Bruce Merchant

Coupling of large haptens to proteins and cell surfaces: Preparation of stable, optimally sensitized erhythrocytes for hapten-specific, hemolytic plaque assays

Abstract Procedures are described for converting tertiary-butyloxycarbonyl (Boc) hydrazides of large haptenic structures to reactive acyl azides and for using these intermediates in the preparation of a sereis of highly specific immunochemical reagents. The synthesis of the initial C-terminal Boc hydrazides, which are tripeptide derivatives, is described in a companion report (Inman et al. (1973) Immunochemistry10, 153). The converted azides react readily and specifically with amino groups in mildly alkaline, aqueous media and thereby provide an excellent means for covalently coupling haptens to immunogen carriers, proteins, or cells. The azide groups have relatively uniform reactivity due to an adjacent, common, glycylglycyl structure. Thus, it has been possible to prepare groups of immunochemical reagents, representing numerous specificities, by use of a single convenient procedure. Erythrocytes bound with large haptens by this method have functioned as highly stable and sensitive indicator cells in hapten-specific hemolytic plaque and hemagglutination assays. Conditions for preparing optimally sensitized erythrocytes for quantitative studies of hapten-specific plaque forming cells have been studies. Maximum numbers of plaque forming cells were detected when about 300,000 hapten molecules were bound per indicator erythrocyte. Plaque counts decreased progressively when indicator red cells had been conjugated with appreciably more or less than this amount of hapten. It has not been previously recognized that excessively hapten derivatization of erythrocytes regularly and profoundly impedes their susceptibility to specific immune lysis. Estimates have been made of the molecular circumstances which may explain this phenomenon. Its generaly implications for complement mediated cytolysis are discussed.

Synthesis of large haptenic compounds having a common functional group that permits covalent linkage to proteins, cell surfaces, and adsorbents.

Abstract Details are given for the synthesis and characterization of two series of tripeptide derivatives that were prepared by joining various, aromatic-ring-based structures to an N-terminal, N-acetyl- l -tyrosyl or β-alanyl residue through an azo or amide linkage, respectively. The remainder of the tripeptide derivative was, in all cases, glycyglycine tertiary-butyloxycarbonyl hydrazide. The latter, C-terminal feature allows these compounds to be stored in a stable form and to be converted readily to reactive acyl azides by methods commonly employed in peptide synthesis. The azides provide a suitable means for covalently linking the derivatized tripeptides to amino groups on carriers and cell surfaces under mild conditions in aqueous media (see Inman et al. , 1973). The linked groups serve very effectively as enlarged or spaced-out haptens in studies of immunological specificity. The design and advantageous features of these compounds and their corresponding haptenic groups are discussed.

Restricted maturation of antibody-binding characteristics for hapten-specific IgM-plaque-forming cells in mice

Abstract Inhibition of plaque formation was used as a method for studying changes in binding characteristics of IgM antibody specific for the hapten, 2,4,6-trinitrophenyl (TNP). By incorporating successive concentrations of TNP ligand into specific hemolytic plaque assays, we were able to obtain inhibition curves and to determine relative ligand binding of anti-TNP antibodies secreted by plaque-forming cell (PFC) populations. The concentration of free hapten required to obtain 50% reduction in IgM PFC decreased with time after immunization, indicating that an increase in average binding affinity of their secreted antibodes occurred. The slopes of the inhibition curves also increased with time after immunization. Maturation with regard to the above two properties was virtually complete for IgM PFC 5–7 days after immunization. The manner by which the slopes of the inhibition curves changed suggested that maturation occurred as the result of a simple selective process, and it appeared to involve primarily precursor cells existing prior to administration of antigen.

Increased multiclonal antibody-forming cell activity in the peripheral blood of patients with SLE.

21 patients with criteria for systemic lupus erythematosus (SLE) and 12 normal controls were studied for their spontaneous circulating IgM and IgG plaque-forming cells (PFCs) reactive against sheep erythrocytes (SRBC) and against a panel of five haptens. Quantitatively defined active and mildly active SLE patients had significantly elevated IgM- and IgG-producing PFCs in their peripheral blood reactive with the panel of five chemically defined haptens. Those patients having inactive SLE also showed increased circulating IgM PFCs. Significant elevations in circulating hapten-reactive PFCs were found to correlate progressively with disease activity in the inactive, mildly active, and active SLE patient groups. Circulating IgM- and IgG-secreting PFC reactive against SRBC were both significantly elevated only in those patients with active SLE. The data support the concept that SLE patients have a generalized increase in B cell activity against a broad repertoire of determinants, even those ostensibly unrelated to natural tissue antigens.

INHIBITION OF THE GRAFT-VERSUS-HOST REACTION BY PRETREATMENT OF DONORS WITH VARIOUS ANTIGENS.

The effect of pretreating mice with diverse antigens on the capacity of their spleen cells to produce fatal graft-versus-host (GVH) disease across an H-2 histocompatibility barrier has been investigated. The mortality rate of midlethally irradiated recipients given spleen cells from untreated donors was a linear function of the number of cells transferred. Equal numbers of spleen cells from antigen-treated donors consistently showed a reduction in their capacity to produce fatal GVH disease. Microbial antigens were ordinarily more potent than soluble protein antigens in producing this effect. Furthermore, intensive daily antigen pretreatment generally caused greater impairment of spleen cell GVH reactivity than pretreatment with lower doses of antigen. Relative dilution of GVH competent cells as a result of splenic hyperplasia in antigen-treated donors accounted for only a portion of the observed effect. The data indicate that when donor cells are actively engaged in processing nontransplantation antigens, there occurs a significant interference in their GVH reactivity.

Effect of donor immunization with somatic polysaccharides on the graft-vs-host reactivity of transferred donor splenocytes.

Summary The effect of immunization of donor mice with bacterial antigens, on the capacity of their spleen cells to product graft-vs-host (GVH) disease has been investigated for irradiated adult recipients differing from donors at the H2 locus. Fatal GVH disease produced by spleen cells from untreated control donors was a function of the number of cells transferred. Antigen pretreatment of donors caused a reduction in the capacity of their spleen cells to produce GVH mortality. Compared with equal numbers of cells from untreated mice, the magnitude of this reduction was proportional to the total quantity of antigen used in pretreatment: 1.4 mg of Serratia marcescens polysaccharide (SMP) decreased donor spleen cell reactivity to 1/5 of control levels whereas 0.12 mg produced a decrease of 1/4 and 0.01 mg to somewhat less than 1/2 that of controls. Salmonella enteritidis polysaccharide (SEP) also provided reduction of donor cell GVH reactivity however when employed in similar dosages (SMP 120 μg, SEP 210 μg) SMP was substantially the more active in inhibiting subsequent GVH reactions. In addition, the very low toxicity of SMP permitted its use at much higher and more effective dosages.

Adaptation of the Hemolytic Plaque Technique for Enumeration of Immune Cells Responding to Heterologous Immunoglobulin Antigens

Summary Rabbit spleen cells producing antibodies against mouse immunoglobulin antigens were detected by a modification of the hemolytic plaque technique. The peak cellular and serologic response occurred 7 to 8 days following primary immunization and had subsided completely by the fifteenth day. The absence of spontaneous background distinguished protein reactive PFC from those reactive with erythrocyte antigens. We thank Mr. B. Harrell for expert technical assistance and Dr. M. Landy for useful suggestions in preparation of this manuscript.

Localization of 125I-Labeled DNP-Ficoll on Lymphoid Cells from the Normal and Defective Hybrid Progeny of CBA/N Mice

CBA/N mice have an X-linked B-cell defect. It prevents them from mounting active immune responses to soluble, nonmitogenic, polysaccharide antigens. The F1 male progeny of defective CBA/N female mice also harbor the same defect. Lymphoid cell suspensions prepared from these defective mice and appropriate controls were examined for their capacity to bind 125I-labeled, dinitrophenylated Ficoll, a representative polysaccharide antigen. In general, lymphoid cells bound substantially greater quantities of this antigen than nonlymphoid cells; however, lymphoid cells from defective F1 male mice differed little, if at all, from the lymphoid cells of normal F1 female mice. Direct binding of this radiolabeled antigen by spleen cell suspensions was minimally affected by depletion of functional T cells or by depletion of glass wool-adherent cells. Lymphoid cells consistently bound only a small fraction of the available radiolabeled antigen at all concentrations tested and progressive differences in direct binding by normal and defective spleen cells were not discernable. In addition, immune spleen cells differed only slightly from unprimed spleen cells in their capacity for direct antigen binding. The data are interpreted to indicate the presence of a high-capacity system for nonspecific association of this radiolabeled Ficoll antigen with lymphoid cell surfaces. The high-capacity system for nonspecific association between antigen and lymphoid cells serves to obscure immunologically significant binding between radiolabeled Ficoll antigens and normal and defective lymphoid cells. In all probability, the failure of lymphoid cells from CBA/N and F1 male mice to respond to dinitrophenylated Ficoll cannot simply be attributed to any intrinsic failure to bind this antigen.

Hapten-specific blockade of CBA/N x C3H/HeN F1 B cell responses in a cell transfer system.

CBA/N mice harbor an X-linked B cell defect which is transmitted by CBA/N female mice to their hybrid male progeny. Hapten-specific plaque-forming cell (PFC) responses to haptenated proteins by B cell-defective CBA/N mice and CBA/N X C3H/HeN F1 male mice can be blockaded by concomitant exposure to polysaccharide agents bearing the same hapten. Various experimental approaches were explored in an attempt to study this phenomenon in a syngeneic cell transfer system. Unprimed donor spleen cells were unable to mount adequate PFC responses to dinitrophenylated-hemocyanin (DNP-KLH) in irradiated, syngeneic recipients. DNP-KLH-primed donor spleen cells produced strong PFC responses after challenge in irradiated recipients, and these secondary responses were subject to hapten-specific blockade by DNP-derivatized polysaccharide agents. Both direct and indirect PFC responses could be blocked. DNP conjugated to bovine serum albumin also produced hapten-specific blockade in this cell transfer system. Under some cell transfer conditions normal CBA/N X C3H/HeN F1 female spleen cells were just as susceptible to hapten-specific blockade as defective F1 male spleen cells.