Immune response pattern varies with the natural history of chronic hepatitis B

Figure 1 Frequencies and absolute number of NK cells, CD4+ and CD8+ T cells in different clinical phases. A: Representative dot plots depicting the gating strategy for CD3-CD56+ NK cells, CD56^bright and CD56^dim subsets; B: Frequencies and absolute number of circulating NK cells in different phases; C: Frequencies of CD56^bright and CD56^dim NK cell subsets in different phases; D: Absolute number of CD56^bright and CD56^dim NK cell subsets in different phases; E: Frequencies of CD4+ and CD8+ T cells in lymphocytes in different phases; F: Absolute number of CD4+ and CD8+ T cells in different phases. All data are presented as mean ± SD. NK: Natural killer; HD: Healthy honors; IT: Immune tolerant; IA: Immune active; IC: Inactive carrier; ENEG: Hepatitis B envelope antigen-negative hepatitis.

Figure 2 Phenotypes of NK cells in different clinical phases. A: Frequency of CD56^bright and CD56^dim NK cells expressing the activating receptor NKp46; B: Frequency of CD56^bright and CD56^dim NK cells expressing the activating receptor NKp44; C: Frequency of CD56^bright and CD56^dim NK cells expressing the activating receptor NKp30; D: Frequency of CD56^bright and CD56^dim NK cells expressing the inhibitory receptor NKG2A; E: Frequency of CD56^bright and CD56^dim NK cells expressing the activating receptor NKG2D; F: Frequency of CD56^bright and CD56^dim NK cells expressing the activating marker CD69. NK: Natural killer; HD: Healthy honors; IT: Immune tolerant; IA: Immune active; IC: Inactive carrier; ENEG: Hepatitis B envelope antigen-negative hepatitis. All data are presented as mean ± SD, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 3 NK cell functions in different clinical phases. For detecting NK cell functions, peripheral blood mononuclear cells were stimulated by IL-12 and IL-18, P/I, K562 target cells or IL-12/18 and K562 target cells. A: Gated on CD3^-CD56⁺ NK cells, representative dot plots depicting the expression of IFN-γ, TNF-α, CD107a, perforin and granzyme B in NK cells; B: Pooled data showing the frequency of NK cells expressing IFN-γ and TNF-α at different phases; C: Pooled data showing the differences in NK cell CD107a degranulation in different phases; D: Summary data showing the frequency of NK cells producing perforin and granzyme B in different phases. IFN-γ: Interferon-gamma; TNF-α: Tumor necrosis factor-alpha; NK: Natural killer; HD: Healthy honors; IT: Immune tolerant; IA: Immune active; IC: Inactive carrier; ENEG: Hepatitis B envelope antigen-negative hepatitis; P/I: Phorbol myristate acetate and ionomycin. All data are presented as mean ± SD, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 4 Global-T cell cytokine production in different clinical phases. Peripheral blood mononuclear cells were stimulated by phorbol myristate acetate and ionomycin. A: Representative dot plots depicting the expression of IFN-γ, IL-2 and TNF-α in global CD3+ T cells; B: Cumulative data showing the frequency of global CD3+ T cells expressing IFN-γ, IL-2 and TNF-α in healthy donors and patients in different clinical phases. IFN-γ: Interferon-gamma; TNF-α: Tumor necrosis factor-alpha; HD: Healthy donors; IT: Immune tolerant; IA: Immune active; IC: Inactive carrier; ENEG: Hepatitis B envelope antigen-negative hepatitis. All data are presented as mean ± SD, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 5 Virus-specific T cell responses after in vitro expansion in different clinical phases. Peripheral blood mononuclear cells were incubated with core or S peptide pools. After 10 d in vitro culture, virus-specific T cell responses were determined by detecting the frequency of T cells producing interferon-gamma (IFN-γ) and IL-2. A: Gated on CD3+ lymphocytes, representative dot plots depict the frequency of CD4+ (upper-left quadrant) and CD8+ (upper-right quadrant) T cells-producing IFN-γ or IL-2; B: IFN-γ production by CD4+ and CD8+ T cells in response to the core peptide pool; C: IL-2 production by CD4+ and CD8+ T cells in response to the core peptide pool; D: IFN-γ and IL-2 production by CD3+ T cells in response to the core peptide pool; E: Positive responses of CD4+ or CD8+ T cells producing IFN-γ or IL-2 in response to the core peptide pool; F: IFN-γ production by CD4+ and CD8+ T cells in response to the S peptide pool; G: IL-2 production by CD4+ and CD8+ T cells in response to the S peptide pool; H: IFN-γ and IL-2 production by CD3+ T cells in response to the S peptide pool; I: Positive responses of CD4+ or CD8+ T cells producing IFN-γ or IL-2 in response to the S peptide pool. IFN-γ: Interferon-gamma; TNF-α: Tumor necrosis factor-alpha; HD: Healthy donors; IT: Immune tolerant; IA: Immune active; IC: Inactive carrier; ENEG: Hepatitis B envelope antigen-negative hepatitis. All data are presented as mean ± SD, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

Figure 6 Longitudinal analysis of NK and T cell responses in representative individuals who transitioned from one phase to another. Patient-1, a patient who transitioned from the immune tolerant to immune active phase. Patient-3, a patient who experienced spontaneous HBeAg clearance. Patient-5, a patient in the clinical phase changed from inactive carrier to HBeAg-negative hepatitis phase. A: Dynamic fluctuations of clinical parameters from one phase to another, including alanine aminotransferase, hepatitis B virus DNA and hepatitis B surface antigen; B: Phenotypic and functional changes of natural killer cells in representative patients who transitioned from one phase to another; C: Changes of interferon-gamma production in global T cells, and core or S peptide pool-stimulated T cells. NK: Natural killer; HBV: Hepatitis B virus; HBsAg: Hepatitis B surface antigen; HBeAg: Hepatitis B envelope antigen; ALT: Alanine aminotransferase; IFN-γ: Interferon-gamma; TNF-α: Tumor necrosis factor-alpha; HD: Healthy honors; IT: Immune tolerant; IA: Immune active; IC: Inactive carrier; ENEG: Hepatitis B envelope antigen-negative hepatitis.