Migratory and adhesive cues controlling innate-like lymphocyte surveillance of the pathogen-exposed surface of the lymph node
- Howard Hughes Medical Institute, University of California, San Francisco, United States
- University of California, San Francisco, United States
- University of Dundee, United Kingdom
Figures
Figure 1
Rapid induction of IL17 expression by IL7RαhiCcr6+ innate-like lymphocytes in a CD169+ macrophage-dependent manner following bacterial and fungal challenge.
(A) Representative FACS plot showing IL7RαhiCcr6+ staining of peripheral LN cells from a Cxcr6GFP/+ mouse, and Cxcr6-GFP intensity on the gated cells. (B) Representative FACS plots showing CD3ε, …
Figure 2
IL7RαhiCcr6+ innate-like lymphocytes are mostly LN resident.
(A) Representative FACS plots showing frequency of IL7RαhiCcr6+ and Vγ4+Ccr6+ cells in LNs and blood. Graphs show summary data for more than 30 mice of each type. (B) FACS analysis of LN IL7RαhiCcr6+…
Figure 3 with 1 supplement
Migration dynamics, sinus exposure and CD169+ macrophage interaction of LN innate-like lymphocytes.
(A) Time series of Cxcr6GFP/+ cell movement with respect to CD169+ SCS macrophages. Upper panels: white arrow indicates a Cxcr6GFP/+ lymphocyte in the LN parenchyma that crosses into the SCS. 300 s …
Figure 3—figure supplement 1
Example of automatically generated tracks for Cxcr6-GFP+ cells in a Cxcr6GFP/+ mouse LN.
Green, Cxcr6-GFP+ lymphocytes; Red, CD11b+ macrophages. 5 min~30 min tracks for Cxcr6-GFP+ lymphocytes are shown in colored lines. LN, Lymph node.
Figure 4 with 4 supplements
Ccr6 promotes innate-like lymphocyte positioning near the SCS.
(A) Ccl20 mRNA abundance in sorted LN lymphatic endothelial cells (LEC), blood endothelial cells (BEC), fibroblastic reticular cells (FRC) and double negative stromal cells (DN) determined by …
Figure 4—figure supplement 1
CCL20 distribution in inguinal LN.
Serial sections were stained with anti-CCL20 (A) or without primary antibody (B, C) (red) and anti-B220 (blue). The arrows in A point to staining in the SCS region adjacent to B cell follicles. The …
Figure 4—figure supplement 2
Movement of Cxcr6-GFP+ cells to SCS location following CCL20 injection.
Cxcr6GFP/+ mice were injected s.c. with saline (A) or CCL20 (B) and 1 hr later inguinal LN sections were stained to detect Cxcr6-GFP (green), and Lyve1 (blue). LN, Lymph node.
Figure 4—figure supplement 3
Ccr6 is required for positioning of Ccr6+ cells at the SCS.
Inguinal LN sections from Ccr6GFP/+ (A) and Ccr6GFP/GFP (Ccr6-deficient) (B) mice were stained to detect GFP (green) and B220 (blue). (C) GFP+B220– cells were counted manually and percentage of …
Figure 4—figure supplement 4
Ccr6 is required for positioning of Scart2+γδT cells at the SCS.
Inguinal LN sections from control (Ccr6+/–) (A) and Ccr6–/– (Ccr6 deficient) (B) mice were stained to detect Scart2 (green) and B220 (blue). (C) Scart2+ cells were counted manually and percentage of …
Figure 5 with 2 supplements
S1pr1 is required for innate-like lymphocyte movement into the SCS.
(A) S1pr1 surface expression on IL7RαhiCcr6+ and Vγ4+Ccr6+ cells from control or FTY720 treated mice. Negative indicates samples stained with no primary antibody. (B) Transwell migration assay …
Figure 5—figure supplement 1
FTY720 treatment depletes SCART2+γδT cells from the SCS.
Mice were treated i.v. with saline (A) or FTY720 (B) and 6 hr later inguinal LN sections were stained to detect Scart2 (green) and Lyve1 (blue). SCS and medulla indicate subcapsular sinus area and …
Figure 5—figure supplement 2
Frequency of Cxcr6GFP/+ cells plotted against their depth from the surface of the LN capsule.
Each individual cell’s location was determined using the Spots tool in Imaris. The depth, or minimum distance from the cell’s center as a Surface object created in Imaris to the capsule, was …
Figure 6 with 1 supplement
LFA1 and ICAM1 control innate-like lymphocyte access to the LN parenchyma from the SCS.
(A) Representative FACS histogram showing LFA1 staining of IL7RαhiCcr6+, Vγ4+Ccr6+ and naïve αβ T cells. (B) ICAM1 staining of WT and ICAM1 KO LN sections. (C) Adhesion of IL7RαhiCcr6+, Vγ4+Ccr6+ …
Figure 6—figure supplement 1
Effects of αL blockade on innate-like lymphocyte distribution.
(A) Frequency of IL7RαhiCcr6+ and Vγ4+Ccr6+ LN cells 6 hr after treatment with saline or αL blocking antibody. (B) Number of IL7RαhiCcr6+ cells in peripheral LNs and blood 3 days after treatment …
Figure 7 with 1 supplement
CD169 mediates SCS retention of innate-like lymphocytes.
(A) CD169-Fc binding of innate-like lymphocytes. (B) Effect of CD169-deficiency or blocking antibody treatment on macrophage bleb acquisition by IL7RαhiCcr6+ cells. Cells were stained to detect the …
Figure 7—figure supplement 1
Effects of CD169 blockade on innate-like lymphocyte properties and distribution.
(A) CD169 CD11b costaining of IL7RαhiCcr6+ cells from control and CD169-deficient LNs. (B) Frequency and Thy1-PE labeling of IL7RαhiCcr6+ and Vγ4+Ccr6+ LN cells in control and CD169-deficient mice. …
Figure 8
Model of requirements for innate-like lymphocyte surveillance of the LN SCS.
(A) Diagram of skin draining LN. (B) Model showing effect of Ccr6-deficiency on innate-like lymphocyte positioning and associated defect in ability to upregulate IL17 in response to IL1-family …
Videos
Video 1
Cxcr6-GFP+ cell shuttling between parenchyma and the SCS.
Representative intravital time-lapse imaging of the popliteal LNs from two Cxcr6GFP/+ mice. Overhead 3D video exemplifies the dynamic movement of Cxcr6-GFP+ cells (green) within the LN. …
Video 2
Representative examples of individual Cxcr6-GFP+ cells crossing into and out of SCS.
Intravital time-lapse imaging of the popliteal LN from a Cxcr6GFP/+ control mouse, highlighting one cell crossing from the parenchyma into the SCS, and one crossing from the SCS into the parenchyma. …
Video 3
Cxcr6-GFP+ cellular dynamics following FTY720 treatment.
One hour time-lapse imaging of a popliteal LN in a Cxcr6GFP/+ mouse 16 hr after treatment with FTY720. Cxcr6-GFP+ cells (green) can be seen accumulated on the parenchymal side of the SCS macrophages …
Video 4
Cxcr6-GFP+ cell fluttering in the SCS following αL blockade.
In Cxcr6GFP/+ mice, four hours after treatment with αL blocking antibody, Cxcr6-GFP+ cells (green) are observed to flutter at the floor of the SCS. Cxcr6-GFP+ cells appear attached to CD11b+ SCS …
Video 5
Increased movement of SCS Cxcr6-GFP+ cells after CD169 blockade and in CD169–/– mice.
Representative time-lapse images of Cxcr6GFP/+ mice 4 hr after treatment with CD169 blocking antibody, as well as Cxcr6GFP/+ CD169–/– mice. In both conditions, there appeared to be an increased …
Video 6
Decreased Cxcr6-GFP+ cell frequency in SCS following αL and CD169 double blockade.
Decreased numbers of Cxcr6-GFP+ cells (green) are observed in the SCS and LN parenchyma following dual antibody blockade of αL and CD169. Time-lapse imaging in a Cxcr6GFP/+ mouse beginning 4 hr post …
Video 7
Addition of CD169 blockade causes release of Cxcr6-GFP+ cells from floor of SCS when pre-treated with αL blocking antibody.
Representative time-lapse imaging beginning 5 min after treatment with CD169 blocking antibody in Cxcr6GFP/+ mice pretreated 4 hr before with αL blocking antibody. Upon addition of CD169 blockade, …
Additional files
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Source code 1
Matlab and R source code files used for computational analysis.
Three files are enclosed with the Matlab and R code used for the compuational analysis in the present paper: 1) spotsMinDistanceToSurface.m - Matlab File encoding an ImarisXT extension that can be used to find the minimum distance between a spot object and a surface object (used in the present paper to find the minimum distance between cells and the lymph node capsule); 2) SCSAnalyzeR_Core.R - R script containing code to import raw Imaris track lists (including depth statistic added by spotsMinDistanceToSurface.m), preform data processing, and create the plots displayed in the main and supplementary figures; 3) SCSAnalyzeR_Core_Functions.R - R script containing functions called by the main SCSAnalyzeR_Core.R script
- https://doi.org/10.7554/eLife.18156.027
