B7-CD28 interaction promotes proliferation but not survival of NKT cells.

<p>5–7-week old WT, B7KO, CD28KO mice received intravenous injection of BrdU. The lymphocytes from the thymus (top) and spleen (bottom) were harvested 4 hours later and stained with antibodies for TCRβ, NK1.1 and BrdU or with Annexin V. A. NKT cells that underwent apoptosis. The numbers represent the percentages (Mean±SD) of apoptotic cells among TCRβ⁺NK1.1⁺ cells and summarizing data from 2 independent experiments involving a total of 6 mice in the WT and CD28 mutant group, and 3 in B7-1/2 mutant mice. B. NKT cells undergo proliferation <i>in vivo</i> as revealed by incorporation of BrdU. The numbers in the panels indicate means and SD (n = 3) of the percentages of NKT cells that have incorporated BrdU in the thymus. FITC conjugated mouse IgG₁, κ was used as isotype control (right). One representative profile from each group is shown.</p

Roles for B7-1/2-CD28 interaction in the accumulation of TCR β⁺NK1.1⁺ NKT cells in the thymus (A), spleen (B) and liver (C).

<p>B7KO, CD28KO and WT C57BL/6 mice were sacrificed at 8-week of age. Total lymphocytes (left column) from viable cells, CD4⁻CD8⁻ DN lymphocytes (middle column) and CD4⁺ lymphocytes (right column) were analyzed for the expression of TCRβ and NK1.1. One representative profile from each group is presented. The numbers in the panels are percentages (Mean±SD) of gated NKT cells (n = 9). The numbers shown on the side of figures are <i>p</i> value between two indicated groups.</p

Proportions of iVα14 NKT cells among TCR β⁺NK1.1⁺ cells isolated from the thymus, spleen and liver of WT, B7-1/2 and CD28 deficient mice.

<p>Total lymphocytes from the thymus (left), spleen (middle) and liver (right) of 8-week old WT (top), B7-1/2- (center) and CD28- (bottom) deficient mice were stained with TCRβ, NK1.1 and α-GalCer/CD1d tetramer. The numbers represent the percentage (Mean±SD, n = 9) of α-GalCer/CD1d⁺ cells in the TCR β⁺NK1.1⁺ population. One representative profile from each group is shown here. The numbers shown on the side of figures are <i>p</i> value between two indicated groups.</p

B7- and CD28-deficiency does not affect the generation of immature iVα14 NKT cells in young mice.

<p>Total lymphocytes from the thymus (upper panel) and liver (lower panel) of 8-day old WT (left), B7-1/2- (middle) and CD28- (right) deficient mice were analyzed. The numbers represent the percentage (Mean±SD, n = 4) of iVα14 NKT cells (A) and the percentage (Mean±SD, n = 4) of individual subsets from gated iVα14 NKT cells (B). One representative profile from each group is shown.</p

The reduction of IFN-γ production among TCR β⁺NK1.1⁺ cells isolated from the liver of B7-1/2 deficient mice.

<p>Eight-week old WT (top) and B7-1/2- (bottom) deficient mice received intravenous injection of Con A (25mg/kg). The mice were sacrificed six hours later. After perfusion the total number of mononuclear cells from the liver and spleen were stained with TCRβ, NK1.1, anti-mouse IFN-γ or IL-4 mAbs, or fluorescence conjugated isotype control. A. The percentage of total NKT cells (NK1.1⁺TCR β⁺) and conventional T cells (NK1.1⁺TCR β⁺) in the spleen and liver from Non-treated (left) and ConA treated mice (right). B. The percentage of cytokine producing cells among the NK1.1⁺TCR β⁺ population. C. The percentage of cytokine producing cells among the NK1.1⁻TCR β⁺ population. The numbers represent the percentage (Mean±SD) of one representative profile from each group is shown. The experiment was repeated twice; 7–8 mice were included in each group. D. Cytokine secretion of purified CD4 T cells from WT and B7KO mice stimulated by KRN7000. The numbers represent the profile of purified CD4 T cells from 3 mice in each group. The experiment was repeated twice.</p

Quantitative and phenotypic variation of iVα14 NKT cells in the thymus, spleen and liver of WT, B7-1/2 and CD28 deficient mice.

<p>Total lymphocytes from the thymus (left), spleen (middle) and liver (right) of 8-week old WT (top), B7-1/2 (center) and CD28 (bottom) deficient mice were analyzed. A. The quantification of iVα14 NKT cells. The numbers represent the percentage (Mean±SD, n = 9) of iVα14 NKT cells. B. The expression of CD44 and NK1.1 among the gated iVα14 NKT cells. The numbers represent the percentage (Mean±SD) of NK1.1⁻ iVα14 NKT cells. C. The expression of CD4 and NK1.1 from gated iVα14 NKT cells. The numbers represent the percentage (Mean±SD) of NK1.1⁻CD4⁺ (upper left) and NK1.1⁺CD4⁻ (lower right) iVα14 NKT cells. One representative profile from each group is shown here. The numbers shown on the side of figures are <i>p</i> value between two indicated groups.</p

Con A induced hepatitis is alleviated in B7- and CD28- deficient mice.

<p>Eight-week old B7KO, CD28KO and C57BL/6 mice received intravenous injection of Con A at a dose of 20 mg/kg. Sixteen hours later, serum and liver tissues from individual mice were harvested. A. Serum ALT and AST levels. B. One representative section from each group showing the pattern of liver injury. C. Average percentages of area with injuries from 3–4 sections per mouse and 4–5 mice per group.</p