TCR peptide-specific T cells inhibit activation of encephalitogenic T cells

Offner, H.; Vainiene, M.; Celnik, B.; Buenafe, Abigail C.; Weinberg, Andrew D.; Vandenbark, Arthur A.

doi:10.1016/0165-5728(94)90468-5

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“…Assembly of scTCR Genes-Reverse transcriptase-PCR analysis for TCR gene usage (26,36) and sequencing analysis demonstrated that only a single V␣ gene and a single V␤ gene were expressed in the A1 hybridoma, derived from the C14 encephalitogenic Lewis rat T cell clone specific for the BP-(69 -89) peptide (29). To construct the scTCR gene, primers containing appropriate restriction sites were designed to flank sequences encoding the V and J regions as follows: V␣ primers, RV␣2NcoI (5Ј-CGACCCATGGCTCAGCAGGTCAAACAAAGTCC) and RJ␣SmaI (5Ј-CGGACCCGGGCTTCACCACCAGTTGCG); V␤ primers, RV␤8Bam (5Ј-ATCAGGATCCGAAGCTGCAGTCACACAAAGC) and RJ␤SacI (5Ј-GATCGAGCTCAACCGTGAGCTTGGTGCCG).…”

Section: Methodsmentioning

confidence: 99%

“…Surface plasmon resonance was used to analyze the binding of recombinant rat C14 scTCRDAK to a soluble rat MHC class II molecule consisting of the ␤ 1 and ␣ 1 domains of the Lewis rat RT1.B molecule with covalently attached MBP-(69 -89) peptide (recombinant TCR ligand or RTL201) (36,37). Coupling of the C14 scTCR or RTL directly to the CM5 chip was tested in initial pilot experiments.…”

Section: Cloning and Modeling Of The Recombinant C14 Sctcr-amentioning

confidence: 99%

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

McMahan

Watson

Meza-Romero

et al. 2003

Journal of Biological Chemistry

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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) ...

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Section: Methodsmentioning

confidence: 99%

Section: Cloning and Modeling Of The Recombinant C14 Sctcr-amentioning

confidence: 99%