Differential Modulation of Human Innate Lymphoid Cell (ILC) Subsets by IL-10 and TGF-β

Abstract

Using multiparameter flow cytometry human innate lymphoid cell (ILC) subsets can be detected in the circulation, in relatively low frequencies. Despite the low frequency of ILCs in circulation, ex vivo experiments have demonstrated that these ILCs release extremely large per cell quantities of signature ILC cytokines following activation. To determine how activated ILC cytokine production is regulated, ILC subsets were activated in the presence or absence of the immunoregulatory cytokines IL-10 and TGF-β. An examination of circulating ILC subsets revealed surface expression of IL-10Rα and mRNA expression of both IL-10Rα and TGF-βR1 for all ILC subsets. Stimulated ILC1 production of IFN-γ was decreased by TGF-β and not IL-10. Interestingly, ILC2s stimulated in the presence of IL-10 had a marked reduction in cytokine production of IL-5 and IL-13 while TGF-β had no effect on ILC2 cytokine production. Ex vivo activated ILC1 and ILC2 subsets were also found to be a source of the immunoregulatory cytokine IL-10, raising the potential for ILC-mediated regulation of immune cells. These findings demonstrate the differential effects of immunoregulatory cytokines IL-10 and TGF-β on activated ILC1 and ILC2 populations ex vivo.

Figure 1

Frequency and distribution of ILC subsets in the peripheral blood of healthy volunteers. (A) Gating strategy for total ILCs ((CD45+ Lin-CD127+), ILC1s (CD45+ Lin-CD127+ cKit-NKp44-), ILC2s (CD45+ Lin-CD127+ CRTH2+) and ILC3s (CD45+ Lin-CD127+ cKit+ NKp44−/+). Panels are representative of multiple independent experiments (n = 89). (B) The frequency of total ILCs per million CD45+ cells and (C) the percentage of total ILCs per million CD45+ cells. Each dot represents an individual donor (n = 89) and the horizontal lines are the GM. ILC surface markers were used to quantify (D) the frequency of ILC1 (blue dots), ILC2 (orange dots) and ILC3 (gray dots) per million CD45+ cells and (E) the percentage of ILC subsets per total ILC cells in 69 donors. (F) ILC3a (CD45+ Lin-CD127+ cKit + NKp44-) and ILC3b (CD45+ Lin-CD127+ cKit+ NKp44+) frequencies per million lymphocytes in the peripheral blood. (G) Stacked bars individual ILC subsets by individual donor (n = 69).

Figure 2

ILC1 and ILC2s produce large quantities of their signature cytokines when activated ex vivo, while ILC3s produce non-signature cytokines when activated. Panel A. The levels of IFN-γ, TNF-α, IL-5, IL-13, IL-17A, and IL-22 produced following stimulation (open circles) under ILC1 conditions (IL-12/IL-15) (top row), ILC2 conditions (IL-15/IL-33) (middle row), or ILC3 conditions (bottom row) or cultured in absence of stimulation (media – closed circles. Data are from multiple experiments with n = 15 for ILC1, n = 19 for ILC2 and n = 8 for ILC3 groups. Levels of significance are indicated by ***p < 0.001, **p < 0.01, *p < 0.05, or ns, not significant, as ascertained using the Wilcoxon pair-wise comparison. Panel B. Sorted ILC subsets from a subset of donors from A (n = 10, 14, and 4 for ILC1s, ILC2s, and ILC3s respectively) were individually cultured in media alone, ILC activating cytokines, or PMA/ionomycin for 5 days. Production of IL-4, IL-5, IL-9, IL-10, IL-13, IL-17A, IL-22, IFN-γ and TNF-α was assessed. (Panel B) Geometric mean cytokine production in pg/ml for unstimulated sorted ILC1 (black bars), ILC2 (white bars), and ILC3 (gray bars). The GM fold change in cytokine production over unstimulated cells for sorted ILC1 (left panel) sorted ILC2 (middle panel) and ILC3 (right panel) after being activated with ILC-subset specific ILC activating cytokines (Panel C) and following PMA/ionomycin stimulation (Panel D).

Figure 3

ILC1 and ILC2s are a source of the immunoregulatory cytokine IL-10 when activated ex vivo. Sorted ILC subsets (2,000 ILCs/well) were stimulated in the presence of 50 ng/mL of ILC activating cytokines (IL-12/IL-15 for ILC1s, IL/25/IL-33 for ILC2s and IL-1β/IL-23 for ILC3 subsets) for 5 days. The culture supernatants were then harvested from ILC subsets in media alone or following stimulation and then evaluated for the immunoregulatory cytokine IL-10 as part of a multiplex Luminex analysis. Data are representative of multiple experiments with n = 15 for ILC1, n = 19 for ILC2 and n = 8 for ILC3 groups. Paired comparisons were accomplished using the Wilcoxon test and levels of significance are indicated as: ***p < 0.001, *p < 0.05, ns, not significant.

Figure 4

IL-10 differentially modulates the cytokine production of activated ILC2s despite expression of IL-10R by all ILC subsets. (A) Sorted ILC subsets (n = 9) and PBMCs (n = 7) were isolated from healthy volunteers and assessed for expression of IL-10R mRNA by qPCR. Data are shown as a combination of three independent experiments. (B,C) IL-10R expression on circulating monocytes and ILC subsets was assessed in the whole blood of healthy volunteers (n = 10) using by flow cytometry for (B) surface expression and (C) per cell intensity assessed by geometric mean fluorescence intensity (geometric MFI). (D,E) ILC1 (n = 13) and ILC2 (n = 10–11) subsets were sorted from healthy donors and stimulated with activating cytokines (IL-12/IL-15 for ILC1s and IL/25/IL-33 for ILC2s) in the presence or absence of 50 ng/mL of IL-10 for 5 days at 2,000 cells/well. The culture supernatants were assessed for production of (D) ILC1 and (E) ILC2 signature cytokines. Paired comparison of stimulated and stimulated + IL-10 conditions was accomplished with the Wilcoxon test and levels of significance were indicated by: ***p < 0.0001, *p < 0.05 and ns, not significant.

Figure 5

ILC1 and ILC2 activity is also differentially regulated by TGF-β. (A) Sorted ILC subsets (n = 9) and PBMCs (n = 7) were isolated from healthy volunteers and assessed for TGF-βRI expression by qPCR. Activated ILC1 (n = 10) and activated ILC2 (n = 7) subsets were stimulated in the presence or absence of 50 ng/mL of TGF-β for 5 days at 2,000 cells/well. The culture supernatants were then analyzed for ILC signature cytokine production (B) IFN-γ and TNF-α for ILC1s and (C) IL-5 and IL-13 for ILC2s. Paired comparison of stimulated and stimulated + TGF-β conditions was accomplished with the Wilcoxon test and levels of significance were indicated by: **p < 0.001 and ns, not significant.