Increased susceptibility of CD137^−/− mice during the resolution phase after 5 days of 2.0% DSS treatment as compared to WT mice.

<p>A, Percentage of body weight loss over the course of 5 days of treatment with 2.0% DSS water followed by 7 days of normal water. B, Kaplan-Meier-Analysis using a combined end-point of weight-loss above 20% of original weight/death in 5 days 2.0% DSS treated mice followed by 7 days of recovery phase with normal water. C, Colon length of mice untreated or treated for 5 days with 2.0% DSS followed by 3 days of normal water. Data are representative of at least three independent experiments, shown here are means ± SEM, n = 10–15 for DSS groups, n = 5 for water control groups (* p<0.05).</p

CD137^−/− and WT mice develop a similar histologic disease severity after 5 days of acute 2.0% DSS exposure, however, resolution of inflammation after withdrawal of DSS is impaired in CD137^−/− mice.

<p>A, Representative histologic colonic cross-sections of mice exposed to normal water (control), 5 days of 2.0% DSS water ((+)DSS Day 5) and 5 days of 2.0% DSS water followed by 3 days normal water ((+)DSS Day 8) stained with Hematoxylin-Eosin (20-fold magnification). B, Histology score of WT (black bars) and CD137^−/− (white bars) mice untreated, treated with 2.0% DSS for 5 days or mice treated with 2.0% DSS for 5 days followed by 3 days of normal water. Data are representative of two independent experiments, shown here are means ± SEM, n = 4 per group (*** p<0.001).</p

CD137^−/− mice have increased numbers of neutrophils in the lamina propria mononuclear cell (LPMNC) subpopulations while presenting lower macrophage numbers in the intraepithelial (IEL) subpopulation.

<p>A, Gating strategy for myeloid (top panel) and lymphocyte (lower panel) populations. B, Quantification of different immune cell populations in the LPMNC cells (B, C) and IEL cells (D, E) during the resolution phase of inflammation 3 days after withdrawal of 2.0% DSS. Data represent means ± SEM of 4 independent experiments pooled together, n = 3–10 per group (* p<0.05). Mo (Monocytes), Neutro (Neutrophils), Macro (Macrophages), DCs (Dendritic cells), NK (Natural Killer cells).</p

2.0% DSS-induced colitis leads to increased local colonic IFN-γ, GM-CSF, CXCL1 and IL-17 secretion in CD137^−/− versus WT mice.

<p>Colon segment culture supernatants of CD137^−/− (white bars) and WT mice (black bars) untreated (day 0), or treated either for 5 days with 2% DSS (day 5) or 5 days with 2% DSS followed by 3 days with normal water (day 8), were examined for IFN-γ (A), GM-CSF (B), IL-2 (C), TGF-§ (D), TNF-α (E), G-CSF (F), IL-17 (G), CXCL1 (H) and CXCL2 (I). Data are representative of two independent experiments, shown here are means ± SEM, n = 4 per group (* p<0.05, ** p<0.01).</p

Impaired HBcAg-specific interferon (IFN)-γ T-cell response in C57BL/6 mice with hepatitis B viral (HBV) persistence was reversed by treatment with anti-PD-1 mAb.

<p>C57BL/6 mice were hydrodynamically injected with WT pAAV/HBV1.2 or HBV core mutant DNA, including HBV-175 or HBV-38 constructs. Ten days after the injection, splenocytes were isolated and HBcAg-specific IFN-γ responses were analyzed by an ELISpot assay. The frequency of HBcAg-specific IFN-γ-secreting cells in the presence of an anti-PD-1 or control antibody were measured. Spot-forming cells per million splenocytes are shown. *<i>p</i><0.05.</p

Increased programmed death (PD)-1-expressing CD8+ and CD4+ T-cells in liver-infiltrating lymphocytes from mice with hepatitis B viral (HBV) persistence.

<p>A. C57BL/6 or BALB/c mice were hydrodynamically injected with the pAAV/HBV1.2 plasmid. Four weeks post-injection, intrahepatic lymphocytes were isolated and PD-1 expression was analyzed by flow cytometry. B and C. C57BL/6 mice were hydrodynamically injected with pAAV/HBV1.2. Four weeks after hydrodynamic injection, intrahepatic lymphocytes from mice which were HBsAg-positive (carrier) or HBsAg-negative (cleared) were isolated, and the PD-1 expressions by CD8⁺ (B) and CD4⁺ (C) T-ells were analyzed by flow cytometry. The data were representative of at least 12 independent experiments. D. C57BL/6 mice were injected with pAAV/HBV1.2 plasmid hydrodynamically. HBsAg-positive carrier were sacrificed at 4 weeks post-injection. Intrahepatic lymphocytes and splenocytes were isolated and the populations of CD4⁺PD-1⁺ or CD8⁺PD-1⁺ were determined by flow cytometry analysis. *<i>p</i><0.05. The data were representative of at least 6 independent experiments.</p

Blockade of the programmed death (PD)-1 pathway by an anti-PD-1 monoclonal antibody (mAb) reduced the hepatitis B viral (HBV) persistence rate and reversed PD-1^hiCD127^low-exhausted CD8+ T cells phenotype in a mouse animal model.

<p>A. C57BL/6 mice were intraperitoneally treated with an anti-PD-1 blocking mAb or isotype control Ab. Antibody administration was initiated every 2 days on day 6 before being injected with an HBV core-truncated (HBV-175) mutant construct. Thereafter, Abs (200 µg) were repeatedly administered every 3∼4 days for a period of 8 weeks. The rates of positive serum HBsAg in the mice receiving anti-PD-1 blocking mAb (•, n = 12) were compared with those in mice receiving isotype control Ab (○, n = 11), The HBsAg level in mice serum was determined by an ELISA. HBsAg-positive mice were defined as having a signal-to-noise (S/N) ratio of ≥2. B. The ALT levels in the mice receiving anti-PD-1 blocking mAb, isotype control Ab, and untreated. The ALT level in mice serum was determined by an ELISA. *<i>p</i><0.05. C and D. Increased CD127 expression and reversed PD-1^hiCD127^low-exhausted CD8+ T cells phenotype in mice treated with an anti-PD-1 mAb. Intrahepatic lymphocytes from C57BL/6 mice hydrodynamically injected with wild-type (WT) pAAV/HBV1.2 and HBsAg-positive 4 weeks after the injection were isolated, and the expressions of PD-1 (C) as well as coexpression of PD-1 and CD127 (D) on CD4+ and CD8+ T cells in the presence of an anti-PD-1 or control antibody were measured by flow cytometry. Serum HBsAg titers were determined by an ELISA. HBsAg-positive mice were defined as having a signal-to-noise (S/N) ratio of ≥2. *<i>p</i><0.05.</p

Liver-infiltrating CD8+ lymphocytes in carrier mice displayed the PD-1^hiCD127^low-exhausted phenotype.

<p>A and B. HBV core mutants induced upregulation of PD-1 expression in intrahepatic T lymphocytes. C57BL/6 mice were hydrodynamically injected with WT pAAV/HBV1.2 or HBV core mutant DNA, including HBV-175 or HBV-38 constructs. Four weeks after the injection, intrahepatic lymphocytes were isolated, and the PD-1 expressions by CD8⁺ (A) and CD4⁺ (B) T-cells were analyzed by flow cytometry. C. C57BL/6 mice were hydrodynamically injected with wild type hepatitis B virus (HBV-wt) and HBV core mutants. Intrahepatic lymphocytes were isolated at 4 weeks, and PD-1 and CD127 expressions by CD8⁺ T cells were analyzed by flow cytometry. D. C57BL/6 mice were hydrodynamically injected with HBV core mutant DNA, HBV-175. Intrahepatic infiltrating lymphocytes and splenic lymphocytes were isolated at 4 weeks, and PD-1 and CD127 expressions by CD8⁺ T cells were analyzed by flow cytometry. *<i>p</i><0.05. The data were representative of at least 6 independent experiments.</p

B7-H4 inhibits phosphorylation of AKT on different T cell subsets similarly.

<p>Naïve CD3⁺, CD4⁺, or CD8⁺ T-cell subsets were stimulated with 0.3 µg/ml of plate-bound anti-CD3 and soluble anti-CD28 (1 µg/ml) for 16 h and then rested for 30 h prior to re-activation. Activated CD3⁺, CD4⁺, or CD8⁺ T-cell subsets were stimulated with plate-bound anti-CD3 (5 µg/ml) and soluble anti-CD28 (2 µg/ml) for 10 min. Representative western blot of protein extracts from CD3⁺, CD4⁺, or CD8⁺ T-cell subsets were detected by phosphorylated AKT Ser473 (A) and quantitated by using Bio-Rad Quantity One program. The y-axis was normalized for the loading control. B7-H4 treatment significantly inhibited AKT phosphorylation at 10 min to a similar degree on different T-cell subsets (B). Data represent 3 independent experiments and are expressed as means ± SD from 3–4 mice per group.</p

B7-H4 inhibits T-cell proliferation.

<p>Naïve (A and B) and pre-activated (C,D, and E) CD3⁺ T cells were stimulated with various concentrations of plate-bound anti-CD3 and soluble anti-CD28 (1 µg/mL) for 72 h. 18 h before harvest, cultures were pulsed with 1 µCi of [3H]-thymidine. B7-H4.Ig or Fc.Ig was added at indicated concentrations (B and D). 10 µg/ml and 30 µg/ml of B7-H4.Ig or Fc.Ig was added for naïve and pre-activated T cells, respectively. Pre-activated CD3⁺, CD4⁺, or CD8⁺ T-cell subsets were stimulated with 0.3 µg/ml of plate-bound anti-CD3 and soluble anti-CD28 (1 µg/ml). Triplicate wells were harvested and counted. Data represent three independent experiments and expressed as means ± SEM. One star (*) indicates p<0.05, two stars (**) indicate p<0.01, and three stars (***) indicate p<0.001.”</p