Molecular analysis of T cell receptor (Ti) variable region (V) gene expression. Evidence that a single Ti beta V gene family can be used in formation of V domains on phenotypically and functionally diverse T cell populations.

Abstract

T cell receptors for antigen and major histocompatibility complex (MHC) ~ determinants have, using anticlonotypic monoclonal antibodies (mAb), been defined on inducer, suppressor, and class I and class II MHC-specific cytotoxic T lymphocytes as T3oassociated molecules of 90 kilodaltons (kD) molecular mass (1-8). These cionotypic structures, termed Ti, are comprised of one 49-54 kD a and one 43 kD/3 subunit, which are disulfide linked. Peptide mapping analysis of isolated Ti a and/3 subunits from clones of differing specificities demonstrated that clonally unique peptides as well as shared peptides existed in each subunit, thus implying that variable (V) as well as constant (C) domains existed within Ti and/3 molecules (5, 9). Subsequently, N-terminal amino acid sequencing and molecular cloning techniques led to identification of the Ti /3 gene structure, and showed that specific V-, D-(diversity), J-(joining), and C-like segments fuse to form an active/3 gene (10-16). These studies also indicated that the N-terminal V domains were encoded by nucleotides derived from V, D, and J segments. Similar characterization of Ti o~ subunits suggests that each contains a unique NH2 V domain created by joining dispersed germline genes (17-21). In addition to the similarities between joining processes of T cell receptor and Ig gene segments, both structures manifest distant but clearcut homology at protein and DNA levels.T cell receptors, like B cell receptors, must accommodate a myriad of different antigenic specificities in order to provide the organism with an efficient system for immunologic recognition. Moreover, since T cells generally recognize antigen in the context of polymorphic MHC determinants, it is reasonable to assume that V region structural diversity of the T cell population will be no less complex than that of B lymphocytes. In the case of B cells, five mechanisms account for Abbreviations used in this paper: C, constant region of Ig; cDNA, complementary DNA; CML, ceil-mediated lympholysis; Con A, concanavalin A; D, diversity-generating segment of Ig; FITC, fluorescein isothiocyanate; IEF, isoelectric focusing; IL-2, interleukin 2;J,joining region of Ig; mAb, monoclonal antibody; MHC, major histocompatibility complex; mRNA, messenger RNA; PAGE, polyacrylamide gel electrophoresis; PHA, phytohemagglutinin; SDS, sodium dodecyl sulfate; TdR, thymidine deoxyribose; Ti, T cell receptor; V, variable region of Ig. 1326J. ExP. MED.

Limited heterogeneity of biased T‐cell receptor Vβ gene usage in lung but not blood T cells in active pulmonary sarcoidosis

“…This unamplified cDNA library was plated and screened using standard methodologies with a Vb8-specific probe (VbREX, kindly provided by E. Reinherz). 25 Hybridizations were performed at 688 and filters washed with 12 SSC, 1% sodium dodecyl sulphate (SDS) to allow detection of all five members of the Vb8 subfamily. 27 Recombinant clones containing Vb8 genes were purified, the inserts subcloned into pUC13 plasmids, and sequenced (see below).…”

“…7 Immunofluorescence analysis Evaluation of lung and blood mononuclear cells for the proportions of CD3 + , CD4 , CD8 + and Vb8 T cells was performed as described previously using flow cytometry and anti-CD3 (Leu-4), anti-CD4 (Leu-3), anti-CD8 (Leu-2) (Becton Dickinson Immunocytometry Systems, Mountain View, CA) and anti-Vb8 (5REX9H5; kindly provided by E. Reinherz, Dana-Farber Cancer Institute, Boston, MA) monoclonal antibodies (mAb) and stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin (Tago Inc., Burlingame, CA). 7,25 Isolation of lung and blood T-cell RNA and preparation of cDNA Total lung cell RNA was extracted using guanidine isothiocyanate and ultracentrifugation through a caesium chloride gradient. 26 Two strategies were then used to evaluate the repertoire of Vb8 mRNA transcripts.…”

Inherited polymorphism of the human T-cell antigen receptor detected by a monoclonal antibody.

“…The same argument applies to the genomic Jet segment pool (<50 gene segments) and the J, segment pool (13 gene segments) (22). Two similar antibodies (12,(23)(24)(25)(26) have been shown to recognize determinants of V,3…”

“…In one case the determinant recognized by this type of mAb was found to be a 13 chain V region determinant (12). We have characterized the small subsets of peripheral blood T cells identified by two such mAbs, designated S511 and C37 (10,11).…”

“…Our group and others have raised monoclonal antibodies (mAbs) against the T-cell antigen receptor, some of which react with small subsets of normal human T cells (9)(10)(11)(12)(13). In one case the determinant recognized by this type of mAb was found to be a 13 chain V region determinant (12).…”