Extended Data Figure 4. Pulmonary ILCs are tissue resident and express markers of migration.

(a) CD69, CD103, CD62L and NKp44 expression on the circulating ILCs in human peripheral blood and lung tissue were measured by flow cytometry. Significance by unpaired Mann-Whitney U test. Percentage of total human ILCs expressing these markers in paired samples of TB participants shown. Significance calculated using a one-way Wilcoxon-matched paired test. (b,c) NKp44, CD56 expression were measured in TB-infected lung tissues in comparison to control samples. Significance by unpaired Mann-Whitney U test (b) and a Kruskal-Wallis test with adjustments for multiple comparisons (c). (d) Percentages of ILC1, ILC2, ILC3, CD56^dim NK cells, and CD56^hi NK cells in human lung tissue were measured by flow cytometry TB⁻HIV⁻ control subjects, TB participants without (TB⁺HIV⁻) and with HIV co-infection (TB⁺HIV⁺). (e) CXCL13 protein levels were measured in the plasma from TB participants without (TB⁺HIV⁻) and with HIV co-infection (TB⁺HIV⁺). Significance calculated by Mann-Whitney U test (no significance after Bonferonni correction). (f) Frequencies of CD103⁺ ILCs were measured by flow cytometry in the blood from TB⁻HIV⁻ control subjects, TB participants without (TB⁺HIV⁻) and with HIV co-infection (TB⁺HIV⁺). Significance by Mann-Whitney U test with Bonferroni corrections (only significant values after correction shown). (g) Representative FACS plots showing two distinct subpopulations of CD103 and CXCR5-expressing ILCs measured in lung tissues from three TB⁺ subjects, where most CXCR5-expressing cells are CD117⁺ ILC3s, and CD103⁺ lung ILCs are a combination of CD117⁺ ILC3s, CRTH2⁺ ILCs and CD117⁻CRTH2⁻ cells. Green = CD117⁺; Red = CRTH2⁺. (h) B6 mice were aerosol infected with ~100 CFU Mtb and lungs were harvested at 14 dpi. Lung ILCs were sorted from single cell suspensions (ILCs: CD45⁺CD127⁺Lin⁻NK1.1⁻). The ability of sorted ILCs to migrate towards media alone or mouse CXCL13 gradient was quantitated in transwell migration assay. n=3–5 biological replicates. Significance by one way ANOVA, *p<0.05, **p<0.01. (i) Human ILC3s sorted from lungs migrated in response to recombinant human CXCL13 in transwell migration assays. Significance by one tailed t-test.