Intravascular Immune Surveillance by CXCR6(+) NKT Cells Patrolling Liver Sinusoids

We examined the in vivo behavior of liver natural killer T cells (NKT cells) by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6(+) cells in liver, were found to crawl within hepatic sinusoids at 10–20 μm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance

Requirement for CXCR6 in Survival of CD1d-Reactive T Cells

<div><p>(A) Flow cytometry analysis of liver leukocytes from GFP knock-in mice after overnight culture. Dot plots represent GFP signal versus CD1d-α-GalCer-tetramer staining. Histogram plots represent Cell Tracker 633 labeling gated on tetramer-positive cells, indicating no cell division during the course of the culture.</p> <p>(B) Survival of liver NKT cells from <i>cxcr6^gfp/+</i> and <i>cxcr6^gfp/gfp</i> mice. Duplicate samples of 5 × 10⁴ CD1d-reactive T cells/well were incubated for each condition and time point. Histograms represent the percentage of viable, CD1d-α-GalCer-tetramer-positive GFP^hi cells, as determined by flow cytometry at the indicated time points. The proportion of CXCL16-cultured cells from control <i>cxcr6^gfp/+</i> mice was set at 100%. Values are mean ± standard deviation from three independent experiments.</p> <p>(C) Flow cytometry analysis of liver leukocyte apoptosis in culture. The percentage of cells binding Annexin V was determined by flow cytometry at the indicated time points. Results are representative of two independent experiments. A representative analysis at 10 h is shown on the right, with the percentage of CD1-αGC-tetramer-positive and Annexin V–positive cells indicated.</p></div

CXCR6⁺ Lymphocytes Stop Patrolling upon TCR Activation

<div><p>(A) Activation of NKT cells delivers stop signal. <i>cxcr6^gfp/+</i> and <i>cxcr6^gfp/gfp</i> mice were imaged before and after intravenous injection of antibody, lectin, or glycolipid antigen. Percentage of immobile cells was determined in 6-min videos either before any injection or 40 min after antigen delivery.</p> <p>(B) Histological examination of hepatic CD1d expression. Liver tissue from a Tie2-GFP transgenic was stained with anti-CD1d antibody (left panel, red) or an isotype control (inset). Green fluorescence indicates sinusoid-lining endothelial cells (expressing GFP) as well as the highly autofluorescent hepatocytes.</p> <p>(C) Flow-cytometric examination of hepatic CD1d expression. Cells isolated from liver tissue were stained with antibodies to endothelial cell markers (CD31 and Tie2), a Kupffer cell marker (F4/80), or a dendritic cell marker (CD11c) while co-stained with anti-murine CD1d (filled-in) or an isotype-matched control (dashed line).</p></div

CXCR6⁺ Lymphocytes Patrol Liver Sinusoids

<div><p>(A) Select confocal microscopic images from intravital videos of liver of an anesthetized <i>cxcr6^gfp/+</i> mouse (40× magnification). CD1d-reactive cells (bright green) can be seen migrating along hepatic sinusoids at an average speed of 16 μm/min. Scale bar is 25 μm. Cell tracks were traced and quantified using Volocity cell-imaging software. Note tracks of cells traveling in opposite directions in the same sinusoid (left side of image, at 0–5 versus 7–12 min).</p> <p>(B) Velocity quantification of GFP⁺ lymphocytes. Videos of liver in <i>cxcr6^gfp/+</i> and <i>cxcr6^gfp/gfp</i> mice (10× magnification) were analyzed for the velocity of GFP⁺ cells in 640 cell migration tracks for <i>cxcr6^gfp/+</i> mice and 574 cell migration tracks for <i>cxcr6^gfp/gfp</i> mice. Results demonstrate similar velocities of cells with the <i>cxcr6^gfp/+</i> (filled bars, average velocity 16.5 ± 8.3 μm/min) and <i>cxcr6^gfp/gfp</i> genotypes (unfilled bars, average velocity 18.4 ± 9.5 μm/min).</p> <p>(C) Analysis of directedness of cell migration. The same cell tracks as in (B) were analyzed for ratio of overall cell displacement to stepwise-summed path length. Results demonstrate similar degrees of directed crawling by cells from <i>cxcr6^gfp/+</i> (filled bars, average 0.44 ± 0.31) and <i>cxcr6^gfp/gfp</i> (unfilled bars, average 0.42 ± 0.30) mice.</p> <p>(D) Analysis of crawling relative to blood flow in peri-central vein areas of <i>cxcr6^gfp/+</i> and <i>cxcr6^gfp/gfp</i> mice. Histograms represent the frequency of cell movements made towards or away from nearby draining areas in likely close proximity to central veins (solid blue curve). The radial step of a cell is the cellular displacement along the axis defined by the cell's initial starting point to the central vein. The same step distances were assigned random orientations and the resultant data were plotted (red dashed curves).</p></div

CXCR6⁺ Liver CD1d-Reactive T Cells Bind CXCL16 and Are Localized within CXCL16⁺ Liver Sinusoids

<div><p>(A) Binding of CXCL16 by splenocytes and liver leukocytes from <i>cxcr6^gfp/+</i> and <i>cxcr6^gfp/gfp</i> mice. Splenocytes and liver leukocytes from <i>cxcr6^gfp/+</i> (+/−) and <i>cxcr6^gfp/gfp</i> (−/−) mice were stained with PE-conjugated CD1d tetramer loaded with α-GalCer (αGC), PerCp-conjugated anti-CD3 antibodies and CXCL16-Fc fusion protein, and Cy5-conjugated goat anti-human Fc. The two black curves represent duplicate samples from two different <i>cxcr6^gfp/+</i> mice.</p> <p>(B) Thymocytes, splenocytes, and liver leukocytes from <i>cxcr6^gfp/+</i> and <i>cxcr6^gfp/gfp</i> mice were stained with PE-conjugated CD1d tetramer loaded with α-GalCer and biotinylated anti-NK1.1 followed by PerCp-conjugated streptavidin and APC-conjugated anti-CD3 antibodies. Left panels are gated on CD3⁺ T cells and represent GFP fluorescence intensity on CD1d/α-GalCer-reactive T cells and on conventional (tetramer-negative) T cells. The right panels are gated on CD3⁻ cells and represent GFP fluorescence intensity on NK1.1⁺CD3⁻ NK cells. Numbers are percent obtained from a representative experiment among six performed.</p> <p>(C) Liver sinusoidal endothelial cells express CXCL16. Sections (10 μm thick) of PFA-fixed liver from wild-type mice (top panel, left), <i>cxcr6^gfp/+</i> mice (top panel, right), and Tie2-GFP mice (bottom panel) were stained with rat monoclonal antibody against mouse CXCL16 (Clone 10H7, IgG2a), or with an isotype control, followed by goat anti-rat antibody (F[ab′]2) conjugated to Cy3.</p> <p>(D) GFP^hi CD1d-restricted T cells within liver sinusoids. Sections (7 μm thick) of PFA-fixed liver from <i>cxcr6^gfp/+</i> mice were stained with rabbit polyclonal serum against caveolin-1 followed by goat anti-rabbit antibody conjugated to Cy3.</p> <p>(E) Flow cytometry analysis of cells harvested by perfusion of <i>cxcr6^gfp/+</i> liver with cold PBS–ethylenediamine tetra-acetic acid.</p></div

CD1d-Reactive NKT Cells Express CXCR6, Are Enriched in Liver, and Are Selectively Reduced in CXCR6-Deficient Mice

<div><p>Splenocytes, thymocytes, and lung, bone marrow, blood, and liver leukocytes from <i>cxcr6^gfp/+</i> (+/−) and <i>cxcr6^gfp/gfp</i> (−/−) mice were stained with one of the following combinations: K1.1 PE-conjugated antibodies and APC-conjugated anti-TCRβ; PE-conjugated CD1d tetramer loaded with α-GalCer (αGC) and APC-conjugated anti-TCRβ; or control unloaded tetramer and APC-conjugated anti-TCRβ.</p> <p>(A) Expression of CD3 and GFP by cells from different organs.</p> <p>(B) Reduction in CD1d -α-GalCer-tetramer-positive liver leukocytes in <i>cxcr6^gfp/gfp</i> mice.</p> <p>(C) Selective reduction of CD1d-reactive T cells in the liver of CXCR6-deficient mice. Results are mean ± standard deviation from four experiments.</p> <p>(D) Reduced ability of CXCR6-deficient NKT cells to accumulate in the liver of recipient mice. Thymocytes from <i>cxcr6^+/+</i> mice were co-transferred with thymocytes from either <i>cxcr6^gfp/+</i> or <i>cxcr6^gfp/gfp</i> littermates into TCRα-deficient mice. The phenotype of hepatic leukocytes 2 d after transfer is shown. Similar results were obtained at 3 days. The results shown are representative of three experiments.</p></div

Decreased Patrolling Efficiency and Decreased ConA Hepatitis Severity in CXCR6-Deficient Mice

<div><p>(A) Serum transaminase levels in <i>cxcr6^gfp/gfp</i>, <i>cxcr6^gfp/+</i>, and <i>cxcr6^+/+</i> littermates 12 h after a challenge with 20 mg/kg ConA IP. Results are pooled from two independent experiments. The horizontal line represents the upper limit for normal serum transaminase levels, as measured in unchallenged mice. Asterisk indicates a two-tailed student's <i>t</i>-test with a <i>p</i> < 0.05 in comparison with littermates.</p> <p>(B) Hematoxylin and eosin staining of paraffin-embedded liver sections from <i>cxcr6^gfp/+</i> and <i>cxcr6^gfp/gfp</i> mice sacrificed 12 h after a challenge with 20 mg/kg ConA IP.</p> <p>(C) Reduced sinusoid patrolling by NKT cells in <i>cxcr6^gfp/gfp</i> mice. By measuring the average inter-nuclei distance of hepatocytes in high-magnification images, the average sinusoidal length of a hepatocyte was determined to be 28.6 μm (data not shown). Utilizing the lymphocyte velocity data (left panel) and assuming that a CD1d-reactive T cell can contact only one hepatocyte at a time, we calculated that each CD1d-reactive T cell can visit 0.58 hepatocytes/min in the <i>cxcr6^gfp/+</i> mice, and 0.64 hepatocytes/min in the CXCR6-deficient mice. The density of GFP⁺ cells in <i>cxcr6^gfp/+</i> and <i>cxcr6^gfp/gfp</i> mice (middle panel) was calculated from the intravital videos used in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030113#pbio-0030113-g003" target="_blank">Figure 3</a> and the flow cytometry data in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030113#pbio-0030113-g002" target="_blank">Figure 2</a>. Combining the similar crawling velocities with the different steady-state densities, we show decreased rate of patrolling by GFP⁺ lymphocytes, expressed as the average time between “visitation” of any single hepatocyte (right panel).</p></div