have reported expression of both RANK-L and RANK on mouse group 3 ILCs (ILC3s).30 We also investigated whether RANK-L is expressed on ILC subsets and found that RANK-L was weakly detected on ILC2s and CRTH2? ILCs which were a mixture of group 1 ILCs (ILC1s) and ILC3s (n = 5, Supplementary Fig. conclusion, RANK-L, together with TSLP, may play an inductive role in the ILC2-mediated type 2 inflammation in CRSwNP. Representative flow cytometric plots for RANK-L+ cells in NPs are shown. We gated on single, live, RANK-L positive (RANK-L+) cells compared to isotype control IgG2b. f, RANK-L+ cells were further separated into CD45+ and CD45? populations and the frequency of RANK-L+ cells in the two populations was calculated (n = 12). *p? ?0.05 and ****p 0.0001 were calculated by Mann-Whitney test (e) and pair-t test (f). As RANK-L is known to be expressed on activated T cells, B cells and basophils in humans, 26C29 we first decided the frequency of RANK-L+ cells in CD3+ T cells, CD19+ B cells, granulocytes (side scatter [SSC] high cells) and CD3-CD19? non-granulocytes (CD3-CD19mid-low cells) (Supplementary Momelotinib Mesylate Fig. 4a). We found that RANK-L was mainly detected on CD3-CD19? cells (median, range; 77.3%, 59.8%?96.1%) and CD3+ T cells (12.5%, 2.6%?24.8%) compared to CD19+ B cells (6.4%, 1.1C11.2%) and granulocytes (1.66%, 0C3.9%) within the population of CD45+ cells (n = 7) (Fig. 4a). The % RANK-L+ cells in each cell type is usually shown in Supplementary Fig. 4b, d. We first focused on CD3+ T cells and found that RANK-L was highly expressed on CRTH2+CD4+ TH2 cells compared to CRTH2-CD4+ T cells and CD4? T cells in NPs (Fig. 4d, ?,e,e, Supplementary Fig. 5a, and not shown). We further analyzed CD3-CD19? cells and found that RANK-L+ cells were mainly CD11c+ cells but not basophils (CD117-FcRIa+), plasmacytoid dendritic cells (DCs) (BDCA4+), macrophages (CD68+), or mast cells (CD117+FcRIa+) (Fig 4b, Supplementary Fig. 4c and not shown). Bando et al. have reported expression of both RANK-L and RANK on mouse group 3 ILCs (ILC3s).30 We also investigated whether RANK-L is expressed on ILC subsets and found that RANK-L was weakly detected on ILC2s and CRTH2? ILCs which were a mixture of group 1 ILCs (ILC1s) and ILC3s (n = 5, Supplementary Fig. 6a). However, the expression of RANK-L on NP ILCs was similar to CRTH2-CD4+ T cells (Supplementary Fig. 6b). We therefore ruled out the importance of RANK-L Momelotinib Mesylate in NP ILCs. Open in a separate windows Fig. 4 Identification of RANK-L expressing cells in NPs. a The frequency of granulocytes (Gran; SSC high and CD45+ cells), CD19+ B cells (CD45+CD19+CD3? cells), CD3+ T cells (CD45+CD3+ cells) and CD3-CD19? cells (SSC mid-low, CD45+CD3-CD19mid-low cells) in RANK-L+CD45+ cells in NPs was calculated (n = 7). b The frequency of CD11c? cells, CD11c+ cells in RANK-L+CD45+ cells is usually shown (n = 7). c The frequency of mDC1 (CD1chighCD141-CD11c+ cells), mDC2 (CD141+CD1c-CD11c+ cells) and CD11c+ Momelotinib Mesylate non-mDCs (CD1cmid-lowCD141? cells) in RANK-L+CD45+ cells in NPs is usually shown (n = 3). d, f Representative histograms of flow cytometric plots and bar graphs for RANK-L on TH2 cells, CRTH2-CD4+ T cells (n = 9), CXCL16-HLADR+CD11c+ cells and CXCL16+HLA-DR+CD11c+ cells (n = 6), in NPs are shown. e, g Comparisons of the gMFI ratio of RANK-L to isotype IgG2b between TH2 cells and CRTH2CD4+ T cells and between CXCL16+ cells-, CXCL16? cells and ILC2s are shown. Not significant Momelotinib Mesylate (NS), *p 0.05, **p? ?0.01 and ****p 0.0001 were calculated by 1-way ANOVA Kruskal-Wallis test (a, g), Holm-Sidaks multiple comparisons test (c), paired-t test (d, f) and Wilcoxon test (b, e). Since we found that a larger subset of RANK-L+CD11c+ cells expressed human leukocyte antigen-D related (HLA-DR) (Supplementary Fig. 4d), we initially hypothesized that RANK-L+CD11c+ cells were myeloid DCs (mDCs). However, the majority of these cells were not classical mDC1s (CD1c+) or mDC2s (CD141+) Rabbit Polyclonal to DP-1 in NPs (n = 3) (Fig. 4c, and Supplementary Fig. 4e). It has been reported that a subset of intestinal DCs express a membrane bound chemokine, CXCL16 around the cell surface.