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. Author manuscript; available in PMC: 2020 Feb 13.
Published in final edited form as: Sci Immunol. 2019 May 31;4(35):eaau8082. doi: 10.1126/sciimmunol.aau8082

Figure 2. ILC frequency in LNs is maintained by balanced ingress and egress.

Figure 2

To further investigate the migratory ILCs identified within the labelled bLN, the effect of photoconversion on the number of ILCs was assessed. (A) Expression of Kaede Green versus Kaede Red protein by ILCs (CD45+ IL-7Rα+ Lin{B220, CD11b, CD11c, CD3, CD5, CD19, Ter119, Gr1, CD49b, F4/80, FcεRI}-) isolated from the bLN of control Kaede mice (no photoconversion) and Kaede mice at 72 hrs post photoconversion. (B) The number of Kaede Green+ and Kaede Red+ ILCs recovered from the bLN of control (n=6) versus photoconverted bLNs at 72 hrs post labelling (n=10). To investigate egress of ILCs from the bLN, Kaede mice were maintained on vehicle control (H2O) or FTY720, the bLN was photoconverted and then analyzed 72 hrs later. (C) Representative flow cytometry plots showing expression of Kaede Green versus Kaede Red protein by ILCs in the bLN 72 hrs after photoconversion. (D) Percentage of Kaede Green+ or Kaede Red+ ILCs in the bLN of Ctrl (n=4) and FTY720 (n=7) mice. (E) Total numbers of Kaede Green+ and Kaede Red+ ILCs in the bLN. To assess whether ILCs recirculate through peripheral LNs, the contralateral LNs of Kaede mice were analyzed at 72 hrs post photoconversion of the bLN, with combined B- and T-cell depletion used to enrich for ILCs. (F) Expression of Kaede Green versus Kaede Red protein by ILCs (CD45+ IL-7Rα+ Lin{B220, CD11b, CD11c, CD3, CD5, CD19, Ter119, Gr1, CD49b, F4/80, FcεRI}-) isolated from contralateral LNs. Percentage of Kaede Red+ ILCs (G) and T cells (H) in WT Kaede (n=14) and CCR7-/- Kaede (n=8) contralateral LNs. (I) Representative flow cytometry plots of Kaede Green+ (left) and Kaede Red+ (right) ILC expression of CD69. (J) Percentage of Kaede Green+ and Kaede Red+ ILCs expressing CD69 (n=7). Data are pooled from 2 independent experiments. Bars on scatter plots represent the median, which is also shown numerically. Statistical significance was tested using an unpaired, non-parametric, Mann-Whitney two-tailed U test: **p≤0.01, ***p≤0.001.