B cell responses to a peptide epitope. VI. The kinetics of antigen recognition modulates B cell-mediated recruitment of T helper subsets

The ability of Ag-primed B cells to recruit distinct Th subsets was examined using two analogous synthetic peptides, G41CT3 and G28CT3, as model Ags. With sequence differences at only two positions, these peptides were identical both with respect to fine specificity of Abs induced and ability to prime T cells. Lymph node cell populations primed with peptide G41CT3, when challenged with the homologous Ag, yielded predominantly Th2 cytokines. In contrast, a challenge with the heterologous Ag, G28CT3, resulted in a markedly increased production of Th1 cytokines. These distinctions derived from altered APC function of Ag-primed B cells due to differential kinetics of recognition of the two Ags by surface Ig receptors, as confirmed by binding studies with a panel of anti-G41CT3 mAbs. A concentration-dependent circular dichroism study revealed differences in the nature of intermolecular associations for these two peptides. Furthermore, the on-rate of peptide G28CT3 binding to Ab also increased with increasing peptide concentration, implying a dependence on intermolecular interactions. This, in turn, correlated well with the ability of peptide G28CT3 to preferentially activate either Th1 or Th2 cells. Thus, the relative proportion of Th1 vs Th2 cells recruited by Ag-primed B cells is governed by the on-rate of Ag binding to surface Ig receptors, with higher on-rates promoting Th1 recruitment. Further, even subtle changes in solution behavior of an Ag can markedly influence the kinetics of recognition by B cells

Signal thresholds and modular synergy during expression of costimulatory molecules in B lymphocytes

We analyzed intracellular pathways modulating surface densities of CD80 and CD86 in B cells activated through ligation of the Ag receptor, and the adhesion molecule CD54. Whereas B cell Ag receptor (BCR) cross-linking alone stimulated increased expression of CD86, up-regulation of CD80 required dual stimulation with anti-IgM and anti-CD54. The principal downstream component contributed by BCR signaling, toward both CD80 and CD86 induction, was the elevated concentration of free cytoplasmic Ca2+, recruited by way of capacitative influx. This alone was sufficient to generate an increase in CD86 levels. However, CD80 enhancement required the concerted action of both intracellular Ca2+ concentration and CD54-initiated pathways. The nexus between anti-IgM and anti-CD54 stimulation, in the context of CD80 regulation, was identified to involve a self-propagating process of sequential synergy. The first step involved amplified accumulation of intracellular cAMP, as a result of cross-talk between BCR-mobilized Ca2+ and CD54-derived signals. This then facilitated a second synergistic interaction between Ca2+ and cAMP, culminating in CD80 expression. Our findings of distinct signal transducer requirements, with the added consequences of cross-talk, offers an explanation for variable modulation of costimulatory molecule expression in response to diverse physiological stimuli. Importantly, these results also reveal how concentration threshold barriers for recruitment of individual second messengers can be overcome by constructive convergence of signaling modules

B cell responses to a peptide epitope. VII. Antigen-dependent modulation of the germinal center reaction

Germinal center responses to two analogous peptides, PS1CT3 and G32CT3, that differ in sequence only at one position within the B cell epitopic region were examined. In comparison with peptide PS1CT3, peptide G32CT3 elicited a poor germinal center response. By demonstrating equal facility of immune complexes with IgM and IgG Ab isotypes to seed germinal centers, we excluded differences in isotype profiles of early primary anti-PS1CT3 and anti-G32CT3 Ig as the probable cause. Quantitative differences in germinal center responses to the two peptides were also not due to either qualitative/quantitative differences in T cell priming or variation in the frequency of the early Ag-activated B cells induced. Rather, they resulted from qualitative differences in the nature of B cells primed. Analysis of early primary anti-PS1CT3 and anti-G32CT3 IgMs revealed that the latter population was of a distinctly lower affinity, implying the existence of an Ag affinity threshold that restricts germinal center recruitment of G32CT3-specific B cells. The impediment in anti-G32CT3 germinal center initiation could be overcome by making available an excess of Ag-activated Th cells at the time of immunization. This resulted in the appearance of a higher affinity population of G32CT3-specific B cells that, presumably, are now capable of seeding germinal centers. These data suggest that the strength of a germinal center reaction generated is Ag dependent. At least one regulatory parameter represents the quality of B cells that are initially primed

Decreased progesterone binding and attenuated progesterone action in cultured human breast carcinoma cells treated with epidermal growth factor

Specific progesterone binding by cultured human breast carcinoma T47D, MCF-7, and ZR75-1 cells was decreased 25-40% by epidermal growth factor (EGF), with a 50% effective dose of 0.1 nm EGF. Studies with the soluble and particulate fractions prepared after homogenization of T47D cells grown in glass roller bottles revealed equivalent EGF-induced decreases in progesterone binding to receptors in both fractions. Equilibrium progesterone binding studies with these soluble and particulate fractions revealed that EGF decreased the receptor number, but had no effect on affinity. With cells grown adherent to plastic dishes, EGF treatment induced a greater decrease in binding to receptors recovered in the particulate fraction, than to receptors recovered in the soluble fraction. The decrease in progesterone binding induced by 20 nm EGF was maximal after 2 min of cellular EGF treatment for receptors recovered in the soluble fraction, but was only half-maximal after 15 min for receptors recovered in the particulate fraction. Decreased progesterone binding persisted for at least 8 days in cells cultured with 1 nm EGF. Either insulin or EGF stimulated T47D cell proliferation by two- to threefold with a 50% effective dose of 100 nm for insulin and 0.1 nm for EGF. The progestin, R5020, decreased T47D cell growth by 30% with a 50% effective dose of 1 nm. Either EGF or insulin antagonized the inhibitory effect of R5020 on cell reproduction, but progestins did not antagonize the growth stimulatory response of cells to EGF. Progestins increased the number of EGF receptors within 12 h of their addition to T47D cells, but this response was lost after 6 days. These data show that EGF or progesterone can regulate the receptor number of the other, but for cell reproduction, the effect of EGF is dominant over that of progestins

Altered glucocorticoid binding and action in response to epidermal growth factor in HBL100 cells

Incubation of adherent human breast epithelial HBL100 cells with epidermal growth factor (EGF) decreased [3H]dexamethasone binding by 35% with no effect on affinity. Maximal inhibition was obtained at 3 nm EGF and the 50% effective dose was 0.2 nm EGF. Decreased dexamethasone binding induced by 3 nm EGF was maximal by 5 min of treatment and, in the continuous presence of EGF, persisted at a constant level over 4 days. The action of EGF was antagonized by 12-O-tetradecanoylphorbol-13-acetate, which did not inhibit dexamethasone binding significantly, and by concanavalin A. In homogenates of EGF-treated cells, decreased dexamethasone binding was observed only in the cytosolic fraction. Saturation dexamethasone binding inhibited the growth rate of HBL100 cells by approximately 50%, but concurrent treatment with EGF overcame this inhibition. The effect of EGF on dexamethasone-inhibited cell growth also was antagonized by 12-O-tetradecanoylphorbol-13-acetate

B cell responses to a peptide epitope. X. Epitope selection in a primary response is thermodynamically regulated

We examine the etiological basis of hierarchical immunodominance of B cell epitopes on a multideterminant Ag. A model T-dependant immunogen, containing a single immunodominant B cell epitope, was used. The primary IgM response to this peptide included Abs directed against diverse determinants presented by the peptide. Interestingly, affinity of individual monomeric IgM Abs segregated around epitope recognized and was independent of their clonal origins. Furthermore, affinity of Abs directed against the immunodominant epitope were markedly higher than that of the alternate specificities. These studies suggested that the affinity of an epitope-specific primary response, and variations therein, may be determined by the chemical composition of epitope. This inference was supported by thermodynamic analyses of monomer IgM binding to Ag, which revealed that this interaction occurs at the expense of unfavorable entropy changes. Permissible binding required compensation by net enthalpic changes. Finally, the correlation between chemical composition of an epitope, the resultant affinity of the early primary humoral response, and its eventual influence on relative immunogenicity could be experimentally verified. This was achieved by examining the effect of various amino-terminal substitutions on immunogenicity of a, hitherto cryptic, amino-terminal determinant. Such experiments permitted delineation of a hierarchy of individual amino acid residues based on their influence; which correlated well with calculated Gibbs-free energy changes that individual residue side chains were expected to contribute in a binding interaction. Thus, maturation of a T-dependant humoral response is initiated by a step that is under thermodynamic control

B cell responses to a peptide epitope. VIII. Immune complex-mediated regulation of memory B cell generation within germinal centers

Using an in vivo reconstitution assay, we examine here the role of immune complexes in both formation of germinal centers (GC) and processes that occur subsequently within. The presence of Ag, as immune complexes, was found not to constitute a limiting requirement for the initiation of GC formation. No detrimental effect either on numbers or sizes of the resulting GC was observed when Ag-containing immune complexes were omitted during reconstitution. Thus, both recruitment and proliferation of Ag-activated B cells within GC appear not to be limited by Ag concentrations. In contrast, the presence of immune complexes was observed to be obligatory for the generation of Ag-specific memory B cells. This optimally required immune complexes to be constituted by IgG-class Abs with epitope specificities that were homologous to those of the GC B cells. The GC reaction was also found to be characterized by an enhancement of Ab specificity for the homologous epitope. Although some improvement in specificity was noted in recall responses from immune complex-deficient GC, the presence of appropriate immune complexes served to further optimize the outcome. Here again, isotype and epitope-specificity of the Ab constituent in immune complexes proved to be important

Crystal structure of an antibody bound to an immunodominant peptide epitope: novel features in peptide-antibody recognition

The crystal structure of Fab of an Ab PC283 complexed with its corresponding peptide Ag, PS1 (HQLDPAFGANSTNPD), derived from the hepatitis B virus surface Ag was determined. The PS1 stretch Gln2P to Phe7P is present in the Ag binding site of the Ab, while the next three residues of the peptide are raised above the binding groove. The residues Ser11P, Thr12P, and Asn13P then loop back onto the Ag-binding site of the Ab. The last two residues, Pro14P and Asp15P, extend outside the binding site without forming any contacts with the Ab. The PC283-PS1 complex is among the few examples where the light chain complementarity-determining regions show more interactions than the heavy chain complementarity-determining regions, and a distal framework residue is involved in Ag binding. As seen from the crystal structure, most of the contacts between peptide and Ab are through the five residues, Leu3-Asp4-Pro5-Ala6-Phe7, of PS1. The paratope is predominantly hydrophobic with aromatic residues lining the binding pocket, although a salt bridge also contributes to stabilizing the Ag-Ab interaction. The molecular surface area buried upon PS1 binding is 756 Å2 for the peptide and 625 Å2 for the Fab, which is higher than what has been seen to date for Ab-peptide complexes. A comparison between PC283 structure and a homology model of its germline ancestor suggests that paratope optimization for PS1 occurs by improving both charge and shape complementarity

Transcription regulation from a TATA and INR-less promoter: spatial segregation of promoter function

The mode of regulation of class II genes that lack the known core promoter elements is presently unclear. Here, we studied one such example, the murine CD80 gene. An unusual mechanism was revealed wherein the pre-initiation complex (PIC) first assembled on an upstream, NF-κB enhancer element. Notably, this assembly occurred independent of contributions from the core promoter domain, and resulted in a PIC that was competent for transcription initiation. Positioning was subsequently achieved by exploiting the intrinsic architecture of the promoter, by virtue of which the tethered PIC was spatially juxtaposed with the transcription initiation site. Bridging interactions then ensued, through protein-protein contacts, which then enabled the elongation phase of CD80 transcription