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

Reversion of anergy signatures in clonal CD21low B cells of mixed cryoglobulinemia after clearance of HCV viremia.

Hepatitis C virus (HCV) causes mixed cryoglobulinemia (MC) by driving clonal expansion of IgM+CD27+ B cells. These cells display both the features of anergy induced by continual engagement of the B cell receptor (BCR), such as high expression of phosphorylated extracellular signal regulated kinase (pERK) and reduced lifespan, and of virus-specific exhaustion such as CD21low phenotype and defective response to ligation of BCR and Toll-like receptor 9 (TLR9). Usually MC regresses after eradication of HCV with interferon, whose immunomodulatory activity might contribute to this effect. We investigated phenotypic and functional changes in clonal B cells of MC patients with sustained virologic responses to direct-acting antivirals (DAA), which lack immunomodulatory properties. We found that high pERK expression and accelerated apoptosis revert within 4 weeks after beginning therapy, whereas clonal B cells unresponsive to TLR9 stimulation persist for at least 24 weeks although they may partially rescue normal CD21 expression. Thus, similar to mouse models, features of anergy in MC B cells rapidly revert after disengagement from HCV, whereas virus-specific exhaustion imparts a durable inhibitory imprint on cell function. Treatment of HCV+ MC with DAA provides a valuable tool for untangling the molecular mechanisms of anergy and exhaustion in human B cells

Dual stimulation by autoantigen and CpG fosters the proliferation of exhausted rheumatoid factor-specific CD21low B cells in hepatitis C virus-cured mixed cryoglobulinemia

Hepatitis C virus (HCV) causes mixed cryoglobulinemia (MC) by driving clonal expansion of B cells expressing B cell receptors (BCRs), often encoded by the VH1-69 variable gene, endowed with both rheumatoid factor (RF) and anti-HCV specificity. These cells display an atypical CD21low phenotype and functional exhaustion evidenced by unresponsiveness to BCR and Toll-like receptor 9 (TLR9) stimuli. Although antiviral therapy is effective on MC vasculitis, pathogenic B cell clones persist long thereafter and can cause virus-independent disease relapses. MethodsClonal B cells from patients with HCV-associated type 2 MC or healthy donors were stimulated with CpG or heath-aggregated IgG (as surrogate immune complexes) alone or in combination; proliferation and differentiation were then evaluated by flow cytometry. Phosphorylation of AKT and of the p65 NF-kB subunit were measured by flow cytometry. TLR9 was quantified by qPCR and by intracellular flow cytometry, and MyD88 isoforms were analyzed using RT-PCR. DiscussionWe found that dual triggering with autoantigen and CpG restored the capacity of exhausted VH1-69pos B cells to proliferate. The signaling mechanism for this BCR/TLR9 crosstalk remains elusive, since TLR9 mRNA and protein as well as MyD88 mRNA were normally expressed and CpG-induced phosphorylation of p65 NF-kB was intact in MC clonal B cells, whereas BCR-induced p65 NF-kB phosphorylation was impaired and PI3K/Akt signaling was intact. Our findings indicate that autoantigen and CpG of microbial or cellular origin may unite to foster persistence of pathogenic RF B cells in HCV-cured MC patients. BCR/TLR9 crosstalk might represent a more general mechanism enhancing systemic autoimmunity by the rescue of exhausted autoreactive CD21low B cells

Late relapses of hepatitis C virus-cured mixed cryoglobulinaemia associated with infection or cancer

not applicable, as letter (case report) in Rheumatology there is no abstrac

Clinico-immunologic outcomes of HCV-cured cryoglobulinemia: lower relapse rate with interferon-based than interferon-free therapy

Sustained virologic response (SVR) obtained with interferon (IFN) or with direct-acting antivirals (DAAs) is commonly followed by response of hepatitis C virus (HCV)-associated mixed cryoglobulinemia vasculitis (MCV), but relapse of MCV despite SVR has been reported in several patients after DAAs and rarely after IFN. Since relapses could have been overlooked in studies with IFN, we retrospectively compared the outcomes of MCV in SVR patients treated with DAAs (n=70) or IFN (n=39) followed-up, respectively, for 30.5 (range 11-51) or 48 months. Groups were comparable for demographics and clinics and response rates of MCV were similar (92% and 86%); however, DAA-treated patients less efficiently reduced cryoglobulins (p=0.006) and circulating B-cell clones (p=0.004), and had more frequently relapses of MCV (18% vs. 3%, p=0.028) and need for rituximab therapy (p=0.01). Although largely inferior on an intention-to-treat basis, IFN may be superior to DAAs on clinico-immunologic outcomes possibly owing to its antiproliferative activity

DEC1/STRA13 is a key negative regulator of activation-induced proliferation of human B cells highly expressed in anergic cells

The transcription factor DEC1/STRA13 (also known as BHLHE40 and SHARP2) is involved in a number of processes including inhibition of cell proliferation and delay of cell cycle, and is a negative regulator of B cell activation and development in mice. We show here that, unlike in mice, DEC1/STRA13 expression is induced in human naïve and memory resting B cells by activation through the B-cell receptor (BCR) or Toll-like receptor 9 (TLR9). siRNA silencing of DEC1/STRA13 increases the capacity of activated B cells to perform a high number of divisions after TLR9 ligation. This identifies DEC1/STRA13 as a critical negative regulator of clonal expansion of activated human B cells. We also show that DEC1/STRA13 is upregulated in human anergic CD21low B cells clonally expanded in patients with HCV-associated mixed cryoglobulinemia, which fail to proliferate in response to BCR or TLR9 ligation. siRNA knockdown of DEC1/STRA13, however, fails to restore responsiveness to stimuli in these cells, although it might improve the proliferative capacity in a subset of anergic cells with less pro- nounced proliferative defect

Hepatitis B virus causes mixed cryoglobulinaemia by driving clonal expansion of innate B-cells producing a VH1-69-encoded antibody

To investigate the expression of a VH1-69-encoded idiotype, and the phenotypic and functional features of monoclonal B-cells from patients with type II mixed cryoglobulinaemia (MC) secondary to chronic hepatitis B virus (HBV) infection

Rheumatoid factor-producing CD21low anergic clonal B-cells in essential mixed cryoglobulinaemia: a model for autoantigen-driven pathogenesis of infectious and non-infectious cryoglobulinaemias

Objective. Essential mixed cryoglo- bulinaemia (EMC) is a disorder of B-cells producing rheumatoid factor (RF), and is clinically and immuno- logically similar to mixed cryoglobuli- naemia (MC) related to hepatitis C vi- rus (HCV-MC). We report here the first comprehensive analysis of B-cell clon- ality, phenotype and function in EMC. Methods. The study population in- cluded 16 patients with EMC and 24 patients with HCV-MC. Molecular analysis was done for the detection of circulating clonal B cells and for B cell receptor sequencing. B-cell phenotype, proliferative response, apoptosis and ERK signaling were analysed by flow cytometry. Results. Molecular analysis of im- munoglobulin genes rearrangements revealed circulating B-cell clones in about half of patients, on average of smaller size than those found in HCV-MC patients. Sequence analy- sis showed usage of the same stereo- typed RF-encoding B-cell receptors frequently expressed in HCV-MC and in primary Sjögren’s syndrome. B-cells with low expression of CD21 (CD21low) and unusual homing and inhibitory re- ceptors were increased in EMC and in HCV-MC, but at a significantly lower extent in the former. The CD21low B- cells of EMC and HCV-MC patients shared functional features of exhaus- tion and anergy, namely reduced pro- liferation upon ligation of Toll-like re- ceptor 9, high constitutive expression of phosphorylated ERK, and proneness to spontaneous apoptosis. Conclusion. Our findings suggest a common pathogenetic mechanism in EMC, HCV-MC and primary Sjögren’ssyndrome, consisting of autoantigen- driven clonal expansion and exhaus- tion of selected RF-producing B-cells. The more massive clonal expansion in HCV-MC may be due to co-stimulatory signals provided by the virus

DataSheet_1_Dual stimulation by autoantigen and CpG fosters the proliferation of exhausted rheumatoid factor-specific CD21low B cells in hepatitis C virus-cured mixed cryoglobulinemia.docx

IntroductionHepatitis C virus (HCV) causes mixed cryoglobulinemia (MC) by driving clonal expansion of B cells expressing B cell receptors (BCRs), often encoded by the VH1-69 variable gene, endowed with both rheumatoid factor (RF) and anti-HCV specificity. These cells display an atypical CD21low phenotype and functional exhaustion evidenced by unresponsiveness to BCR and Toll-like receptor 9 (TLR9) stimuli. Although antiviral therapy is effective on MC vasculitis, pathogenic B cell clones persist long thereafter and can cause virus-independent disease relapses.MethodsClonal B cells from patients with HCV-associated type 2 MC or healthy donors were stimulated with CpG or heath-aggregated IgG (as surrogate immune complexes) alone or in combination; proliferation and differentiation were then evaluated by flow cytometry. Phosphorylation of AKT and of the p65 NF-kB subunit were measured by flow cytometry. TLR9 was quantified by qPCR and by intracellular flow cytometry, and MyD88 isoforms were analyzed using RT-PCR.DiscussionWe found that dual triggering with autoantigen and CpG restored the capacity of exhausted VH1-69pos B cells to proliferate. The signaling mechanism for this BCR/TLR9 crosstalk remains elusive, since TLR9 mRNA and protein as well as MyD88 mRNA were normally expressed and CpG-induced phosphorylation of p65 NF-kB was intact in MC clonal B cells, whereas BCR-induced p65 NF-kB phosphorylation was impaired and PI3K/Akt signaling was intact. Our findings indicate that autoantigen and CpG of microbial or cellular origin may unite to foster persistence of pathogenic RF B cells in HCV-cured MC patients. BCR/TLR9 crosstalk might represent a more general mechanism enhancing systemic autoimmunity by the rescue of exhausted autoreactive CD21low B cells.</p