. All p values calculated by unpaired, two-tailed Mann-Whitney t-test.

Fig. 3

IL-27 tg mice were deficient in T_reg. IL-27 tg mice had very low A. percentages and B. total numbers of Foxp3⁺ T_reg in the spleen, LN, PEC, and thymus at 7–8 wk of age. Plots were gated on CD3⁺CD4⁺ cells. Data represent SEM for 10 WT and 9 IL-27 tg mice from 5 independent experiments (thymus: data represent SEM for 8 WT and 7 IL-27 tg mice, from 4 independent experiments); A. spleen p<0.0001, LN p<0.0001, PEC p<0.0001, thymus p=0.0003; B. spleen p<0.0001, LN p<0.0001, PEC p<0.0001, thymus p=0.0003; all by unpaired, two-tailed Mann-Whitney t-test.

Fig. 4

nT_reg exposed to IL-27 did not downregulate Foxp3. A. Foxp3^GFP+ nT_reg did not downregulate Foxp3 following 72 h of culture with rIL-27, but did downregulate Foxp3 following culture with rIL-6. Plots were gated on CD3⁺CD4⁺ cells. Data represent SEM for IL-2 and IL-2 + rIL-27 from 4 independent experiments, and a representative plot of IL-2 + rIL-6; IL-2 versus IL-2 + rIL-27 p=0.6857. Eleven d after WT CD4⁺CD25⁺ cells were transferred to 7 wk-old WT or IL-27 tg recipients, there was a similar B. percentage of congenic cells that were Foxp3⁺ cells and C. total number of CD45.1⁺CD4⁺Foxp3⁺ T cells in the spleen and LN, regardless of recipient genotype. Plots were gated on CD3⁺CD45.1⁺CD4⁺ cells. Data represent SEM for 7 WT and 8 IL-27 tg mice from 3 independent experiments; B. spleen p=0.2857, LN p=0.7789; C. spleen p=0.1893, LN p=0.0939. All p values calculated by unpaired, two-tailed Mann-Whitney t-test.

Fig. 5

Irradiated WT mice reconstituted with IL-27 tg BM were T_reg deficient. Irradiated WT mice reconstituted with IL-27 tg BM had low A. percentages and B. total numbers of both donor (CD45.2) and host (CD45.1) Foxp3⁺ T_reg in the spleen, LN, and thymus at 5 wk post-irradiation and reconstitution. Plots were gated on CD3⁺CD45.2⁺CD4⁺ or CD3⁺CD45.1⁺CD4⁺ cells. Data represent SEM for 11 WT and IL-27 tg mice from 3 independent experiments; A. spleen donor p=0.005, spleen host p<0.0001, LN donor p<0.0001, LN host p<0.0001, thymus donor p=0.0013, thymus host p=0.0001 by unpaired, two-tailed Mann-Whitney t-test; B. spleen donor p<0.0001, spleen host p=0.0001, spleen total p=0.0001, LN donor p<0.0001, LN host p=0.0002, LN total p=0.0005, thymus donor p<0.0001, thymus host p=0.0878, thymus total p=0.0008 by unpaired, two-tailed Mann-Whitney t-test. C. Irradiated WT mice reconstituted with IL-27 tg BM had inflammatory infiltrates in the liver and pancreas (→) at 5 wk post-irradiation and reconstitution. Images displayed are representative of 8 irradiated WT mice reconstituted with WT or IL-27 tg BM (bar is 100 μm in liver images, and 50 μm for pancreas images). D. Irradiated WT mice reconstituted with IL-27 tg BM died. Data represent 10 WT and IL-27 tg mice; p=0.0012 by Kaplan and Meier logrank test.

Fig. 6

IL-27 tg mice were IL-2 deficient. Following stimulation, there was a lower A. percentage and B. total number of CD4⁺ T cells that produced IL-2 from IL-27 tg mice compared to WT mice in the spleen, LN, and thymus (except for total numbers in the thymus). Plots were gated on CD4⁺ cells. Data represent SEM for 11 WT and 10 IL-27 tg mice from 4 independent experiments; A. spleen p=0.0011, LN p=0.0360, thymus p=0.0125; B. spleen p=0.0001, LN p=0.0003, thymus p=0.5529. Following stimulation, there was a lower C. percentage and D. total number of host and total CD4⁺ T cells that produced IL-2 from irradiated WT mice reconstituted with IL-27 tg BM compared to WT BM in the spleen at 5 wk post-irradiation and reconstitution. Plots were gated on CD4⁺ cells. Data represent SEM for 12 WT and 11 IL-27 tg mice from 3 independent experiments; C. spleen donor p=0.3722, spleen host p=0.0006, spleen total p=0.0107; D. spleen donor p<0.0001, spleen host p=0.0019, spleen total p=0.0003. E. Following stimulation for 48 or 24 h, respectively, with anti-CD3 and anti-CD28, splenocytes from IL-27 tg mice or irradiated WT mice reconstituted with IL-27 tg BM produced less IL-2 than those from WT mice or mice reconstituted with WT BM. Whole mice: data represent SEM for 11 WT and 10 IL-27 tg mice from 4 independent experiments; p=0.0054. Chimeras: data represent SEM for 10 WT and 8 IL-27 tg mice from 3 independent experiments; p=0.0172. All p values calculated by unpaired, two-tailed Mann-Whitney t-test.