In an effort to further elucidate the early cellular events in generation of antibody responses, we have determined the requirements for antigen-specific initiation of the G0 to G1 transition by isolated trinitrophenol (TNP) -binding B lymphocytes. TNP-binding cells were isolated from normal B6D2F1 splenocyte populations using hapten affinity fractionation on disulfide-bonded TNP-gelatin-coated plates. Populations prepared in this way are greater than or equal to 96% immunoglobulin positive and 70-95% antigen binding. Isolated cells were cultured for 48 h in the presence of a variety of TNP conjugates including TNP-Brucella abortus (Ba), TNP-Ficoll, TNP-sheep erythrocytes (SRBC), TNP-human gamma globulin (HGG), or TNP-ovalbumin (OVA) before being harvested and subjected to acridine orange cell cycle analysis. As many as 80% of cells were in cycle by 48 h in response to TNP-Ba, a thymus-independent (TI1 antigen. A smaller proportion (congruent to 40%) were in cycle in response to TNP-Ficoll, a TI2 antigen. Significant activation was not detected in cultures challenged with the thymus-dependent immunogens TNP-SRBC, TNP-HGG, and TNP-OVA. Addition of interleukin 1 (IL-1), IL-2, B cell growth factor, and/or T cell-replacing factor to cultures did not facilitate responses to these immunogens, suggesting a requirement for antigen-specific T cell help for entry into cell cycle induced by thymus dependent antigens. Activation by TNP-Ba was antigen specific and independent of accessory cells, occurring with equal efficiency in bulk and single-cell cultures. Activation by TNP-Ba was inhibitable by anti-Fab and anti-mu antibodies, but not by anti-delta antibodies. Results indicate that activation of TNP-binding cells to enter cell cycle by TNP-Ba is independent of accessory cells and requires interaction of antigen with cell surface IgM. Exposure to thymus-dependent TNP-immunogens plus nonspecific helper factors is insufficient to cause entry of TNP-binding cells into cycle.

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