Figure 4. ILCs mediate iBALT formation and contribute to early protection from Mtb.

Rag1^−/−, Rag2γc^−/− and wild type mice were aerosol infected with ~100 CFU Mtb. ILCs (CD45⁺CD127⁺Lin⁻NK1.1⁻) were isolated from Mtb infected wild type mice and ~5X10³ cells were intratracheally transferred into Rag2γc^−/− mice 1 day before infection. (a) Lung bacterial burden at 14 dpi was determined by plating (n=5/group). (b) Number of ILC1s, ILC2s, total ILC3s and NKp46⁺ ILC3s and (c) AMs were measured by flow cytometry. (d) ILC3 quantification in histological lung sections was carried out by staining with CD3, B220 and Rorγt and the number of Rorγt⁺CD3⁻ ILC3s were counted and shown. (e) Ahr^f/f, Ahr^f/fRorγt^Cre mice were aerosol infected with ~100 CFU Mtb and lung bacterial burden at 14 and 30 dpi was determined by plating (n=7–10/group). (f) Number of ILC1s, ILC2s, total ILC3s, NKp46⁺ ILC3s and AMs were enumerated by flow cytometry. (g) B6 and Il17/Il22^−/− were aerosol infected with ~100 CFU Mtb and lung bacterial CFU were measured by plating (n=12/group). (h) Number of ILC1, ILC2, ILC3, CXCR5⁺ ILC3, and CXCR5⁺NKp46⁺ ILC3 were measured by flow cytometry (n=5/B6, n=8/Il17/Il22^−/−). (i) FFPE lung sections were subjected to ISH with the mouse-Cxcl13 probe and the ratio of Cxcl13 mRNA⁺ area occupied per lung was quantified. (j) B6 mice received IL-23 blocking antibody (i.p.) 1 day prior to infection with ~100 CFU Mtb and the lung bacterial burden and (k) number of AMs, ILC2s and ILC3s were quanified at 14 dpi using plating and flow cytometry respectively (n=5/isotype, n=5–6/anti-IL-23), Iso = Isotype. (l) FFPE lung sections from 30 dpi Mtb infected mice were stained with antibodies to B220 and CD3, and the average size of B cell follicles were quantified in Ahr^f/f, Ahr^f/fRorγt^Cre, B6, Il17/Il22^−/−, isotype-treated B6 and anti-IL-23-treated B6 Mtb-infected mice. All data shown as mean ± SD. Significance by either one way ANOVA (a-d) or Student’s t-test (e-l).