CXCR6 Expression Is Important for Retention and Circulation of ILC Precursors

Abstract

Innate lymphoid cells are present at mucosal sites and represent the first immune barrier against infections, but what contributes to their circulation and homing is still unclear. Using Rag2(-/-) Cxcr6(Gfp/+) reporter mice, we assessed the expression and role of CXCR6 in the circulation of ILC precursors and their progeny. We identify CXCR6 expressing ILC precursors in the bone marrow and characterize their significant increase in CXCR6-deficient mice at steady state, indicating their partial retention in the bone marrow after CXCR6 ablation. Circulation was also impaired during embryonic life as fetal liver from CXCR6-deficient embryos displayed decreased numbers of ILC3 precursors. When injected, fetal CXCR6-deficient ILC3 precursors also fail to home and reconstitute ILC compartments in vivo. We show that adult intestinal ILC subsets have heterogeneous expression pattern of CXCR6, integrin α 4 β 7, CD62L, CD69, and CD44, with ILC1 and ILC3 being more likely tissue resident lymphocytes. Intestinal ILC subsets were unchanged in percentages and numbers in both mice. We demonstrate that the ILC frequency is maintained due to a significant increase of ILC peripheral proliferation, as well as an increased proliferation of the in situ ILC precursors to compensate their retention in the bone marrow.

Figure 1

ILCP and ILC2P in the bone marrow heterogeneously express CXCR6. (a) Flow cytometry of lineage depleted (Lin: CD3ε, CD5, CD8, CD11c, CD19, TCRβ, TCRγδ, Ter119, Gr1, and NK1.1) adult bone marrow (BM) from Cxcr6 ^Gfp/+ Id2 ^Yfp/+ mice. Among Lin⁻ IL-7Rα ⁺ compartments are defined: CLP (filled gray, c-Kit^lo  Sca-1^−/lo Flt3⁺   α ₄ β ₇ ⁻), ILCP (red, c-Kit^lo  Sca-1^−/lo Flt3⁻   α ₄ β ₇ ⁺), and ILC2P (blue, c-Kit⁻  Sca-1^hi). Each compartment is analyzed for CXCR6-GFP and ID2-YFP expression (bottom histograms). (b) Cell culture of BM ILCP, selected as ID2⁺ CXCR6⁻ (upper panels) or ID2⁺ CXCR6⁺ (lower panels). Each population is sorted and cultured at 20 cells per well on OP9-DL4 stromal cells with IL-7, c-KitL, and Flt3L for 10 days and analyzed by flow cytometry. Indicated obtained progenies were analyzed for ID2-YFP, CXCR6-GFP, RORγt, and IL-7Rα expression. Results are representative of at least 3 experiments each ((a): n > 5), or 2 experiments ((b), at least 4 wells of each condition).

Figure 2

CXCR6-deficient precursors are retained in the bone marrow. (a) Percentages in Lin⁻ (upper panels) and absolute numbers (lower panels) of BM CLP, ILCP, and ILC2P as defined in Figure 1(a) from Rag2 ^−/−  Cxcr6 ^+/+ (wt, blue losange), Cxcr6 ^Gfp/+ (HZ, red square), or Cxcr6 ^Gfp/Gfp (KO, green triangle) adult mice. (b) Histograms depicting levels of CXCR6-GFP in ILCP (left panel) or ILC2P (right panel) from Rag2 ^−/−  Cxcr6 ^+/+ (filled gray), Cxcr6 ^Gfp/+ (red line), or Cxcr6 ^Gfp/Gfp (green line) bone marrows. (c) Percentages of CXCR6⁺ cells (upper panels) and absolute numbers of (lower panels) BM ILCP (left panels) and ILC2P (right panels) in Rag2 ^−/−  Cxcr6 ^Gfp/+ (HZ, red square) or Cxcr6 ^Gfp/Gfp (KO, green triangle) adult mice. (d) Cell culture of BM ILCP from Rag2 ^−/−  Cxcr6 ^Gfp/+ (upper panels) or Cxcr6 ^Gfp/Gfp (lower panels) adult mice. Each population is sorted and cultured at 20 cells per well on OP9-DL4 stromal cells with IL-7, c-KitL, and Flt3-L for 7 days and analyzed by flow cytometry. Indicated obtained progenies were analyzed for T-bet, EOMES, RORγt, and GATA-3 expression. (e) Histograms of BM LSK cells (filled gray, selected as Lin⁻ IL-7Rα ⁻  c-Kit^hi  Sca-1⁺) and CLP (red line) for Ki67 expression and DAPI levels. (f) Percentages of Ki67⁺ cells (upper panels) and DAPI⁺ cells (lower panels) among BM CLP, ILCP, and ILC2P in Rag2 ^−/−  Cxcr6 ^+/+ (wt, blue losange), Cxcr6 ^Gfp/+ (HZ, red square), or Cxcr6 ^Gfp/Gfp (KO, green triangle) adult mice. Data are representative of at least 3 experiments ((b), (e): n > 5), or are from one experiment ((d), at least 4 wells of each conditions), or are from 3 pooled experiments ((a), (c), (f), wt: n = 8, HZ: n = 8, KO: n = 5). In ((a), (c), (f)), each dot represents a single mouse. Statistical data are displayed with mean and SEM (Student's unpaired bilateral test, ^∗ p < 0.05; ^∗∗ p < 0.01; ^∗∗∗ p < 0.005).

Figure 3

CXCR6 contributes to fetal ILC3 and ILC3 precursor circulation. (a) Flow cytometry of lineage depleted (Lin: CD3ε, CD11c, CD19, Ter119, Gr1, NK1.1) E15.5 fetal liver (FL). ILC3 precursors are defined as Lin⁻  IL-7Rα ⁺  c-Kit^med RORγt⁺. (b) Percentages of Lin⁻ IL-7Rα ⁺  c-Kit^med (left panel) and absolute numbers (right panel) of FL ILC3 precursors as defined in Figure 2(a) in FL from Cxcr6 ^+/+ (wt, blue losange), Cxcr6 ^Gfp/+ (HZ, red square) or Cxcr6 ^Gfp/Gfp (KO, green triangle) E15.5 embryos. (c) Flow cytometry of Lin⁻ compartment fetal spleen (FS) from Cxcr6 ^Gfp/+ E15.5 embryos. Lin⁻  CD4^hi IL-7Rα ⁺ cells (red) and total Lin⁻ cells (filled gray) are analyzed for CXCR6-GFP (left histogram) and RORγt (right histogram). (d) Scheme depicting injection and reconstitution experiment. (e) Analysis of reconstitution experiment. 1000 to 2000 Ly5.2 Lin⁻  CD4^hi IL-7Rα ⁺ CXCR6⁺ cells were injected in nonlethally irradiated Rag2 ^−/− γc ^−/− Ly5.1 mice. Recipients were killed 4 weeks after injection and intestinal lamina propria (LP, upper panels) and liver (LV, lower panels) were analyzed by flow cytometry. (f) Percentages of reconstitution experiment as explained in Figures 2(d) and 2(e) using Cxcr6 ^Gfp/+ (HZ, red squares) or Cxcr6 ^Gfp/Gfp (KO) E15.5 embryos in intestinal LP (left panel) or LV (right panel). Results are representative of at least 3 experiments each ((a), (c): n > 5), or are from 3 pooled experiments ((b), wt: n = 6, HZ: n = 10, KO: n = 4), or are from at least 2 pooled experiments ((e), (f), LP HZ: n = 2, LP KO: n = 5, LV HZ: n = 4, LV KO: n = 5). In ((b), (f)), each dot represents a single mouse. Statistical data are displayed with mean and SEM (Student's unpaired bilateral test, ^∗ p < 0.05; ^∗∗ p < 0.01).

Figure 4

Intestinal ILC compartments have heterogeneous expression of CXCR6 and egress markers. (a) Flow cytometry of Lin⁻ (Lin: CD3ε, CD5, CD8, CD11c, CD19, TCRβ, TCRγδ, Ter119, and Gr1) adult intestinal LP from Rag2 ^−/−  Cxcr6 ^Gfp/+ mice. ILC subsets are defined as cNK (filled gray, Lin⁻  NK1.1⁺  NKp46⁺ IL-7Rα ⁻), ILC1 (blue, Lin⁻ NK1.1⁺ NKp46⁺ IL-7Rα ⁻), ILC2 (red, Lin⁻  NK1.1⁻  Thy1.2^−/lo  Sca-1^hi  c-Kit⁻  ICOS^hi), ILC3 NCR⁺ (green, Lin⁻ NK1.1⁻  Thy1.2^hi  Sca-1^−/lo  c-Kit⁻ NKp46⁺), ILC3 NCR⁻ (orange, Lin⁻ NK1.1⁻  Thy1.2^hi  Sca-1^−/lo  c-Kit^med NKp46⁺ CD4⁻), and LTi-like (light blue, Lin⁻ NK1.1⁻  Thy1.2^hi  Sca-1^−/lo  c-Kit^med NKp46⁺ CD4⁺). Each compartment is analyzed for CXCR6-GFP expression. (b) Flow cytometry of Lin⁻ adult intestinal LP from Rag2 ^−/−  Cxcr6 ^Gfp/+ mice. ILC subsets are defined as ILC2 (red, Lin⁻ GATA-3⁺ RORγt⁻), ILC3 NCR⁺ (green, Lin⁻ GATA-3⁻ RORγt⁺ NKp46⁺ CD4⁻), ILC3 NCR⁻ (orange, Lin⁻ GATA-3⁻ RORγt⁺ NKp46⁻ CD4⁻), LTi-like (light blue, Lin⁻ GATA-3⁻ RORγt⁺ NKp46⁻ CD4⁺), and ILC1-cNK (blue, Lin⁻ GATA-3⁻ RORγt⁻ NKp46⁺ NK1.1⁺). (c) Histograms depicting levels of CD62L, CD69, CD44, CXCR6-GFP, and α ₄ β ₇ in Lin⁻ (filled gray) or ILC subsets (as defined in Figure 3(b)) in adult LP (upper panels) or mesenteric lymph nodes (mLN, lower panels) from Rag2 ^−/− Cxcr6 ^Gfp/+ mice. Results are representative of at least 3 experiments each ((a), (b), (c): n > 5).

Figure 5

Normal intestinal ILC compartments in CXCR6-deficient mice are compensated by higher proliferative capacities of in situ progenitor cell. (a) Percentages of ILC subsets in Lin⁻ (as defined in Figure 3(b)) in adult LP from Rag2 ^−/−  Cxcr6 ^+/+ (wt, blue losange), Cxcr6 ^Gfp/+ (HZ, red square), or Cxcr6 ^Gfp/Gfp (KO, green triangle) adult mice. (b) Histograms depicting levels of CD62L, CD69, CD44, CXCR6-GFP, and α ₄ β ₇ in Lin⁻ (filled gray) or ILC subsets (as defined in Figure 3(b)) in adult LP (upper panels) or mesenteric lymph nodes (mLN, lower panels) from Rag2 ^−/−  Cxcr6 ^Gfp/Gfp mice. (c) Histograms of total LP Lin⁻ cells (filled gray) or ILC2 cells (red line) for Ki67 expression and DAPI levels. ((d), (e)) Percentages of Ki67⁺ cells (d) and DAPI⁺ cells (e) among ILC subsets (as defined in Figure 4(b)) in LP from Rag2 ^−/−  Cxcr6 ^+/+ (wt, blue losange), Cxcr6 ^Gfp/+ (HZ, red square), or Cxcr6 ^Gfp/Gfp (KO, green triangle) adult mice. (f) Flow cytometry of LP in situ ILCP (upper panel) and CXCR6-GFP and ID2-YFP expression of LP in situ ILCP (lower panels) from Cxcr6 ^Gfp/+  Id2 ^Yfp/+ mice. (g) Percentages of IL-7Rα ⁺ precursors in Lin⁻ (left), Ki67⁺ (middle), and DAPI⁺ (right) cells among IL-7Rα ⁺ precursors in LP from Rag2 ^−/− Cxcr6 ^+/+ (wt, blue losange), Cxcr6 ^Gfp/+ (HZ, red square), or Cxcr6 ^Gfp/Gfp (KO, green triangle) adult mice. Results are from 3 pooled experiments ((a), (c), (d): wt: n = 9, HZ: n = 9, KO: n = 8) or from 2 pooled experiments ((e), (f), GF, wt: n = 8, HZ: n = 8, KO: n = 6) or are representative of 3 experiments (b). In ((a), (d), (e), (g)), each dot represents a single mouse. Statistical data are displayed with mean and SEM (Student's unpaired bilateral test, ^∗ p < 0.05; ^∗∗ p < 0.01).