Proc. Nati. Acad. Sci. USA Vol. 89, pp. 2945-2949, April 1992 Immunology Molecular associations between the T- antigen receptor complex and the surface antigens CD2, CD4, or CD8 and CD5 (signal transduction/tyrosine phosphorylation/T-cell antigen receptor//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 , 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 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 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. In or 0.1% Brij 96]. Immune complexes were incubated in 30 ul previous studies the detergents NP-40 or Triton X-100 were of assay buffer containing 10 mM MnCl2 and 5 pCi (185 kBq) used for cell lysis and washing ofimmune complexes (4, 8-11, of [Y-32P]ATP, with or without 1 ,uM unlabeled ATP and 4 pg 29), and under these conditions more kinase activity is of acid-denatured enolase for 15 min at 25°C. Reactions were associated with CD4 than with CD8 precipitated from thy- stopped with 30 Al of double-strength reducing SDS/PAGE buffer and boiled for 5 min. Products were separated on 10% - CtS C) C)C p6> :0& 9 6> C. SDS/PAGE gels and visualized by autoradiography with Kodak X-Omat film. Alternatively, reactions were stopped with 70 1,u of assay buffer containing 1.43% SDS (final con- " centration, 1%) and boiling as above followed by a 1:9 dilution f) - 4m a. St, in assay buffer and addition of5-20 y1 ofantiserum or 5 pg of .4 , - anti-phosphotyrosine mAb (PY-20) or 5 ,ul ofanti-CD3 ascites (1F4). After rotation for 1.5 hr at 4°C, 100,ul ofa 10% (vol/vol) suspension ofbeads (protein A-Sepharose CL-4B, precoupled 4NM .mw A.Aw ::,... 40MOR to RAM for precipitation of mAbs PY-20 or 1F4) were added I Mr a. and rotated for 1.5 hr at 4°C. Beads were washed as above, - followed by boiling in 60 ,ul of reducing SDS sample buffer. -.1

A OX-21 CD2 CD4 CD8 B CD4 CDt N Br N Br N Br N Br B B. .CD 0 (26>64:6

_ -4 - :

im -,- 11 4

OW INOW -1 'Y 'WI WIP - ": _!I'

FIG. 1. Association of p56lck with CD4 and CD8, precipitated FIG. 2. Kinase assays on immune complexes prepared from rat from NP-40 or Brij 96 lysates of rat thymocytes. Rat thymocytes lymph node cells. Rat lymph node cells (5 x 107 per precipitation) were preincubated with anti-CD2 (OX-34), anti-CD4 (a combination were preincubated for 30 min on ice with mAbs directed against the of W3/25 and OX-70) or anti-CD8a (OX-8) mAbs, lysed in a buffer following antigens: CD4 (a mixture of the mAbs W3/25 and OX-70), containing either 1% NP-40 (lanes N) or 1% Brij 96 (lanes Br), and CD8 (OX-8), afCR (R73), CD3 (1F4), CD2 (OX-34), CD5 (OX-19), the antigens were subsequently precipitated. (A) Immune complexes CD53 (OX-44), OX-47, CD45 (OX-1), CD44 (a mixture ofOX-49 and were washed in the appropriate lysis buffers and then were incubated OX-SO), OX-52, and CD43 (a mixture of W3/13 and OX-56). Cells in 30 ,u of assay buffer containing_~~~~~~~~~~~~~~~~~~~5 ,uCi of [y-32P]ATP, 1 ,uM were then pelleted and lysed in Brij 96 lysis buffer. Kinase reactions unlabeled ATP, and 4 ,ug ofenolase. Phosphoproteins were separated were performed in the absence of unlabeled ATP and enolase as in on a SDS/10%o polyacrylamide gel and visualized by autoradiogra- Fig. 1B. (A) Products were separated by SDS/PAGE and visualized phy. (B) Immune complexes of CD4 and CD8, prepared in Brij 96 by autoradiography. Exposure time was 30 min. (B) Products were detergent, were incubated in 30 p1 of assay buffer containing 5 ,uCi boiled in 1% SDS to dissociate intermolecular associations. The of [y_32P]ATP and 10 mM MnCl2. Products were separated by mixtures were subsequently diluted 1:9, and a second round of SDS/PAGE and visualized by autoradiography. Autoradiography immunoprecipitations was performed with a polyclonal anti-p561ck exposure times of A and B were identical (2 hr). serum. Exposure time was 24 hr. Immunology: Beyers et A Proc. Natl. Acad. Sci. USA 89 (1992) 2947 mocytes (10, 29). Recently it was shown that a greater is seen and a weak band of this apparent molecular mass is amount of kinase activity was associated with CD4 and CD8 present in the CD4, CD8, and CD2 tracks. A 34-kDa band is in Brij 96 lysates than in NP-40 lysates (C. E. Rudd, personal present in CD4 and CD8 tracks. A 105-kDa band is clearly communication), and this prompted us to examine the ratio visible in the TCR and CD3 tracks and is faintly represented of p561ck associated with CD4 compared with CD8. in the CD4, CD8, CD2, and CD5 tracks. Significantly, no Rat thymocytes were lysed in NP40 or Brij 96 detergent, dominant phosphoproteins were seen in the reaction products and immunoprecipitates were made with CD2, CD4, and of kinase reactions on CD53, OX-47, CD45, CD44, OX-52, or CD8. Kinase assays were performed with enolase substrate, CD43 immune complexes prepared under identical conditions. and analysis confirmed that Brij 96 lysates yielded more After prolonged exposure, a 56- to 59-kDa band was detected p56tck associated with CD8 than was seen from NP-40 lysates in the CD44 track. This experiment was repeated three times (Fig. 1A). An additional observation was that phosphorylated with consistent results. In 10 further experiments, kinase bands running at the positions of CD3 E and C chains were assays were performed 18 times on immune complexes of seen in the precipitates from Brij 96. The additional bands CD4, CD8, TCR, CD3, CD2, or CD5, and consistent results were even more clearly revealed in kinase assays done were obtained without failure. without the addition of unlabeled ATP (Fig. 1B). The p56tck The nature ofthe phosphoproteins present in the CD4, CD8, band is seen as a strong 56- to 60-kDa band with additional TCR, CD3, CD2, and CD5 tracks was investigated. After the strong bands at 67, 26, and 18 kDa being precipitated. kinase reaction, samples were boiled in 1% SDS, diluted, and Associations ofSignal Transduction Molecules in T Cels. The then reprecipitated with antibodies directed against p56Ick (a sensitivity of the in vitro immune complex kinase assay with polyclonal serum) (Fig. 2B). Heavy bands of phosphorylated carrier free [y-32P]ATP was used to investigate the possibility p56kck were reprecipitated from the products of the CD4 and of intermolecular associations of various cell-surface antigens CD8 immune complex kinase reactions. A smaller amount of and the Tyr kinases p56ock and p59fyn. Rat lymph node cells p561ck was reprecipitated from CD2 reaction products, and were preincubated for 30 min on ice with mAbs directed weak bands were seen in TCR, CD3, CD5, and CD44 tracks. against CD4, CD8, and OX-47, which are expressed on subsets Reprecipitations were also performed with mAbs directed ofT cells, and with mAbs directed against aI3TCR, CD3, CD2, against phosphotyrosine (PY-20) or CD3 e chain (1F4) or with CD5, CD53, CD45 (OX-1, which recognizes a common deter- polyclonal sera against CD3 E chain, CD3 Cchain, p59y'), or minant of CD45), CD44, OX-52, and CD43, which are ex- CD5. Results are shown in Fig. 3. The anti-phosphotyrosine pressed on virtually all rat T cells. Cells were pelleted and mAb reprecipitated all the proteins that were phosphorylated lysed in Brij 96 buffer, and antigens were immunoprecipitated in the kinase reactions of the particular immune complexes. and analyzed with the kinase assays. Results are shown in Fig. The 67-kDa band was underrepresented in the TCR, CD3, and 2A. Heavily phosphorylated bands of p56tck are seen in the CD5 products after reprecipitation with the anti-phosphoty- CD4 and CD8 tracks, and very weak bands of the same rosine antibody, which may imply that the 67-kDa band was apparent molecular mass are present in the TCR, CD3, and phosphorylated on serine or threonine as well as on tyrosine CD2 tracks. A phosphoprotein of 26 kDa and a broad band of residues. The residues ofCD5 that were phosphorylated in the 16-22 kDa are present in the CD4, CD8, TCR, CD3, CD2 and in vitro reaction and the possibility that a serine/threonine CD5 tracks. In all these tracks a weak band of 24 kDa is also kinase may be present in the immune complexes have not been visible. In TCR, CD3, and CD5 tracks, a strong 67-kDa band further investigated. The anti-CD3 mAb 1F4 and a polyclonal First p~recipilatiofl %sith atnfi-(!) First precipitation ssith anti-('D8g First precipitation ssilh anti-l(T R Reprecipitation Nsitli atnti- Reprecipilation with anti- Reprecipitation Nvilh anti- A A 1 * u&' (: \* ts;' or VI .c5FG V< - G\ C,9 0 <> Cr

_ ) I

() so _. I) .$ j 40. - ;{ sv sC2 GO w +G "I _tU First precipitation with anti-(&I)3 First precipitation with anti-CD2 First precipitation with anti-(D)5 Reprecipitation with anti- Reprecipitation with anti- Reprecipitation with anti- F FIG. 3. Analysis of products irs rv of kinase assays by reimmuno- Eb .,o GiO' o ' \~ CrP Cr - CrP precipitation. Products of ki- nase reactions on immune com- plexes of CD4 (A), CD8 (B), _4 TCR (C), CD3 (D), CD2 (E), I- - {,- and CD5 (F), all obtained as in 1 4_ Fig. 2, were reprecipitated with mAbs directed against phospho- tyrosine (PY-20) or CD3 or with - oz} polyclonal rabbit sera directed against CD3 e and C chains, 2- p59fyn, and CD5. Exposure 8G__b - times were 12-16 hr. ')QA8 Immunology: Beyers et A Proc. Natl. Acad. Sci. USA 89 (1992) anti-CD3 E-chain serum reprecipitated a 26-kDa phosphopro- and acquire CD4 after overnight culture (30). pS6ick message tein from products of TCR, CD4, CD8, CD3, CD2, and CD5 has previously been detected in the most immature (CD4- immune complex kinase reactions. A polyclonal anti-CD3 CD8-) thymocytes, double-positive thymocytes, and mature C-chain serum precipitated a band of 16-22 kDa from each of single-positive thymocytes (31), but a physical association of the above mentioned reaction products. The anti-p59fy serum CD4 or CD8 with p56Ick has only been shown in CD4' CD8' precipitated a 59-kDa band from TCR, CD4C CD8, CD3, and and mature single-positive thymocytes (10). To extend the CD2 reaction products but not from CD5 reaction products. data, we determined whether p561ck is associated with CD8 in Finally, the polyclonal anti-CD5 serum precipitated a 67-kDa immature CD8' CD4- precursors and their prog- band from the CD4, CD8, TCR, CD3, CD2, and CD5 reaction eny produced in vitro. A similar amount of autophosphory- products. lated p56lck was found in CD8 immune complexes of CD4- One candidate for the 105-kDa protein present in immuno- CD8+ CD53- cells compared with that of unfractionated precipitates of the TCR and of CD3, and to a lesser degree in thymocytes (Fig. 4A). After overnight culture, these cells immunoprecipitates of CD4, CD8, CD2, and CD5, is GAP. In expressed both CD4 and CD8, and p561ck was associated with preliminary experiments, a polyclonal anti-GAP serum (PW6) both the CD4 and CD8 molecules (Fig. 4B). did not precipitate any phosphoproteins from products of Expresson in NK Cels and Macrophages. In the rat CD8 is kinase assays on TCR immune complexes (data not shown). expressed on NK cells, and CD4 is expressed on macro- Expression of p56k" in Early Thymocyte Subsets. In rat phages. The presence ofassociated p56Ick with CD8 and CD4 thymopoiesis cells mature in the stages CD4- CD8- to CD4- was analyzed on an NK cell line and on normal peritoneal CD8+ to CD4+ CD8+ to CD4- CD8+ or CD4+ CD8-. macrophages, respectively. In NK cells the kinase was asso- Immature CD4- CD8+ thymocytes lack the CD53 antigen ciated with CD8 but not with CD2, CD53, OX-47, CD45, CD44, CD43, or major histocompatibility complex class I _ il4OiDd which are all on these cells (Fig. 4C). No TCR 0CD053 nfraCtior,.ated CD4 CD8 .trac .o.ri molecules, expressed A ¶hyrnocyles thymocytes B !hyrnocyles lhymocvlTh. p561ck was found with macrophage CD4 (Fig. 4D), and no p56lck was detected by Western blotting in the macrophage e ,

.. w DISCUSSION 0~~~~~~~_0 *^< W~ *~ _ - - v This paper presents evidence for a multimolecular complex between TCR chains and other molecules that is loosely associated on normal T cells such that immunoprecipitation of different molecules in the complex brings down varying proportions of the other chains. In Brij 96 lysates of rat T cells, Tyr kinase activity was associated not only with CD4, and TCR immune complexes but also with CD2 and NK cell line Al 81 CD8, Thy I) TrhVf M. CD5 (Fig. 3). In all these cases, CD3 e and C chains and CD5 ::G.,, - G~ were present in the immunoprecipitates, and these antigens were phosphorylated in vitro on tyrosine residues (Fig. 3). CD2 does not have tyrosine residues in the cytoplasmic Now domain and, thus, cannot be detected in the in vitro kinase _ ! reactions. Ample p561ck was present in CD4 and CD8 immune complexes, smaller amounts were detected in CD2 immune - complexes, and barely detectable amounts were found in TCR, CD3, and CD44 immunoprecipitates (Fig. 2B). p5fYn s - -, was detectable in immunoprecipitates of CD4, CD8, TCR/ CD3, and CD2 but not CD5 (Fig. 3). A recent study showed FIG. 4. Association of p561ck with CD8/CD4 in iDrimature thy- that CD3 e chains were coprecipitated by anti-CD4 mAbs mocytes and NK cells but not in macrophages. (A) CD4- CD8+ from the human T-cell line HPB-ALL (32). The CD4.p561ck TCR- CD53- thymocytes were purified by magnetic cell sortig. association with CD3 E chain could not be demonstrated in Immune complexes of CD2 and CD8 were prepared ifrom purified resting cells, nor were other molecules such as the C chain thymocytes and from unfractionated thymocytes (2 x 106 per pre- coprecipitated. However, in prelabeled microsomal vesicles, cipitation) by using Brij % detergent. Kinase assays were performed an association between CD4p561ck, CD3 E chain, CD3C in the presence of 1 uM ATP and 4 ug of enolase Iper reaction. chain, and a 32- to 34-kDa protein was demonstrated. The Exposure time was 18 hr. (B) After overnight culture ofrCD4 CD8+ differences between these data and that presented in the TCR- CD53- thymocytes, the cells expressed both C be differences in and low amounts ofthe affTCR. Immune complexes of( CD4 and-34) present study may explained by experimen- CD4 (W3/25 and OX-70), and CD8 (OX-8) were prrepared from tal procedures: Burgess et al. lysed transformed cells in cultured cells and from unfractionated thymocytes ((2 x 106 per NP-40 detergent, whereas this study was done on normal precipitation) by using Brij 96. Kinase assays were peirformed as in lymph node cells that were lysed in Brij 96 detergent. A. Exposure time was 22 hr. (C) Kinase assays on iimmune com- A number of different experimental approaches have plexes of CD8 (OX-8) from thymocytes (lane Thy) (5 x 107 per pointed to a physical association of CD4/CD8 with the TCR precipitation) and CD8 (OX-8), CD2 (OX-34), CD53 (O)(-44), OX-47, complex. First, anti-TCR/CD3 mAbs induce comodulation CD45 (OX-1), CD44 (OX-49 plus OX-50), CD43 (W3/13 plus OX-56), of CD4 and vice versa (33-37). Second, TCR/CD3 and CD4 and major histocompatibility complex class 1 (OX-18) from the NK cocluster at the interface between T cells and antigen pre- cell line A181 (5 x 107 cells per precipitation). Immunie complexes senting cells (38), and a clonotypic anti-TCR mAb induces were prepared in Brij 96 buffer, and assays were done Exposure time was 3 hr. (D) Kinase assays on immune 4complexes of coclustering of the TCR and CD4 on T cells (39, 40). Third, CD4 (W3/25), and CD8 (OX-8) from thymocytes (lanes'Thy) (2 x 107 an association of the TCR with CD4 and CD8 in cell lysates per precipitation) and normal rat peritoneal macrophagges (lane MOb) of a CD4+ CD8+ T-cell clone was demonstrated by using (2 x 107 per precipitation). Immunoprecipitations and assays were affinity chromatography and gentle washing conditions (41). performed as in C. Exposure time was 4 hr. Fourth, fluorescence energy transfer experiments provided Immunology: Beyers et al. Proc. Natl. Acad. Sci. USA 89 (1992) 2949 evidence for an interaction of CD4 with the TCR complex 8. Rudd, C. E., Trevillyan, J. M., Dasgupta, J. D., Wong, L. L. & Schloss- (42-44). man, S. F. (1988) Proc. Nat!. Acad. Sci. USA 85, 5190-5194. 9. Veillette, A., Bookman, M. A., Horak, E. M. & Bolen, J. B. (1988) Cell Several studies have indicated that the TCR complex is 55, 301-308. required for efficient signaling via both CD2 (15, 17) and CD5 10. Veillette, A., Zkfiia-Pfldcker, J. C., Bolen, J. B. & Kruisbeek, A. M. (19) in T cells. The very recent demonstration that crosslink- (1989) J. Exp. Med. 170, 1671-1680. ing of CD2 induces the tyrosine phosphorylation ofthe same 11. Glaichenhaus, N., Shastri, N., Littman, D. R. & Turner, J. M. (1991) Cell 64, 511-520. polypeptides as crosslinking of the TCR complex (45) pro- 12. Chalupny, N. J., Ledbetter, J. A. & Kavathas, P. (1991) EMBO J. 10, vided strong evidence that these receptors have common 1201-1207. signaling mechanisms. However, evidence for a physical 13. Samelson, L. E., Phillips, A. F., Luong, E. T. & Klausner, R. D. (1990) association between CD2 and the TCR complex is limited to Proc. Nat!. Acad. Sci. USA 87, 4358-4362. one study showing that anti-CD3 mAbs coprecipitated CD2 14. Altman, A., Coggeshall, K. M. & Mustelin, T. (1990) Adv. Immunol. 48, 227-360. from digitonin lysates of T lymphoblasts or Jurkat cells (21). 15. Moingeon, P., Chang, H. C., Sayre, P. H., Clayton, L. K., Alcover, A., The present study shows a highly reproducible association Gardner, P. & Reinherz, E. L. (1989) Immunol. Rev. 111, 111-144. between CD2 and the TCR complex in resting T 16. Monostori, E., Desai, D., Brown, M. H., Cantrell, D. A. & Crumpton, and provides the first evidence of a physical interaction M. J. (1990) J. Immwnol. 144, 1010-1014. 17. Beyers, A. D., Barclay, A. N., Law, D. A., He, Q. & Williams, A. F. between CD5 and the TCR complex. Recent studies indicate (1989) Immunol. Rev. 111, 59-77. that CD2 is physically associated with CD45 in human and 18. Spruyt, L. L., Glennie, M. J., Beyers, A. D. & Williams, A. F. (1991)J. murine T lymphocytes (46, 47). Furthermore, p561ck was Exp. Med. 174,1407-1415. detected in immunoprecipitations of CD45 and a 32-kDa 19. June, C. H., Rabinovitch, P. S. & Ledbetter, J. A. (1987) J. Immunol. substrate for both and was in 138, 2782-2792. p561ck, CD45 also present the 20. McAteer, M. J., Lagarde, A. C., Georgiou, H. M. & Bellgrau, D. (1988) CD45/p56lck complexes (48). Under these circumstances, it Eur. J. Immunol. 18, 1111-1117. is possible that CD45 might interact with p56lck via CD2, 21. Brown, M. H., Cantrell, D. A., Brattsand, G., Crumpton, M. J. & which would not be detected in in vitro kinase assays. In this Gullberg, M. (1989) Nature (London) 339, 551-553. 22. Jefferies, W. A., Green, J. R. & Williams, A. F. (1985) J. Exp. Med. 162, study, a weakly labeled 32- to 34-kDa protein was also 117-127. detected in CD45 immunoprecipitates, but the kinase in- 23. Paterson, D. J., Green, J. R., Jefferies, W. A., Puklavec, M. & Williams, volved was not identified. Also, very recently it has been A. F. (1987) J. Exp. Med. 165, 1-13. shown that glycosylphosphatidylinositol-anchored cell sur- 24. Beyers, A. D., Davis, S. J., Cantrell, D. A., Izquierdo, M. & Williams, A. F. (1991) EMBO J. 10, 377-385. face molecules are associated with protein tyrosine kinases 25. McMaster, W. R. & Williams, A. F. (1979) Eur. J. Immunol. 9,426-433. (49), but this was not examined in the current experiments. 26. Robinson, A. P., Puklavec, M. & Mason, D. W. (1986) Immunology 57, Taken together, the data presented provide strong evi- 527-531. dence that normal T cells express a multimolecular complex 27. Mason, D. Y., Cordell, J., Brown, M., Pallesen, G., Ralfkiaer, E., Rothbard, J., Crumpton, M. & Gatter, K. C. (1989) J. Clin. Pathol. 42, consisting of CD4 or CD8, TCR/CD3, CD2, and CD5 in 1194-1200. association with the internal Tyr kinases pS6Ick and p59fyn. 28. Kypta, R. M., Hemming, A. & Courtneidge, S. A. (1988) EMBO J. 7, The 34-kDa and 105-kDa proteins associated with this com- 3837-3844. plex have not been identified and the possible involvement of 29. Hurley, T. R., Luo, K. & Sefton, B. M. (1989) Science 245, 407-409. 30. Paterson, D. J. & Williams, A. F. (1987) J. Exp. Med. 166, 1603-1608. other molecules-e.g., molecules with SH2 domains or ser- 31. Reynolds, P. J., Lesley, J., Trotter, J., Schulte, R., Hyman, R. & Sefton, ine/threonine kinases, has not been excluded. The 67-kDa B. M. (1990) Mol. Cell. Biol. 10, 4266-4270. band may consist not only of CD5 but also of ZAP-70, a 32. Burgess, K. E., Odysseos, A. D., Zalvan, C., Druker, B. J., Anderson, 70-kDa tyrosine phosphoprotein that associates with the P., Schlossman, S. F. & Rudd, C. E. (1991) Eur. J. Immunol. 21, 1663-1668. chain after stimulation of the TCR (50). It has been shown 33. Weyand, C. M., Goronzy, J. & Fathman, C. G. (1987) J. Immunol. 138, that the interaction of CD4/CD8:p561ck with major histocom- 1351-1354. patibility complex class II or class I molecules can greatly 34. Saizawa, K., Rojo, J. & Janeway, C. A. (1987) Nature (London) 328, enhance signaling via the TCR complex (reviewed in refs. 11 260-263. 35. Anderson, P., Blue, M. L. & Schlossman, S. F. (1988) J. Immunol. 140, and 14). 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