Production, Characterization, and Immunogenicity of a Soluble Rat Single Chain T Cell Receptor Specific for an Encephalitogenic Peptide

Abstract

The encephalitogenic rat T cell clone C14 recognizes the myelin basic protein 69 -89 peptide in the context of the RT1B major histocompatibility complex (MHC) class II molecule. Modeling of the C14 TCR molecule indicated that previously identified CDR3 motifs are likely to be central to interaction with MHC class II-presented peptide. Here we report the cloning and expression of C14-derived single chain TCR (scTCR) molecules in an Escherichia coli expression system. The recombinant molecule consists of the V␣2 domain connected to the V␤8.2 domain via a 15-residue linker. Soluble C14 scTCR was purified using conventional chromatography techniques and refolded by a rapid dilution procedure. C14 scTCR was able to bind soluble rat MHC class II molecules bearing covalently coupled Gp-BP-(69 -89) peptide, as analyzed using surface plasmon resonance. Immune recognition of the C14 scTCR protein as an antigen revealed that limited regions of the TCR may be more likely to induce responsiveness.Antigen-specific activation of T lymphocytes occurs through the binding of the T cell receptor (TCR).1 In order to initiate an immune response, antigenic peptides must be presented in the context of a major histocompatibility complex (MHC) on the surface of antigen-presenting cells. In addition, co-receptors on the antigen-presenting cells must bind accessory molecules of the T cell in order for activation to occur. The TCR is a heterodimer consisting of ␣ and ␤ subunits containing variable and constant domains that are structurally similar to immunoglobulin molecules. Because TCR interaction with the MHCpeptide complex defines the basis of specific recognition, an understanding of the structural elements of this recognition is of importance. Structural data regarding both human and mouse TCRs, both with (1-9) and without (10 -12) the MHCpeptide complex, has been limited to a small but growing data base of information obtained through crystallographic (1-11) and NMR spectroscopic (12) studies. TCR recognition of specific MHC-peptide complexes occurs through the binding of complementarity-determining regions (CDRs) within the variable (V) regions of the ␣ and ␤ chains. Analysis of the three-dimensional structure suggests that, in general, the CDR1 and CDR2 regions contact the ␣-helices of the MHC molecule, whereas CDR3 regions contact the peptide (3-9).The immunoreactive properties of the TCR molecule as an antigen have been of long standing interest but remain poorly understood. The most compelling evidence that the TCR itself can elicit immune responsiveness has been demonstrated in numerous studies, most notably in models of T cell-mediated autoimmunity. The protective effects of vaccination with attenuated, pathogenic T cells were first demonstrated in the model of experimental autoimmune encephalomyelitis (EAE) and then extended to models of arthritis, thyroiditis, and diabetes (13,14). Protection coincided with T cells expressing TCRs specific for the disease-inducing antigen. Subsequent studies showed that TCR protein (15-17) ...

Gilt required for RTL550-CYS-MOG to treat experimental autoimmune encephalomyelitis

“…This suggests that GILT is required for therapeutic efficacy of the cysteine-tethered, but not the genetically-encoded antigen constructs, and helps clarify the multiple potential in vivo mechanism of RTL therapy. Previous studies using RTLs bearing genetically encoded cognate or non-cognate peptide antigen documented that RTLs bind directly to cognate TCR with low avidity in vitro (McMahan et al 2003). Signal transduction studies using T cell hybridomas and primary human T cell lines further documented that RTLs bind directly to the TCR, with RTL in vitro serving as partial TCR agonists and triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells and without triggering an influx of extracellular calcium (Wang et al 2003).…”

A novel regulatory pathway for autoimmune disease: Binding of partial MHC class II constructs to monocytes reduces CD74 expression and induces both specific and bystander T-cell tolerance

“…HLA-DR2 mice were screened by FACS for the expression of the HLA transgenes [5]. HLA-DR2 positive male and female mice between 8 and 12 weeks of age were immunized s.c. at four sites on the flanks with 0.2 ml of an emulsion of 200 μg immunogenic peptide and complete Freund’s adjuvant containing 400 μg of heat-killed Mycobacterium tuberculosis H37RA (Difco, Detroit, MI) [4,15].…”

“…DR2 for MS) with covalently tethered myelin peptides [3,4]. These constructs could interact directly with the cognate TCR in the absence of co-stimulatory molecules [5], serving as partial TCR agonists and triggering suboptimal downstream signaling [6,7], cytokine shifts and loss of encephalitogenic activity [8]. RTL treatment of EAE has been established previously in two different strains of DR2-Tg mice, including DR*1501-Tg mice (DRA:DRβ1*1501 strain that develops EAE only after injection of mouse (m)MOG-35-55 peptide [9]) and DR*1502-Tg mice (IE b :DRβ1*1502 strain that develops EAE after injection of human (h) MOG-35-55 peptide [10]).…”

Systemic Immunomodulation of Autoimmune Disease Using MHC-Derived Recombinant TCR Ligands

“…Along with "empty" RTLs that can be loaded exogenously with synthetic peptides, we have produced a variety of genetically-encoded variants with antigenic peptide attached by a linker to the N-terminus of the beta-chain (Table I) [64,69,79,80]. RTL molecules have been used for studying binding specificity in vitro [71,81,82], for exploring primary TCR signaling events independent of co-stimulatory input associated with the MHC II α2 and β2 domains or with other molecules expressed by antigen presenting cells [83], and for treating CD4 + T cell-mediated autoimmune disease in an MHC II/epitope-specific manner [68,70,80,83,84].…”

“…What are the molecular mechanisms that control this cytokine switch at the cellular level? In order to comprehensively characterize the effect of RTL binding directly to the TCR, we began a detailed molecular characterization of the signal transduction pathways within T cells that are directly altered following RTL engagement with the TCR that we recently documented by surface plasmon resonance using soluble single chain TCR (SCTCR) [82].…”