Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.T and B cells represent two fundamentally different recognition modes of the specific immune system. Through alternating selection processes T cells are educated to recognize antigenic peptides presented in association with self-molecules of the major histocompatibility complex (MHC) on the surface of antigen-presenting cells. In contrast, B cells are not educated to be self-MHC-restricted and B-cell receptors (antibodies), whether soluble or in membrane-bound form, recognize threedimensional target structures. The distinctly different education of B and T cells explains why antibodies with the MHCrestricted specificity of T cells are rare and why it has been difficult to generate such specificities by conventional B-cell hybridoma techniques. We have taken advantage of the selection power of the phage display technology which makes it possible to test tens of millions of individual clones and have devised a method to generate recombinant antibodies recognizing predetermined peptide/MHC complexes. The speed and feasibility of this method makes it realistic to produce antibodies to a variety of specific peptide/MHC complexes which may be useful in studying MHC-restricted T-cell recognition and may lead to novel approaches in diagnostics and immunotherapy.MATERIALS AND METHODS MHC Purification. The AKR mouse-derived lymphoma RDM-4 was used for Kk production as described (1). In brief, Kk molecules were immunoaffinity purified from detergent cell lysates by using the monoclonal anti-Kk antibody 11.4-1 (American Tissue Type Culture Collection). The affinity columns were washed extensively and bound MHC class I molecules were eluted with 0.05 M diethylamine, pH 11/0.15 M sodium chloride/0.1% sodium azide/0.1% sodium deoxycholate, neutralized, and concentrated by vacuum dialysis. Human 82-microglobulin was obtained from the urine of uremic patients and purified to homogeneity by gel filtration and chromatofocusing (1).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.The influenza virus-derived nucleoprotein peptide NP50-57 (single-letter code, SDYEGRLI) and hemagglutinin peptide Ha255-262 (FESTGNLI) were synthesized manually on a RaMPS synthesizer (DuPont) using standard fluorenylmethoxycarbonyl protection strategy.Generation of Peptide/MHC Cl...

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(2011) Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor, mAbs, 3:6, 584-595,

Due to technical limitations, little knowledge exists on the composition of Ag-specific polyclonal Ab responses. Hence, we here present a molecular analysis of two representative human Ab repertoires isolated by using a novel single-cell cloning approach. The observed genetic diversity among tetanus toxoid-specific plasma cells indicate that human polyclonal repertoires are limited to the order of 100 B cell clones and hypermutated variants thereof. Affinity and kinetic binding constants are log-normally distributed, and median values are close to the proposed affinity ceilings for positive selection. Abs varied a million-fold in affinity but were restricted in their off-rates with an upper limit of 2 × 10−3 s−1. Identification of Abs of high affinity without hypermutations in combination with a modest effect of hypermutations on observed affinity increases indicate that Abs selected from the naive repertoire are not only of low affinity but cover a relatively large span in affinity, reaching into the subnanomolar range.

Superantigens (SAGs) activate T cells by simultaneously binding the Vbeta domain of the TCR and MHC class II molecules on antigen-presenting cells. The preferential expression of certain Valpha regions among SAG-reactive T cells has suggested that the TCR alpha chain may modulate the level of activation through an interaction with MHC. We demonstrate that the TCR alpha chain is required for maximum stabilization of the TCR-SAG-MHC complex and that the alpha chain increases the half-life of the complex to match those of TCR-peptide/MHC complexes. The site on the TCR alpha chain responsible for these effects is CDR2. Thus, the overall stability of the TCR-SAG-MHC complex is determined by the combination of three distinct interactions: TCR-SAG, SAG-MHC, and MHC-TCR.

Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3 (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/ TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency of the SEC3 variants correlated with enhanced binding without any optimum in the binding range covered by native TCR ligands. Comparable studies using anti-TCR antibodies of known affinity confirmed these observations. By comparing the biological potency of the two sets of ligands, we found a significant correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength.T lymphocytes are one of the central components of the acquired immune system. Each T lymphocyte carries a unique T cell receptor (TCR), 1 which recognizes foreign peptides bound to the major histocompatibility complex (MHC) molecules (1, 2). The molecular mechanism by which the ligand-bound TCR transmits a signal across the plasma membrane is not known. Based on the observation that a few peptide/MHC complexes can cause down-modulation of a large number of TCRs, a model for TCR triggering proposes that low affinity (and fast off-rate) is necessary to allow "serial triggering" (3); that is, to permit one ligand to stimulate a large numbers of TCRs (up to 200) within a relatively short period of time. Thus, the model predicts that high affinity TCR ligands are suboptimal activators of T cells and that optimal dissociation kinetics exists (4). Additional models for TCR signaling (5, 6), based on kinetic proofreading principles, suggests that too brief a ligand occupancy, leading to partial assembly of the signaling complex, transmits a negative signal (antagonism) whereas contact times that enable complete assembly of the signaling complex delivers a full positive signal (agonism). Common to these considerations is that the off-rate, which defines the average time of receptor/ligand contact, is thought to determine the signaling strength of the TCR/ligand interaction.Bacterial superantigens, which are involved in serious diseases such as toxic shock syndrome and food poisoning, elicit their biological function by cross-linking TCR and MHC class II molecules. They bind to relative invariant areas of the receptors and can thereby activate large fractions (5-20%) of the T cell population (7). Like peptide/MHC ligands, bacterial superantigens bind only weakly to TCRs with affinities in the M range (8 -11). The SAG-TCR interaction is therefore closely related to the endogenous TCR-antigenic peptide-MHC interaction, and the molecular mechanism by which t...

We have developed efficient methodologies for construction and expression of comprehensive phage display libraries of murine Fab antibody fragments in E. coli cells. Our methods optimize several critical steps of the polymerase chain reaction (PCR) amplification of transcripts of the re-arranged immunoglobulin genes and of their subsequent assembly and expression: Firstly, we have designed exhaustive sets of PCR primers of low degeneracy for the amplification of transcripts of the Fab region of the heavy and light-chain genes. These primers proved effective in amplification of Fab gene fragments from a large panel of hybridoma cell lines of different specificity and family sub-type. Secondly, we have developed a 'jumping PCR' technique that effectively assembled and recombined the amplified heavy and light-chain gene fragments into a bi-cistronic operon. Thirdly, we have constructed expression vectors for insertion of the combinatorial Fab gene-cassette in fusion with a truncated version of the phage surface protein, gIIIp. The heavy chain and the light chain-gIII fusion are transcribed as a polycistronic mRNA from the lacZ promoter and efficient transcriptional control is provided by wildtype lacI present on the vector. The utility of the system was demonstrated by isolating several antigen-binding clones from hybridomas and libraries made from immunized mice.

TCR down-regulation plays an important role in modulating T cell responses both during T cell development and in mature T cells. At least two distinct pathways exist for down-regulation of the TCR. One pathway is activated following TCR ligation and is dependent on tyrosine phosphorylation. The other pathway is dependent on protein kinase C (PKC)-mediated activation of the CD3γ di-leucine-based receptor-sorting motif. Previous studies have failed to demonstrate a connection between ligand- and PKC-induced TCR down-regulation. Thus, although an apparent paradox, the dogma has been that ligand- and PKC-induced TCR down-regulations are not interrelated. By analyses of a newly developed CD3γ-negative T cell variant, freshly isolated and PHA-activated PBMC, and a mouse T cell line, we challenged this dogma and demonstrate in this work that PKC activation and the CD3γ di-leucine-based motif are indeed required for efficient ligand-induced TCR down-regulation.