Activation of naïve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders

Abstract

Infection with hepatitis C virus (HCV), a leading cause of chronic liver diseases, can associate with B lymphocyte proliferative disorders, such as mixed cryoglobulinemia and non-Hodgkin lymphoma. The major envelope protein of HCV (HCV-E2) binds, with high affinity CD81, a tetraspanin expressed on several cell types. Here, we show that engagement of CD81 on human B cells by a combination of HCV-E2 and an anti-CD81 mAb triggers the JNK pathway and leads to the preferential proliferation of the naïve (CD27-) B cell subset. In parallel, we have found that B lymphocytes from the great majority of chronic hepatitis C patients are activated and that naïve cells display a higher level of activation markers than memory (CD27+) B lymphocytes. Moreover, eradication of HCV infection by IFN therapy is associated with normalization of the activation-markers expression. We propose that CD81-mediated activation of B cells in vitro recapitulates the effects of HCV binding to B cell CD81 in vivo and that polyclonal proliferation of naïve B lymphocytes is a key initiating factor for the development of the HCV-associated B lymphocyte disorders.

Fig. 1.

CD81 engagement induces human B cell proliferation. B lymphocytes purified from healthy donor PBMCs were cultured for 5 days in the presence of increasing concentrations of MG81 mAb (0.15 to 10 μg/ml) and 5 μg/ml HCV-E2 (♦), N81 mAb (▪), anti-CD19 (▴), or anti-CD21(•) mAbs (A); MG81 F(ab′)₂ (0.15-10 μg/ml) and 5 μg/ml N81 F(ab′)₂ (B); CD81-LEL (0.03-5 μg/ml) combined with MG81 plus HCV-E2 (□)(5 μg/ml each), goat anti-human IgM F(ab′)₂ fragments (▵) (10 μg/ml), or SAC (○) (1:30,000, vol/vol) (C). Cell proliferation was assessed by [³H]thymidine incorporation. In A and B, data are shown as the mean of cpm × 10³± SD of triplicate wells. In C, data are expressed as the percentage of inhibition of the B cell proliferation rate calculated in the absence of CD81-LEL. Data are representative of at least five different experiments performed with independent donors.

Fig. 2.

CD81 engagement induces the preferential expansion of naïve B lymphocytes. Purified naive (CD27^-) or memory (CD27⁺) B cells were incubated with complete medium (Control), MG81 and N81 mAbs (5 μg/ml each) (CD81), or SAC (1:30,000, vol/vol). (Top) Proliferation was measured as CFSE dilution (x axis) by flow cytometry after 4 days of stimulation. For each sample, 5 × 10⁵ events were acquired. Numbers indicate the percentage of dividing cells. (Middle) Surface expression of CD27 (y axis) and CD71 (x axis) after 3-day stimulation was measured by flow cytometry. For each sample, 1 × 10⁵ events were acquired, and numbers in quadrants represent percentages of CD27- and CD71-positive cells. (Bottom) IgM and IgG secretion in cell-culture supernatants was measured 10 days after stimulation by ELISA. Data are shown as mean ± SD of duplicate wells and are representative of at least five experiments performed with independent donors.

Fig. 3.

CD81-mediated signaling in human primary B cells. (A) Tonsil-derived B cells were stimulated with 5 μg/ml MG81 plus 5 μg/ml N81 (CD81) or with 10 μg/ml anti-IgM (IgM). After the indicated time, equal amounts of protein extracts where subjected to Western blot analysis using anti-phospho-tyrosine 4G10 (pTyr), anti-phospho c-Jun (pJun), anti-phospho JNK1 and -2 (pJNK1 and pJNK2), and anti-total JNK1 (JNK1) antibodies. Protein standards are expressed on the left in kDa. The asterisk indicates an unidentified protein cross-reacting with the anti-phospho JNK1 and -2 antibody. (B) Tonsil-derived B cells were stimulated as described above for the indicated times, and protein extracts were subjected to Western blot analysis using anti-phospho-CD19 (pCD19) and anti-total CD19 (CD19) antibodies. (C) Tonsil-derived B cells were stimulated with 5 μg/ml MG81, 5 μg/ml N81, or 5 μg/ml both MG81 and N81 mAbs. After the indicated time, Western blot analysis was performed by using pJun, anti total c-Jun (Jun), pCD19, and CD19 antibodies. ctr, untreated cells.

Fig. 4.

HCV patients display high percentage of activated naïve B cells. B lymphocytes from HCV patients (HCV) and healthy subjects (Control) were analyzed ex vivo for the surface expression of activation markers (CD69, CD71, and CD86) and CXCR3 by flow cytometry. For each sample, 2 × 10⁵ events were acquired. Data is shown as the percentage of naïve B cells (CD19⁺/CD27^-) or memory B cells (CD19⁺/CD27⁺) expressing the indicated molecules (mean ± SD). Numbers on bars represent the ratio between the level of expression of each marker in HCV patients over control.

Fig. 5.

Eradication of HCV infection associates with a decreased expression of activation markers and CXCR3. Ex vivo expression of activation molecules (CD69, CD71, and CD86) and the chemokine receptor CXCR3 was measured on peripheral B cells derived from 22 HCV-infected patients treated with IFN plus ribavirin therapy (for details, see Materials and Methods) and 21 healthy subjects (Control). Fifteen patients responded to therapy (Responders), whereas seven did not eradicate HCV infection (Non Responders), as demonstrated by HCV-RNA PCR. B lymphocytes from HCV-infected patients were monitored before and soon after the end of therapy. In responders, an additional analysis was performed 12 months after the end of therapy (follow-up). All responders achieved sustained virological response. Data is shown as the percentage of CD19-positive cells expressing the indicated markers (mean ± SD). (*, P < 0.005 as compared with patients before therapy)