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>