Research ArticleINNATE IMMUNITY

Peripheral lymph nodes contain migratory and resident innate lymphoid cell populations

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Science Immunology  31 May 2019:
Vol. 4, Issue 35, eaau8082
DOI: 10.1126/sciimmunol.aau8082
  • Fig. 1 CCR7-dependent entry of ILCs into peripheral LNs.

    To assess the migratory kinetics of different cellular populations within peripheral LNs, the bLN of Kaede mice was photoconverted and analyzed 72 hours later. (A) Expression of Kaede Green (KG) versus Kaede Red (KR) protein by CD45+ cells isolated from the bLN at 72 hours after photoconversion. (B) Percentage of CD45+ cells expressing Kaede Green or Kaede Red protein at 72 hours after photoconversion (n = 10). (C) Characterization of Kaede Green+ CD45+ cells isolated from the bLN, showing αβ T cells (CD45+ CD3+ TCRβ+ TCRγδ), γδ T cells (CD45+ CD3+ TCRβ TCRγδ+), ILCs (CD45+ IL-7Rα+ Lin{B220, CD11b, CD11c, CD3, CD5, CD19, Ter119, Gr1, CD49b, F4/80, FcεRI}), and LCs (CD45+ MHCII+ CD11c+ CD207+). (D) Percentage of αβ T cells (n = 7), γδ T cells (n = 7), ILCs (n = 10), or LCs (n = 5) among CD45+ Kaede Green+ cells. (E) Characterization of Kaede Red+ cells isolated from the bLN, showing αβ T cells, γδ T cells, ILCs, and LCs. (F) Percentage of αβ T cells (n = 7), γδ T cells (n = 7), ILCs (n = 10), and LCs (n = 5) among CD45+ Kaede Red+ cells. Expression of Kaede Green versus Kaede Red protein by αβ T cells, γδ T cells, ILCs, and LCs isolated from the bLN of (G) Kaede and (H) CCR7−/− Kaede mice. (I) Number of αβ T cells (WT, n = 7; CCR7−/−, n = 8), γδ T cells (WT, n = 7; CCR7−/−, n = 8), ILCs (WT, n = 6; CCR7−/−, n = 8), and LCs (WT, = 5; CCR7−/−, n = 7) isolated from the bLN of WT Kaede versus CCR7−/− Kaede mice. (J) Number of Kaede Green αβ T cells (WT, n = 7; CCR7−/−, n = 8), γδ T cells (WT, n = 7; CCR7−/−, n = 8), ILCs (WT, n = 7; CCR7−/−, n = 8), and LCs (WT, n = 5; CCR7−/−, n = 7) isolated from the bLN. Data are pooled from a minimum of two independent experiments. Values on flow cytometry plots represent percentages; bars on scatter plots represent the median, which is also shown numerically. Statistical significance was tested using an unpaired, nonparametric, Mann-Whitney two-tailed U test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

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

    To further investigate the migratory ILCs identified within the labeled 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 hours after photoconversion. (B) The number of Kaede Green+ and Kaede Red+ ILCs recovered from the bLN of control (n = 6) versus photoconverted bLNs at 72 hours after labeling (n = 10). To investigate egress of ILCs from the bLN, Kaede mice were maintained on vehicle control (H2O) or FTY720, and the bLN was photoconverted and then analyzed 72 hours later. (C) Representative flow cytometry plots showing expression of Kaede Green versus Kaede Red protein by ILCs in the bLN 72 hours after photoconversion. (D) Percentage of Kaede Green+ or Kaede Red+ ILCs in the bLN of control (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 hours after photoconversion of the bLN, with combined B and T cell depletion used to enrich 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 two independent experiments. Bars on scatter plots represent the median, which is also shown numerically. Statistical significance was tested using an unpaired, nonparametric, Mann-Whitney two-tailed U test: **P ≤ 0.01, ***P ≤ 0.001.

  • Fig. 3 ILC1s traffic through peripheral LNs from the circulation.

    To test whether all ILC subsets showed an equal ability to traffic through the peripheral LNs, an established panel of surface markers was used to identify which ILC subsets were migrating into the bLN. (A) Representative flow cytometry plots showing identification of ILC1 (CD45+ Lin{B220, CD11b, CD11c, CD3, CD5, CD19, Ter119, Gr1, CD49b, F4/80, FcεRI} IL-7Rα+ CD4 (NK1.1, NKp46)+), ILC2 (CD45+ Lin{B220, CD11b, CD11c, CD3, CD5, CD19, Ter119, Gr1, CD49b, F4/80, FcεRI}) IL-7Rα+ CD4 (NK1.1, NKp46)(KLRG-1, CD25)+), and ILC3 (CD45+ Lin{B220, CD11b, CD11c, CD3, CD5, CD19, Ter119, Gr1, CD49b, F4/80, FcεRI}IL-7Rα+ CD4+] subsets in the bLN. (B) The percentage of each ILC subset identified within the bLN of Kaede mice using the surface marker staining. (C and D) Kaede Green versus Kaede Red protein expression by ILC1s, ILC2s, and ILC3s at 72 hours and 1 week after photoconversion of the bLN. (E) The percentage of Kaede Green+ and Kaede Red+ ILC1s, ILC2s, and ILC3s in the bLN at 72 hours and 1 week after photoconversion. Data are pooled from two independent experiments, n = 7. To investigate the source of ILC1s migrating into the bLN, ILC populations in murine blood were analyzed. (F) Representative flow cytometry plots showing identification of putative ILC1s (CD45+ IL-7Rα+ (NKp46NK1.1)+Lin{B220, CD11b, CD11c, CD3, CD5, CD19, Ter119, Gr1, CD49b, F4/80, FcεRI}) and ILC2s (CD45+ IL-7Rα+ NK1.1 NKp46 KLRG-1+ Lin). (G) The proportion of ILC1s and ILC2s within the ILC population isolated from the blood, n = 8. (H) Representative flow cytometry plots showing expression of T-bet versus GATA-3 by blood ILCs. To isolate sufficient numbers of ILCs, blood samples from three mice were enriched for the CD45+ fraction separately and then pooled for staining and analysis. (I) Representative flow cytometry plots showing expression of CD62L and CCR7 by T-bet+ ILC1s versus T-bet ILCs isolated from blood. All data are pooled from two independent experiments. Values on flow cytometry plots represent percentages; bars on scatter plots represent the median. Statistical significance was tested using an unpaired, nonparametric, Mann-Whitney two-tailed U test: ***P ≤ 0.001.

  • Fig. 4 Minimal ILC migration from skin to the draining LN even under conditions of inflammation.

    To directly test whether ILCs migrate from skin to peripheral LNs, the ears of Kaede mice were photoconverted, and the presence of labeled cells were assessed in the auLN. (A) Expression of Kaede Green versus Kaede Red protein by CD45+ cells isolated from ear skin without (left) or immediately after (right) photoconversion. (B) Percentage of CD45+ cells expressing Kaede Red protein in the ear and auLN immediately after photoconversion (n = 12). (C) Expression of Kaede Green versus Kaede Red protein by CD45+ cells in the auLN at 0, 48, and 72 hours after photoconversion of the ear skin. The percentage (D) and total number (E) of Kaede Red+ CD45+ cells in the auLN over the time course of 0 hours (n = 12), 48 hours (n = 11), and 72 hours (n = 10). (F) Representative flow cytometry plots assessing the phenotype of Kaede Red+ CD45+ cells in the auLN, with identification of T cells (CD3+), ILCs (IL-7Rα+ Lin{B220, CD11b, CD11c, CD3, CD5, CD19, Ter119, Gr1, CD49b, F4/80, FcεRI}), and LCs (CD45+ MHCII+ CD11c+ CD207+). (G) Percentage of Kaede Red+ CD45+ cells in the auLN that were T cells (n = 5), ILCs (n = 12), and LCs (n = 5). To determine whether inflammation in the skin resulted in the migration of ILCs to the draining LN, the ears of mice were treated with vehicle control (VC) or MC903, with photoconversion of the ear after 48 hours of treatment. (H) Representative flow cytometry plots showing ILCs within VC-treated (left) and MC903-treated (right) auLN. Enumeration of CD45+ cells (I) and ILCs (J) recovered from the auLN of mice treated with VC (n = 7) or MC903 (n = 12). (K) Representative flow cytometry plots assessing the phenotype of Kaede Red+ CD45+ cells in the auLN of MC903-treated mice, with the identification of T cells and ILCs. Enumeration of Kaede Red+ (L) CD45+ cells in VC (n = 8) or MC903 (n = 8), (M) T cells in VC (n = 10) or MC903 (n = 11), and (N) ILCs in VC (n = 8) or MC903 (n = 8) within the auLN. (O) Enumeration of ILC subsets including triple negative (TrN) in the auLN of mice treated with VC (n = 6) and MC903 (n = 15). (P) Expression of Ki-67 by ILCs in the auLN of mice treated with VC (n = 6) or MC903 (n = 7). Data are pooled from a minimum of two independent experiments. Values on flow cytometry plots represent percentages; bars on scatter plots represent the median, which is also shown numerically. Pairs of samples were compared using an unpaired Mann-Whitney U test. When comparing more than two sets of data, statistical significance was tested using Kruskal-Wallis one-way analysis of variance (ANOVA) with post hoc Dunn’s test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

  • Fig. 5 CCR6-dependent recruitment of ILCs into the inflamed ear.

    To investigate mechanisms controlling the recruitment of ILCs into the ear, the entire ear CD45+ population was labeled through photoconversion, with Kaede Green+ cells identified at subsequent time points as cells that had newly entered the tissue. (A) Expression of Kaede Green versus Kaede Red protein by CD45+ cells in the ear at 0, 48, and 72 hours after photoconversion of the ear skin. (B) The percentage (left) and total number (right) of Kaede Green+ CD45+ cells in the auLN over the time course of 0 hours (n = 12), 48 hours (n = 11), and 72 hours (n = 11). (C) Representative flow cytometry plots assessing the phenotype of Kaede Green+ CD45+ cells in the ear with identification of T cells (CD3+), ILCs (IL-7Rα+ Lin{B220, CD11b, CD11c, CD3, CD5, CD19, Ter119, Gr1, CD49b, F4/80, FcεRI}) and LCs (CD45+ MHCII+ CD11c+ CD207+). (D) Percentage of Kaede Green+ CD45+ cells in the ear that were T cells (n = 6), ILCs (n = 11), and LCs (n = 6). To assess migration under inflammatory conditions, MC903 was applied to the ears for five consecutive days to induce atopic dermatitis, with photoconversion of the ear 48 hours after treatment was started. Number of Kaede Green+ (E) CD45+ cells in VC (n = 9) or MC903 (n = 7), (F) T cells in VC (n = 10) or MC903 (n = 8), and (G) ILCs in VC (n = 9) or MC903 (n = 7) within the ear. (H) Representative flow cytometry plots of Kaede Green+ ILCs Ki-67 expression within the ear of VC- and MC903-treated mice. To test the requirement for CCR6 in ILC recruitment into the ear, MC903 was applied to the ears of Kaede and CCR6−/− Kaede mice for five consecutive days to induce atopic dermatitis, with photoconversion of the ear 48 hours after treatment was started. (I) Expression of Kaede Green and Kaede Red protein by CD45+ cells within MC903-treated ears. (J) Number of Kaede Green+ CD45+ cells (Kaede, n = 8; CCR6−/− Kaede, n = 9). (K) Representative flow cytometry plots showing Kaede Green+ ILCs. (L) Number of Kaede Green+ ILC (Kaede, n = 8; CCR6−/− Kaede, n = 9). Each data point represents cells isolated from one ear and one auLN. Data are pooled from a minimum of two independent experiments. Values on flow cytometry plots represent percentages; bars on scatter plots represent the median, which is also shown numerically. Pairs of samples were compared using an unpaired Mann-Whitney U test. When comparing more than two sets of data, statistical significance was tested using Kruskal-Wallis one-way ANOVA with post hoc Dunn’s test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

  • Fig. 6 ILC1s in the draining LN are a rapid source of IFN-γ after immunization.

    To investigate the function of ILC1 within peripheral LNs, mice were injected intramuscularly with 20 μg of OVA-2W1S/PBS or 20 μg of OVA-2W1S/AS01 and culled either 6 hours after immunization to assess the early innate IFN-γ response or after 7 days after a second immunization performed 6 hours before analysis. (A) Representative flow cytometry plots showing inguinal LN cells from mice given OVA-2W1S/PBS (top) or OVA-2W1S/AS01 (bottom), with identification of different IFN-γ–producing populations including cNK cells (CD45+ IFN-γ+ CD3ε NK1.1+EOMES+) and ILC1s (CD45+ IFN-γ+ CD3ε NK1.1+ EOMES T-bet+). (B) Total number of IFN-γ+ ILC1s and cNK cells in OVA/ASO1-treated mice (n = 5). (C) Representative flow cytometry plots showing IFN-γ production by 2W1S-specific CD4+ T cells at day 7 after initial immunization with (AS01) or without (PBS) the adjuvant AS01. (D) Total number of IFN-γ producing 2W1S-specific CD4 T cells in the draining inguinal LN of PBS (n = 4) and AS01 mice (n = 6). To test whether both migratory and resident ILC1 subsets were able to produce IFN-γ, the bLN of Kaede mice was photoconverted, and 3 days later, mice were then immunized in the paw pad with either 20 μg of OVA-2W1S/PBS or 20 μg of OVA-2W1S/AS01 and analyzed 6 hours later. (E) Representative flow cytometry plots showing bLN cells from mice given OVA-2W1S/PBS (top) or OVA-2W1S/AS01 (bottom), with identification of ILC1 and cNK populations producing IFN-γ and their expression of Kaede Green versus Kaede Red protein. (F) Percentage Kaede Green (migratory) and Kaede Red (resident) ILC1s and cNK cells among IFN-γ–producing cells. (G) Total numbers of IFN-γ+ ILC1s and cNK cells expressing either Kaede Green (migratory) or Kaede Red (resident). All sets of data are pooled from two independent experiments. Values on flow cytometry plots represent percentages; bars on scatter plots represent the median, which is also shown numerically. Statistical significance was tested using an unpaired, nonparametric, Mann-Whitney two-tailed U test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/35/eaau8082/DC1

    Fig. S1. LN stromal populations remain Kaede Red+ after photoconversion.

    Fig. S2. ILCs continuously traffic into LNs with different kinetics to T cells.

    Fig. S3. Photoconversion does not cause an increase in the number of ILCs within the labeled bLN.

    Fig. S4. Expression of S1P1 by ILC subsets in the bLN.

    Fig. S5. Treatment with FTY720 results in ILC accumulation within the bLN.

    Fig. S6. Analysis of S1PR expression by ILCs.

    Fig. S7. Identification of ILC subsets within the bLN.

    Fig. S8. Analysis of ILC residency in LNs using H2B-Dendra2 mice.

    Fig. S9. Differences in Dendra Red expression by ILC2s dependent on identification by a TF or cell surface marker expression.

    Fig. S10. Expression of Ki-67 by ILC subsets within the bLN.

    Fig. S11. Comparison of ILC1 and NK cell recirculation through peripheral lymph nodes.

    Fig. S12. Induction of atopic dermatitis causes an increase in the frequency of ILC2s in ear skin.

    Fig. S13. CCR6 is expressed by all ILC subsets in the ear and auLN.

    Table S1. Raw data.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. LN stromal populations remain Kaede Red+ after photoconversion.
    • Fig. S2. ILCs continuously traffic into LNs with different kinetics to T cells.
    • Fig. S3. Photoconversion does not cause an increase in the number of ILCs within the labeled bLN.
    • Fig. S4. Expression of S1P1 by ILC subsets in the bLN.
    • Fig. S5. Treatment with FTY720 results in ILC accumulation within the bLN.
    • Fig. S6. Analysis of S1PR expression by ILCs.
    • Fig. S7. Identification of ILC subsets within the bLN.
    • Fig. S8. Analysis of ILC residency in LNs using H2B-Dendra2 mice.
    • Fig. S9. Differences in Dendra Red expression by ILC2s dependent on identification by a TF or cell surface marker expression.
    • Fig. S10. Expression of Ki-67 by ILC subsets within the bLN.
    • Fig. S11. Comparison of ILC1 and NK cell recirculation through peripheral lymph nodes.
    • Fig. S12. Induction of atopic dermatitis causes an increase in the frequency of ILC2s in ear skin.
    • Fig. S13. CCR6 is expressed by all ILC subsets in the ear and auLN.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data.

    Files in this Data Supplement:

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