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Cells may discriminate among ligands with different dwell times for receptor binding through a mechanism called kinetic proofreading in which the formation of an activated receptor complex requires a progression of events that is aborted if the ligand dissociates before completion. This mechanism explains how, at equivalent levels of receptor occupancy, a rapidly dissociating ligand can be less effective than a more slowly dissociating analog at generating distal cellular responses. Simple mathematical models predict that kinetic proofreading is limited to the initial complex; once the signal passes to second messengers, the dwell time no longer regulates the signal. This suggests that an assay for kinetic proofreading might be used to determine which activation events occur within the initial signaling complex. In signaling through the high affinity IgE receptor FcεRI, the transmembrane adaptor called linker for activation of T cells (LAT) is thought to nucleate a distinct secondary complex. Experiments in which the concentrations of two ligands with different dwell times are adjusted to equalize the level of LAT phosphorylation in rat basophilic leukemia 2H3 cells show that Erk2 phosphorylation, intracellular Ca2+, and degranulation exhibit kinetic proofreading downstream of LAT phosphorylation. These results suggest that ligand-bound FcεRI and LAT form a complex that is required for effective signal transmission.

Multivalent DNP-BSA is commonly used to cross-link anti-DNP IgE bound to FcεRI to stimulate cellular responses, although key features of the binding process are unknown. Fluorescence quenching can be used to study the kinetics of DNP-BSA binding to FITC-IgE. We observe that DNP-BSA binds more slowly to IgE than does an equimolar amount of a monovalent DNP ligand, suggesting that the average effective number of DNP groups per BSA is less than one. The binding data are well described by a transient hapten exposure model in which most of the DNP groups are unavailable for binding but have some probability of becoming exposed and available for binding during the time of the binding measurement. Additional experiments indicate that, for suboptimal to optimal concentrations of DNP-BSA, most of the FITC fluorescence quenching on the cell surface is due to cross-linking events. With these concentrations at 15°C, the kinetics of FITC fluorescence quenching by DNP-BSA correlates with the kinetics of DNP-BSA-stimulated tyrosine phosphorylation of FcεRI. At 35°C, the phosphorylation kinetics are biphasic during the time period in which cross-linking continues to increase. Our results establish a quantitative relationship between the timecourse for cross-linking by multivalent Ag and FcεRI-mediated signaling, and they provide the means to predict the kinetics of cross-linking under a wide variety of conditions.

Ligand-induced receptor aggregation is a well-known mechanism for the initiation of intracellular signals, but recent evidence also indicates that oligomerization of distal signaling molecules may be required for signal propagation. Formation of signaling complexes containing oligomers of the transmembrane adaptor protein LAT has recently been identified as a critical step in TCR-mediated signaling [Houtman et al., Nat Struct Mol Biol, 13, 798 (2006)]. Cross-linking of LAT arises from the formation of a 2:1 complex between GRB2 and SOS1, which bridges two LAT molecules through the interaction of an SH2 domain on each GRB2 with a phosphotyrosine of each LAT. We have developed a mathematical model to describe this oligomerization and determined the distribution of LAT oligomers and the kinetics of oligomer formation, which we compare with recent experimental results. The valence of LAT for binding GRB2, i.e., the number of LAT phosphotyrosines that can bind the GRB2 SH2 domain, ranges from 0 to 3 and is a critical factor in determining both the nature and extent of aggregation. A dramatic rise in oligomerization occurs between valence 2 and 3, a phenomenon we call valence switching because the valence is controlled by the extent of phosphorylation of specific LAT tyrosines. Supported by grant R37 GM35556 from the National Institutes of Health and the Department of Energy through contract DE-AC52-06NA25396.

Previous studies with isolated cell systems have shown desensitization (i.e., the induction of immunologic unresponsiveness after exposure to a stimulus under suboptimal conditions) to be antigenically nonspecific. Using pollen antigens and a bivalent penicillin antigen, [BPO]2, we found that, under conditions of passive sensitization, antigen-specific basophil desensitization occurred. Nonspecific desensitization always occurs with anti-IgE, and basophils of most atopic donors show nonspecific desensitization to antigens. Atopic donors were found, however, whose cells could be desensitized to one antigen while responding to noncross-reacting antigens and anti-IgE. Kinetic studies of desensitization revealed no differences between cells undergoing specific vs nonspecific desensitization. In the [BPO]2 system, removal of the antigen from the basophils by washing or by hapten ([BPO]1) competition did not reverse the specific desensitization to [BPO]2; passive replacement of specific IgE antibody did restore the response. Two types of desensitization, therefore, exist: 1) specific, which probably occurs due to the functional loss of some part of the IgE antibody-receptor complex and 2) nonspecific, involving the change noted above as well as an intracellular, biochemical alteration. Our data indicate that basophils with few IgE molecules undergo specific desensitization, whereas nonspecific desensitization occurs in basophils with many surface IgE molecules.

We investigate certain general properties of antigen induced degranulation of sensitized basophils by analyzing two types of experiments: Experiments in which we expose basophils to two antigens sequentially and then determine the fraction of histamine released; and experiments in which we obtain time-dependent release and desensitization curves. To analyze the latter type of experiments we introduce a new way to plot release and desensitization data that depends on the nature of the interactions of histamine-containing units (histamine quanta) with themselves or the cells degranulation apparatus, but not on any specific properties of the antigen. From our analysis we conclude that: 1) A fraction of histamine within a population of basophils is nonreleasable by antigenic stimulation. 2) When a basophil degranulates the initial release of histamine appears to inhibit subsequent release. 3) The rate of histamine release is proportional to the amount of releasable histamine remaining in the cells when the amount remaining is small, as expected if released of histamine granules is a stochastic process. 4) There is no dependence of desensitization on the extracellular calcium concentration.

We describe the characteristics of in vitro histamine release from human basophils passively sensitized with serum from a penicillin-allergic individual. The histamine release is induced by a synthetic bivalent hapten, bis benzylpenicilloyl 1,6 diaminohexane (BPO)2. We present data on the effect of a monovalent hapten, benzylpenicilloyl formyl-l-lysine (BPO)1, on the histamine release. We also examine how histamine release depends on the concentration of serum used for passive sensitization, the source of cells used for passive sensitization, and the time allowed for histamine release. We interpret these experiments in terms of a theory of equilibrium binding of bivalent haptens to cell surface antibody that is presented in the previous paper. The results are consistent with the idea that the amount of histamine release is controlled by the number of cross-linked IgE molecules on the cell surface. In particular, the histamine dose-response curve rises because cross-links rise, has a maximum because the cross-links are a maximum, and falls because the cross-links fall.

Using the mathematical theory of direct plaque growth, we have analyzed the expected variation of plaque size with IgM affinity and secretion rate. We use the theory to comment on recent effector cell blockade experiments and show how the theory can be used to determine the change in the secretion rate of a single antibody-forming cell subjected to blockade by a multivalent antigen. We also argue, using the mathematical theory, that under the usual experimental conditions employed in the plaque assay, cells that produce low affinity IgM antibodies will give rise to smaller plaques than cells that produce high affinity IgM antibodies.

We present a quantitative model of histamine release from human basophils due to the symmetric bivalent hapten, Bis benzylpencilloyl 1,6 diaminohexane, (BPO)2. The major elements of model are: 1) The histamine content of basophils is divided into a large number of discrete and quasi-independent “quanta.” 2) A certain fraction of the quanta in a population of basophils are nonreleasable. 3) Release of the contents of a quanta is a stochastic event. 4) The binding and cross-linking reaction between (BPO)2 and cell surface IgE approaches thermal equilibrium on a time scale that is very short compared to the time scale of histamine release from or desensitization of basophils. 5) The probability per unit time of the release event, PR, is a function of the extracellular calcium concentration [Ca]ex, and of the number of cross-linked IgE molecules per cell, Xpoly. In addition, the functional dependence of PR on Xpoly for fixed [Ca]ex is sigmoid, and the dependence of PR on [Ca]ex is such that PR → 0 as [Ca]ex → 0 for all values of Xpoly. 6) Antigen-specific IgE molecules and/or their associated Fc receptors are inactivated at a rate that is a saturable function of Xpoly. Furthermore, this process of inactivation is not dependent on the extracellular calcium concentration. The model is tested by carrying out a detailed fitting to histamine release data obtained by using cells from a single donor. Good agreement between theory and experiment is obtained, and all the parameters of the model are determined. The ways in which the model can be extended to include systems more complex than the (BPO)2 system are briefly discussed.