Modulation of the NKG2D receptor during acute hepatitis. (A and C) NKG2D expression on the surface of NK1.1⁺ cells from HBV-Env⁺ RAG^−/− (heavy black line) and RAG^−/− (dotted line) before (A) and 3 days after (C) adoptive transfer of 10⁸ splenocytes. (B) Hepatic necrosis in these animals was assessed by the measurement of ALT in the sera of RAG^−/− (○) or HBV-Env⁺ RAG^−/− (●) mice. ALT values are shown as mean ± SEM. (D) Surface expression of NKG2D on intrahepatic NKT (heavy black line) and NK cells (dashed line) from HBV-Env⁺ RAG^−/− mice 3 days after adoptive transfer. The left dot plot depicts the isotype-matched control Ig staining of TCR on NKT cells. (E) Surface expression of NKG2D on NKT cells (heavy black line) and NK cells (dashed line) from the spleen and liver of wild-type mice. The shaded histograms depict staining using an isotype-matched control rat IgG1. All experiments were repeated at least three times, and representative data are shown.

Blocking NKG2D in vivo prevents the liver injury caused by the acute immune response to HBV. (A) Serum ALT levels of HBV-Env⁺ RAG^−/− mice treated with anti-NKG2D mAb (○) or rat IgG (●). The ALT values as mean ± SEM are shown. Student's t test analyses: *, P < 0.02; **, P < 0.01. (B) Hematoxylin and eosin-stained section (magnification ×20) of portal triads (Upper) and hepatic lobular parenchyma (Lower) from HBV-Env⁺ RAG^−/− mice treated with anti-NKG2D mAb (Left) or rat IgG (Right), 4 days after the adoptive transfer of 10⁸ splenocytes. Arrows point to necrotic hepatocytes, and asterisks indicate inflammatory infiltrate. (C) Elispot analyses of IFN-γ- and IL4-producing intrahepatic immune cells from HBV-Env⁺ RAG^−/− mice treated with control rat IgG (black bars) or anti-NKG2D mAb (white bars) at days 3 and 4 after adoptive transfer. Representative data are shown as mean ± SD. Student's t test analyses: *, P < 0.005; **, P < 0.02. All data are representative of at least three independent experiments.

Blocking an NKG2D–ligand interaction in HBV-Replication⁺ RAG^−/− mice prevents liver injury and cytokine production mediated by the acute immune response to HBV. (A) Serum ALT levels of HBV-Replication⁺ RAG^−/− mice treated with anti-NKG2D mAb (○) or rat IgG (●) at 2, 3, and 4 days after adoptive transfer of 1 × 10⁸ syngeneic splenocytes are shown as mean ± SEM. (B) NKG2D surface expression on intrahepatic NK1.1⁺ cells from HBV-Replication⁺ RAG^−/− mice (heavy black line) as compared with RAG^−/− mice (dotted line) at day 3 after the adoptive transfer of syngeneic naïve splenocytes. Shaded histogram depicts staining using the appropriate isotype-matched control Ig (rat IgG1). (C) Surface expression of NKG2D on intrahepatic NKT (heavy black line) and NK cells (lighter black line) from HBV-Replication⁺ RAG^−/− mice 3 days after adoptive transfer. The left dot plot depicts the isotype-matched control Ig staining of TCR on NKT cells. (D and E) Elispot analyses of IFN-γ (D) and IL-4-producing (E) intrahepatic immune cells from HBV-Replication⁺ RAG^−/− mice treated with rat IgG (black bars) or anti-NKG2D mAb (white bars) at day 3 after adoptive transfer. Representative data are shown as mean ± SD. Student's t test analyses: *, P < 0.001. All data are representative of at least two independent experiments.

Constitutive surface expression of the NKG2D ligand RAE-1 on hepatocytes is up-regulated specifically on HBV-Env-expressing hepatocytes, before adoptive transfer. (A) Surface expression of RAE-1 on hepatocytes from HBV-Env⁺ RAG^−/− (solid line) and RAG^−/− (dotted line) mice. Shaded histograms depict staining using the appropriate isotype-matched control Ig. (B) RAE-1 mRNA expression in hepatocytes from HBV-Env⁺ RAG^−/− (black bar) and RAG^−/− (white bar) mice and from intrahepatic (IH) immune cells and splenocytes from RAG^−/− mice in comparison with HPRT expression. All data are representative of at least three independent experiments.

NKG2D⁺ NKT cells are required for efficient disease induction and cytokine production during the acute immune response to HBV. (A) Hepatic injury of HBV-Env⁺ RAG^−/− mice at day 4 after adoptive transfer of 50 × 10⁶ NKG2D-depleted splenocytes (which included 1.25 × 10⁵ NKG2D⁻ NKT cells, and no NK cells) (open diamonds) was compared with hepatic injury in HBV-Env⁺ RAG^−/− mice that received the same total number of unsorted wild-type splenocytes (50 × 10⁶, which included 2.5 × 10⁵ unsorted NKT cells, and 1.25 × 10⁶ NK) (filled circles) (Mann–Whitney test analyses: P < 0.02) or that received the same total number of unsorted NKT cells and NK cells (33 × 10⁶, which included 1.25 × 10⁵ unsorted NKT cells, and no NK cells) (filled squares). (Mann–Whitney test analyses: P < 0.03). (B) Elispot analyses of IFN-γ producing intrahepatic immune cells from HBV-Env⁺ RAG^−/− mice depicted in A. Representative data are shown as mean ± SD. Student's t test analyses: *, P < 0.001.