1. Volume 143, Issue 6, Pages 1586-1596.e8 (December 2012)
Altered Functions of Plasmacytoid Dendritic Cells and Reduced Cytolytic Activity of Natural Killer Cells in Patients With Chronic HBV Infection 
Jeremie Martinet, Tania Dufeu–Duchesne, Juliana Bruder Costa, Sylvie Larrat, Alice Marlu, Vincent Leroy, Joel Plumas, Caroline Aspord 
Gastroenterology 
Volume 143, Issue 6, Pages e8 (December 2012)
DOI: /j.gastro
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2. Figure 1
Higher activation levels in pDCs from blood and liver from patients with chronic HBV compared with noninfected donors. Flow cytometry analysis of pDC frequency and phenotype from PBMCs and liver biopsy specimens from patients with chronic HBV or noninfected donors. (A) pDC gating strategy (representative patient with HBV). (B) Percentage of pDCs. Open circles, noninfected donors (blood, n = 25; liver, n = 8); filled circles, patients with chronic HBV (blood, n = 27; liver, n = 6). (C) Coactivation molecules CD40 and CD86 on pDCs from blood and liver. One representative patient with HBV for each sample (gated on HLA-DR+BDCA2+ cells). (D and E) Percentage of pDCs expressing coactivation molecules CD40 and CD86 in (D) blood (healthy donors, n = 22; patients with chronic HBV, n = 27) and (E) liver (noninfected donors, n = 8; patients with chronic HBV, n = 5).
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3. Figure 2
Impaired pDC activation in response to TLR9-L stimulation in patients with chronic HBV irrespective of viral load. PBMCs from healthy donors or patients with chronic HBV were stimulated with CpGA for 24 hours. IFN-α, IP-10, IL-6, and tumor necrosis factor–α secretion was measured by CBA in culture supernatants. Open symbols, healthy donors (n = 11–18); gray symbols, aviremic patients with chronic HBV (n = 7); black symbols, viremic patients with chronic HBV (n = 10). P values were calculated using the 2-way RM-ANOVA test (straight line) or the Mann–Whitney test (dashed lines).
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4. Figure 3
CpG-matured pDCs from viremic patients with chronic HBV inefficiently trigger NK cell cytotoxicity. pDCs isolated from healthy donors and aviremic and viremic patients with chronic HBV were cocultured with heterologous NK cells for 18 hours in the presence or absence of CpGA. (A and B) CD69 expression measured by flow cytometry on CD56+CD3− NK cells. (A) Representative healthy donor and patient with chronic HBV (gated on NK cells). (B) Percentage of NK cells expressing CD69 (healthy donors [n = 8], aviremic patients with chronic HBV [n = 7], and viremic patients with chronic HBV [n = 5]). (C) IFN-γ and granzyme B secretion as measured by CBA (healthy donors [n = 16], aviremic patients with chronic HBV [n = 28], and viremic patients with chronic HBV [n = 29]). (D and E) NK cells were further cocultured with K562 for 3 hours before measuring CD107 surface expression. (D) Representative flow cytometry profiles (gated on NK cells). (E) Percentages of CD107+ NK cells (healthy donors [n = 16], aviremic patients with chronic HBV [n = 35], and viremic patients with chronic HBV [n = 18]). P values were calculated using the 2-way RM-ANOVA test (straight line) or the Mann–Whitney test (dashed lines).
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5. Figure 4
NKR modulation on NK cells by CpG-matured pDCs. pDCs isolated from healthy donors (HD) and aviremic and viremic patients with chronic HBV were cocultured with heterologous NK cells for 18 hours in the presence of CpGA. Percentages of NKR expression were measured by flow cytometry on CD56+ CD3− NK cells (healthy donors [n = 12), aviremic patients with chronic HBV [n = 14], and viremic patients with chronic HBV [n = 15]). (A) Expression of NKG2A, NKG2C, NKG2D, and NKp30. (B) Expression of CD158ah, CD158b1b2j, and CD158e1e2. P values were calculated using the Mann–Whitney test (dashed lines).
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6. Figure 5
Impaired OX40L expression by pDCs from viremic patients with chronic HBV correlates with viral load and HBsAg. Expression of the costimulatory molecules 4-1BBL, ICOS-L, GITRL, and OX40L by blood pDCs from healthy donors or patients with chronic HBV was determined by flow cytometry. (A) Percentages of pDCs expressing the costimulatory molecules within PBMCs (healthy donors [n = 9], aviremic patients with chronic HBV [n = 11], and viremic patients with chronic HBV [n = 8]). P values were calculated using the Mann–Whitney test (dashed lines). (B and C) Correlation between OX40L expression level on pDCs and (B) viral load or (C) HBsAg (n = 16–18 patients with chronic HBV, Spearman correlation).
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7. Figure 6
IFN-α secretion and OX40L expression by pDCs mediate activation of NK cell cytotoxicity. (A and B) pDCs purified from aviremic or viremic patients with chronic HBV were cocultured with heterologous NK cells for 18 hours. CpGA and/or rhIFNα and sOX40L were added to cultures where indicated. NK cells were further cocultured with K562 before measuring CD107 surface expression. (A) Representative flow cytometry profiles from aviremic and viremic patients with chronic HBV (gated on NK cells). (B) Percentages of CD107+ NK cells obtained from aviremic (n = 22) and viremic (n = 10) patients with chronic HBV. (C–F) pDCs were purified from healthy donors and incubated for 24 hours in plasma from healthy donors and aviremic or viremic patients with chronic HBV before coculture with NK cells for 18 hours. CpGA and/or rhIFNα and sOx40L were added where indicated. NK cells were further cocultured with K562 before measuring CD107 surface expression. (C) Representative flow cytometry profiles (gated on NK cells). (D) Percentages of CD107+ NK cells obtained with plasma from 17 healthy donors, 12 aviremic patients with chronic HBV, and 17 viremic patients with chronic HBV. (E and F) Percentages of CD107+ NK cells according to the presence of (E) HBeAg and (F) HBsAg level in plasma from patients with chronic HBV. P values were calculated using the 2-way RM-ANOVA test (straight line) or the Mann–Whitney test (dashed lines).
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8. Figure 7
High plasma IP-10 concentrations in viremic patients with chronic HBV may explain OX40L down-regulation on circulating pDCs and the subsequent impaired pDC/NK interplay. (A) Plasma IP-10 concentration was measured in healthy donors (n = 25), aviremic patients with chronic HBV (n = 19), and viremic patients with chronic HBV (n = 19). P values were calculated using one-way ANOVA. (B) CXCR3 expression on pDCs within PBMCs from healthy donors (n = 14), aviremic patients with chronic HBV (n = 11), and viremic patients with chronic HBV (n = 5). Statistical significance was determined using Mann–Whitney test. (C) pDCs were purified from healthy donors and cultured for 24 hours in the presence of rhIP-10 (0–5000 ng/mL). (C) CXCR3 and OX40L expression on pDCs was measured by flow cytometry and expressed as a percentage of modulation compared with control conditions (P values calculated using one-way ANOVA).
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9. Supplementary Figure 1
Activation of pDCs in response to TLR7-L stimulation in patients with chronic HBV. PBMCs from healthy donors or patients with chronic HBV were stimulated with inactivated influenza virus (Flu) for 24 hours. IFN-α, IP-10, IL-6, and tumor necrosis factor–α secretion were measured by CBA in culture supernatants. Open symbols, healthy donors (n = 11); gray symbols, aviremic patients with chronic HBV (n = 7); black symbols, viremic patients with chronic HBV (n = 10). P values were calculated using the 2-way RM-ANOVA test (straight line) or the Mann–Whitney test (dashed lines).
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10. Supplementary Figure 2
Correlations between NK cell function on CpG-matured pDC stimulation and clinical parameters. pDCs isolated from aviremic and viremic patients with chronic HBV were cocultured with heterologous NK cells for 18 hours in the presence or absence of CpGA. NK cells were then evaluated for their cytotoxic activity using CD107 degranulation assay. (A and B) NK cytotoxic activity according to (A) the presence or not of HBeAg (n = 48, HBeAg negative; n = 6, HBeAg positive) and (B) the amount of HBsAg in the serum of the patients (n = 7, 17, 22, and 8, respectively). P values were calculated using the 2-way RM-ANOVA test. (C) Correlation between NK cell function and patients' viral load (n = 55). Spearman correlation was used.
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11. Supplementary Figure 3
pDC expression of costimulatory molecules. Expression of the costimulatory molecules 4-1BBL, ICOS-L, GITRL, and OX40L by pDCs from healthy donors (left panels) or patients with chronic HBV (right panels) measured by flow cytometry (gated on HLA-DR+ BDCA2+ cells).
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12. Supplementary Figure 4
IFN-γ and granzyme B secretion by NK cells in response to pDC stimulation. (A and B) pDCs purified from aviremic or viremic patients with chronic HBV were cocultured with heterologous NK cells for 18 hours with or without CpGA and/or rhIFNα and sOX40L where indicated. Supernatants were harvested and assayed by CBA for (A) IFN-γ and (B) granzyme B. (C and D) pDCs were purified from healthy donors and incubated for 24 hours in plasma from healthy donors and aviremic or viremic patients with chronic HBV. Cells were then cocultured with heterologous NK cells for 18 hours with or without CpGA and/or rhIFNα and sOX40L where indicated. Supernatants were harvested and assayed by CBA for (C) IFN-γ and (D) granzyme B. P values were calculated using the 2-way RM-ANOVA test.
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13. Supplementary Figure 5
Correlations between NK cell function induced by pDCs preincubated with plasma from patients with HBV and clinical parameters. pDCs were purified from healthy donors and incubated for 24 hours in the presence of plasma from aviremic or viremic patients with chronic HBV. pDCs were then cocultured with heterologous NK cells for 18 hours in the presence of CpGA. NK cell cytotoxic activity was evaluated by CD107 degranulation assay. (A) Correlation between the percentage of CD107+ NK cells and the amount of HBsAg found in the plasma (n = 24). (B) Correlation between the percentage of CD107+ NK cells and the viral load of the plasma for plasma with a positive viral load (n = 17). Spearman correlation was used.
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14. Supplementary Figure 6
IP-10 found in the plasma of patients with HBV is bioactive. (A) Down-regulation of CXCR3 on pDCs. pDCs purified from healthy donors were cultured for 24 hours in the presence of 5000 ng/mL rhIP-10 (n = 17), 100 ng/mL rhIP-10 (n = 10), or plasma from aviremic (n = 31) or viremic (n = 31) patients with HBV. CXCR3 expression on pDCs was measured by flow cytometry and expressed as a percentage of modulation compared with control conditions; P values were calculated using Kruskal–Wallis test. (B and C) Chemotactic activity. The chemotactic activity of rhIP10 and plasma from healthy donors or patients with chronic HBV for PHA-activated PBMCs was measured in Transwell migration assays and expressed as a fold induction compared with control conditions. (B) Migration index obtained with rhIP10 (n = 9), plasma from healthy donors (n = 15), and plasma from aviremic (n = 15) or viremic (n = 18) patients with HBV. (C) Migration index obtained after preincubation of rhIP10 (n = 8) or plasma from aviremic and viremic patients with HBV (n = 14) with DPP4 to generate the inactive (3-77) form of IP10; P values were calculated using Wilcoxon paired t test.
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