CD1a presentation of endogenous antigens by group 2 innate lymphoid cells

Abstract

Group 2 innate lymphoid cells (ILC2) are effectors of barrier immunity, with roles in infection, wound healing, and allergy. A proportion of ILC2 express MHCII (major histocompatibility complex II) and are capable of presenting peptide antigens to T cells and amplifying the subsequent adaptive immune response. Recent studies have highlighted the importance of CD1a-reactive T cells in allergy and infection, activated by the presentation of endogenous neolipid antigens and bacterial components. Using a human skin challenge model, we unexpectedly show that human skin-derived ILC2 can express CD1a and are capable of presenting endogenous antigens to T cells. CD1a expression is up-regulated by TSLP (thymic stromal lymphopoietin) at levels observed in the skin of patients with atopic dermatitis, and the response is dependent on PLA2G4A. Furthermore, this pathway is used to sense Staphylococcus aureus by promoting Toll-like receptor-dependent CD1a-reactive T cell responses to endogenous ligands. These findings define a previously unrecognized role for ILC2 in lipid surveillance and identify shared pathways of CD1a- and PLA2G4A-dependent ILC2 inflammation amenable to therapeutic intervention.

Fig. 1. Human skin ILC2 express CD1a.

A. Flow cytometry gating strategy for blister fluid derived human ILC2. ILC2 are CD45⁺/CD3^-/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 Reads Per Kilobase of transcript per Million mapped reads (RPKM). C. Whole thickness skin samples were homogenized and analyzed by flow cytometry for presence of ILC2 and expression of CD1a. 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 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⁺/CD3^-/Lineage^-/CRTH2⁺/IL7Rα⁺. B. Flow cytometric analysis of CD1a expression on blood ILC2. Flow cytometry data representative of n = 8 donors. C. Flow cytometric analysis of human serum cultured CD1a expression on blood ILC2 representative of n = 8 donors. D. Multiplex bead array analysis of TSLP concentration in HDM challenged blister fluid of healthy (HC) and atopic dermatitis (AD) patients (p = 0.0374, n = 5 – 6, t-test). E. Multiplex bead array analysis of IL-33 concentration in HDM challenged blister fluid of healthy (HC) and atopic dermatitis (AD) patients (p = 0.6655, n = 8 – 21, t-test). F. Culture of human blood derived ILC2 in the absence of human serum. Flow cytometry analysis of CD1a isotype control, CD1a expression on FCS cultured blood ILC2 and upon TSLP stimulation (plots left to right) Data representative of n = 3 donors and 3 independent experiments. *, P < 0.05; unpaired Student’s t test (mean and 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 fluorescence activated cell sorting and CD3 MACS microbead separation respectively. A. and B. Prior to co-culture with autologous T cells, ILC2 were pulsed with HDM extract (7 µg / ml), and IFNγ (A.) and IL-22 (B.) production was detected by ELISpot in the absence or presence of 10 µg / ml anti-CD1a blocking antibody or isotype control. In addition ILC2 alone were stimulated with PMA (15 ng / ml) and Ionomycin (7.5 ng / ml) (P/I). (A. p < 0.0001, p < 0.0001, p < 0.0001, B. p = 0.0023, p = 0.0158, p = 0.0112, n = 8 donors, one-way ANOVA, and data represent at least 6 independent experiments). C. ILC2 were pulsed in the presence or absence of HDM extract (7 µg / ml) and co-cultured 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 or isotype control, 10 µg / ml. (p = 0.0124, p < 0.0001, n = 3 donors, one-way ANOVA and 3 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 and 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 prior to pulsing with HDM extract and subsequent ELISpot analysis of capacity to activate T cells. A. and B. Effect of the presence or absence of prior stimulation of ILC2 with TSLP on number of IFNγ (A.) or IL-22 (B.) producing T cells induced by co-culture. Fold change was calculated relative to the unpulsed autoreactive baseline response in the absence TSLP (represented by dotted line). Statistics calculated between the baseline response or as indicated in the figure. (A. p = 0.0221, p = 0.0111, B. p = 0.0091, p = 0.0006, n = 8 donors, t-test, data represent at least 6 independent experiments) C. and D. Effect of TSLP concentration upon amplification of CD1a dependent T cell production of IFNγ (C.) or IL-22 (D.). Fold change calculated between cytokine spots produced following T cell culture with unstimulated and TSLP stimulated ILC2. Graph showing two concentration of TSLP 50 ng / ml and 0.5 ng / ml. (C. p = 0.3325, p = 0.4342, D. p = 0.484, p = 0.1, n = 8 donors, t-test and data represents 3 - 6 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 paired Student’s t test (mean and 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 Reads Per Kilobase of transcript per Million mapped reads (RPKM). B. Cytosolic PLA2 activity of recombinant PLA2G4A irreversibly inhibited by 1 µM MAFP measured using a biochemical assay kit. C. and D. Autologous ILC2 and T cells were isolated from donor PBMCs by fluorescence activated cell sorting and CD3 MACS bead separation respectively. Prior to co-culture with autologous T cells, ILC2 were either unpulsed (U) or pulsed with 1 µg / ml PLA2G4A or PLA2G4A inhibited with 1 µM MAFP. IFNγ (C.) (p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001) and IL-22 (D.) (p < 0.0001, p = 0.018, p = 0.0321, p = 0.0007) production was detected by ELISpot in the absence or presence of 10 µg / ml anti-CD1a blocking antibody or isotype control (n = 8 donors, one-way ANOVA, and data represent at least 6 independent experiments). E. and F. ILC2 were cultured in FCS and stimulated in the presence or absence of TSLP for 72 hours prior to pulsing with PLA2G4A. Effect of the presence or absence of prior stimulation of ILC2 with TSLP upon number of IFNγ (E.) (p = 0.0226) or IL-22 (F.) (p = 0.0029) producing T cells induced by co-culture. Fold change was calculated relative to the unpulsed autoreactive response in the absence TSLP (represented by dotted line). Statistics calculated between the baseline response or as indicated in the figure. (n = 8 donors, t-test and data represents at least 6 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 10 µg / ml anti-CD1a blocking antibody or isotype control upon co-culture with ILC2 pulsed or unpulsed (U) with PLA2G4A. (p = 0.041, p = 0.0248, p = 0.0296, n = 3 donors, one-way ANOVA, and 3 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 and SD).

Fig. 6. ILC2 present bacterial lipid ligands derived from Staphylococcus aureus

A. and B. Autologous ILC2 and T cells were isolated from donor PBMCs by flow cytometric sorting and CD3 MACS bead separation respectively. Prior to co-culture with autologous T cells, ILC2 were pulsed with heat-killed S. aureus preparation (HKSA). IFNγ (A.) (p < 0.0001, p < 0.0001, p < 0.0001) and IL-22 (B.) (p < 0.0001, p = 0.0158, p = 0.0002) production was detected by ELISpot in the absence or presence of 10 µg / ml anti-CD1a blocking antibody or isotype control. (n = 8 donors, one-way ANOVA, and data represents at least 6 independent experiments) C. and D. ILC2 were cultured in FCS and stimulated in the presence or absence of TSLP for 72 hours prior to pulsing with HKSA (10⁸ cells / ml). Effect of the presence or absence of prior stimulation of ILC2 with TSLP upon number of IFNγ (C.) or IL-22 (D.) producing T cells induced by co-culture. Fold change was calculated relative to the unpulsed autoreactive response in the absence TSLP (represented by dotted line). Statistics calculated between the baseline response or as indicated in the figure. (p = 0.0021, n = 8 donors, t-test, and data represents at least 6 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 and SD).

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

A. Real-time PCR analysis of PLA2G4A gene expression by ILC2 following stimulation with heat-killed S. aureus preparation (HKSA) (p = 0.0258, n = 3, t-test, data representative of 3 independent experiments). B. Real-time PCR analysis of PLA2G4A gene expression by ILC2 following stimulation with TLR2 (PamCSK 10 µg / ml) and TLR4 (LPS 1 µg / ml) ligands. (p = 0.0009, n = 3, t-test, data representative of 3 independent experiments. C. Cytosolic PLA2 activity was measured in the supernatant of ILC2 stimulated with TLR2 and TLR4 ligands, or HKSA (10⁸ cells / ml) (p = 0.0152, p = 0.0413, p < 0.0001, n = 6, t-test, data representative of 3 independent experiments). D. Real-time PCR analysis of PLA2G4A gene expression by healthy or atopic dermatitis ILC2 following stimulation with TLR2 (PamCSK 10 µg / ml) and TLR4 (LPS 1 µg / ml) ligands or heat-killed S. aureus (HKSA, 10⁸ cells / ml). (p = 0.0329, p < 0.0001, n = 4 donors, one-way ANOVA, and data representative of 4 independent experiments). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (mean and SD).

Fig. 8. TLR stimulation of ILC2 by Staphylococcus 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. Prior to co-culture with autologous T cells, ILC2 were pulsed with HKSA (10⁸ cells / ml) with or without inhibition of cPLA2 (1 µM MAFP) or TLR2 and TLR4 signaling (10 µg / ml anti-TLR2 and anti-TLR4). IFNγ (A.) (p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001) and IL-22 (B.) (p = 0.0051, p = 0.0007, p = 0.0255) production was detected by ELISpot and IL-13 was detected by flow cytometry (C.) (p = 0.0202, p = 0.009, p = 0.017, p = 0.0119) in the absence or presence of 10 µg / ml anti-CD1a blocking antibody or isotype control. ELISpot data represent at least 6 independent experiments and n = 8 donors, one-way ANOVA. IL-13 FACS data represent n = 3 donors, one-way ANOVA and 3 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 and SD).