Figure 3. While early stages of antigen-specific B cell responses are intact during γHV68 infection, proliferation and differentiation are inhibited.

A) Experimental design. B–G) B cells isolated from MD4 mice (CFSE labeled in C–E) were adoptively transferred into γHV68 or sham-infected C57BL/6 mice on day 6 post infection. Twenty-four hours later the mice were immunized with 100μl 10% SRBC-HEL. B) Seven days later serum was collected and the IgM^a anti-HEL response was determined by ELISA (n=5/ group). C) 18 hrs after immunization the cell surface expression of CD69 and CD86 on MD4 B cells transferred into sham-infected (black, unimmunized; green, immunized) or gHV68-infected mice (blue, unimmunized; red, immunized) (n=5/ group) was determined by gating on B220⁺ CFSE⁺ cells. Representative histograms are shown. D) 18 hrs after immunization spleens were collected, sectioned and stained for MD4 B cells (green) and B cells (red) (n=5/ group) Representative sections are shown. E) 72 hrs after immunization spleens were collected and CFSE dilution in MD4 B cells was determined in unimmunized mice (filled) and SRBC-HEL immunized mice (thick line) by gating on B220⁺IgM^a+ cells. Representative histograms are shown. The average % ± SEM of cells which have undergone >1 round of division is show. The difference between the γHV68-infected and sham-infected group is statistically significant (p<0.05) F) 106 hrs after immunization spleens were collected and stained for B220, CD138 and intracellular anti-HEL. The displayed plots are gated on B220⁺ cells (n=4/ group). Representative plots are shown. G) The number of plasmablasts/spleen are plotted, calculated as % CD138+ intracellular HEL^hi cells (F) × total splenocytes. Error bars show SEM. All data is representative of at least 2 independent experiments.