Proc. Nati. Acad. Sci. USA Vol. 89, pp. 2945-2949, April 1992 Immunology Molecular associations between the T-lymphocyte antigen receptor complex and the surface antigens CD2, CD4, or CD8 and CD5 (signal transduction/tyrosine phosphorylation/T-cell antigen receptor/lck/fyn) ALBERTUS D. BEYERS, LOUISE L. SPRUYT, AND ALAN F. WILLIAMS Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, England Communicated by James Gowans, December 20, 1991 ABSTRACT The T-cell antigen receptor (TCR) complex is CD2 and CD5 do not have enzymatic activities, nor are they the key structure involved in signal transduction in T cells. To known to associate with Tyr kinases, but their activities analyze associations between the TCR complex and other might be due to associations with the TCR complex. An molecules, immunoprecipitations were carried out, followed by association between CD2 and CD3 in digitonin lysates of phosphorylation of molecules in vitro by tyrosine kinases asso- human T cells was reported (21), but these findings have not ciated with the precipitated molecules. This provided a sensi- proven to be routinely reproducible (M. H. Brown, personal tive assay for molecular complexes, and associations were communication). demonstrated between the TCR complex and the surface Given (i) the well-documented association of p56Ick with antigens CD2, CD4, or CD8 and CD5 in normal rat T cells. The CD4/CD8 (reviewed in ref. 5), (ii) the association of p59fYn complexes were readily seen in immunoprecipitates from BrU with the TCR complex (13), and (iii) the fact that the ; chain 96 but not Nonidet P40detergent extracts. The multimolecular and the CD3 £ and y chains are substrates for p56lck (4) and complexes are associated with the internal tyrosine kinases p59fy1 (13), we decided to use the sensitivity of in vitro p56kk and p59VY. The presence of p56kk associated with CD4 immune complex kinase assays to analyze associations ofthe or CD8 was also examined in early thymocytes, natural killer TCR complex with other cell-surface molecules. A physical cells, and macrophages. The kinase was present in all cases association ofCD4 and CD8 as well as CD2 and CD5 with the except that of normal macrophages. TCR complex is demonstrated. We propose that this array of transmembrane cell-surface molecules forms a signaling The earliest known events in T-cell activation result in complex in conjunction with the p56lck and p59fyn kinases. phosphorylation oftyrosine residues of cytoplasmic proteins by tyrosine kinases (Tyr kinases) (1). It is believed that phospholipase C,1 is activated by phosphorylation (2, 3) and MATERIALS AND METHODS that another substrate is the ' chain of the T-cell antigen Animals and Antibodies. Thymuses, cervical lymph nodes, receptor (TCR) complex (4, 5). Tyr kinases of the src family and peritoneal exudate cells were from AO-RT1u rats. The rat that are particularly associated with T cells include p56lck and NK cell line A181 arose in a Fischer rat (18). mAbs used (refs. p59fyn (6, 7). The p56lck protein binds to the cytoplasmic 22-24 unless otherwise stated) were W3/25 (IgG1) and OX-70 domains of the CD4 and CD8 molecules (4, 8-10), and this (IgG2a) noncompetitive anti-rat CD4 mAbs; OX-1 (IgG1) association is required for efficient signal transduction via the anti-rat CD45 mAb; OX-2 (IgG1) mAb against an immuno- TCR complex (11, 12). A key role for p59f1n in T-cell signal globulin-related molecule of rat thymocytes and brain (25); transduction is also indicated by the findings that this protein OX-8 (IgG1) anti-rat CD8 a-chain mAb; OX-18 (IgG1) anti-rat is associated with the TCR (13) and that transgenic expres- RT1A mAb (nonpolymorphic); OX-19 (IgG1) anti-rat CD5 sion of p59fyn in thymocytes leads to enhanced signaling in mAb; OX-20 (IgG1) rat anti-mouse immunoglobulin K chain; these cells in response to anti-CD3 monoclonal antibodies OX-21 (IgG1) anti-human C3b inactivator; OX-34 (IgG2a) (mAbs) (6). Taken together, the data suggest that the TCR and anti-rat CD2 mAb; OX-44 (IgG1) anti-rat CD53 mAb; OX-47 the CD4/CD8 coreceptors interact together to transmit sig- (IgG1) against a rat immunoglobulin superfamily glycopro- nals by coupling to the internal Tyr kinases p56lck and p59fyn. tein; OX-49 (IgG2a) and OX-50 (IgGl) anti-rat CD44 mAbs; Crosslinking of the CD2 and CD5 T-cell antigens can also OX-52 (isotype unknown), a rat T-cell marker (26); W3/13 have effects on T-cell activation (14). Certain combinations of (IgG1) and OX-56 (IgG2b) anti-rat CD43 mAbs; PY-20 (IgG1) anti-CD2 mAbs induce mitogenesis in T cells, and the same anti-phosphotyrosine mAb (ICN); R73 (IgGl) anti-rat TCR, activation of signal transduction parameters occurs as is seen pan-a/,B-chain mAb; and 1F4 (IgM) anti-rat CD3 mAb. with activation via the TCR (15, 16). For these events, the Polyclonal rabbit antisera were: anti-CD3 E chain (27) and cytoplasmic domain of CD2 is required (15, 17), and it also anti-CD5 (raised against a 22-mer peptide corresponding to seems that a functional T-cell receptor is required for cells to the C-terminal 21 amino acids of CD5), provided by D. Y. be efficiently activated via CD2 (15, 18). Crosslinking of CD5 Mason (Department of Haematology, John Radcliffe Hospi- antigen by mAbs potentiates T-cell proliferation in response tal, Oxford); anti-CD3 r chain (16), provided by D. A. to mitogens, alloantigens, or anti-CD2 mAbs (14, 17). Again Cantrell (Imperial Cancer Research Fund, London); anti- expression of the TCR complex is required for effects me- p59fyn [a mixture of fynl and fyn2 (28), provided by S. A. diated by CD5 (19), and in turn CD5 may be required for Courtneidge, European Molecular Biology Laboratory, activation via the TCR, since rat cells that were down- Heidelberg]; anti-p56lck, against a peptide corresponding to regulated in expression ofCD5 by administration ofanti-CD5 amino acids 39-64 of murine provided J. B. mAb in vivo did not proliferate in response to alloantigens or p56lck (9), by other mitogenic stimuli (20). The cytoplasmic domains of Abbreviations: Tyr kinase, tyrosine kinase; BSA, bovine serum albumin; GAP, GTPase-activating protein; IL-2, interleukin 2; mAb, The publication costs of this article were defrayed in part by page charge monoclonal antibody; NK, natural killer; RAM, rabbit anti-mouse payment. This article must therefore be hereby marked "advertisement" IgG antibodies; TCR, T-cell receptor for antigen; NP-40, Nonidet in accordance with 18 U.S.C. §1734 solely to indicate this fact. P-40. 2945 2946 Immunology: Beyers et al. Proc. Natl. Acad. Sci. USA 89 (1992) Bolen (Bristol-Myers Squibb, Princeton); anti-GTPase- Magnetic Cell Sorting and Flow Cytometric Analysis. Thy- activating protein (anti-GAP), raised against a glutathione mocytes (2 x 10) were incubated on ice for 45 min in 5 ml S-transferase fusion protein containing the SH2 and SH3 of PBS/BSA containing 2 pg of biotinylated W3/25 (anti- domains ofGAP, provided by J. Downward (Imperial Cancer CD4), 2 pg of biotinylated OX-44 (anti-CD53), and 1 pg of Research Fund); and rabbit anti-mouse immunoglobulin R73 (anti-TCR) mAbs per ml. Cells were washed twice in (RAM), provided by S. Simmonds of the Medical Research PBS containing 0.01% NaN3 and 5 mM EDTA (PBS/NaN3/ Council Cellular Immunology Unit. EDTA) and then incubated on ice for 45 min in 5 ml of Immunoprecipitations and Immune Complex Kinase As- PBS/NaN3/EDTA containing 5 pg offluorescein-conjugated says. Cells (5 x 107, or numbers as in the text) were avidin per ml. After two more washes in PBS/NaN3/EDTA, preincubated with mAbs (10 ,g of immunoglobulin or 10 pJ cells were incubated for 10 min in 5 ml of the same buffer of ascites) in 500 jl of phosphate-buffered saline (PBS) containing 75 Al of biotinylated superparamagnetic micro- containing 0.25% (vol/vol) bovine serum albumin (BSA) for beads (Becton Dickinson). Unlabeled cells were purified by 30 min at 4°C. Cells were pelleted for 15 sec in a microcen- repeated passages over a magnetic column (MACS, Becton trifuge and solubilized in 1 ml of a buffer (10 mM Tris HCl, Dickinson). About 2 x 107 CD4- CD53- TCR- cells were pH 7.4/150 mM NaCl/1 mM EDTA/1 mM phenylmethyl- obtained, and purity was evaluated by flow cytometry (FAC- sulfonyl fluoride/1 mg ofBSA per ml) containing 1% Nonidet SCAN, Becton Dickinson). For purification of CD2- TCR- P-40 (NP-40) (Sigma) or 1% Brij 96 (Sigma) for 30 min on ice. peritoneal cells (1.2 x 108), biotinylated OX-34 (anti-CD2) and Lysates were centrifuged at 12,000 x g for 10 min at 4°C. biotinylated R73 (anti-TCR) mAbs were used, and amounts of Supernatants were precleared by rotation with 100 ,u of reagents were scaled down according to cell numbers. Sepharose CL-4B beads (10% vol/vol) with OX-2 mAb attached for 30 min at 4°C. Supernatants were added to 100 RESULTS y4 ofprotein A-Sepharose CL-4B (1o vol/vol) containing 40 pg of bound RAM and rotated for 1 hr at 4°C. Beads were Effect of Two Detergents on the Association of p563 with washed three times in 1 ml of lysis buffer and twice in 1 ml CD4 and CD8 in Thymocytes. Our initial interest was to study ofassay buffer [25 mM Hepes (pH 7.5) containing 0.1% NP-40 p56 ck associated with CD4 and CD8 in early thymocytes.