Cytokine dependency of human B cell cycle progression elicited by ligands which coengage BCR and the CD21/CD19/CD81 costimulatory complex.

Coengagement of BCR and the C3dg binding CD21/CD19/CD81 costimulatory complex can profoundly reduce the BCR binding threshold for eliciting B cell S phase entry, provided cytokine is present. IL-4 is substantially better than IL-2, IL-13, and TNF-alpha at exhibiting synergy with BCR:CD21 coengaging ligand (anti-IgM:anti-CD21:dextran) in promoting B cell DNA synthesis. Synergy between IL-4 and anti-IgM:anti-CD21:dextran (a) is not explained by the viability-promoting function of IL-4, (b) occurs when the anti-CD21 moiety engages either C3dg binding or non-C3dg binding domains, (c) does not reflect reversal of FcgammaRII-mediated negative regulation, and (d) involves differing temporal requirements for BCR and IL-4R signal transduction during the activation process. The IL-4R signaling pathway appears to synergize directly with the BCR:CD21 signaling pathway(s) in promoting the progression of resting B cells past an early G1 checkpoint, as well as to promote independently the progression of activated B cells past a later G1 to S checkpoint.

Inhibition of retinal angiogenesis by peptides derived from thrombospondin-1.

PURPOSE: Thrombospondin (TSP)1 is a tumor suppressor with activity that is associated with its ability to inhibit neovascularization. Previous studies have mapped this antiangiogenic activity to the type 1 repeats and the amino-terminal portion of the molecule within the procollagen-like domain. The present study was performed to investigate the ability of TSP-1 and peptides derived from the type 1 repeats to inhibit retinal angiogenesis. METHODS: TSP-1 and peptides with tryptophan-rich, heparin-binding sequences and transforming growth factor (TGF)-beta1 activation sequences were evaluated in two models of retinal angiogenesis: a retinal explant assay and a rat model of retinopathy of prematurity (ROP). RESULTS: Platelet-derived TSP-1 inhibited angiogenesis in both experimental models. Peptides from the native TSP-1 sequence, which contained both the tryptophan-rich repeat and the TGF-beta1 activation sequence, were the most potent inhibitors of endothelial cell outgrowth in the retinal explant assay. In contrast, a peptide containing only the tryptophan-rich, heparin-binding sequence was most active in inhibiting neovascular disease in the rat ROP model. CONCLUSIONS: These results indicate that the type 1 repeats of TSP-1 contain two subdomains that may independently influence the process of neovascularization, and that peptides derived from these type 1 repeats may be promising pharmacologic agents for treatment of retinal angiogenesis.

Inactivation of HIV-1 nucleocapsid protein P7 by pyridinioalkanoyl thioesters. Characterization of reaction products and proposed mechanism of action.

The synthesis and antiviral properties of pyridinioalkanoyl thioester (PATE) compounds that target nucleocapsid p7 protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) have been described previously (Turpin, J. A., Song, Y., Inman, J. K., Huang, M., Wallqvist, A., Maynard, A., Covell, D. G., Rice, W. G., and Appella, E. (1999) J. Med. Chem. 42, 67-86). In the present study, fluorescence and electrospray ionization-mass spectrometry were employed to determine the mechanism of modification of NCp7 by two lead compounds, N-[2-(5-pyridiniovaleroylthio)benzoyl]sulfacetamide bromide and N-[2-(5-pyridiniovaleroylthio)benzoyl]-4-(4-nitrophenylsulfonyl )anili ne bromide (compounds 45 and 47, respectively). Although both compounds exhibit antiviral activity in cell-based assays, we failed to detect appreciable ejection of zinc from NCp7 under conditions in which previously described NCp7-active disulfides readily eject zinc. However, upon "activation" by Ag(+), compound 45 reacted with NCp7 resulting in the zinc ejection from both zinc fingers. The reaction followed a two-step mechanism in which zinc was ejected from the carboxyl-terminal zinc finger faster than from the amino-terminal zinc finger. Both compounds covalently modified the protein with pyridinioalkanoyl groups. Compound 45 modified cysteines 36 and 49 of the carboxyl-terminal zinc finger. The results obtained herein demonstrate that PATE compounds can be constructed that selectively target only one of the two zinc fingers of NCp7, thus providing an impetus to pursue development of highly selective zinc finger inhibitors.

A quantitative approach to signal transduction.

The high affinity receptor for IgE (FcepsilonRI), is one of a family of immunoreceptors whose antigen-induced clustering leads to a variety of cellular responses. The signaling pathways are enormously complex but by focusing on only the most initial steps, it is now possible to sketch plausible molecular models that relate the interaction of multivalent antigens with the receptor-bound IgE to the earliest cellular events. In this paper, we describe how we have combined quantitative experimentation and mathematical modeling to probe this system further. We also discuss some of the formidable challenges that remain before we can claim reasonably complete understanding of even these early events.

Synthesis and biological properties of novel pyridinioalkanoyl thiolesters (PATE) as anti-HIV-1 agents that target the viral nucleocapsid protein zinc fingers.

Nucleocapsid p7 protein (NCp7) zinc finger domains of the human immunodeficiency virus type 1 (HIV-1) are being developed as antiviral targets due to their key roles in viral replication and their mutationally nonpermissive nature. On the basis of our experience with symmetrical disulfide benzamides (DIBAs; Rice et al. Science 1995, 270, 1194-1197), we synthesized and evaluated variants of these dimers, including sets of 4,4'- and 3,3'-disubstituted diphenyl sulfones and their monomeric benzisothiazolone derivatives (BITA). BITAs generally exhibited diminished antiviral potency when compared to their disulfide precursors. Novel, monomeric structures were created by linking haloalkanoyl groups to the benzamide ring through -NH-C(=O)- (amide) or -S-C(=O)- (thiolester) bridges. Amide-linked compounds generally lacked antiviral activity, while haloalkanoyl thiolesters and non-halogen-bearing analogues frequently exhibited acceptable antiviral potency, thus establishing thiolester benzamides per se as a new anti-HIV chemotype. Pyridinioalkanoyl thiolesters (PATEs) exhibited superior anti-HIV-1 activity with minimal cellular toxicity and appreciable water solubility. PATEs were shown to preferentially target the NCp7 Zn finger when tested against other molecular targets, thus identifying thiolester benzamides, and PATEs in particular, as novel NCp7 Zn finger inhibitors for in vivo studies.

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.

An unusual mechanism for ligand antagonism.

The ratio of late to early events stimulated by the mast cell receptor for immunoglobulin E (IgE) correlated with the affinity of a ligand for the receptor-bound IgE. Because excess receptors clustered by a weakly binding ligand could hoard a critical initiating kinase, they prevented the outnumbered clusters engendered by the high-affinity ligands from launching the more complete cascade. A similar mechanism could explain the antagonistic action of some peptides on the activation of T cells.

Antiproliferative and antitumor activities of D-reverse peptides derived from the second type-1 repeat of thrombospondin-1.

The extracellular matrix glycoprotein thrombospondin-1 (TSP1) inhibits angiogenesis, endothelial cell growth, motility and adhesion. Peptides from the type I repeats of TSP1 mimic the adhesive and growth inhibitory activities of the intact protein and specifically interact with heparin and transforming growth factor-beta (TGF beta). To define the structural basis for the antiangiogenic activities of these peptides, we prepared analogs of the TSP1 peptide KRFKQDGGWSHWSPWSSC. L-forward, L-reverse, and D-reverse (retro-inverso) analogs displayed identical activities for binding to heparin, demonstrating a lack of stereospecificity for heparin binding. The L-reverse and D-reverse peptides, however, had somewhat decreased abilities to activate latent TGF beta. Conjugation of the forward peptides through a C-terminal thioether and the reverse peptides through an N-terminal thioether to polysucrose abolished the adhesive activity of the peptides and enhanced their antiproliferative activities for endothelial and breast carcinoma cells stimulated by fibroblast growth factor-2. Their antiproliferative activities were independent of latent TGF beta activation, because substitution of an Ala residue for the essential Phe residue in the TSP1 type-1 repeat peptide increased their potency for inhibiting TSP1 binding to heparin and for inhibiting endothelial cell proliferation. Although the conjugated peptides were inactive in vivo, an unconjugated retro-inverso analog of the native TSP peptide inhibited breast tumor growth in a mouse xenograft model. Thus, these TSP-derived peptide analogs antagonize endothelial growth through their heparin-binding activity rather than through activation of latent TGF beta or increasing cell adhesion. These stable analogs may therefore be useful as therapeutic inhibitors of angiogenesis stimulated by fibroblast growth factor-2.

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.

Thrombospondin 1 and type I repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells.

Thrombospondin 1 (TSP1) inhibits angiogenesis and modulates endothelial cell adhesion, motility, and growth. The antiproliferative activity of TSP1 is mimicked by synthetic peptides derived from the type I repeats of TSP1 that antagonize fibroblast growth factor 2 and activate latent transforming growth factor beta. These TSP1 analogues induced programmed cell death in bovine aortic endothelial cells based on morphological changes, assessment of DNA fragmentation, and internucleosomal DNA cleavage. Intact TSP1 also induced DNA fragmentation. The endothelial cell response was specific because no DNA fragmentation was induced in MDA-MB-435S breast carcinoma cells, although TSP1 and the peptide conjugates inhibited the growth of both cell types. Apoptosis did not depend on activation of latent transforming growth factor beta because peptides lacking the activating sequence RFK were active. Apoptosis was not sensitive to inhibitors of ceramide generation but was inhibited by the phosphatase inhibitor vanadate. Induction of DNA fragmentation by the peptides was decreased when endothelial cell cultures reached confluence. Growth of the cells on a fibronectin substrate also suppressed induction of apoptosis by TSP1 or the peptides. Differential sensitivities to kinase inhibitors suggest that apoptosis and inhibition of proliferation are mediated by distinct signal transduction pathways. These results demonstrate that induction of apoptosis by the TSP1 analogues is not a general cytotoxic effect and is conditional on a lack of strong survival-promoting signals, such as those provided by a fibronectin matrix. The antitumor activity of TSP1 may therefore result from an increased sensitivity to apoptosis in endothelial cells adjacent to a provisional matrix during formation of vascular beds in tumors expressing TSP1.

Differential activation requirements of isotype-switched B cells.

In the present studies, we compared the activation requirements of sIgM+/sIgD+ B cells with those of isotype-switched sIgM-/sIgA+ B cells. We found that whereas sIgM+ B cells respond to T cell-independent (TI) and T cell-dependent (TD) Ag with no significant bias toward one stimulus, sIgA+ B cells were deficient in their ability to respond to antigen receptor cross-linking but responded remarkably well to TD stimuli. Thus, dextran-conjugated anti-IgA antibody (anti-IgA-dextran), anti-kappa-dextran, or various immobilized anti-IgA antibodies (Ab) induced only low-level IgA B cell proliferation and no IgA secretion in the presence of various lymphokines; in marked contrast, sIgA+ B cells responded to cognate and noncognate T cell stimulation as well as to stimulation by CD40 ligand-bearing fibroblasts by secreting large amounts of IgA (up to 240 000 ng/ml per 10(5) cells). This pattern of sIgA+ B cell responsiveness was noted with both germinal center peanut agglutininhi (PNAhi) and non-germinal center PNAlo B cells. In confirmation of these results, whole Peyer's patch or lamina propria cell populations containing less than 15% sIgA+ B cells stimulated with a noncognate T cell stimulus or T cell membranes secreted mainly IgA (68%-94% of the total Ig secreted) and relatively little IgM. The strict T cell dependence of IgA B cell activation and differentiation provides important insights into immune responses of mucosal tissues and must be considered in the development of vaccines, particularly those designed to stimulate mucosal tissues containing large numbers of isotype-switched B cells.

Reciprocal regulation of mucosal surface IgA+ B cells by Ig receptor cross-linking and CD40 ligand.

In the present study, we analyze the role of Ig receptor cross-linking in T cell-dependent stimulation of both preswitch (surface IgM+ (sIgM+/sIgD+) B cells and postswitch (sIgA+) B cells. We demonstrate that purified sIgA+ B cells pretreated with anti-IgA-dextran at low concentrations (10 and 100 ng/ml) exhibited an increased response to activated T cells, whereas pretreatment with higher doses (1 and 10 micrograms/ml) led to a profound suppression of IgA secretion (> or = 90%). The suppressive effect of anti-IgA-dextran was accentuated in the presence of IL-2 and attenuated in the presence of IL-4. Anti-IgA-dextran pretreatment had no effect on sIgA+ B cell survival. sIgM+/sIgD+ B cells pretreated with anti-IgD-dextran or anti-IgM-dextran did not show significant inhibition. The increased susceptibility of sIgA+ B cells, but not of sIgM+/sIgD+ B cells, to Ig cross-linking-mediated suppression was confirmed in cross-linking studies with the same Ab (anti-kappa-dextran). Importantly, anti-IgA-dextran-mediated suppression could be reversed by stimulation of sIgA+ B cells with fibroblasts expressing CD40L; such a reversal required persistent exposure to cells expressing high levels of CD40L. These studies imply that Ig receptor cross-linking renders postswitch sIgA+ B cells unresponsive to subsequent stimulation via activated T cells, but this unresponsiveness is overcome by a persistent high level CD40L signal.

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.

Enhanced immunogenicity of protein-dextran conjugates: I. Rapid stimulation of enhanced antibody responses to poorly immunogenic molecules.

In view of our observation that anti-immunoglobulin antibody conjugated to high-molecular-weight dextran stimulates high levels of B-cell activation (Brunswick et al. J. Immunol. 1989, 143, 1239), we coupled T cell-dependent antigens to dextran. When mice were immunized, in the absence of adjuvant, with a BSA-dextran conjugate (BSA-dex), a persistent, high-titre anti-BSA IgG1 response was induced. Titres were dose-dependent and seen with as little as 10 micrograms of conjugated protein. Anti-BSA titres were detected as early as day 7, usually peaked at about day 14 and persisted for at least 4 weeks. Anti-hapten antibodies were also elicited in mice that were immunized with haptenated BSA covalently bound to dextran, and secondary responses could be induced even after inoculation of the unconjugated protein. Covalent attachment of the protein to the polymer was necessary, and the response was specific, as coinjection of BSA-dex and an unrelated antigen, goat IgG, did not elicit detectable anti-goat antibodies. The immunogenic potential of these conjugates did not depend on the ability of the dextran carrier to induce antibody, inasmuch as they stimulated high levels of anti-protein antibody in mice unresponsive to dextran. A minimum size dextran polymer was required for enhanced immunogenicity as conjugates of BSA with dextran of molecular mass 500 or 2000 kDa but not of 70 kDa gave detectable anti-BSA titres.

Differences among various lineages of antigen-presenting cells in processing exogenous antigen internalized through transferrin receptors.

The Ag, pigeon cytochrome c, was coupled to human ferric transferrin by a heteroligation technique to target Ag into the endosomal transport pathway via transferrin receptors. The ability of various types of APC that do or do not express transferrin receptors to process exogenous Ag in their endosomes was investigated by the stimulation of Ag-specific CD4+ T cells with the transferrin-Ag conjugate in a serum-free assay. When two B lymphoma cells were the source of APC, the conjugate was significantly more potent than native Ag in activating the T cells, agreeing with our previous finding using a third B lymphoma cell. The conjugate and Ag were similarly presented by splenic B cells that lack transferrin receptors to the T cells. However, both a macrophage hybridoma and a MHC class II-L cell transfectant hardly elicited a T cell response to the conjugate, although a response to native Ag was readily observed. These findings could not be attributed to an absence of transferrin receptors or receptor-mediated internalization of the conjugate, nor to differential expression of MHC class II molecules or li chain by the APC. The poor presentation of the conjugate by the L cell transfectants was associated with diminished catabolism of the conjugate, however, the macrophage hybridoma rapidly degraded the conjugate, similar to the B lymphoma cell. Peritoneal macrophages, which lack transferrin receptors, and the macrophage hybridoma induced a response to the conjugate only at concentrations that allowed internalization by fluid phase pinocytosis. The lower potency of the conjugate compared with native Ag with non-B-presenting cells suggest that these cell types process the conjugate by a different mechanism than used by B cells. Differences in the mechanism of Ag processing used by APC of distinct cell lineages may possibly influence immune responsiveness.

Antigen presentation by B lymphoma cells. Requirements for processing of exogenous antigen internalized through transferrin receptors.

The Ag, pigeon cytochrome c, was delivered to the early endosomes in a form coupled to human ferric transferrin that entered APC through transferrin receptors. The processing of the transferrin-Ag conjugate by B lymphoma cells was compared with that of unconjugated native Ag that entered APC by a nonreceptor-mediated mechanism. Within 5 min after internalization, catabolized conjugate was detected in isolated early endosomes and did not accumulate in these organelles. Analysis of the rapid catabolism of the conjugate demonstrated that the Ag, not the transferrin, portion of the molecule was degraded by the APC, suggesting that similar proteases may mediate the processing of the conjugate and native Ag. The processing mechanisms of these molecules shared similarities. Treatment of APC with chloroquine or paraformaldehyde interfered with the stimulation of Ag-specific CD4+ T cells by both transferrin-Ag conjugate and native Ag. However, the T cell responses to the conjugate and native Ag were different in two important respects. First, T cell activation by the conjugate began at an earlier time point and occurred at a faster rate than T cell stimulation by the same concentration of native Ag during a 3-h time course. Second, the T cell response to the conjugate, but not to native Ag, was diminished by treating APC with cycloheximide, a reversible protein synthesis inhibitor. This partial inhibition of the conjugate response by cycloheximide could not be attributed to significant effects on transferrin receptor expression, or on internalization, recycling, or degradation of the conjugate. The differential cycloheximide-sensitivity of the T cell responses indicates that the processing pathways of the two molecules are different. Our findings suggest that the early endosomes may function as an Ag-processing compartment, and that more than one pathway may lead to productive processing in B lymphoma cells.

Direct detection of major histocompatibility complex class I binding to antigenic peptides using surface plasmon resonance. Peptide immobilization and characterization of binding specificity.

We have developed model systems in which the binding of purified, genetically engineered, soluble analogues of major histocompatibility complex (MHC) class I molecules to immobilized antigenic peptides can be monitored in real time using surface plasmon resonance (SPR). Synthetic analogues of several peptides known to bind different mouse and human MHC class I molecules were prepared with cysteine residues substituted at appropriate positions. The analogue peptides were immobilized via the bifunctional reagent N-gamma-maleimidobutyryloxy-succinimide to amino groups generated on the dextran-modified gold surface of a biosensor flow cell. Using this approach, each position in the sequence of an H-2Ld-specific viral peptide, pMCMV (YPHFMPTNL), was used for coupling, and the resulting surfaces were tested for binding of the soluble analogue of H-2Ld, H-2Lds. In accord with our previously described H-2Ld/pMCMV three-dimensional structural model, only those residues of the peptide that remain exposed following binding (positions 4-8) can be replaced by cysteine and used for coupling. Stable binding of soluble MHC class I molecules, H-2Lds, H-2Dds, H-2Kbs, and HLA-A2s to their respective immobilized cognate peptides was detected by SPR. Specificity of the peptide/MHC interaction was characterized both by direct binding using immobilized peptides and by competition with peptides in solution, and in general was consistent with known immunological reactivity. Some peptides bound not only their cognate MHC molecule, but others at lower apparent affinity. Measurement of real time binding of MHC class I molecules to peptides immobilized through specific side chains suggests the application of a similar approach to the study of the interaction of peptides with a wide variety of peptide-binding macromolecules.