Through CD5 in Jurkat T Cells CD5 and Differentially Regulate

CD2 and CD3 Associate Independently with CD5 and Differentially Regulate Signaling Through CD5 in Jurkat T Cells

This information is current as Alexandre M. Carmo, Mónica A. A. Castro and Fernando A. of September 24, 2021. Arosa J Immunol 1999; 163:4238-4245; ; http://www.jimmunol.org/content/163/8/4238 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. CD2 and CD3 Associate Independently with CD5 and Differentially Regulate Signaling Through CD5 in Jurkat T Cells1

Alexandre M. Carmo,2*† Mo´nica A. A. Castro,* and Fernando A. Arosa*

In T lymphocytes, the CD2 and CD5 glycoproteins are believed to be involved in the regulation of signals elicited by the TCR/CD3 complex. Here we show that CD2 and CD3 independently associate with CD5 in human PBMC and Jurkat cells. CD5 coprecipitates with CD2 in CD3-deﬁcient cells and, conversely, coprecipitates with CD3 in cells devoid of CD2. In unstimulated CD2؉ CD3؉ Jurkat cells, CD5 associates equivalently with CD2 and CD3 and is as efﬁciently phosphorylated in CD2 as in CD3 immune complexes. However, upon activation the involvement of CD5 is the opposite in the CD2 and CD3 pathways. CD5 becomes rapidly tyrosine phosphorylated after CD3 stimulation, but is dephosphorylated upon CD2 cross-linking. These opposing effects correlate Downloaded from with the decrease in the activity of the SH2 domain-containing protein phosphatase 1 (SHP-1) following CD3 activation vs an enhanced activity of the phosphatase after CD2 triggering. The failure of CD5 to become phosphorylated on tyrosine residues in the CD2 pathway has no parallel with the lack of use of ␨-chains in CD2 signaling; contrasting with comparable levels of association of CD2 or CD3 with CD5, ␨ associates with CD2 only residually and is nevertheless slightly phosphorylated after CD2 stimulation. The modulation of CD5 phosphorylation may thus represent a level of regulation controlled by CD2 in signal

transduction mechanisms in human T lymphocytes. The Journal of Immunology, 1999, 163: 4238–4245. http://www.jimmunol.org/

ajor histocompatibility complex/peptide recognition mAbs can induce T cell activation and proliferation (6, 7). Signal- by the appropriate TCR is central to the process of T ing through CD2 depends on the integrity of its 116-aa-long cy- M lymphocyte activation. The simultaneous binding of toplasmic tail (8, 9), which is responsible for the association with TCR/CD3 and the coreceptors CD4 and CD8 to the same MHC/ the tyrosine kinases Lck and Fyn through proline sequence-SH3 peptide complexes present on APC induces the approximation of domain interactions (10–12). Indeed, activation of CD2 with mAb, the CD4/CD8-associated tyrosine kinase Lck to the CD3 chains. like that of TCR/CD3, has been shown to induce the activation of This results in the phosphorylation of the two conserved tyrosine Lck (13). However, it was reported that signal transduction via residues within immune receptor tyrosine-based activation motifs CD2 fails to phosphorylate ␨-chains and consequently does not use by guest on September 24, 2021 (ITAM)3 contained in CD3 chains and TCR-␨, and recruits through ZAP-70 (14) despite the fact that many features of the CD2 path- SH2 domains the ␨-associated protein ZAP-70, a kinase of the Syk way are similar to those of the pathway originated by the TCR family (1–4). Concomitantly with the binding, ZAP-70 becomes (15–17). phosphorylated on tyrosine residues, and newly formed phospho- In T lymphocytes, CD2 is embodied in a loosely associated tyrosine residues in ZAP-70 then become docking sites for other membrane complex that additionally comprises the TCR/CD3 SH2-containing enzymes (2). chains, CD4 or CD8, Lck and Fyn, and CD5, a membrane Ag CD2 is a 45- to 58-kDa type I integral protein expressed in expressed mainly on T cells (18). CD5 is a 67-kDa type I trans- human T and NK cells (5). Binding of CD2 on T cells to its membrane glycoprotein whose cytoplasmic domain contains mul- counter-receptor CD58 contributes not only to the stabilization of tiple potential sites for the phosphorylation of threonine, serine, interactions between lymphocytes and APC, but also to the trans- and tyrosine residues (5). CD5 is rapidly phosphorylated after duction of activation signals, as CD58 in combination with CD2 stimulation of the TCR/CD3 complex (19, 20), and this may allow Lck to bind through its SH2 domain and, as a result, to increase its catalytic activity (21). This can have some consequences for the *Laborato´rio de Imunologia Molecular, Instituto de Biologia Molecular e Celular, phosphorylation of Lck substrates such as the CD3 chains, because Universidade do Porto, Porto, Portugal; and †Medical Research Council Cellular Im- CD5 is closely associated with the TCR/CD3 complex (22), and, in munology Unit, Sir William Dunn School of Pathology, University of Oxford, Ox- ␨ ford, United Kingdom fact, tyrosine phosphorylation of CD5 precedes that of -chains (19). Given the failure of the CD2 pathway to progress through the Received for publication April 13, 1999. Accepted for publication August 6, 1999. ITAMs present on CD3 chains, and as CD2 constitutively associ- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ates with Lck, which is an effector of CD5, we investigated the role with 18 U.S.C. Section 1734 solely to indicate this fact. of CD5 in signal transduction via CD2. 1 This work was supported by fellowships from the Fundac¸a˜o para a Cieˆncia e a Tecnologia (to A.M.C. and M.A.A.C.) and a fellowship from the American Portu- Materials and Methods guese Biomedical Research Fund (to F.A.A.). Cells 2 Address correspondence and reprint requests to Dr. Alexandre M. Carmo, Labora- to´rio de Imunologia Molecular, Instituto de Biologia Molecular e Celular, Univer- Human PBMC were obtained from buffy coats of normal healthy donors sidade do Porto, Rua do Campo Alegre 823, 4150 Porto, Portugal. E-mail address: after centrifugation over Lymphoprep (Nycomed, Oslo, Norway). The [email protected] Jurkat E6.1 and JRT3-T3.5 cell lines (23) were obtained from A. Weiss ϩ ϩ 3 Abbreviations used in this paper: ITAM, immune receptor tyrosine-based activation (University of California, San Francisco, CA). The Jurkat CD2 CD3 2ϩ motif; RAM, rabbit anti-mouse; [Ca ]i, intracellular calcium concentration; SHP-1, JKHM cell line was donated by D. A. Cantrell (Imperial Cancer Research SH2 domain-containing protein phosphatase 1. Fund, London, U.K.). Cell lines were maintained in RPMI with 10% FCS,

1 mM sodium pyruvate, 2 mM L-glutamine, penicillin G (50 U/ml), and branes (Amersham). Membranes were blocked overnight in Tris-buffered streptomycin (50 ␮g/ml). saline and 0.1% (v/v) Tween 20 (TBS-T) containing 5% (w/v) nonfat dried milk, washed once for 15 min and twice for 5 min each time with TBS-T, Antibodies and incubated for1hatroom temperature with ExtrAvidin peroxidase- conjugated (Sigma, Madrid, Spain; dilution, 1/7500 in TBS-T). Mem- mAbs against cell surface Ags were: CD2-RFT11 (24), given by G. branes were washed again for 15 min and an additional four times for 5 min Jańossy (Royal Free Hospital, London, U.K.), OKT11 (25), obtained from each time with TBS-T, and biotinylated proteins were visualized by en- European Cell Culture Collection (ECACC, Porton Down, U.K.), GT2 hanced chemiluminescence (Amersham) and exposure to Biomax MR-1 (26), donated by D. A. Cantrell, and CD2–300 (27) (a polyclonal Ab rec- films (Eastman Kodak, Rochester, NY). ognizing the C-terminal-conserved end of CD2); CD3-OKT3 (28), and anti-CD3 polyclonal (29), gifts from M. H. Brown (Medical Research Immune complex kinase assays Council Cellular Immunology Unit); CD4-OKT4 (28), obtained from ␮ ECACC; CD5-Y2/178 (11), and a polyclonal anti-CD5 raised against a Brij 96 assay buffer (30 l) containing 10 mM MnCl2,1mMNa3V04,1 peptide sequence of 451–471 aa of human CD5 (18), gifts from D. Y. mM NaF, and 50 ␮Ci (185 KBq) of [␥-32P]ATP was added to the beads Mason (John Radcliffe Hospital, University of Oxford, Oxford, U.K); containing the immune complexes, and in vitro kinase reactions were al- CD45-BMAC-1 (30), donated by J. Fabre (Institute of Child Health, Uni- lowed to occur for 15 min at 25°C. Reactions were stopped by the addition versity of London, London, U.K.); C3bi-OX21 (31); polyclonal anti-Lck, of 30 ␮lof2ϫ SDS buffer, after which the samples were boiled for 5 min. raised against a peptide consisting of 39–64 aa of murine Lck (18), a gift Products were separated on 11% SDS-PAGE gels, and autoradiography of from J. Borst (The Netherlands Cancer Institute, Amsterdam, The Nether- the dried gels was performed with Kodak X-OMAT S films (Eastman lands); anti-Fyn rabbit polyclonal Ab, donated by P. Burn (Hoffmann- Kodak). LaRoche, Basel, Switzerland); polyclonal anti-CD3 ␨ (32), a gift from D. A. Cantrell; anti-protein tyrosine phosphatase 1C, a polyclonal Ab rec- Cell stimulation ognizing 576–595 aa at the C terminus, from Santa Cruz Biotechnology Approximately 3 ϫ 107 Jurkat cells were used per sample. mAbs at 5 Downloaded from (Santa Cruz, CA); anti-phosphotyrosine PY-20, purchased from Transduc- ␮ ␮ ␮ tion Laboratories (Lexington, KY); goat anti-mouse peroxidase conjugate, g/ml and rabbit anti-mouse Ig at 30 g/ml, or PHA at 10 g/ml, were from Transduction Laboratories; rabbit anti-mouse Ig (RAM), from Sero- added to cells maintained at 37°C in RPMI (no FCS) and mixed by vor- tec (Kidlington, U.K.); and RAM conjugated with fluorescein (RAM- texing. After the times indicated, cells were briefly pelleted and lysed in lysis buffer, and the nuclear pellet was removed by centrifugation at FITC), donated by S. Simmonds (Medical Research Council Cellular Im- ϫ munology Unit). 12,000 g for 10 min at 4°C. Immunoblotting Flow cytometry http://www.jimmunol.org/ Cell lysates were denatured in 2ϫ SDS buffer and run on SDS-PAGE. Between 1–5 ϫ 106 cells were resuspended in 50 ␮l of PBS containing 0.25% (w/v) BSA (PBS/BSA) and incubated with mAb (50 ␮l of hybrid- Samples were transferred to Hybond-C-super membranes by electroblot- oma tissue culture supernatant) for 30 min on ice. Cells were washed twice ting. Membranes were blocked overnight in TBS-T containing 5% (w/v) nonfat dried milk, washed once for 15 min and twice for 5 min each time at 4°C with 1 ml of PBS/BSA and 10 mM NaN3 (PBS/BSA/NaN3) and incubated with 50 ␮l of RAM-FITC (10 ␮g/ml) for 30 min on ice. Cells with TBS-T, and incubated for1hatroom temperature with the primary Ab (1/5000 dilution). Membranes were washed again for 15 min and twice were then resuspended in 1 ml of PBS/BSA/NaN3 and analyzed on a FAC- Scan (Becton Dickinson, Mountain View, CA). for 5 min with TBS-T, and incubated with goat anti-mouse or goat anti- rabbit Ig conjugated with peroxidase (1/20,000 dilution) for1hatroom Cell surface biotinylation temperature. Membranes were washed again for 15 min and an additional four times for 5 min each time with TBS-T, and detection was accom- by guest on September 24, 2021 Cell surface biotinylation was performed as previously described (33). plished using enhanced chemiluminescence (Amersham) and exposure to Briefly, cells were washed three times with ice-cold PBS and incubated for Biomax MR-1 films. 10 min at room temperature with PBS containing 0.5 mg/ml of EZ-Link 2ϩ sulfo-NHS-LC-biotin (Pierce, Rockford, IL). Cells were then washed for Measurement of [Ca ]i an additional three rounds with PBS, divided into aliquots of 3.5 ϫ 107 [Ca2ϩ] was determined as previously described (34). Briefly, cells were cells, and lysed for 30 min in ice-cold 1% Brij 96 lysis buffer (10 mM i washed twice in RPMI containing 0.25% BSA and were resuspended at Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 1% (v/v) 2 ϫ 107 Brij 96 or Nonidet P-40). cells/ml. Cells were incubated at 37°C for 10 min in the dark with 2 ␮M fura-2/AM and washed twice at room temperature in HBSS con- Immunoprecipitations taining 1 mM CaCl2, 0.25% BSA, and 10 mM HEPES (pH 7.4). Before fluorometry, cells were diluted to 2 ϫ 106 cells/ml, allowed to equilibrate Aliquots of 3.5 ϫ 107 cells were lysed for 30 min on ice in lysis buffer (10 to 37°C, and stimulated with mAbs at 2 ␮g/ml and with RAM at 25 ␮g/ml mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mg/ml BSA (not or PHA at 10 ␮g/ml. used in cell surface biotinylation), 1 mM PMSF, and 1% (v/v) Brij 96 or Nonidet P-40), the nuclear pellet was removed by centrifugation at Phosphatase assays 12,000 ϫ g for 10 min at 4°C, and the supernatants were precleared by end-over-end rotation with protein A-Sepharose CL-4B (Pharmacia, Ay- Phosphatase assays were performed as described previously (35). Immune lesbury, U.K.) for 30 min at 4°C. Abs (10 ␮g) or antisera (1–3 ␮l) and 100 complexes precipitated by biotinylated anti-SHP-1 Abs and ImmunoPure ␮l of 10% protein A-Sepharose beads were added to the samples and ro- avidin beads (Pierce) were washed three times in Nonidet P-40 lysis buffer tated for 90 min at 4°C. The beads containing the immune complexes were and incubated for 3–4 h at 37°C in 25 mM HEPES (pH 7.5), 100 mM KCl, washed three times in 1 ml of lysis buffer, and in in vitro kinase assays, an 3 mg/ml DTT, and 1 mg/ml p-nitrophenylphosphate (Sigma). Absorbance additional two washes were performed with 1 ml of Brij 96 assay buffer (25 was measured at 413 nm. mM HEPES (pH 7.5) and 0.1% (v/v) Brij 96). All washes were performed at 4°C. Results For reprecipitation experiments, the beads containing the immune com- Independent associations of CD2 and CD3 with CD5 in human plexes were boiled for 5 min in 3% SDS and diluted 8-fold with lysis T cells buffer. The beads were spun, and the supernatants were recovered and precleared for 30 min with 100 ␮l of 10% protein A-Sepharose beads. Human PBMC were separated from whole blood, and surface la- Proteins were precipitated with antisera plus 100 ␮l of 10% protein A- beled with biotin. Following cell lysis using the nonionic deter- Sepharose beads for 90 min. Immunoprecipitates were washed three times gents Brij 96 and Nonidet P-40, immunoprecipitations of CD2, with 1 ml of lysis buffer. Samples were boiled for 5 min in SDS buffer and run on 11% SDS-PAGE. CD3, CD5, and CD45 were conducted. Immune complexes were separated by SDS-PAGE, and biotinylated proteins were visual- Detection of biotinylated cell surface Ags in precipitated ized by enhanced chemiluminescence. When cells were lysed with immune complexes Brij 96, CD5 was coprecipitated with CD3, as previously reported Samples containing immunoprecipitates from surface-biotinylated cells (22), as well as with CD2, as shown in Fig. 1A. A protein of 67 were run on 11% SDS-PAGE and transferred to Hybond-C-super mem- kDa is clearly visible in CD2 and CD3 immune complexes from 4240 CD5 IN CD2 AND CD3 SIGNALING

FIGURE 2. Flow cytometric analysis of Jurkat cell variants. JRT3-T3.5 and a selected clone from E6.1 Jurkat cells were analyzed for cell surface FIGURE 1. CD2 coprecipitates with CD5 from Brij 96 and Nonidet expression of CD2, CD3, and CD5 using RFT11, OKT3, and Y-2/178 Abs, P-40 lysates of human PBMC. A, Human PBMC were surface biotinylated respectively. OX21 Abs were used as the negative control. The FACS Downloaded from and lysed with either 1% Brij 96 lysis buffer (indicated as B on top of the profiles confirm that JRT3-T3.5 are negative for CD3 expression while gel) or with 1% Nonidet P-40 lysis buffer (indicated as N). Immunopre- expressing CD2 at the cell surface. The E6.1/CD2Ϫ cells express CD3, but cipitations were conducted using Abs against the molecules indicated. not CD2. Samples were separated in 11% SDS-PAGE under nonreducing conditions and transferred onto Hybond-C-super membranes. Membranes were incubated with peroxidase-conjugated streptavidin, and biotinylated proteins in CD3, the amount of CD5 coprecipitated was negligible compared immune complexes were visualized by enhanced chemiluminescence and with that of CD5 in CD2 immunoprecipitates, suggesting that CD3 http://www.jimmunol.org/ exposure to Biomax MR-1 films. Molecular masses are shown in kilodal- has no role in CD2-mediated CD5 phosphorylation. Therefore, it tons. B, CD5 was reprecipitated from the immune complexes shown in A, seemed that both CD2 and CD3 have the potential to specifically run in SDS-PAGE, and transferred to Hybond-C-super membranes. Bio- associate with both CD5 and kinases that phosphorylate it. tinylated CD5 was detected by incubation of the membranes with streptavidin, enhanced chemiluminescence, and exposure to Biomax MR-1 films. Divergent patterns of CD5 phosphorylation following CD2 and CD3 cross-linking primary precipitates, and this protein was confirmed to be CD5 by We next wanted to study the possible role of CD5 in signal trans- reprecipitation using an anti-CD5 polyclonal serum (Fig. 1B). duction via the CD3 and the CD2 pathways. For that purpose, we by guest on September 24, 2021 However, when cells were lysed in lysis buffer containing 1% used a Jurkat cell variant, JKHM, that expresses both CD2 and Nonidet P-40, CD5 was detected in CD2, but not in CD3, immu- CD3 at high levels. Through surface biotinylation and immuno- noprecipitates, indicating that the interaction between CD2 and precipitation we determined that in these cells equivalent amounts CD5 in normal human T lymphocytes, although of an apparently of CD5 are precipitated with CD2 and CD3 (Fig. 5, A and B), and lower stoichiometry, is stronger than the interaction between CD3 by in vitro kinase assays that CD5 is effectively phosphorylated by and CD5. These results also indicate that the interaction between CD2 and CD3-associated kinases. Moreover, CD2 and CD3 asso- CD2 and CD5 in normal human T cells can be independent of any ciations with CD5 are independent of each other, as depletion of contribution from CD3.

CD5 associates independently with CD2 and CD3, and is efﬁciently phosphorylated by kinases present in immune complexes of CD3 and of CD2 in Jurkat cells In the Jurkat cell line JRT3-T3.5, which is negative for the expression of CD3 (Fig. 2), immunoprecipitation of CD2 again coprecipitated the CD5 Ag (Fig. 3A). Conversely, the association between CD5 and CD3 did not require the presence of CD2 at the cell surface. In Jurkat cells devoid of CD2 (selected clone from E6.1), CD3 could precipitate CD5 (Fig. 3B). Not only could CD5 be coprecipitated with CD2 and CD3 independently, but it also could be phosphorylated by tyrosine kinases within the different immune complexes. We performed kinase assays on CD2, CD3, CD4, and CD5 immunoprecipitates from Jurkat cells not expressing CD3 complexes at the surface FIGURE 3. CD2 and CD3 associate with CD5 in Jurkat cells indepen- (Fig. 4A) and on immunoprecipitates from CD2Ϫ Jurkat cells (Fig. dently of each other. Immunoprecipitation of CD2, CD3, CD5, CD45, and the control Ab OX21 was performed from 1% Brij 96 lysates of Jurkat cells 4B). CD5 was present on CD2 immune complexes from the CD3- Ϫ Ϫ JRT3-T3.5 (A) and E6.1/CD2 (B), cells that do not express CD3 and CD2, negative cell line and coprecipitated with CD3 in CD2 Jurkat respectively. Detection of biotinylated proteins in immune complexes was cells, as the results from primary immunoprecipitations suggested done by enhanced chemiluminescence after 11% SDS-PAGE under non- and reprecipitations conﬁrmed (Fig. 4). Despite the fact that CD3 reducing conditions and transfer to nitrocellulose membranes. Lower pan- Abs were able to precipitate some kinase activity and phospho- els, CD5 in immune complexes from JRT3-T3.5 and E6.1/CD2Ϫ cells was Ϫ proteins from CD3 cells, possibly from endogenously produced reprecipitated with a polyclonal Ab. The Journal of Immunology 4241 Downloaded from

FIGURE 5. CD5 associates at similar levels with CD2 and CD3 and is efﬁciently phosphorylated by kinases present in immune complexes of CD2 and CD3. CD2ϩ CD3ϩ JKHM Jurkat cells were surface biotinylated, lysed in Brij 96, and subjected to immunoprecipitations with Abs against the molecules indicated. Abs used were RFT11 (CD2), OKT3 (CD3), Y-2/178 (CD5), and BMAC1 (CD45). OX21 was used as a negative control. A, http://www.jimmunol.org/ Molecules in immune complexes were run on SDS-PAGE under nonreducing conditions and visualized by enhanced chemiluminescence. B, Im- mune complexes were disrupted with SDS and boiling, following which CD5 was reprecipitated with a polyclonal Ab. C, In parallel experiment, kinase assays were performed on immunoprecipitations, complexes were disrupted, and phosphorylated CD5 was reprecipitated.

FIGURE 4. CD5 is efficiently phosphorylated in CD3 immune complexes from Jurkat cells devoid of CD2 and in CD2 immune complexes which was faster when using the Ab combinations that induced the by guest on September 24, 2021 from CD3Ϫ Jurkat cells. In vitro kinase assays were performed in immune highest calcium signals. complexes prepared from Brij 96 lysates of JRT3-T3.5 cells (A) and E6.1/ CD2Ϫ cells (B). Following separation on 10% SDS-PAGE under reducing Differential usage of CD5 phosphorylation cannot be explained conditions, phosphorylated products were visualized after exposure of the by differences in stoichiometry of CD5-CD3 association vs CD5- dried gels to X-OMAT Kodak films. Molecular mass markers are indicated CD2 association in kilodaltons. Following kinase reactions, complexes were disrupted, and A number of features that are different between the CD2 and CD3 CD5 from immunoprecipitates of JRT3-T3.5 and E6.1/CD2Ϫ cells was signaling pathways have been reported (14, 36, 37), one of the reprecipitated with a polyclonal Ab (indicated by arrows). most significant being the report that stimulation of T cells via CD2 fails to induce the phosphorylation of CD3 ␨-chains and con- sequent docking of ZAP-70 to the CD3 complex (14). This may CD3 from cell lysates does not abrogate association between CD2 simply be due to the low level of association between CD2 and ␨. and CD5; conversely, preclearing of CD2 from cell lysates does We performed kinase assays on immunoprecipitates of CD2 and not influence the level of CD5 coprecipitated with CD3 (Fig. 6). CD3, following which ␨, CD5, and Lck were reprecipitated from The cells were functional in signaling, as calcium fluxes were the primary complexes. As displayed in Fig. 9, the amount of ␨ generated following different stimulations with PHA and anti-CD3 associating with CD2 was just a tiny fraction of that associating and anti-CD2 Abs. Cross-linking CD2 using the RFT11 Ab gave with CD3. Following CD2 stimulation, the phosphorylation of ␨ the best signal, comparable to that of CD3, and significantly higher was perceptible (Fig. 10), but was so low that it may not have a than the mitogenic combination of anti-CD2 monoclonals OKT11 physiological meaning compared with TCR stimulation. As ex- and GT2 (Fig. 7). Using OKT11 cross-linking alone resulted in pected, Lck was increasingly phosphorylated. By contrast, the low calcium fluxes. Also, stimulation of CD2 was comparable to amount of CD5 coprecipitating with CD2 was comparable to that that emerging from the TCR/CD3 complex in inducing the phos- coprecipitated by CD3 (Fig. 9), so it is striking that following CD2 phorylation of tyrosine residues in a number of substrates (data not triggering, CD5, contrary to ␨, became dephosphorylated (Fig. 10). shown). However, when we investigated the phosphorylation sta- The changes observed in the phosphorylation pattern of CD2 com- tus of CD5 following different stimulations, we were unable to plexes following CD2 cross-linking reflect mainly changes in the detect phosphorylation following cross-linking of CD2 (Fig. 8). phosphorylation level and not in the stoichiometry of associations, Time-course experiments showed a rapid increase in the phosphor- as CD2, Lck, and CD5 were present at similar amounts in both ylation of CD5 following CD3 cross-linking, followed by a decline activated and nonactivated states (Fig. 10, lower panel). However, in the signal. However, none of the combinations of anti-CD2 Abs it seems that more ␨ is associated with CD2 in activated cells, so could produce a similar pattern. On the contrary, there seemed to it is possible that ␨ is recruited to the CD2 complex following be a rapid dephosphorylation of CD5 following CD2 stimulation, activation. Nevertheless, the amount of protein associated with 4242 CD5 IN CD2 AND CD3 SIGNALING Downloaded from

FIGURE 6. CD2 and CD3 associations with CD5 are independent http://www.jimmunol.org/ events in JKHM cells. Cells were surface biotinylated and lysed in Brij 96 ϩ FIGURE 7. Ca2 signaling upon activation of JKHM cells. Cells were lysis buffer. Upper panel, CD2 was depleted from the lysates with five subjected to stimulation with different Abs or PHA. mAbs were used at 2 30-min rounds of depletion using the RFT11 mAb and protein A-Sepha- ␮g/ml, and cross-linking was achieved with rabbit anti-mouse Ig at 25 rose beads, plus two 30-min rounds of incubation with protein A-Sepharose ␮g/ml. PHA was used at 10 ␮g/ml. Addition of reagents to cells is indi- beads (PAS). CD3 was then immunoprecipitated with OKT3. In the CD3 cated by arrows in the calcium profiles. lane, CD5 is clearly detected as well as the TCR ␣␤ heterodimer. Lower panel, Depletion of CD3 using five 30-min rounds of OKT3 immunoprecipitation and two extra rounds using protein A-Sepharose alone, following which CD2 was immunoprecipitated. CD5 is clearly detected in association a similar pattern of phosphopeptides after cross-linking either with CD2. TCR/CD3 or CD2, and activation of the kinase of the Tec family, by guest on September 24, 2021 Itk (13, 15–17). It was therefore commonly accepted that CD2 would transduce signals of the same nature as those initiated by CD2 was so low and difficult to detect by immunoblotting that the stimulation of the TCR/CD3 complex. However, recently it has result could not be considered conclusive. been shown that features as central to CD3-mediated signaling as Changes in the activity of SHP-1 following activation via CD2 utilization of CD3 ␨-chains and ZAP-70 do not seem to be effec- and CD3 tively used in signal transduction via CD2 (14), thus suggesting that CD2 and CD3 pathways may diverge at that level. It has been argued that CD5 can mediate negative or modulatory Although we have detected some phosphorylation of ␨ upon signals, possibly through its association with the protein tyrosine CD2 stimulation, the fact remains that the level of association be- phosphatase 1C/SHP-1 (38, 39). Therefore, we measured the ac- tween CD2 and CD3-␨ in unstimulated cells is minimal, and in- tivity of that phosphatase following activation of Jurkat cells via creases in the phosphorylation of ␨-chains after CD2 cross-linking CD3 or CD2. Cells were stimulated with OKT3 or RFT11 Abs, are so minute that this phosphorylation may be physiologically SHP-1 was specifically precipitated with biotinylated Abs and irrelevant compared with ␨ phosphorylation after TCR/CD3 trig- streptavidin beads, and the phosphatase activity of the immuno- gering. Therefore, we initially considered the hypothesis that ac- precipitates was determined using a synthetic substrate, p-nitro- tivation via CD2, alternatively to using the ITAMs on CD3 chains, phenylphosphate. Time-course experiments showed that there could proceed through the phosphorylation of CD5. Supporting were consistently increments in the activity of SHP-1 following our initial assumption was the finding that CD2 associates with CD2 stimulation and a decline in the activity of the enzyme fol- CD5 in human T cells and cell lines independently of the TCR/ lowing TCR/CD3 triggering (Fig. 11). We therefore investigated CD3 complex, which was previously reported to closely interact whether SHP-1 could be found in association with CD2, which with CD5 (22). Moreover, the CD5 fraction associating with CD2 could explain why cross-linking of CD2 resulted in the enhance- had the potential to be phosphorylated by kinases contained in the ment of SHP-1 activity. Through in vitro kinase assays we were CD2 immune complexes, again independently of the contribution able to detect SHP-1 in immunoprecipitates of CD5 and CD3. of any kinase associated with the TCR/CD3 complex. However, we failed to detect any SHP-1 in CD2 immunoprecipi- Interestingly, however, upon stimulation of T cells via CD2 we tations (Fig. 12). observed not an increase but, rather, a decline in the phosphorylation status of CD5, which was faster as the signal emerging from Discussion CD2 became stronger. This result was striking, as in contrast with Some biochemical events following CD2-mediated activation ap- the difference in the level of association between CD2 and ␨ vs pear to be very similar to those initiated by CD3, including calcium CD3 and ␨, discussed above, CD5 seemed to associate with both mobilization, activation of the tyrosine kinase Lck, appearance of CD2 and CD3 at comparable levels. The Journal of Immunology 4243 Downloaded from FIGURE 10. Unlike Lck or ␨, CD2-associated CD5 shows decreased phosphorylation following CD2 stimulation. Jurkat cells were stimulated for 1 min with RFT11 ϩ RAM Ab (A) or were left unstimulated (R), following which CD2 was immunoprecipitated and subjected to kinase reactions in the presence of radiolabeled ATP and the absence of phosphatase inhibitors. Left panel, Kinase reaction products were separated by

SDS-PAGE under nonreducing conditions and were visualized by expo- http://www.jimmunol.org/ sure to X-OMAT S films. Presumed CD5 (a), Lck (b), and ␨ (c) are indicated by arrows. Right panel, Lck, CD5, and ␨ were reprecipitated from the original kinase reactions in nonactivated (R) or activated (A) cells, run on SDS-PAGE, and exposed to X-OMAT films. Lower panel, CD2, Lck, FIGURE 8. Failure of CD2 stimulation to induce CD5 tyrosine phos- CD5, and ␨ from the original immunoprecipitations were detected by im- phorylation. Following stimulation using different combinations of Abs, munoblotting using specific Abs. CD5 was precipitated from Jurkat cells at different times poststimulation and run on SDS-PAGE. Phosphorylated CD5 was detected by immunoblotting with PY-20. Equivalent amounts of CD5 were loaded on each lane, induce phosphorylation of CD5 on tyrosine residues, although it is as confirmed by immunoblotting using a CD5 polyclonal Ab. functional in other signaling pathways (43, 44). Therefore, it seems by guest on September 24, 2021 that CD5 may have a different role in signal transduction when Previous studies have also reported the absence of phosphory- coupled to the CD3 pathway or in its absence. lation of CD5 or any other protein of similar molecular mass fol- We show that CD2 is constitutively associated with CD5, and lowing CD2 stimulation (14, 40–42). The lack of phosphorylation this association can be detected even under strong lysis conditions. of CD5 following CD2 triggering has a parallel in the cross-linking Therefore, the lack of CD5 phosphorylation after CD2 triggering of CD5 alone, which, in contrast to TCR stimulation, does not may have an unforeseen functional significance, possibly not re- flecting only the lack of involvement in the CD2 pathway, but,

FIGURE 9. CD2 associates with TCR-␨ at a residual level. Jurkat cells FIGURE 11. The activity of SHP-1 is differentially regulated following were lysed in Brij 96, CD2 and CD3 were immunoprecipitated, and im- CD2 and CD3 stimulation. Jurkat cells were stimulated by OKT3 or RFT11 mune complexes were subjected to kinase reactions using [␥-32P]ATP. ϩ RAM Ab and lysed after the indicated times, and SHP-1 was precipi- Reaction products were denatured, and Lck, CD5, and ␨ present in the tated with the use of anti-SHP-1-biotinylated Abs and avidin beads. Im- immune complexes were reprecipitated with the use of speciﬁc Abs, run on munoprecipitated SHP-1-coupled beads were washed three times with lysis SDS-PAGE under reducing conditions, and exposed to autoradiography. buffer and incubated in phosphatase buffer as described in Materials and Contrasting to the comparable levels of CD5 and Lck in CD2 and CD3 Methods. Phosphatase activity was measured at 413 nm by the release of immunoprecipitates, ␨ is signiﬁcantly associated with CD3, but only re- yellow nitrophenol cleaved from the synthetic substrate p-nitrophenylphos- sidually with CD2. phate present in the phosphatase buffer. 4244 CD5 IN CD2 AND CD3 SIGNALING

the increased reactivity of TCRs in the animal models discussed above (45). Alternatively, it is possible that cross-linking of CD2 results in only the partial phosphorylation of the ITAMs present on CD3 chains. It has been suggested that the full positive signal involving FIGURE 12. SHP-1 is coprecipitated with CD5 and CD3, but not with the coupling of ZAP-70 to ITAMs requires both tyrosine residues CD2. CD2, CD3, CD5, and CD45 were immunoprecipitated from Jurkat to be phosphorylated. If only one of the residues becomes phos- Brij 96 lysates. Immune complexes were washed and subjected to in vitro phorylated, the resulting signal may be inhibitory (P. Allen, un- kinase assays. Reaction products were disrupted by boiling in SDS, and published observation). As SHP-1 can be coprecipitated with CD3, SHP-1 from the immune complexes was recovered through precipitation it may be that incomplete ITAM phosphorylation induced by CD2 with a specific Ab. Reprecipitates were run on SDS-PAGE and visualized could result in the recruitment of SHP-1, and not ZAP-70, to the by autoradiography. TCR/CD3 complex. Following TCR-positive stimulation with complete ITAM phosphorylation, full occupancy of CD3 ITAMs by ZAP-70 could displace SHP-1 from the activation motifs, thus instead, a key regulatory event. In this context, it is important to explaining the decrease in the activity of the phosphatase. note that CD2 may functionally associate with CD5 in restraining The present results together with the recent findings of the reg- the physiological activation through the TCR. In a mouse model ulatory role of CD2 and CD5 in signal transduction and the par- where T cells express specific MHC class I-restricted TCRs, the allelism in CD2 and CD5 expression observed during T cell on- absence of CD2 results in enhanced positive selection (45). A sim- togeny and after polyclonal activation (45, 47) support a functional Downloaded from ilar phenotype is observed in CD5 null MHC I-restricted TCR role for the CD2/CD5 association described here in the regulation transgenic mice, which suggests that both CD2 and CD5 contribute of signal transduction in T lymphocytes. to the modulation of signals during thymic selection. Furthermore, in mice deficient in both CD2 and CD5, the effect seems to be Acknowledgments synergistic (45). We thank Dr. S. P. Watson, Oxford University, for help with the measure- The negative role of CD5 is possibly due to its functional as- 2ϩ http://www.jimmunol.org/ ment of [Ca ]i, and Dr. M. H. Brown, Dr. D. Mason (Medical Research sociation with the tyrosine phosphatase SHP-1, as the absence of Council Cellular Immunology Unit, Sir William Dunn School of Pathol- CD5 as well as of SHP-1 results in hyper-responsiveness upon ogy, University of Oxford, Oxford, U.K.), and Prof. M. de Sousa (Instituto TCR stimulation, and also in increased positive selection of thy- de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal) mocytes (38, 39). We had previously detected tyrosine phospha- for useful discussions and continuous support. tase activity associated with CD5 in rat T cells (35), and in the present report a correlation between the phosphatase activity of References SHP-1 following CD2 and CD3 activation and the status of phos- 1. Irving, B. A., A. C. Chan, and A. Weiss. 1993. Functional characterization of a phorylation of CD5 after the different stimuli was found. Although signal transducing motif present in the T cell antigen receptor ␨ chain. J. 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