Proliferation and activation of RF B cells in response to B. burgdorferi are increased in the presence of B. burgdorferi/IgG complexes. (A and B) Splenocytes of Hul Tg mice were cultured with suboptimal concentrations (2.5 μg/ml) of sonicated B. burgdorferi, purified human anti–B. burgdorferi huIgG (1 mg/ml), a mixed solution containing B. burgdorferi and purified anti–B. burgdorferi huIgG, or a mixed solution containing B. burgdorferi and nonspecific huIgG (normal huIgG). (A) Side scatter versus forward scatter plots were used to monitor cell size of 17.109^high or 17.109^low/– B cells (top 2 rows). The next 2 rows of histograms show divisions of CFSE-labeled 17.109^high or 17.109^low/– B cells; percentages indicate proliferating cells. The bottom row of histograms shows surface expression of CD86 on 17.109^high B cells. Thick lines represent the stimulated B cells, and thin lines the nonstimulated B cells. Results shown are representative of 4 separate experiments. Acquisition parameters were determined to analyze at least 2 × 10³ cells in the 17.109^high or 17.109^low/– B cell gates (total numbers of cells: 4 × 10⁴ to 2 × 10⁶). (B) RF supernatant production was measured at day 3. Values ± 1 SEM are shown. *P < 0.05. (C and D) Purified splenic B cells of Hul Tg mice were cultured as in A. (C) CsA (50 ng/ml) was added at culture initiation. Values ± SEM represent the percentage of proliferating IgMa⁺17.109^high cells in the different conditions of stimulation. *P < 0.05. (D) RF supernatant production ± SEM was measured at day 3.