The Accessory Molecules CD5 and CD6 Associate on the Membrane of Lymphoid T Cells
2003; Elsevier BV; Volume: 278; Issue: 10 Linguagem: Inglês
10.1074/jbc.m209591200
ISSN1083-351X
AutoresIdoia Gimferrer, Montse Farnós, Marı́a Calvo, Marı́a Mittelbrunn, Carlos Enrich, Francisco Sánchez‐Madrid, Jordi Vives, Francisco Lozano,
Tópico(s)Cell Adhesion Molecules Research
ResumoCD5 and CD6 are closely related lymphocyte surface receptors of the scavenger receptor cysteine-rich superfamily, which show highly homologous extracellular regions but little conserved cytoplasmic tails. Both molecules are expressed on the same lymphocyte populations (thymocytes, mature T cells, and B1a cells) and share similar co-stimulatory properties on mature T cells. Although several works have been reported on the molecular associations and the signaling pathway mediated by CD5, very limited information is available for CD6 in this regard. Here we show the physical association of CD5 and CD6 at the cell membrane of lymphocytes, as well as their localization at the immunological synapse. CD5 and CD6 co-immunoprecipitate from Brij 96 but not Nonidet P-40 cell lysates, independently of both the co-expression of other lymphocyte surface receptors and the integrity of CD5 cytoplasmic region. Fluorescence resonance energy transfer analysis, co-capping, and co-modulation experiments demonstrate the physical in vivo association of CD5 and CD6. Analysis of T cell/antigen-presenting cells conjugates shows the accumulation of both molecules at the immunological synapse. These results indicate that CD5 and CD6 are structurally and physically related receptors, which may be functionally linked to provide either similar or complementary accessory signals during T cell activation and/or differentiation. CD5 and CD6 are closely related lymphocyte surface receptors of the scavenger receptor cysteine-rich superfamily, which show highly homologous extracellular regions but little conserved cytoplasmic tails. Both molecules are expressed on the same lymphocyte populations (thymocytes, mature T cells, and B1a cells) and share similar co-stimulatory properties on mature T cells. Although several works have been reported on the molecular associations and the signaling pathway mediated by CD5, very limited information is available for CD6 in this regard. Here we show the physical association of CD5 and CD6 at the cell membrane of lymphocytes, as well as their localization at the immunological synapse. CD5 and CD6 co-immunoprecipitate from Brij 96 but not Nonidet P-40 cell lysates, independently of both the co-expression of other lymphocyte surface receptors and the integrity of CD5 cytoplasmic region. Fluorescence resonance energy transfer analysis, co-capping, and co-modulation experiments demonstrate the physical in vivo association of CD5 and CD6. Analysis of T cell/antigen-presenting cells conjugates shows the accumulation of both molecules at the immunological synapse. These results indicate that CD5 and CD6 are structurally and physically related receptors, which may be functionally linked to provide either similar or complementary accessory signals during T cell activation and/or differentiation. scavenger receptor cysteine-rich monoclonal antibody T cell receptor peripheral blood lymphocytes fluorescein isothiocyanate cyanine 3 fluorescence resonance energy transfer glutathione S-transferase fetal calf serum Texas Red poly-l-lysine staphylococcal enterotoxin E horseradish peroxidase chloromethyl derivate of aminocoumarin horseradish peroxidase phosphate-buffered saline antibodies streptavidin supramolecular activation clusters antigen present cell The correct activation and differentiation of T lymphocytes results from the fine-tuning of intracellular signals delivered through co-engagement of the antigen-specific receptor and a series of accessory molecules simultaneously expressed on the cell surface. Among the lymphocyte accessory molecules (CD2, CD4, CD8, CD9, CD28, CD43, CD45, etc.), there are CD5 and CD6, two highly homologous representatives of the scavenger receptor cysteine-rich (SRCR)1 superfamily (1Aruffo A. Bowen M.A. Patel D.D. Haynes B.F. Starling G.C. Gebe J.A. Bajorath J. Immunol. Today. 1997; 18: 498-504Google Scholar, 2Vilà J.M. Padilla O. Arman M. Gimferrer I. Lozano F. Immunologı́a. 2000; 19: 105-121Google Scholar). The SRCR superfamily includes a heterogeneous group of soluble and/or membrane-associated receptors involved in the development of the immune system and in the regulation of both innate and adaptive immune responses (1Aruffo A. Bowen M.A. Patel D.D. Haynes B.F. Starling G.C. Gebe J.A. Bajorath J. Immunol. Today. 1997; 18: 498-504Google Scholar). This family is characterized by the presence of one or several repeats of a well conserved extracellular domain named SRCR, which was first reported at the C terminus of the macrophage type I scavenger receptor (SR-AI) (3Freeman M. Ashkenas J. Rees D.J. Kingsley D.M. Copeland N.G. Jenkins N.A. Krieger M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8810-8814Google Scholar). CD5 and CD6 are the two representatives of the SRCR superfamily reported to be expressed on human lymphocytes. Both lymphocyte receptors are type I transmembrane glycoproteins with extracellular regions composed of three SRCR domains and cytoplasmic domains devoid of intrinsic catalytic activity but well adapted for signal transduction (4Jones N.H. Clabby M.L. Dialynas D.P. Huang H.J. Herzenberg L.A. Strominger J.L. Nature. 1986; 323: 346-349Google Scholar, 5Aruffo A. Melnick M.B. Linsley P.S. Seed B. J. Exp. Med. 1991; 174: 949-952Google Scholar). The two molecules show a similar cell expression pattern, being expressed on thymocytes, mature T cells, B1a cells, and B chronic lymphocytic leukemia cells (2Vilà J.M. Padilla O. Arman M. Gimferrer I. Lozano F. Immunologı́a. 2000; 19: 105-121Google Scholar), and their surface expression levels are also up-regulated by similar stimuli (6Carrera A.C. Cardenas L. Tugores A. Alonso M. Sanchez-Madrid F. de Landazuri M.O. J. Biol. Chem. 1989; 264: 15650-15655Google Scholar, 7Bott C.M. Doshi J.B. Li L.L. McMurtry S.A. Sanders J.L. Fox D.A. J. Immunol. 1994; 153: 1-9Google Scholar). The genes for CD5 and CD6 map to contiguous regions of human chromosome 11q12.2 and are supposed to have arisen from duplication of a common ancestral gene (8Padilla O. Calvo J. Vila J.M. Arman M. Gimferrer I. Places L. Arias M.T. Pujana M.A. Vives J. Lozano F. Immunogenetics. 2000; 51: 993-1001Google Scholar, 9Bowen M.A. Whitney G.S. Neubauer M. Starling G.C. Palmer D. Zhang J. Nowak N.J. Shows T.B. Aruffo A. J. Immunol. 1997; 158: 1149-1156Google Scholar). CD5 has been shown to behave as a dual receptor, which provides either positive or negative co-stimulatory signals depending on the cell type and the developmental stage (10Lozano F. Simarro M. Calvo J. Vila J.M. Padilla O. Bowen M.A. Campbell K.S. Crit. Rev. Immunol. 2000; 20: 347-358Google Scholar). To achieve this, engagement of different signaling molecules and cascades by CD5 (phosphatidylcholine-specific phospholipase C (PC-PLC), acidic sphingomyelinase (ASMase), casein kinase II (CKII), and phosphatidylinositol 3-kinase, Cbl, RasGAP, extracellular signal-regulated protein kinase/mitogen-activated protein kinase, etc.) have been reported (10Lozano F. Simarro M. Calvo J. Vila J.M. Padilla O. Bowen M.A. Campbell K.S. Crit. Rev. Immunol. 2000; 20: 347-358Google Scholar). Initial positive co-stimulatory properties on peripheral T cells were first reported for CD5, by using either soluble or solid phase-bound monoclonal antibodies (mAbs) (11Ceuppens J.L. Baroja M.L. J. Immunol. 1986; 137: 1816-1821Google Scholar, 12Ledbetter J.A. Norris N.A. Grossmann A. Grosmaire L.S. June C.H. Uckun F.M. Cosand W.L. Rabinovitch P.S. Mol. Immunol. 1989; 26: 137-145Google Scholar, 13Alberola-Ila J. Places L. Cantrell D.A. Vives J. Lozano F. J. Immunol. 1992; 148: 1287-1293Google Scholar, 14Verwilghen J. Vandenberghe P. Wallays G. de Boer M. Anthony N. Panayi G.S. Ceuppens J.L. J. Immunol. 1993; 150: 835-846Google Scholar). Later, data from CD5-deficient mice have shown that CD5 may also negatively regulate the signaling through the antigen-specific receptor complex on thymocytes and B1a cells (15Tarakhovsky A. Kanner S.B. Hombach J. Ledbetter J.A. Muller W. Killeen N. Rajewsky K. Science. 1995; 269: 535-537Google Scholar, 16Bikah G. Carey J. Ciallella J.R. Tarakhovsky A. Bondada S. Science. 1996; 274: 1906-1909Google Scholar). Subsequent studies (17Pena-Rossi C. Zuckerman L.A. Strong J. Kwan J. Ferris W. Chan S. Tarakhovsky A. Beyers A.D. Killeen N. J. Immunol. 1999; 163: 6494-6501Google Scholar, 18Azzam H.S. DeJarnette J.B. Huang K. Emmons R. Park C.S. Sommers C.L. El-Khoury D. Shores E.W. Love P.E. J. Immunol. 2001; 166: 5464-5472Google Scholar, 19Gary-Gouy H. Bruhns P. Schmitt C. Dalloul A. Daeron M. Bismuth G. J. Biol. Chem. 2000; 275: 548-556Google Scholar) have highlighted the importance of CD5 in thymocyte development and of its cytoplasmic domain for the inhibitory function. It seems, however, that CD5 could exert negative effects on only one branch (Ca2+influx following phospholipase C-γ activation) of the T cell receptor (TCR)-mediated signal, during CD5-mediated differentiation of double positive thymocytes (20Cibotti R. Punt J.A. Dash K.S. Sharrow S.O. Singer A. Immunity. 1997; 6: 245-255Google Scholar, 21Zhou X.Y. Yashiro-Ohtani Y. Toyo-Oka K. Park C.S. Tai X.G. Hamaoka T. Fujiwara H. J. Immunol. 2000; 164: 1260-1268Google Scholar). The influence of CD5 in the selection process of developing thymocytes has been shown to depend on the TCR/major histocompatibility complex/ligand affinity (15Tarakhovsky A. Kanner S.B. Hombach J. Ledbetter J.A. Muller W. Killeen N. Rajewsky K. Science. 1995; 269: 535-537Google Scholar, 17Pena-Rossi C. Zuckerman L.A. Strong J. Kwan J. Ferris W. Chan S. Tarakhovsky A. Beyers A.D. Killeen N. J. Immunol. 1999; 163: 6494-6501Google Scholar, 18Azzam H.S. DeJarnette J.B. Huang K. Emmons R. Park C.S. Sommers C.L. El-Khoury D. Shores E.W. Love P.E. J. Immunol. 2001; 166: 5464-5472Google Scholar). This, together with the fact that CD5 surface expression is developmentally regulated by TCR signals and TCR avidity (22Azzam H.S. Grinberg A. Lui K. Shen H. Shores E.W. Love P.E. J. Exp. Med. 1998; 188: 2301-2311Google Scholar), has led to the notion that CD5 functions to fine-tune TCR signaling (18Azzam H.S. DeJarnette J.B. Huang K. Emmons R. Park C.S. Sommers C.L. El-Khoury D. Shores E.W. Love P.E. J. Immunol. 2001; 166: 5464-5472Google Scholar). Whether this tuning is achieved by CD5 alone or in conjunction with other lymphocyte receptors remains to be ascertained. Recently, the synergistic effect on thymocyte-positive selection of the null mutations of CD5 and CD2, two associated molecules (23Carmo A.M. Castro M.A. Arosa F.A. J. Immunol. 1999; 163: 4238-4245Google Scholar), has been reported (24Teh S.J. Killeen N. Tarakhovsky A. Littman D.R. Teh H.S. Blood. 1997; 89: 1308-1318Google Scholar). The identity and the role played by the CD5 ligand(s) are also a matter of controversy (2Vilà J.M. Padilla O. Arman M. Gimferrer I. Lozano F. Immunologı́a. 2000; 19: 105-121Google Scholar, 25Bhandoola A. Bosselut R. Yu Q. Cowan M.L. Feigenbaum L. Love P.E. Singer A. Eur. J. Immunol. 2002; 32: 1811-1817Google Scholar). A good candidate is, however, gp150 (26Calvo J. Places L. Padilla O. Vila J.M. Vives J. Bowen M.A. Lozano F. Eur. J. Immunol. 1999; 29: 2119-2129Google Scholar), a broadly distributed receptor that is expressed on thymic epithelial cells. The precise function of CD6 in lymphocyte activation and differentiation still remains elusive. Available evidence indicates that it is an accessory molecule capable of providing co-stimulatory signals in mature T cells (27Morimoto C. Rudd C.E. Letvin N.L. Hagan M. Schlossman S.F. J. Immunol. 1988; 140: 2165-2170Google Scholar, 28Gangemi R.M. Swack J.A. Gaviria D.M. Romain P.L. J. Immunol. 1989; 143: 2439-2447Google Scholar, 29Swack J.A. Gangemi R.M. Rudd C.E. Morimoto C. Schlossman S.F. Romain P.L. Mol. Immunol. 1989; 26: 1037-1049Google Scholar, 30Bott C.M. Doshi J.B. Morimoto C. Romain P.L. Fox D.A. Int. Immunol. 1993; 5: 783-792Google Scholar, 31Osorio L.M. Garcia C.A. Jondal M. Chow S.C. Cell. Immunol. 1994; 154: 123-133Google Scholar, 32Osorio L.M. Rottenberg M. Jondal M. Chow S.C. Immunology. 1998; 93: 358-365Google Scholar). The signaling pathway used by CD6 to influence T cell activation is, however, mostly unknown. It has been shown that CD6 becomes hyperphosphorylated on Ser and Thr residues (29Swack J.A. Gangemi R.M. Rudd C.E. Morimoto C. Schlossman S.F. Romain P.L. Mol. Immunol. 1989; 26: 1037-1049Google Scholar,33Cardenas L. Carrera A.C. Yague E. Pulido R. Sanchez-Madrid F. de Landazuri M.O. J. Immunol. 1990; 145: 1450-1455Google Scholar) and transiently tyrosine-phosphorylated after CD3 stimulation alone or by co-cross-linking of CD3 with either CD2 or CD4 (34Wee S. Schieven G.L. Kirihara J.M. Tsu T.T. Ledbetter J.A. Aruffo A. J. Exp. Med. 1993; 177: 219-223Google Scholar, 35Kobarg J. Whitney G.S. Palmer D. Aruffo A. Bowen M.A. Eur. J. Immunol. 1997; 27: 2971-2980Google Scholar). In B chronic lymphocytic leukemia cells, CD6 ligation protects from anti-IgM-mediated apoptosis through Bcl-2 induction (36Osorio L.M. De Santiago A. Aguilar-Santelises M. Mellstedt H. Jondal M. Blood. 1997; 89: 2833-2841Google Scholar). A role for CD6 in thymocyte development has also been suggested. CD166/activated leukocyte cell adhesion molecule, a CD6 ligand, is expressed on thymic epithelium and mediates adhesion of thymocytes to it (37Bowen M.A. Patel D.D. Li X. Modrell B. Malacko A.R. Wang W.C. Marquardt H. Neubauer M. Pesando J.M. Francke U. Barton F.H. Aruffo A. J. Exp. Med. 1995; 181: 2213-2220Google Scholar, 38Patel D.D. Wee S.F. Whichard L.P. Bowen M.A. Pesando J.M. Aruffo A. Haynes B.F. J. Exp. Med. 1995; 181: 1563-1568Google Scholar, 39Whitney G.S. Starling G.C. Bowen M.A. Modrell B. Siadak A.W. Aruffo A. J. Biol. Chem. 1995; 270: 18187-18190Google Scholar). The correlation of CD6 expression with thymocyte-positive selection and resistance to apoptosis has also been reported (40Singer N.G. Fox D.A. Haqqi T.M. Beretta L. Endres J.S. Prohaska S. Parnes J.R. Bromberg J. Sramkoski R.M. Int. Immunol. 2002; 14: 585-597Google Scholar). Mice deficient for CD6 are not available yet, and the possibility that CD6 possesses negative regulatory properties similar to those reported for CD5 on thymocyte development still has to be evaluated. CD5 has been found physically associated with the antigen-specific receptor present on B1a (B cell receptor) and T (TCR) cells (41Lankester A.C. van Schijndel G.M. Cordell J.L. van Noesel C.J. van Lier R.A. Eur. J. Immunol. 1994; 24: 812-816Google Scholar, 42Osman N. Lazarovits A.I. Crumpton M.J. Eur. J. Immunol. 1993; 23: 1173-1176Google Scholar), as well as with the surface receptors CD4 or CD8, CD2, and CD9 (23Carmo A.M. Castro M.A. Arosa F.A. J. Immunol. 1999; 163: 4238-4245Google Scholar, 43Beyers A.D. Spruyt L.L. Williams A.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2945-2949Google Scholar,44Toyo-oka K. Yashiro-Ohtani Y. Park C.S. Tai X.G. Miyake K. Hamaoka T. Fujiwara H. Int. Immunol. 1999; 11: 2043-2052Google Scholar). This positions CD5 as an important regulatory molecule relevant for T and B cell-mediated responses. In the CD6 case, no associations with other lymphocyte surface receptors have been reported up to now. The fact that CD5 and CD6 are closely related molecules, not only at structural but also at expression and functional level (2Vilà J.M. Padilla O. Arman M. Gimferrer I. Lozano F. Immunologı́a. 2000; 19: 105-121Google Scholar), prompted us to investigate a possible physical association between them. Here we present strong biochemical and microscopic evidence on the in vivo association and co-localization of CD5 and CD6 on the membrane of T lymphocytes. This suggests that they may constitute a functional unit subserving lymphocyte activation and differentiation processes. Lymphocytes were obtained either from peripheral blood (PBL) samples subjected to centrifugation on a standard Ficoll gradient (d = 1,077) or from lymph node suspensions. Thymocytes were obtained by disruption of human thymus specimens from children undergoing cardiac surgery. The human T cell lines HUT-78 and Jurkat JE6.1, and the lymphoblastoid B cell line Raji were obtained from the American Tissue Culture Collection (ATCC). COS-7 cells were from the European Collection of Cell Cultures (ECACC 87021302). Cell lines were grown in RPMI with 10% fetal calf serum (FCS), 1 mm sodium pyruvate, 2 mm l-glutamine, 50 units/ml penicillin G, and 50 μg/ml streptomycin. The mouse Cris-1 (anti-CD5, IgG2a), 148.1C3 (anti-CD43, IgG2a), 33-2A3 (anti-CD3, IgG2a) and 161.8 (anti-CD6, IgG1) mAbs were produced in our laboratory by R. Vilella (Hospital Clı́nic, Barcelona, Spain). Affinity-purified Leu-1 (anti-CD5, IgG2a) mAb was purchased from BD Biosciences. The SPV L14.2 (anti-CD6, IgG1) mAb was from Immunotech (Marseille, France), and the W6/32 (anti-HLA class I, IgG2a) was from the ATCC (HB-95). The fluorescein isothiocyanate (FITC)-labeled anti-CD5 (UCTH2, IgG1), anti-CD6 (M-T605, IgG1), and anti-CD3 (UCHT1, IgG1) mAb were from Pharmingen. The 148.1C3 mAb was conjugated to FITC (Sigma) as described previously (45Goding J.W. J. Immunol. Methods. 1976; 13: 215-226Google Scholar). The Leu-1 and 161.8 mAbs were conjugated to cyanine 3 (Cy3) using the Cy3 mAb labeling kit (Amersham Biosciences). The FITC-conjugated goat anti-mouse polyvalent immunoglobulins (GAMIg-FITC) were from Sigma. Horseradish peroxidase (HRP)-conjugated streptavidin (SAv) was from Dako (Denmark). Tricolor- and Texas Red (TR)-conjugated SAv were from Caltag (Burlingame, CA). The generation of the rabbit polyclonal antiserum against the extracellular region of human CD5 has been reported elsewhere (46Calvo J. Places L. Espinosa G. Padilla O. Vila J.M. Villamor N. Ingelmo M. Gallart T. Vives J. Font J. Lozano F. Tissue Antigens. 1999; 54: 128-137Google Scholar). The anti-CD6 polyclonal antiserum was produced in our laboratory by immunizing rabbits for 2 weeks with four intramuscular injections (50 μg each) of glutathioneS-transferase (GST)-CD6cy in complete (first) and incomplete (next) Freund's adjuvant (Invitrogen). Biotinylation of mAbs and cell surface proteins was performed with EZ-Link sulfo-NHS-LC-LC-Biotin following the manufacturer's instructions (Pierce). Mowiol 4-88 was from Calbiochem, and poly-l-lysine (PLL) was from Sigma. The blue fluorescent cell tracker chloromethyl derivative of aminocoumarin (CMAC) was from Molecular Probes (Eugene, OR). Staphylococcal enterotoxin E (SEE) was from Toxin Technology (Sarasota, FL). The cDNA used to amplify the cytoplasmic region of CD6 was obtained by retrotranscription of total mRNA from PBL with SuperscriptTM II RNase H− Reverse Transcriptase (Invitrogen) following the manufacturer's instructions. The GST-CD6cy construct codes for a fusion protein was composed of the GST protein fused in-frame to the CD6 cytoplasmic region (from Lys-427 to Ala-688). This was done by PCR amplification of CD6 cDNA with the sense/antisense oligonucleotide pair, 5′-AGCAGTCGACGAAAGGAAAATATGCCCTCCCCGTA-3′ and 5′-AAGAATGCGGCCGCTAGGCTGCGCTGATGTCATCGT-3′, and further cloning intoSalI- and NotI-restricted (Fermentas MBI, Vilnius, Lithuania) pGEX 4T2 expression vector (Amersham Biosciences AB). The expression construct coding for wild-type CD6 (CD6.WT) was obtained by cloning SalI/EcoRI- andEcoRI/BamHI-restricted (Fermentas MBI) fragments corresponding to the extracellular and cytoplasmic regions of CD6, respectively, into SalI/BamHI-restricted pHβAPr-1-neo mammalian expression vector. The extracellular portion of CD6 was obtained by PCR amplification using the 5′-TCTCGTCGACATGTGGCTCTTCTTCGGGAT-3′ and 5′-AACTTCTTTGGGGATGGTGATGGG-3′ primers and the CD6-PB1 cDNA sequence cloned into pBJneo as a template (47Robinson W.H. Neuman de Vegvar H.E. Prohaska S.S. Rhee J.W. Parnes J.R. Eur. J. Immunol. 1995; 25: 2765-2769Google Scholar). The intracellular region of CD6 was obtained by PCR amplification of HUT78 cDNA with the 5′-GTCACTATAGAATCTTCTGTG-3′ and 5′-AAAGGATCCCTAGGCTGCGCTGATGTCATC-3′ primers. The generation of expression constructs coding for CD5.WT and CD5.K384stopmolecules has been described elsewhere (48Simarro M. Pelassy C. Calvo J. Places L. Aussel C. Lozano F. J. Immunol. 1997; 159: 4307-4315Google Scholar). Aliquots of surface biotinylated cells were lysed for 30 min on ice with a buffer containing 10 mmTris-HCl, pH 7.6, 140 mm NaCl, 5 mm EDTA, 140 mm NaF, 0.4 mm orthovanadate, 5 mmpyrophosphate, 1 mm phenylmethylsulfonyl fluoride, protease inhibitor mixture tablets (CompleteTM, Roche Molecular Biochemicals), and either 1% Nonidet P-40 (Nonidet P-40) (Roche Molecular Biochemicals) or 1% Brij 96 (Fluka). After centrifugation at 12,000 × g for 15 min at 4 °C, the cell lysates were precleared by end-over-end rotation with 50 μl of 50% protein A-Sepharose CL-4B beads (Amersham Biosciences AB). Immunoprecipitations were carried out by adding 3 μg of mAb plus 20 μl of 50% protein A-Sepharose beads and by rotating for 2 h at 4 °C. The immune complexes were washed three times in lysis buffer with 1% detergent. For re-precipitation, the immune complexes were boiled for 5 min in lysis buffer containing 3% SDS. The eluate was recovered and diluted 9-fold with lysis buffer and then pre-cleared with 50 μl of 50% protein A-Sepharose beads for 30 min. Proteins were re-precipitated with 5 μl of anti-CD6 or -CD5 polyclonal rabbit antiserum plus 20 μl of protein A-Sepharose beads for 90 min at 4 °C. Re-precipitates were washed three times with 200 μl of lysis buffer, eluted by boiling for 5 min in SDS sample buffer, and run on 8% SDS-PAGE. For immunodepletion experiments, surface-biotinylated HUT-78 cells were lysed with 1% Brij 96 and then sequentially immunoprecipitated (eight times) with anti-CD5 (Cris-1) or anti-CD6 (161.8) mAbs. Once depleted, the lysates were immunoprecipitated with the reverse mAb and re-precipitated as above with rabbit polyclonal antisera (5 μl) against the depleted molecule. Transient transfection into COS-7 cells was performed by the diethylaminoethyl-dextran/Me2SO method as previously described (49Ausubel F.M. Brent R. Kingston R.E. Moore D.T. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1987Google Scholar). Briefly, 2 μg of plasmid DNA per 2 × 105 cells in a 9-cm2 dish were used. The cells were harvested ∼48 h after transfection and then surface-biotinylated and lysed with lysis buffer containing 1% Brij 96 as indicated above. Samples resolved by 8% SDS-PAGE were electrophoretically transferred (at 0.4 A, 100 V for 1 h) to nitrocellulose membranes (Bio-Rad). Filters were blocked for 30 min at 37 °C with 5% non-fat milk powder in phosphate-buffered saline (PBS) and then incubated for 30 min at room temperature with a 1/1000 dilution of HRP-SAv in blocking solution. After three washes with PBS plus 0.1% Tween 20, the membranes were developed by chemiluminescence with SuperSignal West Dura Extended Duration Substrate (Pierce) and exposure to X-OmatTM films (Eastman Kodak). All procedures were performed at 4 °C unless otherwise indicated. 1 × 106 PBL were incubated for 10 min with saturating amounts of biotinylated anti-CD6 (SPV L14.2) or anti-CD5 (Cris-1) mAbs. After washing with ice-cold PBS plus 0.1% sodium azide, cells were incubated with saturating doses of TR-SAv for 10 min at 4 °C. For capping to proceed, cells were then incubated at 37 °C for 30 min. The reaction was stopped by adding ice-cold PBS/azide. Next, the cells were transferred onto PLL-coated coverslips and stained for 30 min with FITC-conjugated anti-CD5, -CD6, or -CD43 mAbs before fixation with PBS 2% paraformaldehyde. After washing twice, the coverslips were transferred onto Mowiol-treated glass slides, and visualized in a confocal spectral microscope (Leica Microsystems Heidelberg GmbH, Mannheim, Germany). The images were analyzed with the Image Processing Leica confocal software and Photoshop 4.0 (Adobe Systems). 1 × 106 PBL were incubated for 30 min on ice with 500 μl of RPMI medium supplemented with 10% FCS in the presence or absence of 3 μg of Cris-1 or 161.8 mAbs. After washing twice with cold PBS, the cells were incubated on ice with 500 μl of RPMI 10% FCS containing 3 μl of FITC-GAMIg for 30 min. Then the cells were washed and left overnight at 37 °C in order to allow the immune complexes to be internalized. At this point, part of the cells were analyzed by flow cytometry (FACScan, BD Biosciences), and the remaining cells were incubated with biotinylated mAb plus Tricolor-SAv before flow cytometry analysis. Cells left overnight in RPMI 10% FCS alone were stained with 1 μg of Cris-1 or 161.8 mAb and 3 μl of FITC-GAMIg and used as a control to monitor CD5 and CD6 expression on unmodulated cells. 1 × 106 PBL (in 300 μl of PBS) were transferred onto PLL-coated coverslips for 30 min at room temperature. After blocking with 1% heat-inactivated rabbit serum for 10 min at room temperature, cells were incubated for 15 min at 4 °C with saturating amounts of FITC- (donor) and Cy3-labeled (acceptor) antibodies, either alone or mixed. Cells were then rinsed twice for 5 min at 4 °C with PBS and fixed with PBS, 2% paraformaldehyde for 10 min at room temperature. After washed twice, the coverslips were transferred onto Mowiol-treated glass slides. FRET measurements were based on the sensitized emission method (50Sorkin A. McClure M. Huang F. Carter R. Curr. Biol. 2000; 10: 1395-1398Google Scholar, 51Gordon G.W. Berry G. Liang X.H. Levine B. Herman B. Biophys J. 1998; 74: 2702-2713Google Scholar) with minor modifications for the confocal microscope. A Leica TCS SL laser scanning confocal spectral microscope (Leica Microsystems) equipped with argon and green HeNe lasers, ×100 oil immersion objective lens, and triple dichroic filter (488/543/633 nm) was used. To measure FRET, three images were acquired in the same order in all experiments as follows: 1) the FITC channel (absorbance 488 nm and emission 500–555 nm), 2) the FRET channel (absorbance 488 nm and emission 590–700 nm), and 3) the Cy3 channel (absorbance 543 nm and emission 590–700 nm). Background was subtracted from images before FRET calculations. Control and experiment images were taken under the same conditions of photomultiplier gain, offset, and pinhole aperture. The crossover of donor and acceptor fluorescence through the FRET filter is a constant proportion between the fluorescence intensity levels of donor and acceptor and their bleed through. In order to calculate the spectral bleed through of the donor and acceptor through the FRET filter, images of cells labeled only with FITC-conjugated mAb and cells labeled only with Cy3-conjugated mAb were also taken under the same conditions as for the experiments. As a control, images of FITC-GAMIg combined with an unlabeled anti-CD5 mAb (Leu-1) were included. The fraction of bleed through of FITC (A) and Cy3 (B) fluorescence through the FRET filter channel was calculated for different labeling conditions: FITC-anti-CD3, 0.13 ± 0.024; FITC-anti-CD43, 0.13 ± 0.020; FITC-anti-CD6, 0.13 ± 0.022; anti-CD5 plus FITC-GAMIg, 0.12 ± 0.015; and Cy3-anti-CD5, 0.56 ± 0.043. Corrected FRET (FRETc) was calculated on a pixel-by-pixel basis for the entire image by using Equation 1,FRETc=FRET−(A×FITC)−(B×Cy3)Equation 1 where FRET, FITC, and Cy3 correspond to background-subtracted images of cells labeled with FITC- and Cy3-conjugated antibodies acquired through the FRET, FITC, and Cy3 channels, respectively. Images of FRETc intensity were renormalized according to a lookup table where the minimum and maximum values are displayed as blue and red, respectively. Mean FRETc values were calculated from mean fluorescence intensities for each of the 10 regions of interest (ROI) selected from 5 different cells according to Equation 1. The apparent efficiency (E a) of FRET was calculated according to Equation 2,Ea=FRETc/Cy3Equation 2 where FRETc and Cy3 are the mean intensities of FRETc and Cy3 in the selected regions of interest. These calculations allowed E a to be <0. All calculations were performed using the Image Processing Leica confocal software and Microsoft Excel. The statistical analysis was performed by SPSS statistical software (Chicago, IL). The results are graphed showing the mean ± S.D. and percentiles 25 and 75. Statistical differences between groups were tested using the non-parametric Mann-Whitney test. A value of p < 0.001 was taken to indicate statistical significance. T cell-APC cell conjugates were generated by using Vβ8 TCR-expressing Jurkat cells (J77c120) and the human B cell line Raji in the presence or absence of SEE as described previously (52Niedergang F. Dautry-Varsat A. Alcover A. J. Immunol. 1997; 159: 1703-1710Google Scholar). Jurkat J77 cells were loaded with 10 μm CMAC for 20 min at 37 °C. Raji cells (5 × 106 cell/ml) were resuspended in Hanks' balanced salt solution and incubated for 20 min in the presence or absence of 5 μg/ml of SEE. J77 cells (5 × 104cell/well) were mixed with an equal number of Raji cells in a final volume of 600 μl and incubated for 30 min before plating onto PLL-coated slides in flat-bottomed 24-well plates (Costar Corp.). Cells were allowed to settle for 10 min at 37 °C, fixed for 5 min in PBS 2% formaldehyde, and blocked with 100 μg/ml human IgG (Sigma) before staining with the appropriated mAb plus FITC-GAMIg. Cells were observed by a DMR photomicroscope (Leica) with ×63 and 100 oil immersion objectives. Images were acquired using the Leica QFISH 1.0 software. Conjugates were first identified by directly observing both cell morphologies under differential interference contrast and blue fluorescent CMAC-labeled J77 cells. The possible membrane association of CD5 and CD6 was explored by co-immunoprecipitation experiments of biotin-labeled surface proteins solubilized under different detergent conditions (either 1% Brij 96 or 1% Nonidet P-40). The presence of CD6 in CD5 immunoprecipitates w
Referência(s)