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.

Dual stimulation by autoantigen and CpG fosters the proliferation of exhausted rheumatoid factor-specific CD21low B cells in hepatitis C virus-cured mixed cryoglobulinemia / Del Padre, Martina; Marrapodi, Ramona; Minafò, Ylenia A; Piano Mortari, Eva; Radicchio, Giovanna; Bocci, Chiara; Gragnani, Laura; Camponeschi, Alessandro; Colantuono, Stefania; Stefanini, Lucia; Basili, Stefania; Carsetti, Rita; Fiorilli, Massimo; Casato, Milvia; Visentini, Marcella. - In: FRONTIERS IN IMMUNOLOGY. - ISSN 1664-3224. - 14:(2023). [10.3389/fimmu.2023.1094871]

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

Del Padre, Martina;Marrapodi, Ramona;Piano Mortari, Eva;Radicchio, Giovanna;Bocci, Chiara;Colantuono, Stefania;Stefanini, Lucia;Basili, Stefania;Fiorilli, Massimo;Casato, Milvia;Visentini, Marcella
2023

Abstract

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.
2023
BCR/TLR9 crosstalk; CD21low B cells; autoantigen; exhaustion; mixed cryoglobulinemia; rheumatoid factor
01 Pubblicazione su rivista::01a Articolo in rivista
Dual stimulation by autoantigen and CpG fosters the proliferation of exhausted rheumatoid factor-specific CD21low B cells in hepatitis C virus-cured mixed cryoglobulinemia / Del Padre, Martina; Marrapodi, Ramona; Minafò, Ylenia A; Piano Mortari, Eva; Radicchio, Giovanna; Bocci, Chiara; Gragnani, Laura; Camponeschi, Alessandro; Colantuono, Stefania; Stefanini, Lucia; Basili, Stefania; Carsetti, Rita; Fiorilli, Massimo; Casato, Milvia; Visentini, Marcella. - In: FRONTIERS IN IMMUNOLOGY. - ISSN 1664-3224. - 14:(2023). [10.3389/fimmu.2023.1094871]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1685784
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