A method for production of antibodies to human T-cell receptor beta-chain variable regions.

Abstract

Mouse T-cell hybridomas bearing human Vp elements were produced by transfection of human/mouse hybrid T-cell receptor 18-chain genes into a mouse T-cell hybridoma lacking an endogenous #-chain gene. These hybridomas were entirely mouse in origin except for the human Vp region. These cells were used to immunize mice against human V13 elements. Mouse monoclonal antibodies have thus been generated against human Vp13.1 and -13.2. We expect that the method outlined in this paper will be useful in the production of monoclonal antibodies specific for other human V13 or V.elements.The receptor for antigen on most peripheral T cells is made of a and 13 chains. Usually these T-cell receptors (TCRs) engage antigen in the form of peptides, derived from the antigen, bound to cell surface major histocompatibility complex (MHC) molecules (1-4). The specificity of the TCR for this complex ligand is determined by the five variable elements (Va, Ja, V13, Do, and JO3) of the a and 13 chains as well as non-germ-line-encoded junctional regions (5-8). Exceptions to this rule have been documented by us and others. These are proteins that stimulate T cells bearing the appropriate V13 element almost independently ofcontributions from other parts of the TCRs borne by the T cells (9-22). Such proteins, called superantigens because they stimulate large numbers ofT cells, are encoded by microorganisms including retroviruses, bacteria, and mycoplasma (22-29).There is considerable interest in the control and consequences of the T-cell repertoire in humans and other species. Obviously a detailed understanding of the T-cell repertoire would require knowledge of all the V,, Ja, V13, D13, and Jo combinations present in a given individual. Such knowledge will not be available in the foreseeable future. However, much has already been learned from studies of the distribution between animals of just one of the TCR variable elements, V13. Examination of V13 expression in mice has been used to study positive selection, T-cell tolerance, autoimmunity, and the consequences of attack by superantigens (22).Similarly, V13 expression in humans has been used to study the effects of superantigens and also to examine the repertoires of T cells involved in inflammatory and autoimmune disease (18,19,29,30 MATERIALS AND METHODSPreparation of RNA and cDNA and Amplification by QPCR. Total RNA was prepared from phytohemagglutinin-, antihV,013

Epstein-Barr Virus Latent Membrane Protein LMP-2A Is Sufficient for Transactivation of the Human Endogenous Retrovirus HERV-K18 Superantigen

“…We simultaneously assessed activation of TCRBV8 T-cell hybridomas as a specificity control. Hybridoma assays were performed as described previously (52,53) by using the TCRBV13 T-cell hybridoma, hV␤13.1-1 (11,12) and the TCRBV8 T-cell hybrid YL␤8#24 (53). Figure 1A depicts the results of a representative hybridoma assay with two lymphoblastoid cell lines (LCL), Mg68 and 253.30, that were derived by transformation with EBV deletion mutants lacking the majority of the lytic genes.…”

Regional Variation in the Lamina Propria T Cell Receptor Vβ Repertoire in Normal Human Colon

“…V␤ ϩ expansions in the colonic lamina propria were defined in reference to the percentage of CD4 ϩ or CD8 ϩ T cells staining positive for the same V␤ in the same subject's peripheral blood. This corrects for bias introduced by the influence of MHC and TCR genes on the level of expression of different V␤s in different individuals (27,31,33,35,36,40). In individuals where a particular CD4 ϩ of CD8 ϩ V␤ expansion was present in the peripheral blood, expansions in the same V␤ ϩ in the LP could not be defined by reference to the level of expression of that V␤ in the individual's peripheral blood.…”

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

“…Studies of T-cell clones that recognize the HLA-A2-restricted influenza A matrix peptide-(58-66) (16) and of clones that recognize the HLA-B27-restricted influenza A nucleoprotein peptide-(383-391) (15) concluded that use of TCR Vp, NDNs, V0, and Ja Recently mouse T-cell hybridomas transfected with human V,3 elements have been used as a source of immunogen (9,11,14). Although these methods overcome many of the probGap-A3 lems, B-cell hybridomas derived with this protocol may show a low frequency of reactivity with the human Vp element.…”

“…Monoclonal antibodies (mAbs) to the V chains of the TCR have been extremely difficult to generate, despite the availability of T-cell clones for >10 yr. The existing panel of mAbs recognizes only 20-25% of the Vp gene segment products and only three of the Va products (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Studies of the T-cell repertoire have suggested a conservation of TCR Va and Vp gene use in major histocompatibility complex-restricted antigen recognition (15,16) and also that limited TCR Va and V gene use may occur in some autoimmune diseases (17)(18)(19).…”