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

Front Immunol. 2023 Feb 8:14:1094871. doi: 10.3389/fimmu.2023.1094871. eCollection 2023.

Abstract

Introduction: 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.

Methods: Clonal 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.

Discussion: We 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.

Keywords: BCR/TLR9 crosstalk; CD21low B cells; autoantigen; exhaustion; mixed cryoglobulinemia; rheumatoid factor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autoantigens
  • B-Lymphocytes / immunology
  • Cell Proliferation
  • CpG Islands
  • Cryoglobulinemia* / etiology
  • Hepacivirus
  • Hepatitis C*
  • Humans
  • Myeloid Differentiation Factor 88 / metabolism
  • NF-kappa B / metabolism
  • Phosphatidylinositol 3-Kinases / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism
  • Receptors, Complement 3d / immunology
  • Rheumatoid Factor
  • Toll-Like Receptor 9 / metabolism

Substances

  • Autoantigens
  • Myeloid Differentiation Factor 88
  • NF-kappa B
  • Phosphatidylinositol 3-Kinases
  • Proto-Oncogene Proteins c-akt
  • Rheumatoid Factor
  • Toll-Like Receptor 9
  • Receptors, Complement 3d

Grants and funding

This work was supported by grants from the Istituto Pasteur Italia-Fondazione Cenci Bolognetti 2017-76 to MV, from the Intramural Research Program of Sapienza University of Rome RM11715C7D005D19 to MC and from the Italian Ministry of Education and Research PRIN-2017WJBKKW to SB.