Separation of human B lymphocytes on Helix pomatia A haemagglutinin into two major fractions differing in responsiveness to T-dependent mitogen (pokeweed mitogen) or antigen (tetanus toxoid)

Abstract

A B-cell fraction consisting of 70% of cells carrying the B-cell-associated B1 antigen, 15-20% of M1+ non-B cells, and less than 3% of T cells was prepared from the peripheral blood of healthy human donors, previously vaccinated with tetanus toxoid (TT). As assessed by immunofluorescence after treatment with neuraminidase, approximately 40-50% of the B cells had surface structures binding to Helix pomatia A haemagglutinin (HP). The cells were separated into three fractions by affinity chromatography on HP conjugated to Sepharose (P, non-retained cells; EI, cells eluted with 0.1 mg/ml N-acetyl-D-galactosamine (D-GalNac); EII, cells eluted with 1 mg D-GalNac/ml). The majority of B cells in fraction EII were HP+ and were rich in cells expressing the B2 differentiation antigen. Sixty per cent of the B cells in this fraction also expressed the major HP-binding glycoprotein, gp 150. In the presence of autologous T cells, these B cells were strongly responsive to activation by either pokeweed mitogen (PWM) or antigen (TT), as reflected by differentiation into plasma cells, secretion of polyclonal IgG and IgM, or IgG anti-TT antibodies. In contrast, fraction P, which contained more than 90% HP-B cells, and which was partially depleted of B2+ cells, responded poorly or not at all to both PWM and TT. Fraction EI was a mixed fraction that responded in an intermediate fashion. When the preparations were depleted of contaminating non-B cells carrying the monocyte or large granular lymphocyte associated M1 antigen, their response to the two stimulating agents did not alter. The results suggest that HP+ B cells differ from HP-B cells in their responsiveness to T-cell signals. Fractionation on unsolubilized HP offers a simple and efficient way of separating B cells into at least two subsets differing in their responsiveness to T-cell-derived differentiation and maturation signals.