Reversion of anergy signatures in clonal CD21low B cells of mixed cryoglobulinemia patients after clearance of HCV viremia with direct-acting antivirals

Introduction Type II Mixed cryoglobulinemia (MC) is an autoimmune and benign lymphoproliferative disorder caused by Hepatitis C virus (HCV) and characterized by the expansion of monoclonal CD27+ IgM+ B cells producing a rheumatoid factor often encoded by the VH1-69 and VK3-20 genes. These cells display peculiar phenotypic and functional features: in particular, they commonly express low levels of CD21 (CD21low B cells), an array of inhibitory and apoptosis-related genes and a distinctive pattern of homing receptors, fail to proliferate in response to the stimulation of BCR or of TLR9 and, similarly to murine B cells made anergic by continual antigenic stimulation, overexpress pERK and are prone to apoptosis. Usually MC regresses after eradication of HCV with interferon (IFN), whose immunomodulatory activity might contribute to this effect; the newly available direct-acting antivirals (DAAs) rapidly suppress HCV viremia in HCV+MC patients and lack the immunomodulatory properties of IFN. Aim To investigate phenotypic and functional changes in clonal B cells of MC patients with sustained virologic response to DAAs, untangling the effects of BCR disengagement in a human model of virus-driven anergy and exhaustion. Results In patients treated with DAA, B cell phenotype, constitutive and BCR-induced ERK signaling, spontaneous apoptosis and cell proliferation were analyzed before and after HCV eradication, using healthy donors as control. Immunophenotyping studies were performed with combinations of fluorochrome-labeled monoclonal antibodies, using the VH1-69-specific G6 mAb (which recognize an epitope of the VH1-69-encoded protein) to identify VH1-69+ B cells; spontaneous apoptosis was assessed by staining cells with annexin V and 7- aminoactinomycin D; the intracellular pERK content was measured by the BD PhosFlow system and represented as pERK-specific Mean Fluorescence Intensity (MFI); finally, cell proliferation was evaluated at day 5 of in vitro culture by the carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay. All these experiments have been done by flow cytometry. Phenotypic and functional analyses in untreated patients were in agreement with previous data showing an anergic status and an exhausted behavior of VH1-69+ CD21low B cells. After treatment with DAA, when all patients were negative for HCV RNA, it was observed a slow reduction of VH1-69+ CD21low B cells in peripheral blood; moreover, these cells showed a significantly reduced constitutive ERK phosphorylation and a significative decrease in spontaneous apoptosis after eradication of the virus. To investigate whether reduced lifespan was related to ERK signaling, MC B cells were treated with the MEK/ERK inhibitor U0126; no effect on spontaneous in vitro apoptosis was observed, suggesting that ERK signaling is not directly involved in the pro-apoptotic pathway of these cells. Despite phenotypic changes, clonal B cells failed to restore their capacity to proliferate in response to TLR9 stimulation. Conclusions Clonal B cells of HCV+MC display signatures of anergy induced by continual BCR occupancy and of exhaustion driven by chronic viral infection. Anergy features (pERK overexpression and accelerated apoptosis) rapidly revert after disengagement from HCV; phenotypic and functional features of exhaustion persist for several months. The rapid clearance of HCV viremia with DAAs offers a unique model for untangling the interplay of virus-driven anergy and exhaustion in human B cells.