Table 1.
Clinical characteristics of HCV+ patients and healthy controls in study.
Figure 1.
Gating strategy for flow cytometry analysis of memory and naïve B cell activation.
Lymphocytes were gated by FSC/SSC properties and memory B cells (CD19+CD27+) and naïve B cells (CD19+CD27−) were analyzed within the lymphocyte gate for the expression of 6 different markers described in the Materials and Methods. Histograms show a representative example of the expression of CD183 (numbers represent geometric mean fluorescent intensity) on memory and naïve B cells from a healthy control (HCV-) and chronic HCV patient (HCV+).
Figure 2.
B cell subset frequencies are unchanged in HCV patients compared to controls.
(A–C) 7.5×105 freshly isolated PBMCs from chronically infected HCV patients (n = 54) or healthy controls (n = 50) were incubated with fluorescently labeled antibodies to CD19, CD27 and CD5 for multi-color flow cytometry analysis. (A) The total percent of CD19+ B cells within the lymphocyte gate and number of B cells per ml of blood based on our isolations. (B) The percent of memory (CD19+CD27+) and naïve (CD19+CD27−) and number of each subset per ml of blood. (C) The percent and number per ml of CD5+CD19+ B cells. Horizontal lines on graphs represent means +/− SEM (percentages) or median (number of cells/ml) values. In (C) only 48 HCV and 47 healthy control samples were tested for CD5. *, P<0.05; n.s., not significant.
Figure 3.
Memory, but not naïve B cells are significantly activated in chronically infected HCV patients.
PBMCs were freshly isolated and analyzed by flow cytometry as in Figure 2. Antibodies to CD183, CD71, CD86, CD69, HLA-DR and CD40 were added in combinations with CD19 and CD27 to gate on memory (CD19+CD27+) and naïve (CD19+CD27−) B cells. The percent positive was calculated based on mouse isotype controls and the geometric mean fluorescent intensities (MFI) for each marker were determined. HLA-DR and CD40 are expressed by all B cells therefore only the MFI is shown. Horizontal lines on graphs represent median values. For CD71 analysis, only 48 HCV and 47 healthy control samples were stained. ***, P<0.001, **, P<0.01, *, P<0.05, n.s., not significant.
Table 2.
Geometric mean fluorescent intensities of each marker on memory and naïve B cells.
Table 3.
Summary of significant correlations between B cell activation marker expression and liver clinical tests.
Figure 4.
PBMC and B cell quantification and clinical characteristics of cryoglobulin positive versus cryoglobulin negative HCV patients.
(A–B) PBMCs were freshly isolated and analyzed by flow cytometry as in Figure 2 except that this analysis compares cryoglobulin+ (n = 20, cryo+) and cryoglobulin- (n = 34, cryo-) HCV patients. (A) The total percent of CD19+ B cells within the lymphocyte gate and number of B cells per ml of blood based on our isolations. (B) The percent of memory (CD19+CD27+) and naïve (CD19+CD27−) and number of each subset per ml of blood. (C) The number of total PBMCs isolated per ml of blood. (D) The alanine aminotransferase (ALT) and gamma-glutamyl transpeptidase (GGT) levels were measured in the serum in units/L (U/L) for 54 (ALT) or 48 (GGT) HCV patients. (E) Serum HCV RNA titers were quantified by qRT-PCR as described in the Materials and Methods for all 54 HCV patients. Horizontal lines on graphs represent mean +/− SEM (A-B, percentages, C) or median (A–B numbers, D, E) values. *, P<0.05, n.s., not significant. IU/ml, international units/ml.
Figure 5.
Memory, but not naïve B cells from cryoglobulin positive HCV patients express higher levels of activation markers compared to B cells from cryoglobulin negative HCV patients.
PBMCs were freshly isolated and analyzed by flow cytometry as in Figure 3 except that this analysis compares cryoglobulin+ (n = 20, cryo+) and cryoglobulin- (n = 34, cryo-) HCV patients. Antibodies to CD86, CD71, HLA-DR and CD183 were added in combinations with CD19 and CD27 to gate on memory (CD19+CD27+) and naïve (CD19+CD27−) B cells. The percent positive was calculated based on mouse isotype controls and the geometric mean fluorescent intensities (MFI) for each marker were determined. Horizontal lines on graphs represent median values. For CD71 analysis, 18 cryo+ and 30 cryo- HCV samples were stained. ***, P<0.001, **, P<0.01, *, P<0.05, n.s., not significant.
Figure 6.
HCV patients with advanced fibrosis have increased expression of CD86 on memory B cells.
Fibrosis scores were determined as outlined in the Materials and Methods, where scores of 3–4 are considered advanced fibrosis/cirrhosis (F4). PBMCs were freshly isolated and analyzed by flow cytometry as in Figure 3. The expression level and percent positive of cells expressing CD86 (geometric mean fluorescent intensity (MFI) or percent positive compared to mouse isotype controls) were calculated for memory (CD19+CD27+) and naïve (CD19+CD27−) B cells. Horizontal lines represent median values. *, P<0.05; n.s., not significant.