Evidence for an upper affinity threshold for anti-IgM-induced apoptosis in a human B-cell lymphoma.

The influence of ligand:receptor affinity on B-cell antigen receptor (BCR)-induced apoptosis in the IgM+ Burkitt lymphoma line, Ramos, was evaluated with a group of affinity-diverse murine monoclonal antibodies (MoAbs) specific for human B-cell IgM. The studies showed not only a minimal affinity threshold for the induction of apoptosis, but, interestingly, also a maximal affinity threshold above which increases in affinity were associated with diminished apoptosis. The lesser capacity of high-affinity MoAb to induce apoptosis was paralleled by a lesser capacity to induce receptor cross-linking. At high ligand concentration, high MoAb affinity was also associated with a diminished capacity to induce early protein tyrosine phosphorylation. The compromised capacity of two high-affinity MoAbs to trigger apoptosis may be, at least in part, explained by two separate phenomena that can impair the formation of mIgM cross-links: (1) more stable univalent binding and (2) a tendency for monogamous binding of both MoAb Fab to two Fab epitopes on mIgM. These in vitro studies suggest that the use of the highest affinity MoAbs for antireceptor immunotherapies that depend on receptor cross-linking might, on occasion, be contraindicated.

Membrane IgM-stimulated human B lymphocytes succumb to activation-related apoptosis at a G1-->S transition: influence of ligand affinity and valency.

Culture of human B lymphocytes with polyclonally activating surrogates for type II T-cell-independent antigen, i.e., anti-IgM mAb and anti-IgM:dextran, resulted in both membrane IgM (mIgM)-triggered S/G2/M entry and apoptosis. Although high ligand valency could compensate for low affinity, and high affinity could compensate for low valency, in achieving mIgM-triggered apoptosis, the phenomenon was most pronounced when the soluble "antigen" had both high binding site affinity and valency. Most of the mIgM-triggered apoptosis may represent B cells which progress into G1 but fail to receive a sufficient level of continuous mIgM-mediated signaling during G1 for passage through a G1 --> S phase restriction point(s). This was supported by the findings that (a) a lesser proportion of mIg-triggered cells enter S phase than G1; (b) maximal mIgM-triggered apoptosis was noted at 48-72 h of culture and surrounding activated cell clusters; (c) mIgM-triggered apoptosis was not inhibited by pharmacologic blockers of S phase; and (d) a high proportion of viable mIgM-triggered B cell blasts in G1 succumb to apoptosis rather than enter S phase, if high-affinity multivalent ligand is washed from the cultures. In addition to quantitative aspects of initial receptor engagement, the potential for a protracted period of recurrent mIgM signaling events may influence whether apoptosis or cell cycle progression is the functional outcome of B cell encounter with a multivalent antigen.

The affinity threshold for human B cell activation via the antigen receptor complex is reduced upon co-ligation of the antigen receptor with CD21 (CR2).

The present studies have examined whether the potential of an Ag to co-ligate the complement (C3d)-binding CD21 receptor complex with the membrane IgM (mIgM) receptor complex can reduce the mIgM:Ag affinity threshold for triggering human B cell S phase entry. A series of Ab:dextran conjugates consisting of affinity-diverse anti-IgM mAb, with and without anti-CD21 mAb, were synthesized as polyclonally reactive, moderately multivalent ligands that mimic C3d-bearing and non-C3d-bearing Ag. Co-ligation of mIgM and CD21 significantly diminished both the ligand concentration threshold and the IgM:ligand affinity threshold for eliciting S phase entry in the presence of IL-4. Furthermore, such co-engagement ablated the triggering bonus associated with high mlgM:ligand affinity, suggesting that B cells with a high affinity for Ag are not preferentially activated over B cells of intermediate affinity upon encountering a multivalent Ag with bound C3d. The enhancing effects of mIgM:CD21 co-ligation were restricted to low concentrations of ligand; at high concentrations, a decrease in B cell DNA synthesis was often observed. The findings suggest that the ability a moderately multivalent Ag substrate to engage B cells through both mIgM and CD21 is critical for B cell activation at limiting Ag concentrations, and furthermore, that mIgM:CD21 co-engagement may be particularly important in eliciting an immune response to such Ags in unprimed individuals in whom the majority of specific B cells are of low affinity.

Human B cell activation. Effect of T cell cytokines on the physicochemical binding requirements for achieving cell cycle progression via the membrane IgM signaling pathway.

Given the range of mIg-binding affinities expressed by Ag-specific B cells, the ligand:receptor affinity threshold for achieving full B cell activation via the mIgM-mediated signaling pathway is quite high. Several recombinant, or semi-purified, cytokines were found to reduce the very high mIgM:ligand affinity threshold for induction of human B cell S phase entry by bivalent, affinity-diverse anti-IgM mAbs without notably affecting the lower affinity threshold for G1-related RNA synthesis. Two-stage culture experiments suggested that one major means by which IL-4, IL-2, and low m.w. B cell growth factor lower the affinity threshold for S phase entry is an indirect one, i.e., rescue of B cells whose mIg engagements with Ag are of sufficient affinity for achieving G1 entry, but of insufficient affinity for initiating the late-phase mIgM-mediated signals needed for the G1-->S phase transition. IL-4 had additional effects in early G1. In contrast to the above cytokines, IFN-gamma, did not function as an independent cell cycle progression factor, but rather required the concomitant presence of mIgM-cross-linking ligand for enhancement. A greater potential of multivalent anti-IgM-dextran conjugates to trigger S phase entry in the absence of cytokines was found to reflect a greater potential for initiating mIgM signals during the late phase in B cell activation. The results indicate that progression of mIgM receptor-activated B cells past a G1-->S phase restriction point is dependent upon continued signal transduction via either the mIgM receptor and/or a cytokine receptor signaling pathway. When mIgM-engaging ligands are ineffective at initiating late-phase signals, due to limited size and binding site valency and/or affinity, ancillary signal transduction through cytokine receptors becomes most relevant.

Membrane IgM-mediated signaling of human B cells. Effect of increased ligand binding site valency on the affinity and concentration requirements for inducing diverse stages of activation.

The potential for ligand-initiated signal transduction through B cell membrane IgM is assessed in terms of ligand concentration, binding site valency, and binding site affinity for membrane Ig. Estimates of the physicochemical requirements for achieving G0* enhancement of class II MHC expression, G1 entry, and S phase entry in human B cells were made by comparing the stimulatory effects of three affinity-diverse anti-Cmu2 mAb when in bivalent (unconjugated) form, or as mAb-dextran conjugates with low binding site valency (oligovalent ligands) or high binding site valency (multivalent ligands). An increase in binding site number (and concomitant molecular mass) caused a profound reduction in both the minimal concentration and affinity requisites for B cell activation. The enhancing effect of increased binding site valency was most evident for the signaling of those most distal stages in B cell activation, i.e., G1 and S phase, which were difficult to induce with bivalent ligands. The results suggest that highly multimeric TI-2 Ag may be good immunogens because they are able to elicit a full activation response not only from infrequent high affinity B cells, but also from a substantial proportion of the many lower affinity Ag-specific B cells in virgin B cell populations. Interestingly, the activation of B cells by ligands with binding sites of high intrinsic affinity (Ka = 5 x 10(8) M-1) was less influenced by increases in binding site valency than was B cell activation by ligands with intermediate binding site affinity (Ka = 2 x 10(7) M-1). This suggests that the minimal epitope valency requirement for T cell-independent B cell activation by mIg cross-linking Ag may be dependent on the intrinsic affinity with which membrane Ig molecules on a given B cell interact with the redundantly expressed epitopes.

Synthesis of N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine: its use in peptide synthesis for placing a bromoacetyl cross-linking function at any desired sequence position.

A new amino acid derivative, N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine (BBAL), has been synthesized as a reagent to be used in solid-phase peptide synthesis for introducing a side-chain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group subsequently serves as a sulfhydryl-selective cross-linking function for the preparation of cyclic peptides, peptide conjugates, and polymers. BBAL is synthesized by condensation of N-bromoacetyl-beta-alanine with N alpha-Boc-L-lysine and is a white powder which is readily stored, weighed, and used with a peptide synthesizer, programmed for N alpha-Boc amino acid derivatives. BBAL residues are stable to final HF deprotection/cleavage. BBAL peptides can be directly coupled to other molecules or surfaces which possess free sulfhydryl groups by forming stable thioether linkages. Peptides containing both BBAL and cysteine residues can be self-coupled to produce either cyclic molecules or linear peptide polymers, also linked through thioether bonds. Products made with BBAL peptides may be characterized by amino acid analysis of acid hydrolyzates by quantification of beta-alanine, which separates from natural amino acids in suitable analytical systems. Where sulfhydryl groups on coupling partners arise from cysteine residues, S-(carboxymethyl)cysteine in acid hydrolyzates may also be assayed for this purpose. Examples are given of the use of BBAL in preparing peptide polymers and a peptide conjugate with bovine albumin to serve as immunogens or model vaccine components.

T cell activation by purified, soluble, class I MHC molecules. Requirement for polyvalency.

To examine the nature of the interaction of the TCR with the MHC class I Ag, we have studied the stimulation requirements of an H-2Dd-reactive T cell hybridoma, using a homogeneous, purified preparation of a molecularly engineered soluble counterpart of the class I Ag, H-2Dd/Q10b. We demonstrate that this monovalent, soluble MHC Ag is incapable of stimulating the release of IL-2 from this T cell hybridoma. However, the same preparation of the purified protein can elicit a dose-dependent response when made multivalent either by covalent coupling to soluble, high m.w. dextran or to agarose beads, or by adsorption to polystyrene tissue culture plates.

Picogram quantities of anti-Ig antibodies coupled to dextran induce B cell proliferation.

To investigate the properties which enable type 2 Ag, as exemplified by dextran and Ficoll, to stimulate high levels of antibody responses in the relative absence of T cells, we conjugated anti-IgD and anti-IgM mAb to both dextran and Ficoll and examined their B cell-activating properties. Such conjugated anti-Ig antibodies stimulated both early and later stages of B cell activation at picogram concentrations, which are at least 1000-fold lower than that required for B cell stimulation by unconjugated anti-Ig antibodies, and the level of proliferation they stimulated was on average 10-fold greater. Furthermore, concentrations of anti-Ig dextran (100 pg/ml) which modulated little sIgD from the B cell surface were strong inducers of enhanced B cell expression of MHC class II molecules. Conjugation of Fab fragments of anti-IgD or nonmitogenic anti-IgM mAb to dextran rendered them as mitogenic as dextran conjugated to strongly stimulatory anti-IgD or anti-IgM antibodies. The ability of dextran and Ficoll to serve as effective carrier molecules for anti-IgD was not related solely to their large m.w., because anti-IgD coupled to polymerized BSA (m.w. 1.5 X 10(6), was only 10- to 50-fold more potent than unconjugated anti-IgD antibodies at stimulating B cell DNA synthesis. These results suggest, therefore, that the unique ability of picogram concentrations of haptenated type 2 Ag to stimulate Ig secretion in the absence of T cells may be a function of their ability to promote effective cross-linking without resulting in the modulation of sIg. This would enable such Ag to mediate repetitive B cell signaling, a situation that cannot be achieved by unconjugated anti-Ig antibodies which result in modulation of sIg at their mitogenic concentrations. These compounds therefore may be employed to study B cell activation stimulated by sIg cross-linking at concentrations that may more closely reflect those which are achieved under physiologic conditions by type 2 Ag.

Characterization and localization of human renal kininogen.

Kininogen was isolated from human urine by batch adsorption with immobilized antibody to the immunologically identical heavy (H) chains of both high molecular weight (HMW) and low molecular weight (LMW) human plasma kininogens. All releasable kinin in the guanidinium chloride eluate was associated with kininogen antigen in gel filtration fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the eluate gave major stained and antigenic bands corresponding to the major form of plasma LMW kininogen. Also, the staining patterns and antigenic profiles obtained upon alkaline disc gel electrophoresis of the urinary and plasma LMW kininogens were strikingly similar. When antibody to H chain was used in an indirect immunofluorescence technique, cytoplasmic staining was observed in cells of distal tubules and c cortical and medullary collecting ducts of human kidneys. No fluorescence was observed using antibody to the unique light (L) chain of plasma HMW kininogen and no intact HMW kininogen was found in urine by radioimmunoassay. We conclude that the kidney is a source of urinary kininogen, while the L chain antigen in urine probably represents filtered degradation products of plasma HMW kininogen.

Effects of dietary phytol and phytanic acid in animals.

Feeding of phytol in large doses (2-5% by weight in the diet) led to accumulation of phytanic acid in the mouse, rat, rabbit, and chinchilla, the degree of accumulation depending upon the level of dietary intake. The relative concentration of phytanic acid, expressed as a percentage of the total fatty acids, was as high as 20-60% in liver and 30-40% in serum. Phytenic acid, which may be an intermediate in the conversion of phytol to phytanic acid, also accumulated. When phytol was withdrawn from the diet, tissue and serum concentrations of phytanic acid fell rapidly, which indicates the ability of the normal animal to metabolize phytanic acid readily. At high dosages in the diet, phytol inhibited growth and caused death within 1-4 weeks. In the mouse, dietary phytanic acid and dietary phytol fed in equivalent amounts were of comparable toxicity. Accumulation of tissue phytanic acid occurred more rapidly when phytanic acid was fed than when phytol was fed in equal amounts. In none of the animals fed either phytol or phytanic acid were there any signs of neurological defects. Histologic examination of rats fed phytol showed some fat accumulation, glycogen depletion, and karyokinesis in the liver. There were no pathologic changes in the retina or in the peripheral and central nervous system such as those described in Refsum's disease.