In 1972, Neils K. Jerne stated in the First Annual Report of the Basel Institute for Immunology, “For bursa cells and B lymphocytes, it is relatively easy to demonstrate that receptors are antibody-like molecules which enable the cell to recognize antigens. Membrane fluidity permits the receptors to move over the cell and, in certain conditions, to aggregate and to form a cap over one pole of a lymphocyte…. These studies lead up to the question by which mechanism the receptors transmit excitatory or inhibitory signals to the cell, resulting in stimulation or paralysis.”
At that time, the burgeoning field of cellular immunology was abuzz with riddles posed by the newly embraced clonal selection paradigm. These ignited intense research activity, including programs aimed at understanding Ag receptor diversity and its genetic basis, as well as efforts concentrated on how this theoretical construct could explain acquired tolerance and immunologic memory. As evidenced by Jerne’s remarks from the Basel Institute for Immunology’s First Annual Report, basic principles governing signaling via Ag receptors also remained mysterious. Indeed, although the necessity of receptor occupation for lymphocyte activation was a central tenet of clonal selection, even rudimentary knowledge of lymphocyte signaling mechanisms was lacking. Furthermore, the manner through which activation thresholds might be established, as well as how this could accommodate affinity and kinetic differences between primary and secondary responses, was unexplored territory. The Journal of Experimental Medicine paper published by Norman Klinman in that year (1), which is this month’s Pillars of Immunology selection, shed light on each of these questions.
Within this heady atmosphere, Klinman and his colleagues developed and refined a system that allowed study of normal B cell responses at a clonal level in vitro, including detailed analyses of the secreted Ab molecule (2, 3). Although nowadays it is difficult to envisage the in vitro generation and examination of mAbs as a technical tour de force, this had only been possible previously through either the in vivo amplification of adoptively transferred splenocytes or the study of Ab products from plasma cell tumors. Klinman’s approach involved the adoptive transfer of B cells at limiting dilution to carrier-primed adoptive hosts, and the subsequent in vitro stimulation of splenic organ fragments prepared from host spleens (see Fig. 1). By combining adoptive transfer with in vitro organ culture, this system afforded several analytic advantages. First, although B cell limiting dilution had previously been achieved in vivo (4), the extension of this scheme to an in vitro stimulation system allowed close control over the nature, level, and timing of Ag stimulation. Second, when combined with the sensitivity of newly developed radioimmunoassay and isoelectric focusing methodologies, it allowed detailed analyses of the Ab products generated in vitro.
By varying the molarity and form of the haptenic determinants used for stimulation in vitro, and then comparing these with the affinities of the elicited Abs, the studies highlighted here provided the first definitive demonstration of an affinity threshold for lymphocyte activation. Indeed, a close reading of the discussion on these points (see pages 252–253) reveals the remarkably powerful conclusions drawn when rigorous physical chemical analyses—skills cultivated during Klinman’s training under the tutelage of Karush (5)—were applied to clonal stimulation and the resulting Ab products. First, it was noticed that, beyond a certain hapten molarity, the frequency of responding precursors reached a plateau. Moreover, affinity analyses of the Ab products from primary clones revealed that few fell below affinities of ∼106 liters/mol, and that raising the molarity of stimulating hapten beyond that level did not yield responding clones of lesser affinity. Together, these two observations yielded the conclusion that ligand binding per se was insufficient for activation, and that instead primary B cells display an affinity threshold below which activation does not occur.
The question remaining was as follows: what biological algorithm might underlie such a threshold? The answer to this riddle emerged from the experiment summarized in Table VI, which showed that as little as a 2-fold molar excess of monovalent ligand could thwart primary B cell activation by multiply substituted hapten carrier complexes. This result offered a profound conceptual insight: receptor cross-linking and the resultant aggregation mediated by multivalent ligand must be the ultimate determinant of activation. This would afford an affinity threshold, inasmuch as low-affinity ligand-receptor interactions would not persist long enough to achieve aggregation. Although surface Ig capping and patching had been described (6, 7), visualizing this aggregation as a means to impose activation thresholds on receptor-ligand interactions governed by reversible chemical bonds was fresh conceptual ground. In addition to this fundamental principle, the paper goes on to reveal that memory B cells differ from their naive counterparts in activation thresholds, suggesting a fundamental shift in the stringency of activation requisites. This observation added an important facet to the problem of immunologic memory in the context of clonal selection—showing that it is not simply an increase in the Ag-responsive precursor frequency due to clonal expansion, but also reflects a physiologic switch in how receptor ligation is achieved or processed.
The fundamental concepts established by these experiments remain robust. Indeed, 30 years hence, these ideas form core principles of contemporary research programs in lymphocyte signal transduction and immunologic memory.