A number of cell surface structures are thought to belong to the Ig superfamily (IgSF) t because they contain at least one domain with a characteristic folding pattern, called the Ig fold (reviewed in reference 1). Several of these molecules have critical functions in immune responses . In addition to ensuring specific antigen recognition (Ig, TCR), they may function as monomorphic ligands critical in cell-cell interactions (e .g., ICAM, CD4, CD8), receptors for viruses (e .g., CD4, ICAM), or lymphokine receptors (e .g., IL-1-R, IL-6-R).We report here the characterization of a novel human gene, termed lymphocyte activation gene 3 (LAG-3), selectively transcribed in activated NK and T lymphocytes . It codes for a membrane protein with four extracellular IgSF domains . The sequence data, the compared exon/intron organization, and the chromosomal localization revealed that LAG-3 is closely related to CD4. Materials and MethodsCell Lines. The isolation and growth of the fetal CD3 -CD2`F55111E5 (or F5) cloned cell line has been described elsewhere (2) . For mass production, the cell suspensions were plated on a feeder layer composed of irradiated allogeneic PBL plus the EBV transformed B cell line Laz388 in Vbottomed 96-well plates at 3,000 cells per well with rIL-2 and lymphocyte-conditioned medium . 200 plates were harvested at a concentration of 3 x 106 cells/ml after 12 d in culture to give 6 x 109 cells . For the Northern blot analyses, similar culture conditions were used to produce the relevant cells .

We report here the characterization of a series of T cell receptor (TcR) V alpha or V beta subfamily-specific oligonucleotide primers. Criteria that have guided the design of each oligonucleotide include appropriate thermodynamic parameters as well as differential base-pairing scores with related and unrelated target sequences. The specificity of the oligonucleotides for each V alpha or V beta subfamily was tested by polymerase chain reaction (PCR) on both a series of TcR encoding plasmid DNA and clonal T cell populations. Unexpected cross-reactivities were observed with plasmid cDNA sequences corresponding to unrelated subfamily gene segments. This led to the synthesis of additional series of oligonucleotides to obtain a relevant panel. A series of V alpha 1-w29/V beta 1-w24 TcR subfamily-specific oligonucleotides was eventually selected which generates little, if any, cross-reactivity. The use of C alpha or C beta primers for the amplification of internal positive control templates (i.e. C beta for the V alpha series and C alpha for the V beta series) has been tested in PCR performed with cDNA derived from peripheral blood lymphocytes; it was shown not to alter the amplification of the V subfamily-specific DNA fragments. This panel of oligonucleotides will be helpful in the study of TcRV gene segment usage and, thus, may lead to a better characterization of T cell responses in physiological and pathological situations.

Invest. 1993Invest. . 91:1183Invest. -1190

We have characterized a functional T cell receptor (TcR) delta transcript in a Ti gamma A+ human cloned cell line derived from peripheral blood. This cDNA includes a novel V gene (V-AB12), whose expression was initially studied in a series of TcR gamma/delta+ clones. Nine Ti gamma A+ clones derived independently from distinct donors have been tested: each of them was found to possess a unique V-AB12/J-IDP2 5.5-kb Eco RI rearrangement, which was constantly transcribed. Surface expression of the protein encoded by this unique rearranged gene was demonstrated by immunoprecipitations performed on three Ti gamma A+ polyclonal cell lines using a specific rabbit heteroantiserum. Further analysis strongly suggested that a monoclonal antibody (mAb), designated anti-BB3, detects a V-AB12-encoded antigenic determinant on the cell surface. Double-color immunofluorescence analysis of peripheral blood lymphocytes from ten donors indicated that most BB3+ cells are recognized by anti-Ti gamma A mAb. In previous studies, we have shown that a majority of TcR gamma/delta+ peripheral T cells expresses a gamma chain including V9 (Ti gamma A) and most frequently JP-encoded peptides. Given the present results on the delta chain, it can be concluded that, in many individuals, a predominant fraction (V gamma 9+/V-AB12+) of circulating CD3+ TcR alpha/beta- T lymphocytes expresses a receptor with little, if any, combinatorial diversity.