In vitro anamnestic immune responses and modulating factors.

The term “anamnestic” refers to the specific and enhanced immune responses of antigen-immunized (primed) lymphoid memory cells to secondary challenge with a foreign substance (antigen). These responses include the accelerated and quantitatively greater syntheses of antibody and other macromolecules than upon primary challenge of such cells. Rabbits were primarily immunized with keyhole limpet hemocyanin (KLH). Six days later their memory lymph node cells (LNC) were removed, and upon culture with KLH, responded with the synthesis of antibody, immunoglobulin (Ig), protein, DNA and RNA, as well as with active transport of dibutryl cyclic AMP (DbcAMP). Purified thymus-derived (T) LNC were prepared on anti-rabbit Ig affinity columns. Bursal-equivalent (B) cells were prepared by binding to a complex of sheep erythrocytes (SRBC)-antibody to SRBC-complement and centrifugation of these complexes on suitable gradients. When these T and B KLH-primed LNC were mixed and challenged with KLH the aforementioned macromolecular syntheses and active transport occurred. Indeed, by a variety of criteria, the reconstituted anamnestic immune responses were indistinguishable from these responses of unfractionated LNC. Antigenic stimulation of KLH-primed T cells induced the synthesis of proteins and DNA, but not antibody, but antigenic challenge of KLH-primed B cells did not evoke these syntheses. However, added KLH induced a mixture of T and B antigen-primed LNC to synthesize more protein, Ig, DNA than either population alone and more antibody than T cells per se; B cells required help for all of these responses. The thymus (T) cell-dependent phase of in vitro anamnestic antibody response lasted the first 24–36 hr. The antibody response was regulated by antigen-concentration. One μg KLH evoked maximal antibody synthesis, 10 and 100 μg KLH much less. Challenge of the separated T and B cell populations with different KLH concentrations, followed by recombination and eventual assay of antibody synthesis revealed different optima. The optimal concentration for T cell help was 0.01–0.1 μg KLH; higher amounts induced much less antibody production. The optimum for B cells was 1–10 μg KLH; 100 μg inhibited antibody formation. The antibody response to KLH and human serum albumin (HSA) was regulated nonspecifically utilizing LNC from rabbits immunized simultaneously with these two antigens. Thus stimulation of LNC from these rabbits with either antigen induced the synthesis of antibodies to both antigens. HSA and KLH did not cross-react either serologically or cellularly. Cross-stimulation of antibody synthesis also was observed when rabbit LNC were primed with KLH and Mb. However, in this instance, cross-reaction between KLH and sperm-whale myoglobulin (Mb) was observed at the cellular, presumably the T cell, level, although not at the antibody (B cell) level. The antibody response could also be modulated by exogenous cholera enterotoxin (CT), dibutyryl cyclic AMP (DbcAMP) and prostaglandins of the E series. The addition of each substance together with 1–100 μg KLH to KLH-primed LNC enhanced the antibody response many-fold. CT-induced non-immunized LNC to produce soluble factor(s) (SF) which, when added to KLH-primed LNC together with KLH, enhanced antibody synthesis significantly. The addition of Indomethacin, an inhibitor of PGE synthesis to KLH-immunized cells together with KLH inhibited antibody production, suggesting that PGE was involved in this response. Evidence was adduced that neither cyclic AMP nor PGE was required for the antibody response: Ca2+ was not required for induction of this response by KLH, but only its regulation by cAMP. Moreover, when KLH-primed LNC were fractionated on Nylon columns, the effluent cells were induced by KLH to synthesize antibody, but this synthesis was not enhanced by added DbcAMP or PGE; presumably, regulatory cells were removed on the column. Added KLH induced PGE synthsis in these cultures; this synthesis required macrophages. In all of the LNC cultures — including cultures from rabbits immunized with KLH, HSA, and MB months or a year earlier — much antibody synthesis occurred even when antigen was not added to the cultures. This “spontaneous” antibody was anamnestic, thymus (T cell)-dependent and involved the interaction of residual immunogen on dendritic cells with T and B memory cells. This spontaneous antibody response provides a model for the study of the factors involved in the longterm maintenance of humoral immunity. Mb was employed as a source of more refined antigenic determinants. Rabbits were immunized with Mb in complete Freunds adjuvant. The addition of small synthetic peptides corresponding to the five antigenic sites of Mb to the Mb-primed LNC induced the synthesis of antibody, Ig, protein, DNA, RNA, and macrophage migration inhibitory factor (MIF). The N terminal 1–6 peptide, which is not antigenic, i.e. does not combine with antibody to Mb, also induced all of these syntheses, except MIF. These peptide-induced responses appeared to be thymus-dependent.