Research ArticleHUMAN IMMUNOLOGY

CD1a presentation of endogenous antigens by group 2 innate lymphoid cells

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Science Immunology  22 Dec 2017:
Vol. 2, Issue 18, eaan5918
DOI: 10.1126/sciimmunol.aan5918
  • Fig. 1 Human skin ILC2 express CD1a.

    (A) Flow cytometry gating strategy for blister fluid–derived human ILC2. ILC2 are CD45+/Lineage/CRTH2+/IL7Rα+. (B) CD1a and CD1d gene expression of skin- and blood-derived ILC2 and T cells determined by RNA sequencing and measured in RPKM. (C) Whole-thickness skin samples were homogenized and analyzed by flow cytometry for the presence of ILC2 and expression of CD1a. SSC, side scatter. (D) CD1a expression by epidermal CD11c+ cells was analyzed by flow cytometry. (E) CD1a expression by epidermal ILC2 was analyzed by flow cytometry. (F) Summary of CD1a expression on ILC2 as a proportion of total ILC2 derived from whole-thickness skin and epidermis. (G) CD1a expression by epidermal T cells was analyzed by flow cytometry. Flow cytometry data are representative of at least 10 independent experiments and n = 12 donors.

  • Fig. 2 Human blood ILC2 could be stimulated to express CD1a.

    (A) Flow cytometry gating strategy for blood-derived human ILC2. ILC2 are CD45+/Lineage/CRTH2+/IL7Rα+. FSC, forward scatter. (B) Flow cytometric analysis of CD1a expression on blood ILC2. Flow cytometry data are representative of n = 8 donors. (C) Flow cytometric analysis of CD1a expression on human serum–cultured blood ILC2 representative of n = 8 donors. (D) Multiplex bead array analysis of TSLP concentration in HDM-challenged blister fluid of healthy control (HC) and atopic dermatitis (AD) patients (n = 5 to 6; P = 0.0374, t test). (E) Multiplex bead array analysis of IL-33 concentration in HDM-challenged blister fluid of healthy control and atopic dermatitis patients (n = 8 to 21, P = 0.6655, t test). (F) Culture of human blood-derived ILC2 in the absence of human serum. Flow cytometry analysis of anti-CD1a isotype control, of CD1a expression on FCS-cultured blood ILC2, and upon TSLP stimulation (plots from left to right). Data are representative of n = 3 donors and three independent experiments. *P < 0.05, unpaired Student’s t test (mean + SD).

  • Fig. 3 ILC2 present HDM-derived lipid ligands to CD1a-responsive T cells.

    Autologous ILC2 and T cells were isolated from donor PBMCs by FACS and CD3 magnetic cell separation (MACS), respectively. (A and B) Before coculture with autologous T cells, ILC2 were pulsed with HDM extract (7 μg/ml), and IFN-γ (A) and IL-22 (B) production was detected by ELISpot in the presence or absence of anti-CD1a blocking antibody (10 μg/ml) or isotype control (10 μg/ml). In addition, ILC2 alone were stimulated with PMA (10 ng/ml) and ionomycin (500 ng/ml) (P/I; n = 8 donors; one-way analysis of variance (ANOVA); data represent at least six independent experiments). U, unpulsed. (C) ILC2 were pulsed in the presence or absence of HDM extract (7 μg/ml) and cocultured with autologous T cells. Intracellular staining for flow cytometry was used to assess the proportion of T cells expressing IL-13 in the presence or absence of anti-CD1a blocking antibody (10 μg/ml) or isotype control (10 μg/ml) (n = 3 donors; one-way ANOVA; data represent three independent experiments). *P < 0.05; **P < 0.01; ****P < 0.0001, repeated-measures (RM) one-way ANOVA with Tukey’s post hoc test (mean + SD).

  • Fig. 4 TSLP further enhances the ability of ILC2 to present CD1a ligands to T cells.

    ILC2 were cultured in 10% FCS and stimulated in the presence or absence of TSLP for 72 hours before pulsing with HDM extract and subsequent ELISpot analysis of capacity to activate T cells. (A and B) Effect of the presence or absence of previous stimulation of ILC2 with TSLP on the number of IFN-γ–producing (A) or IL-22–producing (B) T cells induced by coculture. Fold change was calculated relative to the unpulsed autoreactive baseline response in the absence of TSLP (represented by dashed line). Graphs show the effect of TSLP on the unpulsed (P = 0.0221) and HDM-induced (P = 0.0111) IFN-γ responses (A) and the effect of TSLP on the unpulsed (P = 0.0091) and HDM-induced (P = 0.0006) IL-22 responses (B) (n = 8 donors; t test; data represent at least six independent experiments). (C and D) Effect of TSLP concentration upon amplification of CD1a-dependent T cell production of IFN-γ (C) or IL-22 (D). Fold change was calculated between cytokine spots produced after T cell culture with unstimulated and TSLP-stimulated ILC2. Graphs show the two concentrations of TSLP (50 and 0.1 ng/ml) (n = 8 donors; t test; data represent three to six independent experiments). *P < 0.05; ***P < 0.001, paired Student’s t test (mean ± SD).

  • Fig. 5 ILC2 express PLA2G4A, which generates CD1a ligands.

    (A) PLA2 gene expression analysis of skin- and blood-derived ILC2 and T cells determined by RNA sequencing and measured in RPKM. (B) cPLA2 activity of recombinant PLA2G4A irreversibly inhibited by 1 μM MAFP measured using free thiol detection. (C and D) Autologous ILC2 and T cells were isolated from donor PBMCs by FACS and CD3 MACS bead separation, respectively. Before coculture with autologous T cells, ILC2 were either unpulsed (U) or pulsed with PLA2G4A (1 μg/ml) or PLA2G4A (1 μg/ml) inhibited with 1 μM MAFP. IFN-γ (C) and IL-22 (D) production was detected by ELISpot in the presence or absence of anti-CD1a blocking antibody (10 μg/ml) or isotype control (10 μg/ml) (n = 8 donors; one-way ANOVA; data represent at least six independent experiments). (E and F) ILC2 were cultured in FCS and stimulated in the presence or absence of TSLP for 72 hours before pulsing with PLA2G4A. Effect of the presence or absence of previous stimulation of ILC2 with TSLP upon the number of IFN-γ–producing (P = 0.0226; E) or IL-22–producing (P = 0.0029; F) T cells induced by coculture. Fold change was calculated relative to the unpulsed autoreactive response in the absence of TSLP (represented by dashed line). Statistics calculated between the baseline response or as indicated in the figure (n = 8 donors; t test; data represent at least six independent experiments). (G) Intracellular staining for flow cytometry was used to assess the proportion of T cells expressing IL-13 in the presence or absence of anti-CD1a blocking antibody (10 μg/ml) or isotype control (10 μg/ml) upon coculture with ILC2 pulsed or unpulsed with PLA2G4A (n = 3 donors; one-way ANOVA; three independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, RM one-way ANOVA with Tukey’s post hoc test (mean + SD).

  • Fig. 6 ILC2 present bacterial lipid ligands derived from S. aureus.

    (A and B) Autologous ILC2 and T cells were isolated from donor PBMCs by flow cytometric sorting and CD3 MACS bead separation, respectively. Before coculture with autologous T cells, ILC2 were pulsed with heat-killed S. aureus (HKSA) preparation. IFN-γ (A) and IL-22 (B) production was detected by ELISpot in the presence or absence of anti-CD1a blocking antibody (10 μg/ml) or isotype control (10 μg/ml) (n = 8 donors; one-way ANOVA; data represent at least six independent experiments). (C and D) ILC2 were cultured in FCS and stimulated in the presence or absence of TSLP for 72 hours before pulsing with heat-killed S. aureus (108 cells/ml). Effect of the presence or absence of previous stimulation of ILC2 with TSLP upon the number of IFN-γ–producing (C) or IL-22–producing (D) T cells induced by coculture. Fold change was calculated relative to the unpulsed autoreactive response in the absence of TSLP (represented by dashed line). Statistics calculated between the baseline response or as indicated in the figure (n = 8 donors; t test; data represent at least six independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, RM one-way ANOVA with Tukey’s post hoc test (mean ± SD).

  • Fig. 7 Bacterial components can stimulate ILC2 to produce cPLA2.

    (A) Real-time PCR analysis of PLA2G4A gene expression by ILC2 after stimulation with heat-killed S. aureus preparation (n = 3; P = 0.0258, t test; data representative of three independent experiments). (B) Real-time PCR analysis of PLA2G4A gene expression by ILC2 after stimulation with TLR2 (PamCSK; 10 μg/ml) and TLR4 (LPS; 1 μg/ml) ligands (n = 3; t test; data representative of three independent experiments). (C) cPLA2 activity was measured in the supernatant of ILC2 stimulated with TLR2 and TLR4 ligands or heat-killed S. aureus (108 cells/ml) (n = 6; t test; data representative of three independent experiments). (D) Real-time PCR analysis of PLA2G4A gene expression by ILC2 from healthy controls or atopic dermatitis patients after stimulation with TLR2 (PamCSK; 10 μg/ml) and TLR4 (LPS; 1 μg/ml) ligands or heat-killed S. aureus (108 cells/ml) (n = 5 donors; one-way ANOVA; data representative of four independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (mean ± SD).

  • Fig. 8 TLR stimulation of ILC2 by S. aureus induces PLA2G4A and generation of lipid ligands, which can be presented to T cells by CD1a.

    Autologous ILC2 and T cells were isolated from donor PBMCs by flow cytometric sorting and CD3 MACS microbead separation, respectively. Before coculture with autologous T cells, ILC2 were pulsed with heat-killed S. aureus (SA; 108 cells/ml) with or without inhibition of cPLA2 (1 μM MAFP) or TLR2 and TLR4 signaling [anti-TLR2 (10 μg/ml) and anti-TLR4 (10 μg/ml)]. (A to C) IFN-γ (A) and IL-22 (B) production was detected by ELISpot, and IL-13 was detected by flow cytometry (C) in the presence or absence of anti-CD1a blocking antibody (10 μg/ml) or isotype control (10 μg/ml). ELISpot data represent at least six independent experiments and n = 8 donors. IL-13 FACS data represent n = 3 donors and three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, RM one-way ANOVA with Tukey’s post hoc test (mean + SD).

Supplementary Materials

  • Supplementary Materials

    Supplementary Material for:

    CD1a presentation of endogenous antigens by group 2 innate lymphoid cells

    Clare S. Hardman, Yi-Ling Chen, Maryam Salimi, Rachael Jarrett, David Johnson, Valtteri J. Järvinen, Raymond J. Owens, Emmanouela Repapi, David J. Cousins, Jillian L. Barlow, Andrew N. J. McKenzie, Graham Ogg*

    *Corresponding author. Email: graham.ogg{at}ndm.ox.ac.uk

    Published 22 December 2017, Sci. Immunol. 2, eaan5918 (2017)
    DOI: 10.1126/sciimmunol.aan5918

    This PDF file includes:

    • Fig. S1. Gating strategy for analysis of ILC2.
    • Fig. S2. Stimulation of CD1a expression by blood-derived ILC2.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (.pdf format). Source data.
    • Table S2 (Microsoft Excel format). RPKM expression values for RNA sequencing data.

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