B cell homeostasis in chronic hepatitis C virus-related mixed cryoglobulinemia is maintained through naïve B cell apoptosis

Abstract

Mixed cryoglobulinemia (MC) is the most common extrahepatic manifestation of chronic hepatitis C virus (HCV) infection. Although the formation of inflammation-triggering immune complexes is driven by clonal expansions of autoreactive B cells, we found total B cell numbers paradoxically reduced in HCV-infected patients with MC. HCV patients with MC (n = 17) also displayed a reduced number and a reduced frequency of naïve B cells compared with HCV-infected patients without MC (n = 19), hepatitis B virus-infected patients (n = 10), and uninfected controls (n = 50). This was due to an increased sensitivity of naïve B cells to apoptosis resulting in a reduction in the size of the naïve B cell subset. In addition, 4-fold expansion and skewing (lower T1/T2-ratio) of the immature B cell subset was noted in MC patients, suggesting that apoptosis of naïve B cells triggered the release of B cell precursors from bone marrow in an attempt to maintain normal B cell numbers. Following treatment of MC with the B cell-depleting antibody rituximab, the size of all B cell subsets, the T1/T2-ratio, and the cyroglobulin levels all normalized. Cryoglobulin levels correlated with in vivo proliferation of T2 B cells, suggesting a link between the skewing of the T1/T2 ratio and the formation of immune complexes.

Conclusion: This study provides insight into the mechanisms maintaining B cell homeostasis in HCV-induced MC and the ability of rituximab therapy to restore normal B cell compartments. (HEPATOLOGY 2012;56:1602-1610).

Fig. 1. Analysis of B-cells by flow cytometry

(A) B-cells were identified using time, single cell and lymphocyte and CD19+ gates. Dead cells, T-cells, NK cells and macrophages were excluded. (B) CD19+ B-cells were grouped into CD10− mature and CD10+ immature subsets. Mature B-cells were classified into plasmablasts (PB), activated (Act), tissue-like memory (TLM), naïve (N) and resting memory (RM) B-cells based on CD21 and CD27 expression. Immature transitional (CD10+CD27−) B-cells were classified as CD21− T1 or CD21+ T2 cells. Gates were based on fluorescence-minus-one controls.

Fig. 2. HCV-infected patients with MC display reduced percentages of CD19+ B-cells and CD19+ CD10− mature B-cells

(A) Percentage of CD19+ B-cells in PBMC. (B) Percentage of CD10− mature B-cells in the CD19+ B-cell population. Mean±SEM are shown. *p<0.05, ***p<0.001.

Fig. 3. FACS dot plots displaying mature B-cell subsets of representative patients

Gates and abbreviations as in Fig. 1.

Fig. 4. HCV-infected patients with MC display reduced a percentage of naïve B-cells and an increased percentage of activated B-cells with differential sensitivity to apoptosis

(A–B) Percentage of CD21+CD27− naive B-cells (A) and CD21−CD27+ activated B-cells (B) in the mature B-cell subset. (C–D) MFI of the antiapoptotic protein Bcl-2 in naive (C) and activated/memory B-cells (D). (E–F) Percentage of caspase 3/caspase 8-double-positive and D4-GD1-positive B-cells in naive (E) and activated/memory B-cell subsets (F) following overnight culture in the absence of growth factors and cytokines. Mean±SEM are shown. *p<0.05, **p<0.01, ***p<0.001. There is a trend towards a lower frequency of naïve B-cells in HCV-infected patients with MC as compared to HCV-infected patients without MC (p=0.06).

Fig. 5. HCV-infected patients with MC display an increased percentage of immature transitional B-cells with an altered T1/T2-ratio

(A) Percentage of immature transitional CD19+CD10+CD27− B-cells in the CD19+ B-cell subset. (B) Percentage of T1 (CD21−) and T2 (CD21+) cells within the immature transitional B-cell subset. Mean±SEM are shown. *p<0.05, ***p<0.001.

Fig. 6. Rituximab restores the T1/T2-ratio in MC patients

HCV-infected patients with MC were studied prior to, during and after treatment with Rituximab for changes in HCV titer and percentage of B-cells (A), the kinetics of cryoglobulin and RF levels (B), the percentage of immature transitional B-cells within CD19+ B-cells (C), the percentage of T1 and T2 immature transitional B-cells (D), Ki-67 levels in T1 and T2 immature transitional B-cells (E), and the correlation of Ki-67 MFI of T2 immature transitional B-cells with the percentage of cryoglobulins (F). Mean±SEM are shown (n=9 patients). *p<0.05, **p<0.01, ***p<0.001 compared to pretreatment.

Fig. 7. Rituximab treatment normalizes mature B-cell subsets

Paired analysis of the frequency of naïve (A) and activated B-cells (B) within the CD19+ B-cell population prior to and 12 months after Rituximab treatment, compared to the corresponding B-cell percentages in uninfected controls. *p<0.05, ***p<0.001.