A method for producing monoclonal antibodies to human T-cell-receptor beta-chain variable regions.

Callan, Margaret; Reyburn, Hugh; Bowness, Paul; Ottenhoff, Tom H. M.; Engel, Isaac; Klausner, Richard D.; Bell, John I.; McMichael, Andrew

doi:10.1073/pnas.90.22.10454

Cited by 14 publications

(5 citation statements)

References 29 publications

(33 reference statements)

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“…To obtain A k molecules uniformly occupied with individual HEL peptides, we made constructs encoding A k ␤ molecules with amino-terminal extensions corresponding to the desired sequence (13). We also replaced the transmembrane and the cytoplasmic regions of both the ␣ and ␤ chains of A k with those of the mouse TCR chain because immunization with RBL cells expressing chimeric molecules of poorly immunogenic proteins fused to TCR has been reported to elicit strong antibody responses to those proteins (18). As shown in Fig.…”

Section: Generation Of Mabs To Hel Peptides Bound To I-amentioning

confidence: 99%

Production, specificity, and functionality of monoclonal antibodies to specific peptide–major histocompatibility complex class II complexes formed by processing of exogenous protein

Zhong

Sousa

Germain

1997

Proc. Natl. Acad. Sci. U.S.A.

136

145

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Several unanswered questions in T cell immunobiology relating to intracellular processing or in vivo antigen presentation could be approached if convenient, specific, and sensitive reagents were available for detecting the peptide-major histocompatibility complex (MHC) class I or class II ligands recognized by ␣␤ T cell receptors. For this reason, we have developed a method using homogeneously loaded peptide-MHC class II complexes to generate and select specific mAb reactive with these structures using hen egg lysozyme (HEL) and I-A k as a model system. mAbs specific for either HEL-(46-61)-A k or HEL-(116-129)-A k have been isolated. They cross-react with a small subset of I-A k molecules loaded with self peptides but can nonetheless be used for f low cytometry, immunoprecipitation, Western blotting, and intracellular immunof luorescence to detect specific HEL peptide-MHC class II complexes formed by either peptide exposure or natural processing of native HEL. An example of the utility of these reagents is provided herein by using one of the anti-HEL-(46-61)-A k specific mAbs to visualize intracellular compartments where I-A k is loaded with HEL-derived peptides early after antigen administration. Other uses, especially for in vivo tracking of specific ligand-bearing antigen-presenting cells, are discussed.The majority of ␣␤ receptor-bearing T cells recognizes antigen as peptide-major histocompatibility complex (MHC) class I or class II complexes displayed on the surface of antigenpresenting cells (APCs), and the biochemistry of peptide-MHC molecule interaction, the cell biology of peptide generation from intact protein antigen, and the site(s) of intracellular association of these peptides with MHC molecules have been explored in great detail (for review, see refs. 1 and 2). Despite this progress in understanding antigen processing and presentation, it is still not known exactly where MHC class II molecules are loaded with particular peptides or whether all APCs process antigen in the same way or with the same efficiency. Past studies have necessarily relied on cells that can be obtained in large numbers for biochemical analysis. Rare APCs, such as dendritic cells, could have specialized antigenprocessing mechanisms not easily amenable to study by current techniques, and for many antigens and most cells, a kinetic and spatial analysis of the formation of class II complexes involving specific peptide has not been possible.In vivo antigen presentation is also incompletely understood. It is not clear which cells capture antigen and present it in immunogenic or tolerizing form to T cells after antigen introduction by various routes. Likewise, we are only beginning to understand the dynamics of antigen-specific immune cell A major limitation in studying these issues is the lack of reagents able to identify and quantitate T cell receptor (TCR) ligands on individual cells, to detect such complexes within intact cells, or to characterize cells bearing these ligands by methods such as flow cytometry or immunohis...

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Section: Generation Of Mabs To Hel Peptides Bound To I-amentioning

confidence: 99%

Production, specificity, and functionality of monoclonal antibodies to specific peptide–major histocompatibility complex class II complexes formed by processing of exogenous protein

Zhong

Sousa

Germain

1997

Proc. Natl. Acad. Sci. U.S.A.

136

145

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“…Use of this vector results in expression of a chimeric TCR-CD3´fusion protein promoting §´-g´heterodimer formation and permitting cytoplasmic signaling through´phosphorylation [3]. The GRb heterodimer, expressed in RBL cells (see below), was used previously to generate the mAb 3C5 specific for HVB7S1 (formerly known as V g 7.1) [10]. Table 2 shows the amino acid sequences of the CDR3 regions of clone GRb and of the § chain mutants generated.…”

Section: Tcr Mutagenesis and Expression In Rbl Cellsmentioning

confidence: 99%

“…TCR of one CTL clone (GRb) expressed in the rat basophil leukemia cell line RBL-2H3 [10]. Mutant TCR function was assessed firstly by direct binding of PE-labeled tetrameric complexes of HLA B*2705 with influenza virus nucleoprotein residues 383-391 peptide (B27/NP tetramer), and secondly by the induction of RBL degranulation in the presence of peptide-pulsed HLA B27-expressing APC.…”

Section: Introductionmentioning

confidence: 99%

Importance of a conserved TCR J α-encoded tyrosine for T cell recognition of an HLA B27/ peptide complex

et al. 1998

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Human HLA B27-restricted cytotoxic T lymphocytes (CTL) specific for the influenza A epitope NP383-391 use similar TCR alpha and beta chains, with two closely related J alpha segments used by six of nine CTL clones from three unrelated donors (Bowness et al., Eur J. Immunol. 1993. 23: 1417-1421). The role of TCR complementarity-determining region (CDR)3alpha residues 93 and 100-102 was examined by site-directed mutagenesis, following expression of the TCR alpha and beta extracellular domains from one clone as a TCR zeta fusion heterodimer in rat basophil leukemia (RBL) cells. For the first time we have measured direct binding of tetrameric HLA B*2705/NP383-391 complexes to transfected TCR. Independently peptide-pulsed antigen-presenting cells (APC) were used to induce TCR-mediated degranulation of RBL transfectants. Our results show a key role for the conserved TCRalpha CDR3 J alpha-encoded residue Y102 in recognition of HLA B27/NP383-391. Thus the Y102D mutation abolished both tetramer binding and degranulation in the presence of peptide-pulsed APC. Even the Y102F mutation, differing only by a single hydroxyl group from the native TCR, abolished detectable degranulation. Further mutations F93A and S100R also abolished recognition. Interestingly, the N101A mutation recognized HLA B27/NP in functional assays despite having significantly reduced tetramer binding, a finding consistent with "kinetic editing" models of T cell activation. Modeling of the GRb TCR CDR3alpha loop suggests that residue Y102 contacts the HLA B*2705 alpha1 helix. It is thus possible that selection of germ-line TCRAJ-encoded residues at position 102 may be MHC driven.

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“…TcR usage was studied by "anchored" PCR of lymphocyte cDNA, followed by M13 cloning and sequencing, as reported previously [lo]. TcR usage was confirmed where possible by staining with the monoclonal antibodies MX6 (anti-Vg8) [13], 3C5 (anti-VP7.1) [14] and 6D6 (anti-Va12) 1151.…”

Section: Cytotoxic T Cell Clones and Cytotoxicity Assaysmentioning

confidence: 99%

Identification of T cell receptor recognition residues for a viral peptide presented by HLA B27

1994

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The fine specificity of T cell recognition of peptide analogues of the influenza nucleoprotein epitope, NP 383-391 SRYWAIRTR, was studied using HLA B27-restricted influenza-specific cytotoxic T cell (CTL) clones, of defined T cell receptor (TcR) usage, derived from unrelated individuals following natural infection. Even conservative amino acid substitutions of the peptide residues P4, P7 and P8 influenced CTL recognition. These side chains are probably directly contacted by the TcR. CTL clones which used the TcR V alpha 14 gene segment (but not those using TcR V alpha 12) were also sensitive to P1 substitutions, suggesting that the TcR alpha chain of these clones lies over the N terminus of bound peptide, and that the "footprint" of certain TcR can span all exposed residues of a peptide bound to a major histocompatibility complex class I molecule. These results, taken together with previous structural and functional data, suggest that, for nonamer peptides bound to HLA B27, P1, P4 and P8 are "flag" residues with TcR-accessible side chains.

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A method for producing monoclonal antibodies to human T-cell-receptor beta-chain variable regions.

Cited by 14 publications

References 29 publications

Production, specificity, and functionality of monoclonal antibodies to specific peptide–major histocompatibility complex class II complexes formed by processing of exogenous protein

Production, specificity, and functionality of monoclonal antibodies to specific peptide–major histocompatibility complex class II complexes formed by processing of exogenous protein

Importance of a conserved TCR J α-encoded tyrosine for T cell recognition of an HLA B27/ peptide complex

Identification of T cell receptor recognition residues for a viral peptide presented by HLA B27

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