CD1d-mediated activation of group 3 innate lymphoid cells drives IL-22 production

Abstract

Innate lymphoid cells (ILCs) are a heterogeneous family of immune cells that play a critical role in a variety of immune processes including host defence against infection, wound healing and tissue repair. Whether these cells are involved in lipid-dependent immunity remains unexplored. Here we show that murine ILCs from a variety of tissues express the lipid-presenting molecule CD1d, with group 3 ILCs (ILC3s) showing the highest level of expression. Within the ILC3 family, natural cytotoxicity triggering receptor (NCR)^-CCR6⁺ cells displayed the highest levels of CD1d. Expression of CD1d on ILCs is functionally relevant as ILC3s can acquire lipids in vitro and in vivo and load lipids on CD1d to mediate presentation to the T-cell receptor of invariant natural killer T (iNKT) cells. Conversely, engagement of CD1d in vitro and administration of lipid antigen in vivo induce ILC3 activation and production of IL-22. Taken together, our data expose a previously unappreciated role for ILCs in CD1d-mediated immunity, which can modulate tissue homeostasis and inflammatory responses.

Keywords: ILC; CD1d; IL‐22; NKT cell.

Figure 1. CD1d expression on ILCs

1. Flow cytometry for CD1d expression (top) and CD1d mean fluorescence intensity (MFI; bottom) on CD45⁻ cells, ILCs (Lin⁻CD45⁺CD127⁺), DCs (CD11c⁺) and B cells (B220⁺) of the depicted tissues from WT mice (n = 4).

2. Flow cytometry profiles showing gating strategy (dot plots), CD1d expression (histograms) and CD1d MFI (bars) in T‐bet⁺ (empty profile), GATA‐3⁺ (dark grey), RORγt⁺ (light grey) and T‐bet⁺RORγt⁺ (dotted line) ILCs in mLN, spleen, PP and SI‐LP (n = 5). Numbers indicate percentage of cells in the depicted gates.

3. Quantitative RT–PCR analysis of mRNA encoding CD1d in freshly isolated ILC3s (from mLN of CD1d‐deficient mice and mLN and spleen of WT mice), B cells and DCs (n = 3). Results are normalized to those of GAPDH; RE, relative expression. **P < 0.01 (two‐tailed unpaired t‐test).

4. Flow cytometry profiles showing NKp46 and RORγt expression in Lin⁻CD45⁺ cells; CD1d and CCR6 expression in NCR⁻ ILC3s; and CD1d and CD4 expression in NCR⁻CCR6⁺ ILC3s from mLN and SI‐LP (n = 4). Numbers indicate percentage of cells in the depicted gates.

5. CD1d expression (histograms) and CD1d MFI in NCR⁺ (empty profile), NCR⁻CCR6⁻ (dark grey) and NCR⁻CCR6⁺ (light grey) ILC3s from mLN and SI‐LP (n = 4).

Data information: Graphs represent mean ± SEM. Data are from three (A, C–E) or five (B) independent experiments.

Figure EV1. CD1d expression on ILCs

1. Flow cytometry profiles showing gating strategy and CD1d expression (histogram) in Lin⁻CD45⁺CD127⁺ cells in the depicted tissues from WT (grey filled, n = 4) and CD1d‐deficient (empty profile, n = 4) mice.

2. Flow cytometry profiles showing gating strategy and CD1d expression (histograms) in T‐bet⁺ (empty profile), T‐bet⁺RORγt⁺ (dotted line), GATA‐3⁺ (dark grey) and RORγt⁺ (light grey) ILCs in colonic lamina propria (n = 3). Numbers indicate percentage of cells in the depicted gates.

3. Flow cytometry profiles showing expression of CD1d and T‐bet in Lin⁻CD45⁺CD127⁺RORγt⁺ cells from mLN, spleen and SI‐LP as indicated (n = 4); numbers indicate percentage of cells. Right panel, percentage of CD1d^hi ILC3s in the depicted tissues. ***P < 0.001 two‐tailed unpaired t‐test.

4. Relative expression (RE) of lymphotoxin‐alpha (LTA) mRNA (qPCR, normalized to GAPDH) in sort‐purified CD1d^hiCCR6^hi (light grey) and CD1d^lowCCR6^low (dark grey) ILC3s isolated from mLN or spleen; **P < 0.01, two‐tailed unpaired t‐test.

Data information: Graphs represent mean ± SEM. Data are from three independent experiments.

Figure EV2. Phenotype of CD1d⁺ ILCs in mLN

Flow cytometry profiles showing expression of the depicted markers in Lin⁻CD45⁺CD127⁺ cells from mLN. Numbers indicate percentage of cells in the depicted gates. Data represent 2–4 independent experiments.

Figure EV3. Phenotype of RORγt⁺ ILCs

Flow cytometry profiles showing expression of the depicted markers in RORγt⁺Lin⁻CD45⁺CD127⁺ ILC3s from mLN, spleen and SI‐LP as indicated. Numbers indicate percentage of cells in the depicted gates. Data represent 2–5 independent experiments.

Figure 2. ILC3s internalize and present lipid antigens to iNKT cells

1. A

   Cells were cultured in the presence or absence of BODIPY‐αGalCer at 4 or 37°C as indicated. Flow cytometry profiles and percentages of BODIPY‐αGalCer⁺ DC or ILC3 from the indicated tissues incubated at 4°C (white bars) or 37°C (grey bars) are shown (n = 3).

2. B

   WT mice were intravenously injected with BODIPY‐αGalCer or PBS (control); lipid uptake was analysed by flow cytometry in the depicted populations from the spleen 16 h after lipid injection (n = 3). Numbers indicate percentage of cells in the depicted gates.

3. C

   IL‐2 secretion by DN32.D3 cells co‐cultured with ILC3 sort‐purified from mLN or spleen as indicated, pulsed (+) or not (−) with αGalCer and with or without αCD1d‐blocking antibody.

4. D–F

   Sort‐purified ILC3s were preincubated with αGalCer (ILC3(αGal)) or PBS (ILC3(cont)) and adoptively transferred into WT recipients. (D) Experimental protocol; (E) fold change in mRNA expression for the indicated cytokines in iNKT cells sort‐purified from spleen, mLN or SI‐LP of mice injected with αGalCer‐loaded ILC3s. Gene expression was measured by qPCR, normalized to GAPDH and presented as expression relative to iNKT cells sort‐purified from mice injected with control ILC3s (n = 3–5). (F) Intracellular cytokine staining (left) and percentage of cytokine⁺ cells (right) in splenic iNKT cells from ILC3‐recipient mice (n = 3). Numbers indicate percentage of cells in the depicted gates. **P < 0.01, two‐tailed unpaired t‐test.

Data information: Graphs represent mean ± SEM. Data are from more than two independent experiments.

Figure 3. Engagement of CD1d on ILC3s induces IL‐22 production

1. A, B

   Flow cytometry plots showing gating strategy (A) and percentage of ILC populations (from Lin⁻CD45⁺CD127⁺ cells, B) in mLN from WT (n = 4) and CD1d‐deficient (n = 3) mice.

2. C

   Fold change in mRNA expression for the indicated cytokines in ILC3s after antibody‐mediated CD1d cross‐link (n = 3). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. *P < 0.05 two‐tailed unpaired t‐test.

3. D

   Fold change in IL22 mRNA expression in ILC3s after antibody‐mediated CD1d cross‐link and/or IL‐23 stimulation (n = 4). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. **P < 0.01 two‐tailed unpaired t‐test.

4. E

   IL‐22 detection by ELISA in the supernatant of ILC3s cultured in the presence of αCD1d and/or IL‐23 (n = 3). *P < 0.05 two‐tailed unpaired t‐test.

5. F, G

   Intracellular cytokine staining (F) and percentage of IL‐22⁺ ILC3s (G) after stimulation with αCD1d, IL‐23 and/or IL‐1β. *P < 0.05, **P < 0.01 two‐tailed unpaired t‐test (n = 4–8)

6. H

   WT mice were i.v. injected with αGalCer or PBS (control) and activation of splenic ILC3s was assessed by flow cytometry as CD69 up‐regulation at 6 or 16 h after injection. Grey filled profile, ILC3 from αGalCer‐injected mice; empty profile, ILC3 from PBS‐injected mice; dotted line, CD45⁻ cells. Right graph, percentage of CD69⁺ ILC3s 6 h after injection of αGalCer (grey) or PBS (white) (n = 3). *P < 0.05 two‐tailed unpaired t‐test.

7. I, J

   Fold change in mRNA expression for the indicated cytokines in ILC3s sort‐purified from spleen (I) or SI‐LP (J) 6 h after intravenous (I) or oral (J) αGalCer administration. Gene expression was measured by qPCR and normalized to GAPDH and to the expression levels in ILC3s sort‐purified from PBS‐injected mice (n = 3). *P < 0.05, **P < 0.01 two‐tailed unpaired t‐test.

Data information: Graphs represent mean ± SEM.

Figure EV4. ILCs in WT and CD1d‐deficient mice

Flow cytometry profiles showing ILC populations from SI‐LP and spleen in WT and CD1d‐deficient mice as indicated. Data represent four independent experiments.

Figure EV5. Effect of CD1d cross‐link on ILC3s

1. Fold change in mRNA expression for the indicated cytokines in ILC3s after antibody‐mediated CD1d cross‐link (n = 4). Gene expression was measured by qPCR and normalized to GAPDH and to the mRNA expression levels in control ILC3s. Graphs represent mean ± SEM.

2. Flow cytometry profiles showing MHC‐II expression on ILC3s in after cross‐link with αCD1d antibody (grey) or control (white).

Data information: Data are from four independent experiments.