Isolation of Human Peripheral B Cells from PPBL Samples.
Purified B cells from PPBL patients and controls (~2.5 x [10.sup.5] cells/mL) were seeded in Primaria plates (BD Biosciences, Mountain View, CA) in the presence of gamma-irradiated (75 Gy/7500 rad) L4.5 cells in a ratio of either 3 or 25 B cells per L4.5 cell, corresponding to high and low stimulations, respectively .
The six patients studied were all female and displayed the clinical features associated with PPBL (Table 1).
All B cells from PPBL patients also expressed CD40 (data not shown).
Evolution of PPBL B Cells following Long-Term CD40 Stimulation.
PPBL B Cells Are Able to Switch to and to Secrete IgG.
Indeed, patients with PPBL showed elevated serum IgM and polyclonal B cell proliferation.
The following interesting observation was made: the [CD19.sup.+][IgG.sup.+] cell population, encompassing globally less than 5% of the cell population at the beginning of the culture in PPBL patients, increased beyond 25% on day 14.
The high proportion of [IgM.sup.+][IgD.sup.+] B cell population and increased IgM levels in PPBL patients suggest a difficulty in normally completing isotype switching.
In summary, we have shown throughout this study that PPBL B cells could proliferate in a CD40-CD154 culture system under proper conditions and that proliferation also results in IgM and IgG secretion, all of which indicating an adequate CD40 signaling pathway.
Weimann et al., "Combined automated cell and flow cytometric analysis enables recognition of persistent polyclonal B-cell lymphocytosis (PPBL), a study of 25 patients," Annals of Hematology, vol.