Fig. 7: Clustering analysis of tonsil single-cell multiplexed histology data uncovers IRF4 expression in ILC3s. | Nature Communications

Fig. 7: Clustering analysis of tonsil single-cell multiplexed histology data uncovers IRF4 expression in ILC3s.

From: Multiplexed histology analyses for the phenotypic and spatial characterization of human innate lymphoid cells

Fig. 7

a Heat-map representation of the relative expression level of 14 relevant markers analyzed for every clustered ILC identified in (Fig. 6a, b). b Overlay of MELC fluorescence images depicting relevant markers for the identification of ILCs (white arrows). Three representative examples of CD3CD127+CD161+/−c-Kit+CCR6+/-CD7+/− ILC3s are shown in two different regions of interest of the tonsil (upper and lower panel). These ILC3 cells were allocated in the ILC cluster as shown in (a), and express IRF4 and/or CD138. CD3 (red), CD127 (green), and DAPI (blue) are shown in the first column. IRF4 (cyan), CD138 (magenta), and CD161 (yellow) are shown in the middle column. CD7 (cyan), CCR6 (magenta), and c-Kit (yellow) are shown in the right column. Scale bar: 10 µm in the upper panel and 20 µm in the lower panel. a, b (n = 5). c Dot plot depicting IRF4 normalized transcript reads in sorted DAPILinCD94CD127hiCD56+ ILC3s, CD34+c-Kit+ hematopoietic progenitor cells (HPC), and CD34hic-KitHPCs, extracted from the published microarray data41. Data are shown as mean ± SD and analyzed by one-way ANOVA (F = 21.78 and 2 df) with Bonferroni´s multiple comparison test, where **p < 0,005 and ***p < 0.0005 (n = 7). d Representative complete gating strategy for flow cytometry analysis of tonsillar ILCs after magnetic depletion of CD3+ and CD19+ cells (n = 3) (see “Methods”). Lin gate contains CD14CD19CD20CD123CD141FcεR1α cells. LinCD3CD45+CD94+CD56+ are defined as NK cells (black). LinCD3CD45+CD127+CD161+ are defined as helper ILCs (green), which are subclassified as c-Kit+CRTH2 ILC3s (98.6 %, cyan) or CRTH2+ ILC2s (0.96 %, magenta). IRF4 is shown to be co-expressed with the ILC3 markers c-Kit and NKp44 within the ILC population. See also Supplementary Fig. 4. Source data are provided as a Source Data file.

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