FIG. 4.

Detection of transitional B cells expressing mLANA⁺ during chronic infection by using a recombinant marker virus. For these experiments, B6 mice were infected i.n. with 10⁴ PFU of MHV68.ORF73βla, and spleens were harvested at various times postinoculation. For each sample group in each experiment (n = 3 experiments), splenocytes from 3 mice were pooled. Following blocking, cells were stained with anti-CD19-allophycocyanin/Cy7 and anti-AA4-allophycocyanin. Subsequently, cells were loaded with the β-lactamase substrate CCF2/AM (Invitrogen). Flow cytometry was used to identify transitional B cells (CD19⁺ AA4⁺) that expressed mLANA/β-lactamase, as previously described (25). (A) Representative flow cytometry plots from samples at 16 days postinoculation. Plots have been pregated on CD19⁺ AA4⁺ cells. The inner box indicates the gate for mLANA⁺ cells. (B) Reciprocal frequency of mLANA⁺ transitional B cells at 7, 16, 28, 42, and 90 days postinoculation calculated from flow cytometric analyses.