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• W2081929153 abstract "Engagement of antigen receptors on the surface of T-cells with peptides bound to major histocompatibility complex (MHC) proteins triggers T-cell activation in a mechanism involving receptor oligomerization. Receptor dimerization by soluble MHC oligomers is sufficient to induce several characteristic activation processes in T-cells including internalization of engaged receptors and up-regulation of cell surface proteins. In this work, the influence of intermolecular orientation within the activating receptor dimer was studied. Dimers of class II MHC proteins coupled in a variety of orientations and topologies each were able to activate CD4+ T-cells, indicating that triggering was not dependent on a particular receptor orientation. In contrast to the minimal influence of receptor orientation, T-cell triggering was affected by the inter-molecular distance between MHC molecules, and MHC dimers coupled through shorter cross-linkers were consistently more potent than those coupled through longer cross-linkers. These results are consistent with a mechanism in which intermolecular receptor proximity, but not intermolecular orientation, is the key determinant for antigen-induced CD4+ T-cell activation. Engagement of antigen receptors on the surface of T-cells with peptides bound to major histocompatibility complex (MHC) proteins triggers T-cell activation in a mechanism involving receptor oligomerization. Receptor dimerization by soluble MHC oligomers is sufficient to induce several characteristic activation processes in T-cells including internalization of engaged receptors and up-regulation of cell surface proteins. In this work, the influence of intermolecular orientation within the activating receptor dimer was studied. Dimers of class II MHC proteins coupled in a variety of orientations and topologies each were able to activate CD4+ T-cells, indicating that triggering was not dependent on a particular receptor orientation. In contrast to the minimal influence of receptor orientation, T-cell triggering was affected by the inter-molecular distance between MHC molecules, and MHC dimers coupled through shorter cross-linkers were consistently more potent than those coupled through longer cross-linkers. These results are consistent with a mechanism in which intermolecular receptor proximity, but not intermolecular orientation, is the key determinant for antigen-induced CD4+ T-cell activation. major histocompatibility complex T-cell receptor Stokes radius polyacrylamide gel electrophoresis interleukin phycoerythrin streptavidin Helper (CD4+) T-cells play a key role in the adaptive immune response, by detecting and responding to foreign antigens bound to class II major histocompatibility complex (MHC)1 proteins found on the surface of B cells, macrophages, and other specialized antigen presenting cells of the immune system (1Germain R.N. Cell. 1994; 76: 287-299Abstract Full Text PDF PubMed Scopus (1268) Google Scholar). T-cells express on their surface clonotypic antigen receptors (TCR), which bind complexes of MHC protein and specific antigenic peptides (2Hennecke J. Wiley D.C. Cell. 2001; 104: 1-4Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). TCR engagement by MHC-peptide complexes induces T-cell signaling cascades. Once triggered, helper T-cells elicit a variety of characteristic processes, including cytokine secretion, up-regulation of adhesion and costimulatory molecules, and T-cell proliferation, which leads to activation of effector functions in antigen presenting cells, recruitment of other immune cells, and eventually to clearance of the foreign material (3Ullman K.S. Northrop J.P. Verweij C.L. Crabtree G.R. Annu. Rev. Immunol. 1990; 8: 421-452Crossref PubMed Scopus (488) Google Scholar, 4Glimcher L.H. Singh H. Cell. 1999; 96: 13-23Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 5Qian D. Weiss A. Curr. Opin. Cell Biol. 1997; 9: 205-212Crossref PubMed Scopus (286) Google Scholar). Some characteristic T-cell activation responses can be triggered by soluble agents that multivalently engage TCR, including anti-TCR antibodies (6Bekoff M. Kubo R. Grey H.M. J. Immunol. 1986; 137: 1411-1419PubMed Google Scholar, 7Yoon S.T. Dianzani U. Bottomly K. Janeway Jr., C.A. Immunity. 1994; 1: 563-569Abstract Full Text PDF PubMed Scopus (88) Google Scholar) and oligomeric MHC-peptide complexes (reviewed in Ref. 8Cochran J.R. Aivazian D.A. Cameron T.O. Stern L.J. Trends Biochem. Sci. 2001; 26: 304-310Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Monomeric reagents generally are not able to induce a response in T-cells, although some exceptions have been reported, particularly for CD8+ T-cells (7Yoon S.T. Dianzani U. Bottomly K. Janeway Jr., C.A. Immunity. 1994; 1: 563-569Abstract Full Text PDF PubMed Scopus (88) Google Scholar, 9Delon J. Gregoire C. Malissen B. Darche S. Lemaitre F. Kourilsky P. Abastado J.P. Trautmann A. Immunity. 1998; 9: 467-473Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). These considerations have led to the understanding that T-cells can be triggered by oligomerization of their surface TCR, although the full cellular activation response requires additional (co-stimulatory) signals from the antigen-presenting cell in addition to the antigenic signal transduced by the TCR (10Chambers C.A. Allison J.P. Curr. Opin. Cell Biol. 1999; 11: 203-210Crossref PubMed Scopus (347) Google Scholar). Several studies have pointed to formation of a TCR dimer as the key event for triggering T-cell activation. Quantitative analyses of the dependence of T-cell activation on the surface density of MHC-peptide complexes presented on the surface antigen presenting cells (11Bachmann M.F. Ohashi P.S. Immunol. Today. 1999; 20: 568-576Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 12Sousa J. Carneiro J. Eur. J. Immunol. 2000; 30: 3219-3227Crossref PubMed Scopus (34) Google Scholar) or incorporated into planar lipid bilayers (13Fox B.S. Quill H. Carlson L. Schwartz R.H. J. Immunol. 1987; 138: 3367-3374PubMed Google Scholar), have suggested a crucial role of dimer formation in triggering a T-cell response. For soluble class II MHC oligomers, the minimal MHC valency required to initiate signaling in CD4+ T-cells appears to be a dimer (14Boniface J.J. Rabinowitz J.D. Wulfing C. Hampl J. Reich Z. Altman J.D. Kantor R.M. Beeson C. McConnell H.M. Davis M.M. Immunity. 1998; 9: 459-466Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 15Cochran J.R. Cameron T.O. Stern L.J. Immunity. 2000; 12: 241-250Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Higher valency MHC oligomers can activate more potently than dimers, but this is due only to their increased binding avidity (15Cochran J.R. Cameron T.O. Stern L.J. Immunity. 2000; 12: 241-250Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). The mechanism by which dimerization of TCR triggers cytoplasmic signaling cascades is unknown. Receptor dimerization as the proximal activating stimulus is consistent with several potential molecular mechanisms, including formation of a specific TCR dimer in an activating conformation (as observed for receptor tyrosine kinases (16Hubbard S.R. Till J.H. Annu. Rev. Biochem. 2000; 69: 373-398Crossref PubMed Scopus (879) Google Scholar)), molecular rearrangement of a pre-existing receptor dimer (as observed for the bacterial aspartate receptor (17Falke J.J. Bass R.B. Butler S.L. Chervitz S.A. Danielson M.A. Annu. Rev. Cell Dev. Biol. 1997; 13: 457-512Crossref PubMed Scopus (422) Google Scholar)), or nonspecific co-localization of receptor cytoplasmic domains (18Klemm J.D. Schreiber S.L. Crabtree G.R. Annu. Rev. Immunol. 1998; 16: 569-592Crossref PubMed Scopus (275) Google Scholar). Experimental discrimination between these potential mechanisms can be difficult, and recent evidence has suggested that some systems originally thought to occur through a mechanism of receptor oligomerization may in fact involve allosteric rearrangements in a pre-existing receptor oligomer (reviewed in Ref. 19Jiang G. Hunter T. Curr. Biol. 1999; 9: R568-571Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In support of a nonspecific dimerization model, T-cell activation has been induced by many different anti-receptor antibodies (6Bekoff M. Kubo R. Grey H.M. J. Immunol. 1986; 137: 1411-1419PubMed Google Scholar, 7Yoon S.T. Dianzani U. Bottomly K. Janeway Jr., C.A. Immunity. 1994; 1: 563-569Abstract Full Text PDF PubMed Scopus (88) Google Scholar), and by oligomerization of a variety of chimeric TCR cytoplasmic domains (20Romeo C. Amiot M. Seed B. Cell. 1992; 68: 889-897Abstract Full Text PDF PubMed Scopus (264) Google Scholar, 21Letourneur F. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8905-8909Crossref PubMed Scopus (243) Google Scholar, 22Irving B.A. Weiss A. Cell. 1991; 64: 891-901Abstract Full Text PDF PubMed Scopus (624) Google Scholar). The exact stoichiometry of the unliganded form of the TCR complex is unknown, but has been proposed to contain two αβ TCRs (23San Jose E. Sahuquillo A.G. Bragado R. Alarcon B. Eur. J. Immunol. 1998; 28: 12-21Crossref PubMed Scopus (86) Google Scholar, 24Fernandez-Miguel G. Alarcon B. Iglesias A. Bluethmann H. Alvarez-Mon M. Sanz E. de la Hera A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1547-1552Crossref PubMed Scopus (116) Google Scholar, 25Exley M. Wileman T. Mueller B. Terhorst C. Mol. Immunol. 1995; 32: 829-839Crossref PubMed Scopus (60) Google Scholar), raising the possibility that molecular rearrangement of a pre-existing receptor oligomer may be a potential activation mechanism. However, it is possible that the presence of two antigen-binding domains could serve only to facilitate the large-order clustering and oligomerization of receptors on the cell-surface that has been observed physiologically (26Grakoui A. Bromley S.K. Sumen C. Davis M.M. Shaw A.S. Allen P.M. Dustin M.L. Science. 1999; 285: 221-227Crossref PubMed Scopus (2529) Google Scholar). To investigate these possible mechanisms, dimeric MHC-peptide complexes were prepared in a variety of intermolecular orientations and topologies, and used to probe the effects of receptor orientation and topology on T-cell triggering. Receptor orientation was not critical for T-cell signaling, as a variety of conformationally constrained MHC dimers coupled through either the α- or β-subunit each were able to induce T-cell activation processes. However, efficient T-cell triggering was dependent on receptor proximity, as activation was diminished when MHC dimers were coupled through longer cross-links. Collectively, these results suggest that T-cells are triggered by a mechanism of generalized intermolecular receptor proximity that does not depend on the intermolecular receptor orientation. HLA-DR1 (A*0101, B1*0101) α- and β-subunits, or modified versions carrying a C-terminal cysteine residue (Cysα,βCys) (27Cochran J.R. Stern L.J. Chem. Biol. 2000; 7: 683-696Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), were expressed as insoluble inclusion bodies in E. coli BL21(DE3) cells as described (28Frayser M. Sato A.K. Xu L. Stern L.J. Protein Expr. Purif. 1999; 15: 105-114Crossref PubMed Scopus (87) Google Scholar). Subunits included the peptide binding and membrane-proximal immunoglobulin domains (α1–182 and β1–190). In some experiments a longer version of the β-subunit (βL-Cys) was used, which included also the “connecting peptide” β191–198 (RSESAQSK). Recombinant MHC-peptide complexes were folded by dilution of urea-solubilized subunits in the presence of peptide and redox buffers, and isolated by ion-exchange chromatography, as previously described (28Frayser M. Sato A.K. Xu L. Stern L.J. Protein Expr. Purif. 1999; 15: 105-114Crossref PubMed Scopus (87) Google Scholar). HLA-DR1 complexes carried antigenic peptide Ha (resiudes 306–318) (PKYVKQNTLKLAT), derived from influenza virus hemagglutinin (29Roche P.A. Cresswell P. J. Immunol. 1990; 144: 1849-1856PubMed Google Scholar), or control endogenous peptide A2 (residues 103–117) (VGSDWRFLRGYHQYA), derived from class I MHC HLA-A2 (30Chicz R.M. Strominger J.L. Nature. 1992; 358: 764-768Crossref PubMed Scopus (671) Google Scholar). HLA-DR1 used in preparation of MHC dimers carried a cysteine residue at either the α or β C terminus. To prevent oxidation of the introduced cysteine residues, MHC-peptide complexes containing introduced cysteines were purified in 5 mm dithiothreitol, which was removed immediately prior to cross-linking. The introduced cysteines undergo facile reaction with thiol-specific reagents, allowing specific cross-linking at the α- or β-subunit termini. Polypeptide-based cross-linkers were synthesized by Fmoc chemistry on a solid-phase peptide synthesizer as previously described (27Cochran J.R. Stern L.J. Chem. Biol. 2000; 7: 683-696Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and verified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. All cross-linker peptides were capped at their N termini by reaction with fluorescein isothiocyanate. To introduce thiol-reactive maleimide groups, purified peptides (2–5 mg) were reacted through their lysine ε-amino groups withN-(ε-maleimidocaproyloxy)succinimide ester (Pierce), by dissolving 5-fold molar excess in N,N-dimethylformamide and adding it to peptide in 10 mm Na-phosphate buffer (pH 7), 150 mm NaCl. After 1.5 h at room temperature, the modified peptides were purified by reverse phase high performance liquid chromatography using a C18 column (Vydac), and the presence of both maleimide functional groups was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. For direct disulfide bond formation between the introduced thiols, 0.25 mmCuSO4 and 1.3 mm 1,10-phenanthroline (Sigma-Aldrich) were added to MHC-peptide complexes in 50 mm HEPES, (pH 8) (31Bubis J. Khorana H.G. J. Biol. Chem. 1990; 265: 12995-12999Abstract Full Text PDF PubMed Google Scholar), for at least 1 h at room temperature. For dimerization through the thiol-reactive maleimide groups on the synthetic peptide-based cross-linkers X3X, X9X, and X14X (Fig. 1A), the cross-linker was added in small batches to MHC-peptide complexes in 10 mm Na-phosphate (pH 7), 150 mm NaCl, 5 mm EDTA over ∼5 h at room temperature, to a final cross-linker:MHC ratio of 1:2. Cross-linked MHC-peptide complexes were isolated by gel filtration chromatography on Superdex-200, using two HR 10/30 FPLC columns (Amersham Pharmacia Biotech) linked in series. The integrity of covalent thiol linkages and the presence of bound antigenic peptide were confirmed by SDS-PAGE (12.5%). Maleimide-to-maleimide distances for cross-linkers in extended conformations were estimated using molecular models. ApparentM r (M r,app) values for MHC-peptide dimers were determined from elution volumes obtained by gel filtration chromatography, by reference to a calibration curve obtained from the elution volume of knownM r standards (Bio-Rad). The Stokes radii (R S) of the MHC dimers were derived from theM r,app as previously described (32Zarutskie J.A. Sato A.K. Rushe M.M. Chan I.C. Lomakin A. Benedek G.B. Stern L.J. Biochemistry. 1999; 38: 5878-5887Crossref PubMed Scopus (74) Google Scholar), except the following equation was used to calculate the hydrated volume (V H), VH=Mr,app(psv+(hydprotein)(ρwater))N(Eq. 1) where hydprotein is the estimated extent of hydration in the protein (0.35 g of water/g of protein), ρwater is the density of water at 20 °C (0.998 g/cm3), N is Avogadro's number, and psv is the partial specific volume of HLA-DR1-Ha (0.738 cm3/g) calculated from the amino acid composition. Confidence intervals (±ς) reported in Table Ireflect the standard deviation from the mean of replicate samples in separate experiments.Table IHydrodynamic properties of MHC dimers as determined by gel filtrationComplexCross-linkR s1-aStokes radius estimated from gel filtration data as described (32).ÅCysαβS-S34.3 ± 0.11CysαβX3X34.9 ± 0.15CysαβX9X35.8 ± 0.06CysαβX14X36.3 ± 0.15αβCysS-S35.6 ± 0.12αβCysX3X36.2 ± 0.15αβCysX9X37.0 ± 0.06αβCysX14X37.4 ± 0.06αβL-CysX14X38.7 ± 0.141-a Stokes radius estimated from gel filtration data as described (32Zarutskie J.A. Sato A.K. Rushe M.M. Chan I.C. Lomakin A. Benedek G.B. Stern L.J. Biochemistry. 1999; 38: 5878-5887Crossref PubMed Scopus (74) Google Scholar). Open table in a new tab HA1.7 (33Lamb J.R. Eckels D.D. Lake P. Woody J.N. Green N. Nature. 1982; 300: 66-69Crossref PubMed Scopus (163) Google Scholar) is a well studied human CD4+ T-cell clone that responds to the Ha peptide bound to HLA-DR1. The HLA-DR1-restricted, Ha peptide-specific polyclonal T-cell line (designated 1H) was raised from the mononuclear cell fraction of peripheral blood from an HLA-DR1 homozygous individual, by repeatedin vitro stimulation with Ha peptide. HA1.7 and 1H were maintained in RPMI containing 5% human AB serum (Sigma-Aldrich) and 5% fetal bovine serum (Sigma-Aldrich), with antigenic stimulation every 2 weeks using 120 IU/ml IL-2 (BIOSOURCE) and an irradiated mixture of nonspecific peripheral blood lymphocytes and EBV1.24, a DR1+ B-cell line, pulsed with 1 µmHa peptide. T-cells were rested for a minimum of 7 days after stimulation before use in activation and binding assays. Soluble MHC-peptide complexes were added to 5 × 104 T-cells in complete medium in round-bottom polypropylene 96-well plates. After the desired incubation time at 37 °C and 7% CO2, cells were placed on ice and levels of cell surface markers were determined using the following fluorescent monoclonal antibodies: phycoerythrin (PE)-labeled anti-CD3 (clone UCHT-1, Pharmingen), or allophycocyanin (APC)-labeled anti-CD25 (M-A251, Pharmingen), APC-anti-CD69 (FN50, Pharmingen), and APC-anti-CD71 (T56/14, Leinco Technologies). After 1 h at 4 °C, cells were washed with phosphate-buffered saline containing 1% fetal bovine serum and 0.1% sodium azide, and analyzed by flow cytometry. Fluorescence data were obtained with a Becton-Dickinson FACS Calibur flow cytometer and analyzed using Cell Quest software. The number of CD3 molecules per cell was determined from the mean PE fluorescence using SPHERO rainbow calibration particles (Spherotech) containing known amounts of PE equivalents. Experiment to experiment variation was observed in the overall time course and extent of activation, which appeared to be dependent on the length of time the T-cells had been in culture, but relative levels of activation induced by the various dimers were the same in different experiments. Competitive binding assays were performed as previously described (15Cochran J.R. Cameron T.O. Stern L.J. Immunity. 2000; 12: 241-250Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 27Cochran J.R. Stern L.J. Chem. Biol. 2000; 7: 683-696Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Phycoerythrin-labeled, streptavidin-coupled MHC oligomers (SA-PE), prepared by binding biotinylated MHC monomers to streptavidin preparations (34Cameron T.O. Cochran J.R. Yassine-Diab B. Sekaly R.-P. Stern L.J. J. Immunol. 2001; 166: 741-745Crossref PubMed Scopus (64) Google Scholar), were used as a binding probe, with competition by unlabeled or fluorescein-labeled MHC-peptide oligomers. These SA-PE oligomers exhibit a strong fluorescence from the R-phycoerythrin conjugate and bind to T-cells in an antigen-specific manner (34Cameron T.O. Cochran J.R. Yassine-Diab B. Sekaly R.-P. Stern L.J. J. Immunol. 2001; 166: 741-745Crossref PubMed Scopus (64) Google Scholar). Various concentrations of MHC dimers and monomer were incubated with a constant amount of SA-PE oligomer ([MHC]= 4 µg/ml) and 5 × 104 HA1.7 T-cells in 96-well round-bottomed plates for 3 h at 37 °C, 7% CO2. Cells were washed and fluorescence arising from bound SA-PE oligomer was measured by flow cytometry as described above. To investigate the orientation requirements of T-cell triggering, we prepared soluble dimers of the human class II MHC protein HLA-DR1, with a variety of different intermolecular orientations and conformational constraints. The oligomerization strategy involved specific cross-linking at the sulfhydryl moiety of a cysteine residue introduced either at the C terminus of the α-subunit immunoglobulin domain (α182), the C terminus of the β-subunit immunoglobulin domain (β190), or after the β-subunit connecting peptide region (β191–198), which immediately precedes the native transmembrane domain (Fig.1 B). Proteins carrying the introduced cysteine residues (Cysαβ, αβCys, or αβL-Cys, respectively) were produced by in vitro folding in the presence of antigenic (Ha) or endogenous (A2) peptides, using previously described protocols (27Cochran J.R. Stern L.J. Chem. Biol. 2000; 7: 683-696Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 28Frayser M. Sato A.K. Xu L. Stern L.J. Protein Expr. Purif. 1999; 15: 105-114Crossref PubMed Scopus (87) Google Scholar). MHC-peptide complexes were dimerized either by using a direct disulfide bond between the introduced cysteine thiols (31Bubis J. Khorana H.G. J. Biol. Chem. 1990; 265: 12995-12999Abstract Full Text PDF PubMed Google Scholar), or using a sulfhydryl-reactive synthetic cross-linking reagent of varying length (Fig. 1 A). The synthetic cross-linkers X3X, X9X, and X14X are based on a flexible peptide scaffold containing glycine, serine, and glutamic acid residues, and each carry an N-terminal fluorophore and two maleimidylcaproyl (X) groups, attached as amides to lysine residues. MHC-peptide dimers coupled by direct disulfide bonds (S-S) or by the synthetic cross-linkers, through either the α- or β-subunit, each had the desired covalent linkage (Fig. 1 C, right panel). Each of the dimers retained the ability to tightly bind peptide antigen, as demonstrated by resistance to SDS-induced chain dissociation (35Stern L.J. Wiley D.C. Cell. 1992; 68: 465-477Abstract Full Text PDF PubMed Scopus (286) Google Scholar) (Fig. 1 C, left panel), and exhibited the expected apparent molecular weight with no tendency to aggregate, as determined by gel filtration chromatography (see below). The orientation dependence for T-cell activation was investigated by comparing the dose-response curves of MHC dimers linked through the α- or β-subunit. MHC dimers were tested for their ability to trigger T-cell activation processes in HA1.7, a well characterized human T-cell clone (33Lamb J.R. Eckels D.D. Lake P. Woody J.N. Green N. Nature. 1982; 300: 66-69Crossref PubMed Scopus (163) Google Scholar) specific for an antigenic peptide derived from influenza virus hemagglutinin (Ha) as presented by the class II MHC protein HLA-DR1 (29Roche P.A. Cresswell P. J. Immunol. 1990; 144: 1849-1856PubMed Google Scholar). T-cells down-regulate engaged TCR (CD3), as part of the activation process (36Itoh Y. Hemmer B. Martin R. Germain R.N. J. Immunol. 1999; 162: 2073-2080PubMed Google Scholar, 37Valitutti S. Muller S. Cella M. Padovan E. Lanzavecchia A. Nature. 1995; 375: 148-151Crossref PubMed Scopus (984) Google Scholar). The down-modulation of TCR in response to the soluble MHC dimers was measured by flow cytometry using a PE-labeled antibody against the TCR CD3ε subunit. Disulfide-linked dimers of MHC proteins, complexed with Ha peptide and cross-linked through either the α- or β-subunit (Cysαβ S-S and αβCys S-S), induced TCR down-regulation in a dose-dependent manner (Fig.2A, open and closed circles). Dimers coupled with a long, flexible cross-linker (Cysαβ X14X and αβCys X14X,open and closed squares), or dimers carrying the additional flexible connecting peptide linker on the β-subunit (αβL-Cys X14X, open diamonds) also induced CD3 down-regulation. The level of activation induced by dimers linked through either the α- or β-subunit was similar for complementary pairs of disulfide-bonded dimers, and for complementary pairs of dimers coupled through the peptide-based cross-linkers. As previously observed (15Cochran J.R. Cameron T.O. Stern L.J. Immunity. 2000; 12: 241-250Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 27Cochran J.R. Stern L.J. Chem. Biol. 2000; 7: 683-696Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), neither MHC-peptide monomers (Fig. 2 A, ×), nor oligomers carrying the non-antigenic, endogenous peptide A2 (data not shown), induced significant T-cell triggering in the concentration range tested. Down-regulation of CD3 induced by the MHC dimers was observed as early as 2 h and continued to increase over time, with similar time courses for complementary dimers linked through the α- or β-subunits (Fig. 2 B). Collectively, these data demonstrate that receptor orientation is not crucial for signaling, as in each case dimers linked through the α- or β-subunit induced T-cell activation to an equivalent extent. Similar responses were observed for other measures of T-cell activation processes, including up-regulation of the early T-cell activation marker CD69 (38Testi R. D'Ambrosio D. De Maria R. Santoni A. Immunol. Today. 1994; 15: 479-483Abstract Full Text PDF PubMed Scopus (120) Google Scholar) (Fig. 3A), up-regulation of the low-affinity IL-2 receptor α-subunit (CD25) (39Waldmann T.A. Annu. Rev. Biochem. 1989; 58: 875-911Crossref PubMed Google Scholar) (Fig. 3 B), and up-regulation of transferrin receptor (CD71) (40Ponka P. Lok C.N. Int. J. Biochem. Cell Biol. 1999; 31: 1111-1137Crossref PubMed Scopus (445) Google Scholar) (Fig. 3 C). In these assays, dimers with a range of cross-linker lengths were used. For each T-cell activation marker studied, MHC dimers coupled through the α- or β-subunit with direct disulfide bonds (S-S) induced the most potent response. As the dimer cross-linker length was increased, an incremental decrease in the extent of T-cell activation was observed. Dimers coupled through MHC-peptide complexes carrying the additional flexible connecting peptide regions on the β-subunit (LX14X) exhibited the least potent activation. These data indicate a systematic activation dependence on linker length, with only a slight dependence on inter-molecular orientation. To address whether the observed activation responses were due to idiosyncratic aspects of the long-term T-cell clone HA1.7, we repeated the activation experiments using a polyclonal T-cell line raised from the peripheral blood of a DR1+individual (designated 1H). The 1H polyclonal T-cell line exhibited CD3 down-regulation in response to MHC dimers, with dose-response curves for disulfide-linked and X14X-linked MHC dimers (Fig.4A) that were similar to those observed for the HA1.7 T-cell clone. Activation of CD3 down-regulation (Fig. 4 B) and CD69 up-regulation (Fig. 4 C) in the 1H polyclonal line were relatively independent of intermolecular orientation, but were dependent on cross-linker length, with the shortest cross-links (S-S) exhibiting the most potent signal, as observed for the HA1.7 clone. The observed dependence of T-cell triggering on cross-linker length was investigated in more detail. To evaluate the actual intermolecular spacing in the intact oligomers, an apparent hydrodynamic radius was characterized for each of the various dimers by gel filtration chromatography (Fig. 5A). For the Cysαβ dimer series, a systematic dependence of apparent hydrodynamic radius (R S) on cross-linker length was observed (Table I), with dimers coupled through short disulfide cross-links (S-S) exhibiting the most compact conformation. Similar hydrodynamic behavior was observed for the αβCys dimer series (Table I). Dimers linked through the β-subunit exhibited slightly but systematically larger apparent hydrodynamic radii than the corresponding dimers linked through the α-subunit. Plots of the apparent hydrodynamic radiusversus the T-cell response induced by the MHC dimers exhibited a striking linear dependence. This dependence was observed in the HA1.7 T-cell clone for CD3 down-regulation (Fig. 5 B), CD69 up-regulation (Fig. 5 C), and CD25 up-regulation (Fig.5 D), and was also evident in the 1H polyclonal line (not shown). Thus, T-cell activation is more efficiently triggered by more compact dimers, for each of the responses studied. The observed dependence of T-cell triggering on linker length could possibly be due to decreased binding for the dimers coupled through longer cross-links. To address this possibility the relative binding of the MHC dimers was measured, using a competitive binding assay in which unlabeled dimers compete for the T-cell surface with streptavidin-linked, phycoerythrin-labeled (SA-PE) MHC oligomers (34Cameron T.O. Cochran J.R. Yassine-Diab B. Sekaly R.-P. Stern L.J. J. Immunol. 2001; 166: 741-745Crossref PubMed Scopus (64) Google Scholar). MHC dimers linked through the α-subunit with the X3X, X9X, and X14X cross-linkers each exhibited essentially identical competition curves (Fig.6A), with half-maximal inhibitions of ∼0.3 µm. Dimers linked though the α-subunit with a direct disulfide bond (S-S) exhibited slightly weaker binding, with a half-maximal inhibition of ∼0.8 µm (Fig. 6 A, filled circles). MHC dimers linked through the β-subunit with the X3X, X9X, and X14X cross-linkers or direct disulfide bonds (S-S) bound similarly (IC50 ∼0.3 µm) (Fig. 6 B). Overall, the MHC dimers competed for binding more efficiently than monomeric MHC-peptide complexes, which exhibited an IC50 of ∼2.5 µm (Fig. 6 A, ×), as previously observed (15Cochran J.R. Cameron T.O. Stern L.J. Immunity. 2000; 12: 241-250Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 27Cochran J.R. Stern L.J. Chem. Biol. 2000; 7: 683-696Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Since the competition curves for all of the MHC dimers are similar, the decre" @default.
• W2081929153 created "2016-06-24" @default.
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• W2081929153 date "2001-07-01" @default.
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• W2081929153 title "Receptor Proximity, Not Intermolecular Orientation, Is Critical for Triggering T-cell Activation" @default.
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