Intravascular Immune Surveillance by CXCR6+ NKT Cells Patrolling Liver Sinusoids (original) (raw)
Figure 1
CXCR6+ Liver CD1d-Reactive T Cells Bind CXCL16 and Are Localized within CXCL16+ Liver Sinusoids
(A) Binding of CXCL16 by splenocytes and liver leukocytes from cxcr6gfp/+ and cxcr6gfp/gfp mice. Splenocytes and liver leukocytes from cxcr6gfp/+ (+/−) and cxcr6gfp/gfp (−/−) 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 cxcr6gfp/+ mice.
(B) Thymocytes, splenocytes, and liver leukocytes from cxcr6gfp/+ and cxcr6gfp/gfp 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.
(C) Liver sinusoidal endothelial cells express CXCL16. Sections (10 μm thick) of PFA-fixed liver from wild-type mice (top panel, left), cxcr6gfp/+ 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.
(D) GFPhi CD1d-restricted T cells within liver sinusoids. Sections (7 μm thick) of PFA-fixed liver from cxcr6gfp/+ mice were stained with rabbit polyclonal serum against caveolin-1 followed by goat anti-rabbit antibody conjugated to Cy3.
(E) Flow cytometry analysis of cells harvested by perfusion of cxcr6gfp/+ liver with cold PBS–ethylenediamine tetra-acetic acid.