Phenotypic and functional changes in clonal B cells of HCV⁺MC patients after clearance of HCV infection by DAA therapy. (A) Constitutive pERK expression by whole B cells, expressed as mean fluorescence intensity (MFI), is higher in untreated MC patients than in healthy donors (HD), whereas (B) the relative increase of pERK induced by BCR ligation with anti-immunoglobuliln, expressed as fold increase compared with constitutive pERK MFI, is reduced in MC B cells. Bars denote the means. (C) MC B cells are more prone to spontaneous apoptosis than HD B cells. (D) pERK constitutive expression in MC B cells decreases significantly 4 weeks after beginning DAA compared with pretherapy (pre-th), whereas (E) the BCR-induced fold-increase of pERK remains unmodified at this time point. (F) Spontaneous apoptosis is significantly reduced in MC B cells 4 weeks after beginning DAA therapy and at the end of treatment (EOT). (G) Changes in the proportions of CD21^low B cells among circulating B cells in a cohort of 24 MC patients treated with DAA. (H) Changes in the proportions of V_H1-69⁺ B cells in 5 MC patients treated with DAA. (I) In one MC patient, V_H1-69⁺ B cell expansion persists unmodified after DAA therapy, but a large proportion of clonal B cells rescue a CD21^high phenotype at SVR24. (J) MC V_H1-69⁺ B cells remain unable to proliferate efficiently in response to TLR9 ligation at SVR24. (Ji) Electronic gating of V_H1-69⁻ and V_H1-69⁺ B cells; the CD20^dim cells are plasmablasts. (Jii-Jiii) Analysis of the proliferative responses of (ii) V_H1-69⁻ and (iii) V_H1-69⁺ B cells. Percentages denote the number of cells that started dividing (precursor cohort); numbers denote cell divisions as calculated by the FlowJo software. CFSE, carboxyfluorescein diacetate succinimidyl ester.