Fig 1: In vitro generated ILC3 produce high amounts of IL-22, but not IL17a. At day 28 on MSCs, NKp44+ILC3s were freshly sorted and stimulated either with medium alone or with IL1β/IL-23 (50 ng/ml each) with or without IL-2 (1000 U/ml). Supernatant was taken at day 5 (A). IL-22 (B), IL-17A (C), GM-CSF, and LIF (D) levels in the supernatant were measured after a 5-day stimulation with medium alone, IL1β/IL-23 (blue bar), and IL1β/IL-23/IL-2 (blue-white striped bar). CD34+ HSPCs were sorted and 2,000 cells were seeded in parallel in wells with MSC (+MSC) or without MSC (-MSC). Line graphs showing total cell counts (E) and ILC3 (NKG2A-CD56+) cell counts (F) in cultures with MSC (+MSC, filled dots, n = 5–7) and without MSC (-MSC, open dots, n = 3) from day 0 to day 28. CD34+ HSPCs were cultured on EL08. Representative dot plots of NKG2A and CD56 expression (left panels) and quantification of ILC3 (right panel) cultured on EL08 (light blue, n = 3) or on MSC (dark, blue, n = 7–9) (G). Representative dot plots showing RORγT and EOMES expression (left panels) and quantification of RORγTneg, RORγTint, and RORγThigh frequency distribution (right panels, bar graphs) for cells cultured on EL08 (light blue, n = 3) or on MSC (dark blue, n = 7) (H). Cultures derived on MSC or EL08 were re-stimulated with IL1β/IL-23 and stained for intracellular IL-22 secretion. Bar graphs showing IL-22 secretion for MSC-derived ILC3 (dark blue, n = 5) and EL08-derived ILC3 (light blue, n =3). The data are representative of one (EL08 and culture -MSC) and two different experiments (+MSCs) with each dot representative of an individual donor. The height of the bar graphs represents the mean ± SEM. Levels of significance were calculated with a nonparametric ANOVA (Kruskal–Wallis with a Dunn`s post-test) (B–D) and Mann–Whitney tests (E–I), *p < 0.05, **p < 0.01, ****p < 0.0001.
Fig 2: Cord blood (CB) innate lymphoid cell subpopulation (ILC1)‐ and ILC3‐like cells are functionally immature, whereas CB ILC2 are fully functional. A, Normalized RNA sequencing read counts are displayed as violin plots for the signature transcription factors EOMES, GATA3, RORC, and T‐BET(Tbx21) for the indicated cell types. B, Histograms showing intranuclear staining for the signature TFs T‐bet, Eomes, Gata‐3, and RORC for ILC1‐like (filled yellow), ILC2 (filled green), ILC3‐like (filled blue), CD56bright natural killer (NK) cells (filled red), and CD56dim NK cells (taken as reference unfilled red). C, Normalized read counts of ex vivo sorted CB ILC1‐like, ILC2, ILC3‐like, CD56bright NK cells, and tonsillar ILC3 for IFNγ, IL‐5, IL‐13, IL‐4, IL‐17A, and IL‐22. D, CB ILC1‐like, ILC2, and ILC3‐like cells were sorted and specifically stimulated with IL‐12/IL‐18 (for ILC1‐like and CD56bright NK cells), IL‐2/IL‐33 (for ILC2), and IL1𝛽/IL‐23 ± IL‐2 (for ILC3‐like cells) for 5 days. IFNγ, IL‐4, IL‐5, IL‐13, IL‐22, IL‐17a, IL‐10 and E, LIF secretion was analyzed within the supernatant via multi‐cytokine assay at day 1 and 5 (LegendPlex©), n = 5 (ILC1‐like cells), n = 2 (CD56bright NK cells), n = 4 (ILC2), and n = 3‐7 (ILC3‐like cells). The height of the bars represents the mean ± SEM. Levels of significance were calculated with a nonparametric ANOVA (Kruskal‐Wallis test) with a multiple comparison post‐test (Dunn's), *P‐value <.05. Data represent at least three donors from three different experiments
Fig 3: Gene expression patterns defining the identity of each cord blood (CB) innate lymphoid cell (ILC) subtype. A, A four‐way plot with a log2 fold change cut off at ±1 (dotted lines) and adjusted P‐values of .05 showing differentially expressed genes of CB ILC1‐like (“control”) compared ILC2 (“y”) and ILC3‐like cells (“x”). Blue dots represent genes with an adjusted P‐value <.05 with a fold change >1. Green dots represent genes with an adjusted P‐value <.05 with a fold change between >1 (x‐axis) and <1 (y‐axis). Gray dots represent genes with an adjusted P‐value >.05. Red dots represent genes with an adjusted P‐value <.05 with fold rates <1 (x‐axis) and >1 (y‐axis). Selected genes differentially expressed between ILC1‐like cells, ILC2, and ILC3‐like cells are highlighted. B‐D, MA plots showing all differentially expressed genes and heatmaps showing differential expressed genes between (B) ILC1‐like vs ILC2, (C) ILC3‐like vs ILC1‐like, and (D) ILC3‐like vs ILC2. In the MA plots, dotted lines represent a log2 fold change cut off of 1. Blue dots represent a P‐value <.05 and a log2 fold change of >1. Green dots represent a P‐value of <.05 and a log2 fold change of <1. Gray dots represent a P‐value >.05 and a log2 fold change of <1. Numbers of blue or green genes are indicated next to the MA plot. Selected blue genes are highlighted. E, Violin plots showing normalized read counts for LIF (left hand side) and ZBTB46 (right hand side) for CB ILC1‐like, ILC2, and ILC3‐like cells. Levels of significance were calculated with a nonparametric ANOVA (Kruskal‐Wallis test) with a multiple comparison post‐test (Dunn's), *P‐value <.05, **P‐value <.005. Data represent at least three donors from three different experiments
Fig 4: Cord blood (CB) innate lymphoid cell subpopulation (ILC3)‐like cells are sensing TLR2:1 ligands. A, Normalized read counts of RNAseq data of CB ILC1‐like, ILC2, and ILC3‐like cells for TLR 1‐9. B, Bar graphs showing the frequency of CB ILC3‐like cell survival in medium alone, with Pam3CSK4 alone, with IL‐2 alone, and a combination of IL‐2 and Pam3CSK4. C, Cryopreserved CB samples were stained for ILCs. Representative dot plots and quantification for CD117 and CD25 expression are shown for ILC1‐like cells, ILC2, ILC3‐like cells, CD56bright natural killer (NK) cells, and CD56dim NK cells. D, Normalized read counts for IL12RA encoding CD25 are shown for CB ILC3‐like cells and tonsillar ILC3 (E/F) Fresh CB MNCs were isolated, depleted of CD3, CD14, CD19, and CD66b‐expressing cells and subsequently purified by sorting for ILC3‐like cells (Lin−CD94−CD127+CD117+CRTH2−). ILC3‐like cells were stimulated for 5 days with IL‐2 with or without the TLR2:1 ligand Pam3CSK4. E, Expansion of ILC3‐like cells (n = 7); and F, TNFα, IFNγ, IL‐13, IL‐5, IL‐10, IL‐22, LIF, and GM‐CSF secretion is shown (n = 6). The height of the bars represents the mean ± SEM. Levels of significance were calculated with a nonparametric ANOVA (Kruskall‐Wallis with a Dunn's post‐test) (B,C) and t test (Mann‐Whitney) (E,F), *P‐value <.05, **P‐value <.005
Supplier Page from BioLegend for LEGENDplex™ HU Hematopoietic Stem Cell Panel (12-plex) w/ VbP