FIGURE 2.

Kaposi’s sarcoma-associated herpesvirus enhances class-switching efficiency in primary mouse B cells. (A) Representative FACS plots of LPS/ IL-4 stimulated splenocytes showing the percentage cells expressing GFP⁺ and RFP⁺ (dot plot and histogram) at 48 h post-infection. The top-right inset of each dot plot shows GFP or RFP values for the mock-infected control. Histograms depict the dot plot data, where the dotted line represents the mock-infected control and the solid green or red line represents the shift in GFP or RFP expression, respectively. (B) 5 × 10⁵ cell/mL of naïve mouse splenic B cells were cultured with LPS (control) and purified rKSHV.219 (KSHV) or UV-irradiated rKSHV.219 (UV-KSHV). After 24 h, IL-4 was added to the cultures to stimulate IgG1-switching. Surface IgG1 expression was measured by FACS after 4 days of IL-4 stimulation. Values on the graph show the percentage of IgG1⁺ (% IgG1) cells relative to the control. Below, (A) representative FACS plot displays the raw values splenocytes that class-switched to IgG1⁺ (inside red polygon). (C) 5 × 10⁵ cell/mL of naïve mouse splenic B cells were cultured with LPS (control) and/ or purified rKSHV.219 (KSHV) or UV-irradiated rKSHV.219 (UV-KSHV). After 24 h, TGFβ was added to the cultures to stimulate IgG2b-switching. Surface IgG2b expression was measured by FACS after 4 days of stimulation. Values on the graph show the percentage of IgG2b⁺ (% IgG2b) cells relative to the control. Below, (A) representative FACS plot displays the raw values of splenocytes that class-switched to IgG2b⁺ (inside red rectangle). (D) AID and (E) Sμ germline mRNA transcripts were assessed using qPCR from splenic B cells cultured with LPS/IL-4 (control) or LPS/IL-4 /rKSHV.219 (KSHV). Values on the graph are calculated relative to the control and are depicted as arbitrary units. (F) Representative FACS plot evaluating cell proliferation using a CFSE dilution assay in splenic B cells cultured with LPS/IL-4 (control) or LPS/IL-4/rKSHV.219 (KSHV).