Differential expression of functional chemokine receptors on human blood and lung group 2 innate lymphoid cells (ILC2s)

lymphocyte precursors within the bone marrow but is only maintained on mature ILC1 and ILC3s, in particular subsets of ILCs found in the spleen and lymph nodes (8). Our data suggests that CCR7 does not play a role in the trafficking of mature ILC2s in the blood or lung tissues. CXCR5 and CXCR6 were only expressed on a small subset of blood T-cells and even fewer (<5%) lung T-cells. Furthermore, <5% of ILC2s displayed CXCR5 or CXCR6 81 receptor in either compartment. These results are consistent with the notion that CXCR5 is 82 largely involved in B-cell homing and that CXCR6 is important for the retention of T-cells in 83 the liver, and for the emigration of ILC3s from the bone marrow to the small intestine (8). CCR9, thought to direct cells to the small intestine was only detectable on 10% of ILC2s lung tissue. (CCL2, CCL17, CCL3L1, CCL20, all Biotechne) to activate their cognate receptor was then tested through measurement of chemokine-induced increases in the filamentous (F)-actin content of both CD3 + T-cells and ILC2s. The method has previously been demonstrated as a marker for T-cell activation (1-2) Briefly, 100 µ l of cells per 10-point dose response curve were taken, Fc receptors blocked and stained with FITC conjugated CD123 and lineage cocktail, AF647 conjugated CD294, EF450 conjugated CD3 and the required PE/cy7 conjugated chemokine receptor antibodies for 20min at room temperature. Cells were washed in PBS containing 2% FBS and then resuspended in pre-warmed (37 o C) RPMI 1640 (ThermoFisher) containing 2% FBS before stimulation for 10sec with the appropriate chemokine. The reaction was terminated by the addition of an equal volume of fixation buffer from the intracellular fixation and permeabilisation buffer set (Affymetrix) for 30min at room temperature. Cells were washed (1200 g , 5min) 3 times with 1x permeabilisation buffer (from the intracellular fixation and permeabilisation buffer set Affymetrix) and stained with AF555 conjugated phalloidin (Life Technologies) (30min at room temperature) before washing and resuspension in PBS containing 2%FBS. Data was acquired using an Attune NxT cytometer running NxT software v2.2. Chemokine-induced changes in F-actin content were quantified as an increase in the mean fluorescence intensity (MFI) in the AF555 channel. To account for photobleaching and other non-specific effects on AF555 fluorescence the MFI for chemokine receptor positive cells was expressed as a fraction of that observed in the population not displaying the receptor for each sample. Data analysis. Analysis of flow cytometry data was performed using FlowJo v10 (Tree star, Oregon, USA). Concentration-response curves were fitted using the three-parameter logistic equation in Graph Pad PRISM v6 (California, USA) to obtain EC 50 values. Statistical analysis was performed using a two-way ANOVA and Tukey’s multiple comparison test with a probability ( p ) <0.05 being considered significant.


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Although highly detectable on blood derived T-cells, CCR7 was not detected on ILC2s 75 isolated from the blood or lung. CCR7 expression has previously been described on ILC3s, in particular subsets of ILCs found in the spleen and lymph nodes (8). Our data 78 suggests that CCR7 does not play a role in the trafficking of mature ILC2s in the blood or 79 lung tissues. CXCR5 and CXCR6 were only expressed on a small subset of blood T-cells and 80 even fewer (<5%) lung T-cells. Furthermore, <5% of ILC2s displayed CXCR5 or CXCR6 81 receptor in either compartment. These results are consistent with the notion that CXCR5 is 82 largely involved in B-cell homing and that CXCR6 is important for the retention of T-cells in 83 the liver, and for the emigration of ILC3s from the bone marrow to the small intestine (8).

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CCR9, thought to direct cells to the small intestine was only detectable on around 10% of 85 ILC2s in lung tissue.

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Several receptors were significantly (p <0.05) upregulated in lung tissue derived ILC2s 87 compared to the cells isolated from the blood including CCR3 and CXCR4 ( Figure 4). CCR3 88 in combination with CCR4 has been shown in multiple T-cell studies (7) to regulate 89 recruitment to the lung. Furthermore, the potent inflammatory ligand (CXCL12) acting via 90 CXCR4 appears to coordinate with CCL11 activation of CCR3 and CCL22 stimulation of 91 CCR4 to recruit lymphocytes to the lung and generate an inflammatory reaction (7). Our data 92 indicate that a similar mechanism could be used to activate or recruit ILC2s to the airways 93 thereby driving inflammation.

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The receptors displayed on the highest proportion of ILC2s isolated from both blood and lung 95 tissues were CCR2, CCR4, CCR5 and CCR6. We therefore wished to determine if these 96 receptors could be used to activate blood ILC2s via various chemokine ligands. Since ILC2s 97 are only found as a low percentage of human PBMCs traditional chemotaxis assays would 98 have been difficult to reliably perform. We therefore used an actin polymerization assay as a  and tissue recruitment as it is thought that a higher receptor number is required to achieve cell 113 chemotaxis than. In T-cells CCR5 does not direct tissue specific recruitment but is required in 114 combination with different panels of receptors to enable migration (7). Therefore it may be 115 that initial activation of ILC2s by the more highly expressed receptors occurs before 116 chemotaxis is enhanced through the binding of CCR5 specific ligands 117 Given that a higher proportions of "activated" IL-5 + , IL-13 + ILC2s have been shown to 118 correlate with asthma severity (9) and that mouse models of asthma demonstrate that an 119 increase in ILC2 number is sufficient for airway hyper responsiveness, targeting the 120 activation and recruitment of these cells is an attractive treatment strategy. Our data provide   The authors wish to thank all research participants for taking part in this study and 136 Tracy Thornton and Sarah Glover for their assistance with volunteer recruitment. We 137 wish to thank Dr Adam Wright and members of the Cousins lab for helpful 138 discussions. We also wish to thank Prof Andrew Wardlaw, Paige Tongue, Malgorzata 139

Rekas, Will Monteiro, Dr Amanda Sutcliffe, Beverley Hargadon, Sarah Parker and 140
Hilary Marshall for their assistance in the procurement and processing of lung tissue.