Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure.

Abstract

A cDNA encoding the CD2 antigen has been isolated by a highly efficient technique based on transient expression in COS cells and adherence of cells expressing surface antigen to antibody-coated dishes. COS cells expressing a CD2 cDNA isolated by this method readily formed rosettes with sheep as well as human and other mammalian erythrocytes. Pretreatment of transfected COS cells with anti-CD2 antibody, or pretreatment of human erythrocytes with anti-LFA-3 antibody, abolished rosette formation.The ability of human thymocytes and peripheral T lymphocytes to form spontaneous rosettes with sheep erythrocytes (1-3) has been a curiosity of substantial practical, but unknown physiological, significance. Several T-cell-specific monoclonal antibodies that block erythrocyte rosette formation have been developed (4-7), and a subset of these have been shown to inhibit T-cell activation (8-10) and cytolysis (9). Conversely, an antibody identified by inhibition of cytolytic function has been shown to block rosette formation (11). All of these antibodies recognize a 50-kDa surface antigen present on thymocytes and peripheral T lymphocytes, now called CD2. Some pairwise combinations of monoclonal antibodies recognizing the CD2 molecule (but not necessarily blocking rosette formation) have been shown to cause T-cell activation (12-14). Recently, purified preparations of the potent mitogen phytohemagglutinin were shown to cause calcium influx in T cells or T leukemia cells bearing CD2 but not in cells lacking CD2 antigen or pretreated with anti-CD2 antibodies (15,16).In (17) by inserting a synthetic transcription unit between the suppressor tRNA gene and the simian virus 40 (SV40) origin. The transcription unit consisted of a chimeric promoter composed of human cytomegalovirus AD169 immediate early enhancer sequences fused to the human immunodeficiency virus (HIV) long terminal repeat (LTR) -67 to +80 sequences. Immediately downstream from the LTR +80 sequence was inserted a polylinker containing two BstXI sites separated by a 350-base-pair (bp) "stuffer." The BstXI sites were flanked by Xba I and Xho I sites, which could also be used to excise the insert. Downstream from the polylinker were placed the SV40 small tumor (t) antigen splice and early region polyadenylylation signals derived from pSV2. The nucleotide sequence of the vector is available upon request.cDNA Library Construction. RNA was prepared from HPB-ALL T-cell-leukemia cells by the guanidinium thiocyanate/CsCl method, and cDNA was synthesized from the poly(A)+ fraction by the method of Gubler and Hoffman (18). The cDNA was inserted into the vector using non-selfcomplementary BstXI adaptors, as will be described elsewhere. The ligated DNA was transformed into Escherichia coli MC1061/p3 made competent by the protocol of Mike Scott (Department of Neurology, University of California, San Francisco; personal communication). Laboratory protocols for these and subsequent steps are available from the authors.Recovery of cDNA Clones by Panning. Bacteriological cultu...

Expression cloning of a human Fc receptor for IgA.

“…We used this mAb as the basis for immunoselection of an FcaR cDNA clone, using a modification of the technique previously described by Seed and Aruffo (21). The U937 cell line represented a convenient source ofmRNA for cDNA library construction, since FcaR expression as measured by My43 binding is inducible by PMA treatment .…”

“…Plasmid DNA from the cDNA library was used to transfect a subconfluent layer ofmonkey COS-7 cells using DEAF dextran followed by chloroquine treatment, as described (20). FCaR`cells were selected by incubation in the My43 anti-FcciR mAb (14), followed by panning on anti-IgMcoated plates according to established procedures (21). Episomal DNA was prepared from the panned cells, amplified, and reintroduced into COS cells.…”

Phenotypic and functional similarities between 5‐azacytidine‐treated t cells and a t cell subset in patients with active systemic lupus erythematosus

“…Controls included a 2-kilobase Barn HI cDNA fragment of pactin, donated by Dr. Gary Nabel, and a full-length CD2 cDNA (31). Autoradiograms were exposed for 1, 5, or more hours.…”

Homophilic interaction of NTBA, a member of the CD2 molecular family: induction of cytotoxicity and cytokine release in human NK cells

“…Molecular cloning revealed that NTBA is a member of the Ig superfamily characterized by structural features that allowed its assignment to the CD2 family. This family also includes CD2 [3] as well as CD48 [4], CD58 [5], CD84 [6], CD150 (signaling lymphocyte activation molecule, SLAM) [7], CD229 (hLy9) [8], CD244 (2B4) [9], CS1 (19A, CD2-like receptor activating cytotoxic cells, CRACC) [10][11][12], B lymphocyte activator macrophage-expressed (BLAME) [13], and CD84H1 [14]. These surface glycoproteins are characterized by similar structures consisting of a membrane distal IgV that lacks the typical disulfide bond, and a membrane proximal IgC2 domain, with the exception of CD229 that contains an IgV-IgC2 domains tandem repeat.…”