Direct triggering of B lymphocytes by insolubilized antigen

The in vitro immune response of mouse spleen cells to trinitrophenylated keyhole limpet hemocyanin (TNP‐KLH) conjugated onto Sepharose beads, TNP‐KLH(B), was investigated. Both primed and unprimed spleen cells responded well to this antigen. TNP‐KLH‐primed spleen gave a greater IgM, but a lesser IgG response to TNP‐KLH(B) than to soluble TNP‐KLH (TNP‐KLH(S)), regardless of its concentration. Spleen cell populations depleted of T cells or macrophages responded well to TNP‐KLH(B), but not to TNP‐KLH(S). Thus, TNP‐KLH(B) directly activates B cells, and the conjugation of a thymus‐dependent antigen, TNP‐KLH, to a particle, Sepharose, rendered it thymus independent. Varying the amount of TNP‐KLH conjugated per bead markedly affected their stimulatory capacity, independently of the total amount of antigen added per culture. No responses occurred with very low or very high substitution levels. In addition, it was found that TNP directly conjugated to aminoethylated polyacrylamide beads at low substitution levels is immunogenic for unprimed spleen cells. The immunogenicity of TNP‐KLH(B) and trinitrophenylated polyacrylamide beads indicates that binding of antigen at the surface of a cell is sufficient to stimulate it, and that interiorization of intact antigen is not necessary in this process. Furthermore, these results can be interpreted on the basis of a matrix mechanism of B cell triggering, which involves the simultaneous binding of multiple antigenic determinants to B cell receptors.