A reappraisal of "T-independent" antigens. I. Effect of lymphokines on the response of single adult hapten-specific B lymphocytes.

Four supposedly T-independent antigens, fluorescein (FLU)-E. coli lipopolysaccharide (LPS), FLU-Brucella abortus (FLU-BA), FLU-Ficoll, and FLU-polymerized flagellin (FLU-POL), were tested for their capacity to stimulate B cell proliferation and antibody formation. Single, isolated FLU-specific adult murine splenic B lymphocytes were used as the unequivocal target cell in 10-microliter cultures unsupported by accessory, feeder or filler cells. The stimulatory capacity of four supposed mitogens, LPS, dextran sulfate (DXS), BA, and Ficoll, and of one T-dependent antigen, FLU-keyhole limpet hemocyanin (FLU-KLH), was also studied. Stimuli were used over a wide range of concentrations with or without added T cell-derived, antigen-nonspecific factors promoting B cell growth and differentiation (BGDA). The results allowed triggering stimuli to be divided into four groups: 1) FLU-KLH failed to stimulate single B cells even in the presence of BGDA as did unconjugated Ficoll; 2) FLU-POL was stimulatory only when BGDA was also present, as was unconjugated BA; 3) FLU-BA was slightly stimulatory in the absence of BGDA, but much more so in the presence of BGDA, as was a low concentration of LPS; 4) FLU-LPS and FLU-Ficoll were powerfully stimulatory over a 100,000-fold range of concentrations, and at no concentration did BGDA affect their capacity. LPS or LPS + DXS at high concentrations behaved similarly. The differing behavior of the various conjugates did not correlate either with their supposed "TI-1" or "TI-2" status or with the mitogenic properties of the carrier portion of the antigen. For example, FLU-Ficoll at 0.01 microgram/ml caused 28% of FLU-specific B cells to form proliferating clones, 70% of which secreted immunoglobulin, but a 1000-fold higher concentration of Ficoll failed to stimulate single cells. It is tempting to regard stimuli in group 4 as truly T-independent. However, the intentional addition of thymus filler cells to single B cells triggered by these agents markedly raised antibody formation. Until the nature of this further "helper" effect is understood, it would be unwise to ignore the possibility that T cells or accessory cells are involved.