Impact of cytomegalovirus infection on B cell differentiation and cytokine production in multiple sclerosis

Fig. 1. Gating strategy for B cell subset analysis. Peripheral blood mononuclear cells were gated based on forward (FSC) and side scatter (SSC) (a). After exclusion of DAPI(+) dead cells (b) and selection of CD45(+) lymphocytes (c), CD19+ B cell subsets (d) were defined as plasmablasts-plasma cells (PB-PC: CD38hiCD10−) and transitional (TB: CD38hiCD10+) (e), gating in the CD38lowCD10− B cells (f) to define naive (NB: CD38low/negCD10−IgD+CD27−), unswitched memory (UMB: CD38low/negCD10−IgD+CD27+), switched memory (SMB: CD38low/negCD10−IgD−CD27+), and double negative (DN: CD38low/negCD10−IgD−CD27−). A representative case is illustrated in the figure, showing proportions of B cell subsets

Fig. 2. B cell subsets in controls and untreated MS patients according to clinical form and HCMV. a Percentages of B cell subsets within total B cells (CD19+) in healthy controls (HC) and MS patients classified based on clinical form. b HC and MS patients according to HCMV serostatus. RRMS: relapsing-remitting MS; PMS: progressive MS; PB-PC: plasmablasts-plasma cells; TB: transitional B cells; NB: naive B cells; UMB: unswitched memory B cells; SMB: memory switched B cells, DN: double negative B cells. p values: *< 0.05, ***< 0.001

Fig. 3. Impact of HCMV on the B cell compartment in IFNβ-treated MS patients. a B cell subsets in untreated (n = 27), IFNβ-treated (n = 23), and glatiramer-acetate-treated (n = 5) RRMS patients. b Untreated and IFNβ-treated MS patients classified based on HCMV serostatus. PB-PC: plasmablasts-plasma cells; TB: transitional B cells; NB: naive B cells; UMB: unswitched memory B cells; SMB: memory switched B cells; and DN: double negative B cells. p values: *< 0.05, **< 0.01, ***< 0.001, ****< 0.0001

Fig. 4. B cell cytokine production in controls and MS patients. a Representative cases illustrating the gating strategy and staining of pro-inflammatory (GM-CSF, IL-6, TNFα) and anti-inflammatory cytokines (IL-10) in B cells in resting conditions and stimulated using CpG and PMA/ionomycin. b B cell cytokine production in controls and MS patients according to MS clinical form. c Impact of HCMV on B cell cytokine production in controls and MS patients classified based on clinical form. p values: *< 0.05, **< 0.01, ***< 0.001