Antigen binding to receptors on immunocompetent cells. I. Simple models and interpretation of experiments

We have developed simple mathematical models for treating the kinetics of binding of multivalent antigen to immune cell receptors when the binding may be in competition with nonspecific binding of antigen to cell surfaces and with the binding of hapten to the receptors. All three kinds of binding are treated as reversible bimolecular reactions. In general, the resulting equations must be solved numerically. When, however, the antigen and hapten concentrations are large compared to receptor concentrations, approximate algebraic solutions are found. It is shown that the most important effect of the hapten-receptor binding and of the nonspecific antigen binding is to slow down the antigen-receptor association; this may be viewed as a decrease in the antigen-receptor association rate constant.We have applied these models to analyze experiments of Davie and Paul on the binding of antigen to receptors on immunocompetent cells. Many difficulties have been found to arise from nonspecific binding. In particular, the association rate constant and equilibrium constant will appear reduced by nonspecific binding and the association rate constant will appear anomalously temperature dependent. We interpret hapten inhibition of antigen binding as a nonequilibrium effect in which hapten reduces the rate of antigen-cell association. In this way the concentration of hapten required to give 50% inhibition of antigen binding is found to decrease, as observed, with time after immunization. If equilibrium were to be achieved we predict that the required concentrations of hapten would be found to increase with time.     

Extended half-life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells

The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coli beta-galactosidase conferred by the single-chain Fv (scFv) fragment 13R4 equipped with nuclear import/export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short-lived or destabilized intrabodies could affect the steady-state level of target by redirecting it to the proteasomes. In the absence of antigen, the half-life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with beta-galactosidase. Analysis of destabilized site-directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of beta-galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.     

Antigen binding to lymphoid cells from unimmunized mice: IV. Shedding and reappearance of multiple antigen binding Ig receptors of T- and B-lymphocytes

Patterned antigen-binding cells (ABC) can bind two antigens and show “islands” of Ig receptor with mixed specificity. However, these cells, when unfixed, lose most of their bound fluorescent antigen within minutes upon warming above 0 °C. Residual antigen moves to one pole of the cell forming a “cap” within 5 min at room temperature. If such patterned ABC are capped with a single antigen, receptors to a second antigen can be detected on a portion of the capped cells, but only in the cap. The frequency of capped, “double” ABC approximated the frequency of patterned “double” ABC originally present.If lymphoid cells are mixed with fluorescent antigens at 0 °C and then incubated for 4 hr at 37 °, no ABC are found. When the cells are then fixed and the fluorescent antigens readded, new antigen-binding Ig receptors can be shown to have reappeared on the cell surface during the 4-hr incubation. The reappearance of antigen receptor could be inhibited by prior addition of either 10^?2M sodium azide or 50 μg/ml cycloheximide, implying that the receptors were actively synthesized by the cell. These inhibitors did not prevent shedding, but azide did inhibit the capping process. Both B-cells (bone marrow or spleen cells) and T-cells (splenic T-cells or 99.5% pure cortisone-resistant T-cells) were shown to regenerate multispecific ABC to the frequency found prior to incubation.     

Papillomavirus capsid binding and uptake by cells from different tissues and species.

The inability of papillomaviruses (PV) to replicate in tissue culture cells has hampered the study of the PV life cycle. We investigated virus-cell interactions by the following two methods: (i) using purified bovine PV virions or human PV type 11 (HPV type 11) virus-like particles (VLP) to test the binding to eukaryotic cells and (ii) using different VLP-reporter plasmid complexes of HPV6b, HPV11 L1 or HPV11 L1/L2, and HPV16 L1 or HPV16 L1/L2 to study uptake of particles into different cell lines. Our studies showed that PV capsids bind to a broad range of cells in culture in a dose-dependent manner. Binding of PV capsids to cells can be blocked by pretreating the cells with the protease trypsin. Penetration of PV into cells was monitored by using complexes in which the purified PV capsids were physically linked to DNA containing the gene for beta-galactosidase driven by the human cytomegalovirus promoter. Expression of beta-galactosidase occurred in < 1% of the cells, and the efficiency of PV receptor-mediated gene delivery was greatly enhanced (up to 10 to 20% positive cells) by the use of a replication-defective adenovirus which promotes endosomal lysis. The data generated by this approach further confirmed the results obtained from the binding assays, showing that PV enter a wide range of cells and that these cells have all functions required for the uptake of PV. Binding and uptake of PV particles can be blocked by PV-specific antisera, and different PV particles compete for particle uptake. Our results suggest that the PV receptor is a conserved cell surface molecule(s) used by different PV and that the tropism of infection by different PV is controlled by events downstream of the initial binding and uptake.     

Characterization of T and B antigen-binding cells for beta-galactosidase. II. T antigen-binding cells.

The results reported in this paper suggest that the specific Z-binding cells of the normal mouse include a large portion of T lymphocytes. Depletion of T cells with anti-theta serum and cortisone indicates that the majority of the ZBC of the thymus, which occur at frequencies of about 150/10(6), are indeed T cells. Similar treatment of spleen cells suggests that approximately half the binding cells in that organ are contributed by the T lymphocyte population. T-and B-enriched populations obtained from the spleen by using differential adherence to nylon wool contained equal numbers of ZBC and bound equivalent amounts of the antigen. Hence, there appears to be a high frequency of T lymphocytes that can be shown to bind beta-galactosidase specifically in both the thymus and spleen of normal mice.     

Beta-galactosidase decreases the binding affinity of the insulin-like-growth-factor-II/mannose-6-phosphate receptor for insulin-like-growth-factor II

The insulin-like growth-factor-II/mannose-6-phosphate (IGF-II/Man6P) receptor binds two classes of ligands, insulin-like growth factors and lysosomal enzymes. We have examined the ability of the lysosomal enzyme, beta-galactosidase, to modulate the binding of 125I-IGF-II to the receptor. beta-Galactosidase purified from bovine testis was fractionated on a DEAF-Sephacel ion-exchange column. Column fractions were assayed for enzymatic activity and for ability to inhibit the binding of 125I-IGF-II to the IGF-II/Man6P receptor. Enzyme fractions eluting at higher NaCl concentrations which had previously been shown to exhibit greater uptake by cells in culture, exhibited greater potency in inhibiting the binding of 125I-IGF-II to the receptor. A pool of these fractions from the DEAE-Sephacel column inhibited 125I-IGF-II binding to pure receptor by 80% with the concentration required for half-maximal inhibition being 25 nM. The inhibition of binding by beta-galactosidase was completely blocked by simultaneous incubation with Man6P. Inhibition of the enzymatic activity of beta-galactosidase with D-galactonic acid gamma-lactone did not affect the ability of beta-galactosidase to inhibit the binding of 125I-IGF-II to the receptor. Scatchard analysis of IGF-II binding to pure receptor in the presence and absence of beta-galactosidase showed that beta-galactosidase decreased the binding affinity for IGF-II (Kd 0.26 nM versus 1.0 nM in the presence of 57 nM beta-galactosidase). We confirmed the observations of others that Man6P alone actually increases the binding of 125I-IGF-II to the IGF-II/Man6P receptor, but we found that this phenomenon was dependent upon the method of preparation of the IGF-II/Man6P receptor. Microsomal membrane preparations, solubilized membranes, and receptors purified on an IGF-II-Sepharose column all exhibited stimulation of 125I-IGF-II binding by Man6P, whereas receptors purified on lysosomal enzyme affinity columns showed little or no stimulation of 125I-IGF-II binding by Man6P. We conclude that beta-galactosidase decreases the binding affinity of the IGF-II/Man-6-P receptor for IGF-II by binding with high affinity to the Man6P-recognition site.     

Inability of human alveolar macrophages to stimulate resting T cells correlates with decreased antigen-specific T cell-macrophage binding

Alveolar macrophages (AM) from the majority of human volunteers are defective antigen presenting cells (APC) in T cell proliferation assays despite the display by the cells of HLA-D region antigens. We have confirmed that AM secrete relatively little interleukin 1 (IL 1), but addition of exogenous IL 1 did not improve the capacity of AM to initiate antigen-induced T cell proliferation. Thus, the presence of HLA-D region antigens and IL 1 is not sufficient to enable an accessory cell to act as an APC. We developed a T cell-accessory cell binding assay to investigate early events in T cell activation. AM demonstrated a diminished capacity as compared with monocytes to bind antigen-specific T cell clones. Nevertheless, AM often induced proliferation of T cell clones as effectively as monocytes, indicating that antigen display was intact. The inefficiency of AM in bind T cell clones correlated with their reduced capacity to induce resting T cells to express IL 2 receptors, secrete IL 2, and proliferate in response to antigen. Indirect immunofluorescence established that similar percentages of AM and monocytes expressed LFA molecules, but the density of the molecules was greater on monocytes than AM. A role for LFA antigens in the physical binding of T cells to monocytes was demonstrated by blocking antigen-specific binding with a monoclonal antibody to LFA-1 antigen. LFA-1 antibody also blocked the low levels of specific binding between AM and T cell clones, indicating that LFA-1-ligand interactions were operative between these two cell types. These studies indicate that there are critical cell membrane characteristics that promote binding of T cells to APC in addition to T cell receptor-antigen interactions. This combination of nonspecific and specific interactions leads to avid T cell-APC binding that may be essential for activation of resting T cells. Furthermore, we postulate that the failure to AM to act as effective APC results from an inability to bind T cells efficiently.     

Characterization of T and B antigen-binding cells for beta-galactosidase. III. Independence of antigen-binding cells in normal animals from antigenic stimulation.

Antigen-binding cells to beta-galactosidase were enumerated in thymus and spleen of mice of different ages and found to remain at a constant frequency throughout life. Thymic beta-galactosidase-binding cells (ZBCs),6 representing T-binding cells, showed no appreciable fluctuation, whereas splenic ZBCs, a mixture of T- and B-binding cells, were slightly depressed at birth but found at normal frequencies by 1 week of age. In addition, germfree mice, both within the 1st week after birth and after maturity, had nearly as many binding cells as did conventionally reared age-matched mice. These results considered together suggest that the ability of cells of both T and B origin to recognize antigen, as revealed by their ability to bind beta-galactosidase, arise independently of specific or nonspecific antigenic stimulation, as part of the normal ontogenic sequence of steps of differentiation. This is consistent with the theory of clonal precommitment of T and B cells.     

Specific insulin binding in bovine chromaffin cells; demonstration of preferential binding to adrenalin-storing cells

Insulin binding was studied in subpopulations of bovine chromaffin cells enriched in adrenalin-producing cells (A-cells) or noradrenalin-producing cells (NA-cells). Binding of 125I-insulin was carried out at 15 degrees C for 3 hrs in the absence or presence of excess unlabelled hormone. Four fractions of cells were obtained by centrifugation on a stepwise bovine serum albumin gradient. The four fractions were all shown to bind insulin in a specific manner and the highest binding was measured in the cell layers of higher densities, containing mainly A-cells. The difference in binding of insulin to the four subpopulations of chromaffin cells seemed to be related to differences in numbers of receptors as opposed to receptor affinities. We conclude that bovine chromaffin cells possess high affinity binding sites for insulin and that these binding sites are mainly confined to A-cells.     

The reactivity of canine red cells with heterophile agglutinins having anti-A activity*

Heterophile A-reactive agglutinins, obtained from the seeds of Dolichos biflorus and from the snails Helix aspersa, Helix pomatia and Cepaea nemoralis, were tested for reactivity with the saliva and red cells of a random series of dogs. Saliva from dogs with the A-like Tr red cell antigen inhibited the agglutination of human A red cells by each of the reagents. However, none of the heterophile agglutinins distinguished Tr+ from Tr— red cells: the Dolichos agglutinin was non-reactive, while all of the snail reagents agglutinated both Tr+ and Tr— cells. It is concluded that the canine red cell bears a series of heterophile receptors which mask reactivity with the canine homologue of the red cell A-antigen, and that the restricted size of the binding sites of the agglutinins limits the informational significance of cross-reactivity obtained with these reagents.     

Extracellular HSP70 binding to surface receptors present on antigen presenting cells and endothelial/epithelial cells

Extracellular HSP70 has been found to participate in both innate and adaptive immune responses. However, little is known about the molecular mechanisms that mediate this process. Previous reports suggest that HSP70 interacts with antigen presenting cells (APC) through a plethora of surface receptors. In this study, we have examined the relative binding of potential HSP70 receptors and found high affinity binding to LOX-1 but not other structures with a role in HSP70-APC interactions such as LRP/CD91, CD40, TLR2, TLR4 or another c-type lectin family member (DC-SIGN) closely related to LOX-1. In addition to APC, HSP70 can avidly bind to non-APC cell lines, especially those from epithelial or endothelial background.     

Specific endothelin binding sites in renal medullary collecting duct cells: lack of interaction with ANP binding and cGMP signalling.

The diverse biological actions of endothelins (ET) appear to be mediated by specific cell-surface receptors. Autoradiography and membrane binding studies have shown abundant ET binding sites in the kidney. However, their expression in specific types of renal cells is unclear. We studied the binding of 125I-labelled endothelin-1 in freshly isolated cell suspensions from canine inner medullary collecting duct. Competition binding experiments revealed the presence of specific high-affinity binding sites: unlabelled ET-1 and ET-2 compared with the radioligand with an IC50 of 135 and 83 pM, respectively, while the IC50 of ET-3 and big ET-1 were 2 and 4 orders of magnitude higher, indicating the presence of ETA-type receptor. Angiotensin II, vasopressin, and atrial natriuretic peptide (ANP) did not compete for ET binding even at a concentration of 10(-6) M. Saturation binding experiments showed a single class of binding sites of high density (Bmax = 56.7 +/- 10.3 fmol/10(6) cells) and high affinity (Kd = 69.8 +/- 10 pM). In contrast, ANP receptors in the same cell preparations appeared as two classes of binding sites with widely different affinity and density. The high-affinity ANP site (Kd = 311 +/- 48 pM) was compatible with ANP-B (guanylate cyclase-coupled) receptor. ET-1 did not compete for this receptor. ET-1 (10(-7) M) did not alter ANP-induced cGMP generation in these cells (3.8-fold increase at 10(-7) M ANP), nor basal levels of cGMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin binding ability of B-chronic lymphocytic leukaemia cells.

The binding of five fluorescein-labelled lectins: peanut agglutinin (PNA), lentil agglutinin (LEN), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and asparagus pea agglutinin (ASP) to human B-cell chronic lymphocytic leukaemia (B-CLL) and B lymphocytes of normal donors was studied. The specificity of the fluorescence was demonstrated by inhibition with appropriate saccharides. The proportion of B cells was estimated using anti-B cell monoclonal antibody. Both leukaemic and normal B cells showed the binding ability of all except of one (ASP) studied lectins. We have found the differences in surface carbohydrate patterns between B-CLL and normal B lymphocytes. B-CLL cells showed the considerably lower ability to bind SBA and slightly higher expression of PNA and LEN receptors in comparison to normal B cells. The analysis of WGA binding allowed for recognizing two groups of CLL patients: one with high and the second one with low WGA receptor expression. The double marker studies revealed that B cells could simultaneously react with anti-B cell monoclonal antibody and fluorochrome labelled lectins.     

Epidermal growth factor receptors on PC12 cells: Alteration of binding properties by lectins

The PC12 cell line displays cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). It has been previously shown that the lectin wheat germ agglutinin (WGA) alters the properties of NGF receptors on these cells. We now report that preincubations with either WGA or concanavalin A (Con A) decrease the binding of ¹²⁵I-EGF to PC12 cells by greater than 50%. The inhibition of binding occurred at 37°C and 4°C and could be blocked or reversed by the addition of sugars which bind specifically to WGA or Con A. Scatchard analysis revealed that these lectins decreased binding primarily by lowering the affinity of the receptor and to a lesser extent by decreasing receptor number. Succinylalion of Con A (sCon A) produced a derivative that was less effective than the native lectin in decreasing EGF binding; however, addition of an antibody against Con A restored the ability of sCon A to decrease binding. Similar to results obtained with ¹²⁵I-NGF binding, WGA but not Con A was found to increase, by scveralfold; the proportion of ¹²⁵I-EGF binding that is resistant to solubilization by Triton X-100 detergent. A potential association of the EGF receptor with cytoskeletal elements is discussed which could account for such results.     

Thymocyte binding to human thymic epithelial cells is inhibited by monoclonal antibodies to CD-2 and LFA-3 antigens

With the use of cultured human thymic epithelial (TE) cells, we have previously shown that thymocytes bind to TE cells in suspension in a rosette-forming assay. To identify cell surface molecules involved in human TE-thymocyte rosette formation, we assayed a large panel of monoclonal antibodies for their ability to inhibit rosette formation. We found anti-CD-2 (LFA-2, T11), and anti-LFA-3 antibodies all inhibited binding of TE cells to thymocytes. By using indirect immunofluorescence assays, we determined that cultured TE cells were 90% LFA-3 positive and CD-2 negative, whereas thymocytes were 10% LFA-3 positive and 98% CD-2 positive. Pretreatment of TE cells with anti-LFA-3 but not anti-LFA-2 inhibited TE-thymocyte binding. In contrast, pretreatment of thymocytes with anti-CD-2 but not anti-LFA-3 antibodies inhibited TE-thymocyte binding. Thus TE cell-thymocyte binding is blocked by antibodies to the CD-2 (T11) antigen on thymocytes and by an antibody to the LFA-3 antigen on TE cells. Because the CD-2 antigen has been implicated in T cell activation, these data suggest that a natural ligand for T cell activation via the CD-2 molecule is present on human thymic epithelial cells.     

Non-natural cell surface receptors: synthetic peptides capped with N-cholesterylglycine efficiently deliver proteins into Mammalian cells

Protein toxins such as shiga toxin and cholera toxin penetrate into cells by binding small molecule-based cell surface receptors localized to cholesterol and sphingolipid-rich lipid raft subdomains of cellular plasma membranes. Molecular recognition between these toxins and their receptors triggers endocytic protein uptake through endogenous membrane trafficking pathways. We report herein the synthesis of functionally related non-natural cell surface receptors comprising peptides capped with N-cholesterylglycine as the plasma membrane anchor. The peptide moieties of these receptors were based on high-affinity epitopes of anti-hemaglutinin antibodies (anti-HA), anti-Flag antibodies, and a moderate-affinity Strep Tag II peptide ligand of the streptavidin protein from Streptomyces avidini. These non-natural receptors were directly loaded into plasma membranes of Jurkat lymphocytes to display peptides from lipid rafts on the cell surface. Molecular recognition between these receptors and added cognate anti-HA, anti-Flag, or streptavidin proteins resulted in rapid clathrin-mediated endocytosis; fluorescent target proteins were completely internalized within 4-12 h of protein addition. Analysis of protein uptake by epifluorescence microscopy and flow cytometry revealed intracellular fluorescence enhancements of 100-fold to 200-fold (10 microM non-natural receptor) with typically >99% efficiency. This method enabled intracellular delivery of a functional Escherichia coli beta-galactosidase enzyme conjugated to Protein A from Staphylococcus aureus. We termed this novel delivery strategy "synthetic receptor targeting", which is an efficient method to enhance macromolecular uptake by decorating mammalian cells with chemically defined synthetic receptors that access the molecular machinery controlling the organization of cellular plasma membranes.     

Roles of antiestrogen binding sites in human endometrial cancer cells

Estrogen-noncompatible antiestrogen binding sites (AEBS) as well as estrogen receptors (ER), and the growth-inhibitory effect of tamoxifen were investigated in two human endometrial cancer cell lines, IK-90 and HEC-IA cells. IK-90 cells contained specific AEBS, but no ER was found in these cells. Scatchard plot analysis of AEBS in 12,000 g supernatant from IK-90 cells showed a high affinity binding site for tamoxifen (Kd:5.6 +/- 1.0 nM) with the maximum binding site of 457 +/- 47 fmol/mg protein. However, no measurable ER or AEBS was found in HEC-IA cells. The effect of tamoxifen on the growth of cells was found to be identical in both cell lines; the addition of 10 microM tamoxifen to culture medium was cytocidal whereas tamoxifen at lower concentrations (1 nM-1 microM) did not significantly affect the growth of both IK-90 and HEC-IA cells. These results demonstrate for the first time the presence of AEBS in human endometrial cancer cells. The present results also suggest that AEBS does not play a fundamental role in mediating the growth-inhibitory effect of tamoxifen in endometrial cancer cells.     

High affinity binding of reovirus type 3 to cells that lack beta adrenergic receptor activity

A previous report demonstrated both immunological crossreactivity and structural similarity between the mammalian beta adrenergic receptor and the cell surface receptor for the reovirus type 3 (14). We now demonstrate that reovirus type 3 can bind selectively and with high affinity to cells that lack beta adrenergic receptor activity (L-cells). The present study was also designed to determine what effect reovirus binding has on beta adrenergic receptor function in cells (DDT1) that possess an intact ligand binding site. Based on computer analysis of reovirus competitive inhibition curves, the apparent dissociation binding constants (Kd) for reovirus binding to DDT1 and L-cells are 0.1 nM and 0.25 nM, respectively. High affinity [125I]-iodocyanopindolol (CYP) binding to beta adrenergic receptors can also be demonstrated in DDT1 cells but not in L-cells. In agreement with these ligand binding studies, adenylate cyclase activity is stimulated by the beta receptor agonist isoproterenol in DDT1 cell membranes but not in L-cell membranes. In addition, isoproterenol increases cAMP levels in DDT1 cells but not in L-cells. Neither reovirus serotype stimulates cAMP levels in either cell line, nor do they influence beta-adrenergic agonist stimulation of cAMP in DDT1 cells. These results argue against identity of the receptors for reovirus type 3 and beta adrenergic ligands.