Antibody production and in vitro behavior of CD27-defined B-cell subsets: persistent hepatitis C virus infection changes the rules

Abstract

There is growing interest in the tendency of B cells to change their functional program in response to overwhelming antigen loading, perhaps by regulating specific parameters, such as efficiency of activation, proliferation rate, differentiation to antibody-secreting cells (ASC), and rate of cell death in culture. We show that individuals persistently infected with hepatitis C virus (HCV) carry high levels of circulating immunoglobulin G (IgG) and IgG-secreting cells (IgG-ASC). Thus, generalized polyclonal activation of B-cell functions may be supposed. While IgGs include virus-related and unrelated antibodies, IgG-ASC do not include HCV-specific plasma cells. Despite signs of widespread activation, B cells do not accumulate and memory B cells seem to be reduced in the blood of HCV-infected individuals. This apparent discrepancy may reflect the unconventional activation kinetics and functional responsiveness of the CD27+ B-cell subset in vitro. Following stimulation with T-cell-derived signals in the absence of B-cell receptor (BCR) engagement, CD27+ B cells do not expand but rapidly differentiate to secrete Ig and then undergo apoptosis. We propose that their enhanced sensitivity to BCR-independent noncognate T-cell help maintains a constant level of nonspecific serum antibodies and ASC and serves as a backup mechanism of feedback inhibition to prevent exaggerated B-cell responses that could be the cause of significant immunopathology.

FIG. 1.

Circulating nonspecific and virus-specific antibody levels in HD, PI, and SR. Sera were analyzed by either nephelometry or ELISA. (A) Ig concentrations (means ± standard errors of the means [SEM]). (B) Correlation between total Ig concentration and plasma viral load in PI. (C) Correlation between IgG concentration and plasma viral load in PI. (D) Plasma viral loads in PI with negative or positive RF test. (E and F) Diphtheria and tetanus antitoxin IgG concentrations (means ± SEM). (G and H) Endpoint titers of IgG against HCV core and NS3 proteins. (I and L) Correlation between titers of IgG against core and NS3 proteins and plasma viral load in PI.

FIG. 2.

Circulating nonspecific and virus-specific plasma cell frequencies in HD, PI, and SR. Freshly isolated PBMC were assayed by ELISPOT assay. (A and B) IgG- and IgM-ASC. (C) IgM-ASC in PI with negative or positive RF test. (D) Correlation between IgG-ASC number and plasma viral load in PI. (E and F) Proportions of DT- and TT-specific ASC among total IgG-ASC (means ± standard errors of the means). (G and H) Proportions of HCV core- and NS3-specific ASC among total IgG-ASC in PI and SR.

FIG. 3.

Circulating B-cell subsets in HD, PI, and SR. Whole-blood samples were immunostained and examined by flow cytometry. Lymphocytes were distinguished by forward and orthogonal light scatter characteristics. (A and B) Percentages of CD19⁺ and CD21⁺ cells among gated lymphocytes. (C) Representative flow cytometry analyses of CD19/CD27-stained PBMC. (D) CD27 expression on B cells. The numbers assigned to the patients are shown at left. (E) Absolute numbers of CD19⁺, CD19⁺ CD27⁻, and CD19⁺ CD27⁺ B cells (means ± standard errors of the means). (F) Correlation between CD27⁺ B-cell percentage and plasma viral load in PI.

FIG. 4.

In vitro proliferation of CD27⁻ and CD27⁺ B cells from HD, PI, and SR. B cells were separated into CD27⁺ and CD27⁻ fractions, labeled with CFSE, cultured with the indicated stimuli for 5 days, and analyzed by flow cytometry. (A) Representative immunomagnetic purification of CD27⁺ and CD27⁻ B cells. (B) Representative CFSE profiles. Sequential peaks of decreased fluorescence intensity identify subsequent generations of proliferating daughter cells. (C) Percentages of CD27⁺ B cells undergoing one or more divisions after stimulation (means ± standard errors of the means).

FIG. 5.

In vitro antibody secretion of CD27⁻ and CD27⁺ B cells from HD, PI, and SR. Purified CD27⁺ and CD27⁻ B cells were cultured with the indicated stimuli. Five days later, supernatants were analyzed by ELISA. (A) Nonspecific Ig concentrations. (B) HCV-specific antibody levels (optical density [OD]). The dashed horizontal line indicates the cutoff.

FIG. 6.

In vitro survival kinetics of B cells from HD, PI, and SR. Purified CD19⁺ cells were cultured with CD40L plus IL-4, stained with annexin V and anti-CD27 antibody, and analyzed by flow cytometry at different time points. Five thousand events were acquired for each sample, and dot plots represent one of eight individuals from each group. Numbers indicate the percentage of cells in each quadrant.

FIG. 7.

Apoptotic signals in cultured CD27⁺ B cells from HD, PI, and SR. Purified CD27⁺ cells were cultured with CD40L plus IL-4, stained with either JC-1 or DAPI, and examined by epifluorescence microscopy. (A) Representative micrographs of cells stained with JC-1 (top and middle rows) and DAPI (bottom row) after 2 and 5 days of stimulation, respectively. JC-1 fluoresces red (under green excitation) and yellow-orange (under blue excitation) in cell areas with high mitochondrial potential and green (under blue excitation) in areas of lower potential. DAPI forms blue fluorescent complexes with double-stranded DNA. Apoptotic cells can be identified by a decrease in JC-1 red fluorescence and an increase in JC-1 green fluorescence in the cytoplasm. Apoptotic nuclei can be identified by a condensed chromatin gathering at the periphery of the nuclear membrane or a total fragmented morphology of nuclear bodies. (B) High magnification of apoptotic CD27⁺ B cells from PI. Cytoplasmic accumulation of green monomeric JC-1 (left panel) and nuclear fragmentation (right panel) are shown.