Characterizing the functionality of recombinant T-cell receptors in vitro: A pMHC tetramer based approach (original) (raw)
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A diverse set of oligomeric class II MHC-peptide complexes for probing T-cell receptor interactions
Chemistry & Biology, 2000
Background: T-cells are activated by engagement of their clonotypic cell surface receptors with peptide complexes of major histocompatibility complex (MHC) proteins, in a poorly understood process that involves receptor clustering on the membrane surface. Few tools are available to study the molecular mechanisms responsible for initiation of activation processes in T-cells. Results: A topologically diverse set of oligomers of the human MHC protein HLA-DR1, varying in size from dimers to tetramers, was produced by varying the location of an introduced cysteine residue and the number and spacing of sulfhydryl-reactive groups carried on novel and commercially available crosslinking reagents. Fluorescent probes incorporated into the cross-linking reagents facilitated measurement of oligomer binding to the T-cell surface. Oligomeric MHC-peptide complexes, including a variety of MHC dimers, trimers and tetramers, bound to T-cells and initiated T-cell activation processes in an antigen-speci¢c manner. Conclusion: T-cell receptor dimerization on the cell surface is suf¢cient to initiate intracellular signaling processes, as a variety of MHC-peptide dimers differing in intramolecular spacing and orientation were each able to trigger early T-cell activation events. The relative binding af¢nities within a homologous series of MHC-peptide oligomers suggest that T-cell receptors may rearrange in the plane of the membrane concurrent with oligomer binding.
T Cell Activation by Purified, Soluble, Class I MHC Molecules. Requirement for Polyvalency
The Journal of …, 1988
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. The Ag-specific TCR of effector T lymphocytes recognizes nominal Ag on APC in the context of self MHC class I or class I1 Ag (1.2). Similarly, allospecific T lymphocytes identify allogeneic (MHC disparate) cells by direct interaction of their TCR with the MHC Ag of the target cell. Despite recent progress in the study of the interaction of the MHC class I1 Ag with antigenic peptides . the molecular basis of the interaction of TCR with MHC class I Ag in the allogeneic reaction is poorly understood. In particular, little is known about the valence, topologic requirements, or the intrinsic affinity of TCR interaction with MHC Ag required for successful T cell stimulation. A major experimental difficulty in studying this problem results from the multivalent nature of the TCR and the MHC molecules as displayed on their respective cell surfaces. As a step in the general approach to the study of such interactions between multivalent components, we have devised a strategy for the production of soluble counterparts of the otherwise membrane-bound class I molecules . To examine the nature of these TCR/ MHC interactions in more detail, 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 H-2Dd MHC class I Ag.
Covalent assembly of a soluble T cell receptor-peptide-major histocompatibility class I complex
Proceedings of the National Academy of Sciences, 1996
We used stepwise photochemical crosslinking for specifically assembling soluble and covalent complexes made of a T-cell antigen receptor (TCR) and a class I molecule of the major histocompatibility complex (MHC) bound to an antigenic peptide. For that purpose, we have produced in myeloma cells a single-chain Fv construct of a TCR specific for a photoreactive H-2Kd-peptide complex. Photochemical cross-linking of this TCR single-chain Fv with a soluble form of the photoreactive H-2Kd-peptide ligand resulted in the formation of a ternary covalent complex. We have characterized the soluble ternary complex and showed that it reacted with antibodies specific for epitopes located either on the native TCR or on the Kd molecules. By preventing the fast dissociation kinetics observed with most T cell receptors, this approach provides a means of preparing soluble TCR-peptide-MHC complexes on large-scale levels. Most T lymphocytes recognize antigenic peptides bound to products of the major histocompatibility complex (MHC). This joint specificity is accounted for by a single membrane-bound receptor composed of two chains, a and J, each consisting of an N-terminal variable (V) region and a C-terminal constant (C) region. The resolution of the three-dimensional structure of the extracellular portion of a T-cell antigen receptor (TCR) 13 chain has recently confirmed the prediction that the Vf3 and Co domains are structurally related to the V and C domains of immunoglobulins (Ig). Peptide loops homologous to Ig complementarity-determining regions (CDR) have been identified at the membrane-distal end of the V,B domain where they most likely form part of the peptide-MHC binding site (1). The exact contribution of each of the TCR CDRs to the
PLoS ONE, 2012
Class I Major Histocompatibility Complex (MHC) molecules evolved to sample degraded protein fragments from the interior of the cell, and to display them at the surface for immune surveillance by CD8 + T cells. The ability of these lymphocytes to identify immunogenic peptide-MHC (pMHC) products on, for example, infected hepatocytes, and to subsequently eliminate those cells, is crucial for the control of hepatitis B virus (HBV). Various protein scaffolds have been designed to recapitulate the specific recognition of presented antigens with the aim to be exploited both diagnostically (e.g. to visualize cells exposed to infectious agents or cellular transformation) and therapeutically (e.g. for the delivery of drugs to compromised cells). In line with this, we report the construction of a soluble tetrameric form of an ab T cell receptor (TCR) specific for the HBV epitope Env 183-191 restricted by HLA-A*02:01, and compare its avidity and fine-specificity with a TCR-like monoclonal antibody generated against the same HLA target. A flow cytometry-based assay with streptavidin-coated beads loaded with Env 183-191 /HLA-A*02:01 complexes at high surface density, enabled us to probe the specific interaction of these molecules with their cognate pMHC. We demonstrate that the TCR tetramer has similar avidity for the pMHC as the antibody, but they differ in their fine-specificity, with only the TCR tetramer being capable of binding both natural variants of the Env 183-191 epitope found in HBV genotypes A/C/D (187Arg) and genotype B (187Lys). Collectively, the results highlight the promiscuity of our soluble TCR, which could be an advantageous feature when targeting cells infected with a mutation-prone virus, but that binding of the soluble oligomeric TCR relies considerably on the surface density of the presented antigen.
Journal of Experimental Medicine, 1997
Understanding the regulation of cell surface expression of specific peptide-major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide-MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR-Ig) that have high affinity for their cognate peptide-MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR-Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus-1 presented by human histocompatibility leukocyte antigens-A2. Further, using 2C TCR-Ig, a more detailed analysis of the interaction with cognate peptide-MHC complexes revealed several interesting findings. Soluble divalent 2C TCR-Ig detected significant changes in the level of specific antigenic-peptide MHC cell surface expression in cells treated with ␥ -interferon ( ␥ -IFN). Interestingly, the effects of ␥ -IFN on expression of specific peptide-MHC complexes recognized by 2C TCR-Ig were distinct from its effects on total H-2 L d expression; thus, lower doses of ␥ -IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide-MHC complexes. Analysis of the binding of 2C TCR-Ig for specific peptide-MHC ligands unexpectedly revealed that the affinity of the 2C TCR-Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8-H-2 K bm3 , is very low, weaker than 71 M. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca-H-2 L d , is ف 1000-fold higher. Thus, negatively selecting peptide-MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis, indicate the importance of using high affinity biologically relevant cognates, such as soluble divalent TCR, in furthering our understanding of immune responses.
The Journal of Immunology
CD8 ؉ T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Additionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8 ؉ T cells directed against Ags presented by H-2K b class I molecules. When H-2K b contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the K b -binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate.
Molecular Immunology, 1997
Immune activation is mediated by a specific interaction between the T-cell receptor (TCR) and an antigenic peptide bound to the major histocompatibility complex (MHC). T-cell activation can also be stimulated by superantigens which bind to germline-encoded variable domain sequences of certain TCR p-chains. We have used a surface plasmon resonance biosensor to characterize the molecular interactions between a class II-restricted xfi TCR and its superantigen and MHCipeptide ligands. The extracellular domains of the murine DlO TCR (Vct2, VB8.2) were expressed in insect cells and secreted as a disulfide-linked heterodimer. In the absence of MHC class II, purified soluble DIO TCR bound to Stuphylococcus uurws enterotoxin C2 with an association rate of 1.69&0.12x 104M-'se& and a dissociation rate of 1.9kO.47 x IO 'set ', giving a dissociation constant of 1. I PM. Binding of the TCR to S. uureus enterotoxin B was barely detectable and could not be measured accurately due to the rapid dissociation rate. Soluble D10 TCR also bound to a soluble murine MHC class II I-Ak molecule containing a fused antigenic conalbumin peptide and complementary leucine zipper sequences to facilitate efficient chain pairing. The purified I-Ak chimera specifically stimulated proliferation of the D IO T-cell clone, and bound to immobilized soluble D 10 TCR with an association rate of I .07 *O. 19 x IO" Mm ' set-' and a dissociation rate of 3.2&0.65x IO 'set '. giving a dissociation constant of 2. I PM.
Nature Immunology, 2012
The binding of T cell antigen receptors (TCRs) to specific complexes of peptide and major histocompatibility complex (pMHC) is typically of very low affinity, which necessitates the use of multimeric pMHC complexes to label T lymphocytes stably. We report here the development of pMHC complexes able to be crosslinked by ultraviolet irradiation; even as monomers, these efficiently and specifically stained cognate T cells. We also used this reagent to probe T cell activation and found that a covalently bound pMHC was more stimulatory than an agonist pMHC on lipid bilayers. This finding suggested that serial engagement of TCRs is dispensable for activation when a substantial fraction of TCRs are stably engaged. Finally, pMHC-bound TCRs were 'preferentially' transported into the central supramolecular activation cluster after activation, which suggested that ligand engagement enabled linkage of the TCR and its associated CD3 signaling molecules to the cytoskeleton. T cells express T cell antigen receptors (TCRs) that recognize antigenic peptides bound to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells 1. As the interaction between a TCR and a complex of peptide and MHC (pMHC) is generally very weak and has a dissociation constant (K d) of 1-100 µM and a half-life (t 1/2) of 0.5-10 s, a soluble pMHC monomer cannot in most cases stably adhere to the surface of the T cell. This has led to the development of pMHC tetramers 2 and other multimeric forms for the detection and isolation of specific T cells in mixed populations 3. Such methods have been widely used in both basic and clinical T cell immunology; however, they also have many considerable limitations. For example, pMHC multimers aggregate TCRs, causing
Cellular Immunology, 1999
T cell receptors (TCR) and major histocompatibility complex (MHC) molecules are integral membrane proteins that have central roles in cell-mediated immune recognition. Therefore, soluble analogs of these molecules would be useful for analyzing and possibly modulating antigen-specific immune responses. However, due to the intrinsic low-affinity and inherent solubility problems, it has been difficult to produce soluble high-affinity analogs of TCR and class II MHC molecules. This report describes a general approach which solves this intrinsic low-affinity by constructing soluble divalent analogs using IgG as a molecular scaffold. The divalent nature of the complexes increases the avidity of the chimeric molecules for cognate ligands. The generality of this approach was studied by making soluble divalent analogs of two different classes of proteins, a TCR (2C TCR 2 Ig) and a class II MHC ( MCC I-E k 2 Ig) molecule. Direct flow cytometry assays demonstrate that the divalent 2C TCR 2 Ig chimera retained the specificity of the native 2C TCR, while displaying increased avidity for cognate peptide/MHC ligands, resulting in a high-affinity probe capable of detecting interactions that heretofore have only been detected using surface plasmon resonance. TCR 2 IgG was also used in immunofluorescence studies to show ER localization of intracellular peptide-MHC complexes after peptide feeding. MCC I-E k 2 Ig chimeras were able to both stain and activate an MCC-specific T cell hybridoma. Construction and expression of these two diverse heterodimers demonstrate the generality of this approach. Furthermore, the increased avidity of these soluble divalent proteins makes these chimeric molecules potentially useful in clinical settings for probing and modulating in vivo cellular responses.