Peptide-Major Histocompatibility Complex Dimensions Control Proximal Kinase-Phosphatase Balance during T Cell Activation
2009; Elsevier BV; Volume: 284; Issue: 38 Linguagem: Inglês
10.1074/jbc.m109.039966
ISSN1083-351X
AutoresKaushik Choudhuri, Mathew Parker, Anita Milicic, David K. Cole, Michael K. Shaw, Andrew K. Sewell, Guillaume B. E. Stewart-Jones, Tao Dong, Keith G. Gould, P. Anton van der Merwe,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoT cell antigen recognition requires binding of the T cell receptor (TCR) to a complex between peptide antigen and major histocompatibility complex molecules (pMHC), and this recognition occurs at the interface between the T cell and the antigen-presenting cell. The TCR and pMHC molecules are small compared with other abundant cell surface molecules, and it has been suggested that small size is functionally important. We show here that elongation of both mouse and human MHC class I molecules abrogates T cell antigen recognition as measured by cytokine production and target cell killing. This elongation disrupted tyrosine phosphorylation and Zap70 recruitment at the contact region without affecting TCR or coreceptor binding. Contact areas with elongated forms of pMHC showed an increase in intermembrane distance and less efficient segregation of CD3 from the large tyrosine phosphatase CD45. These findings demonstrate that T cell antigen recognition is strongly dependent on pMHC size and are consistent with models of TCR triggering requiring segregation or mechanical pulling of the TCR. T cell antigen recognition requires binding of the T cell receptor (TCR) to a complex between peptide antigen and major histocompatibility complex molecules (pMHC), and this recognition occurs at the interface between the T cell and the antigen-presenting cell. The TCR and pMHC molecules are small compared with other abundant cell surface molecules, and it has been suggested that small size is functionally important. We show here that elongation of both mouse and human MHC class I molecules abrogates T cell antigen recognition as measured by cytokine production and target cell killing. This elongation disrupted tyrosine phosphorylation and Zap70 recruitment at the contact region without affecting TCR or coreceptor binding. Contact areas with elongated forms of pMHC showed an increase in intermembrane distance and less efficient segregation of CD3 from the large tyrosine phosphatase CD45. These findings demonstrate that T cell antigen recognition is strongly dependent on pMHC size and are consistent with models of TCR triggering requiring segregation or mechanical pulling of the TCR. T cell antigen recognition requires the engagement of the TCR 8The abbreviations used are: TCRT cell receptorMHCmajor histocompatibility complexpMHCcomplex between peptide antigen and MHC moleculeAPCantigen-presenting cellSCTsingle-chain trimerCHOChinese hamster ovaryTAPtransporter associated with antigen processingPBSphosphate-buffered salineILinterleukinELISAenzyme-linked immunosorbent assayIFNinterferonDDAO7-hydroxy-9H-(1,3-dichloro-9,9- dimethylacridin-2-one). 8The abbreviations used are: TCRT cell receptorMHCmajor histocompatibility complexpMHCcomplex between peptide antigen and MHC moleculeAPCantigen-presenting cellSCTsingle-chain trimerCHOChinese hamster ovaryTAPtransporter associated with antigen processingPBSphosphate-buffered salineILinterleukinELISAenzyme-linked immunosorbent assayIFNinterferonDDAO7-hydroxy-9H-(1,3-dichloro-9,9- dimethylacridin-2-one). with peptide antigen presented on cell surface MHC molecules (pMHC) (1Davis M.M. Chien Y.H. Paul W.E. Fundamental Immunology. Lippincott Williams & Wilkins, Philadelphia, PA2003: 227-258Google Scholar). "Accessory" T cell surface receptors modulate T cell antigen recognition by binding to cell surface ligands on antigen-presenting cells (APCs) (2Weiss A. Samelson L.E. Paul W.E. Fundamental Immunology. Lippincott Williams & Wilkins, Philadelphia, PA2003: 321-364Google Scholar). The dimensions of the TCR·pMHC complex dictate that TCR binding to pMHC takes places within close contact areas in which the membranes are ∼15 nm apart (3Springer T.A. Nature. 1990; 346: 425-434Crossref PubMed Scopus (5851) Google Scholar, 4van der Merwe P.A. McNamee P.N. Davies E.A. Barclay A.N. Davis S.J. Curr. Biol. 1995; 5: 74-84Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 5Barclay A.N. Brown M.H. Law S.K. McKnight A.J. Tomlinson M.G. van der Merwe P.A. The Leucocyte Antigen Facts Book. Academic Press, London1997Google Scholar). Many accessory receptor·ligand complexes span similar dimensions to the TCR·pMHC complex and can therefore colocalize with the TCR in such close contact areas (3Springer T.A. Nature. 1990; 346: 425-434Crossref PubMed Scopus (5851) Google Scholar, 4van der Merwe P.A. McNamee P.N. Davies E.A. Barclay A.N. Davis S.J. Curr. Biol. 1995; 5: 74-84Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 5Barclay A.N. Brown M.H. Law S.K. McKnight A.J. Tomlinson M.G. van der Merwe P.A. The Leucocyte Antigen Facts Book. Academic Press, London1997Google Scholar). Conversely, many cell surface molecules, including two of the most abundant, CD43 and CD45, have much larger ectodomains and would therefore be expected to be excluded or depleted from these close contact areas (3Springer T.A. Nature. 1990; 346: 425-434Crossref PubMed Scopus (5851) Google Scholar, 6Davis S.J. van der Merwe P.A. Immunol. Today. 1996; 17: 177-187Abstract Full Text PDF PubMed Scopus (342) Google Scholar). T cell receptor major histocompatibility complex complex between peptide antigen and MHC molecule antigen-presenting cell single-chain trimer Chinese hamster ovary transporter associated with antigen processing phosphate-buffered saline interleukin enzyme-linked immunosorbent assay interferon 7-hydroxy-9H-(1,3-dichloro-9,9- dimethylacridin-2-one). T cell receptor major histocompatibility complex complex between peptide antigen and MHC molecule antigen-presenting cell single-chain trimer Chinese hamster ovary transporter associated with antigen processing phosphate-buffered saline interleukin enzyme-linked immunosorbent assay interferon 7-hydroxy-9H-(1,3-dichloro-9,9- dimethylacridin-2-one). Signal transduction by the TCR is mediated by the associated CD3 subunits (7Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1952) Google Scholar). The earliest event that is known to be required for signaling is tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs in the cytoplasmic portion of these TCR-associated CD3 subunits. This phosphorylation, which is mediated by Src-related kinases such as Lck, is followed by recruitment and activation of the tyrosine kinase Zap70 (which binds doubly phosphorylated immunoreceptor tyrosine-based activation motifs via tandem SH2 domains). Zap70 then phosphorylates downstream proteins, including adaptor proteins such as LAT and SLP-76, leading to the recruitment and activation of a cascade of adaptor and effector proteins (2Weiss A. Samelson L.E. Paul W.E. Fundamental Immunology. Lippincott Williams & Wilkins, Philadelphia, PA2003: 321-364Google Scholar). Although the downstream events in TCR signal transduction are fairly well characterized, the mechanism by which TCR binding to pMHC leads to increased phosphorylation of CD3 immunoreceptor tyrosine-based activation motifs, a process termed TCR triggering, remains relatively poorly understood and controversial (8Davis M.M. Krogsgaard M. Huse M. Huppa J. Lillemeier B.F. Li Q.J. Annu. Rev. Immunol. 2007; 25: 681-695Crossref PubMed Scopus (119) Google Scholar, 9Davis S.J. van der Merwe P.A. Nat. Immunol. 2006; 7: 803-809Crossref PubMed Scopus (361) Google Scholar, 10Kuhns M.S. Davis M.M. Garcia K.C. Immunity. 2006; 24: 133-139Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 11Schamel W.W. Risueño R.M. Minguet S. Ortíz A.R. Alarcón B. Trends Immunol. 2006; 27: 176-182Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 12Trautmann A. Randriamampita C. Trends Immunol. 2003; 24: 425-428Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 13van der Merwe P.A. Immunity. 2001; 14: 665-668Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). A number of models have been proposed for TCR triggering. These can be classified into three groups depending on whether the signal transduction mechanism involves aggregation, conformational change, or segregation of the TCR·CD3 complex upon pMHC binding (reviewed in Ref. 14Choudhuri K. van der Merwe P.A. Semin. Immunol. 2007; 19: 255-261Crossref PubMed Scopus (53) Google Scholar). Models based on aggregation have difficulty accounting for TCR triggering by very low densities of agonist pMHC, so recent versions postulate a role for self-pMHC, which is present at higher densities (8Davis M.M. Krogsgaard M. Huse M. Huppa J. Lillemeier B.F. Li Q.J. Annu. Rev. Immunol. 2007; 25: 681-695Crossref PubMed Scopus (119) Google Scholar). Models postulating conformational change within TCRαβ have not generally been supported by structural studies (15Rudolph M.G. Stanfield R.L. Wilson I.A. Annu. Rev. Immunol. 2006; 24: 419-466Crossref PubMed Scopus (925) Google Scholar) and so have been adapted by proposing conformational changes of the entire TCRαβ complex with respect to other components or the plasma membrane (14Choudhuri K. van der Merwe P.A. Semin. Immunol. 2007; 19: 255-261Crossref PubMed Scopus (53) Google Scholar, 16Choudhuri K. Kearney A. Bakker T.R. van der Merwe P.A. Curr. Biol. 2005; 15: R382-385Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). A version of these models proposed that conformational change may be the result of pMHC binding subjecting the TCR to a mechanical "pulling" force toward the APC membrane (14Choudhuri K. van der Merwe P.A. Semin. Immunol. 2007; 19: 255-261Crossref PubMed Scopus (53) Google Scholar, 16Choudhuri K. Kearney A. Bakker T.R. van der Merwe P.A. Curr. Biol. 2005; 15: R382-385Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 17Ma Z. Janmey P.A. Finkel T.H. FASEB J. 2008; 22: 1002-1008Crossref PubMed Scopus (62) Google Scholar). However, very recently evidence has been presented that binding to agonist pMHC may indeed trigger a conformational change within the constant domain of the TCRαβ (18Beddoe T. Chen Z. Clements C.S. Ely L.K. Bushell S.R. Vivian J.P. Kjer-Nielsen L. Pang S.S. Dunstone M.A. Liu Y.C. Macdonald W.A. Perugini M.A. Wilce M.C. Burrows S.R. Purcell A.W. Tiganis T. Bottomley S.P. McCluskey J. Rossjohn J. Immunity. 2009; 30: 777-788Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), so that models based on conformational change need to be reassessed. In addition, conformational changes in the cytoplasmic domains of the TCR-associated CD3 polypeptides may help to regulate TCR activation (19Xu C. Gagnon E. Call M.E. Schnell J.R. Schwieters C.D. Carman C.V. Chou J.J. Wucherpfennig K.W. Cell. 2008; 135: 702-713Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Finally, TCR triggering models based on segregation postulate that TCR binding to pMHC functions to retain the TCR·CD3 components within a region of the plasma membrane within which tyrosine kinases such as Lck are enriched and receptor tyrosine phosphatases are depleted. The kinetic segregation model postulates that this segregation is the result of the large size of the ectodomain of tyrosine phosphatases CD45 and CD148 with respect to the TCR·pMHC complex, which leads to physical exclusion from close contact areas (6Davis S.J. van der Merwe P.A. Immunol. Today. 1996; 17: 177-187Abstract Full Text PDF PubMed Scopus (342) Google Scholar, 9Davis S.J. van der Merwe P.A. Nat. Immunol. 2006; 7: 803-809Crossref PubMed Scopus (361) Google Scholar, 20van der Merwe P.A. Davis S.J. Shaw A.S. Dustin M.L. Semin. Immunol. 2000; 12: 5-21Crossref PubMed Scopus (248) Google Scholar). To explore the mechanism of TCR triggering, we have examined whether the small size of the TCR·pMHC complex is functionally significant. We showed previously that elongation of one mouse pMHC class I complex abrogated recognition by cognate T cells (21Choudhuri K. Wiseman D. Brown M.H. Gould K. van der Merwe P.A. Nature. 2005; 436: 578-582Crossref PubMed Scopus (252) Google Scholar). The present study extends this previous work in several important ways. First, we extend this analysis to other pMHC complexes and cognate T cells, including human CD8 T cells. Second, we test directly whether the inhibitory effect could be the result of decreased TCR or coreceptor binding to elongated pMHC class I. Third, we look at the effect of pMHC elongation on early signaling events and segregation of CD45 from TCR·CD3 within the contact area. Our results conclusively demonstrate the importance of pMHC size in T cell antigen recognition and are consistent with the kinetic segregation model of TCR triggering. The H-2Db and HLA-A2 single-chain trimer (SCT) constructs are analogous to the SCT we have described previously for H-2Kb (21Choudhuri K. Wiseman D. Brown M.H. Gould K. van der Merwe P.A. Nature. 2005; 436: 578-582Crossref PubMed Scopus (252) Google Scholar). The H-2Db SCT presents the peptide epitope ASNENMDAM (NT60), residues 366–374 of influenza virus A/NT/60/68 nucleoprotein (22Palmowski M.J. Parker M. Choudhuri K. Chiu C. Callan M.F. van der Merwe P.A. Cerundolo V. Gould K.G. J. Immunol. 2009; 182: 4565-4571Crossref PubMed Scopus (16) Google Scholar), and the HLA-A2 SCT presents the peptide epitope SLYNTVATL, residues 77–85 of human immunodeficiency virus, type 1 Gag p17. The H-2Db SCT construct uses murine β2-microglobulin, and the HLA-A2 SCT uses human β2-microglobulin. The SCT constructs were expressed in transfected cells from the SV40 early promoter, using expression vectors pKG4 or pKG5. To obtain cells with low levels of surface SCT expression, a tetracycline-inducible expression system consisting of the vector pcDNA5/TO and T-Rex-CHO (Chinese hamster ovary) cells (Invitrogen) was used in the absence of tetracycline. The strategy to generate elongated SCT chimeras was exactly as described (21Choudhuri K. Wiseman D. Brown M.H. Gould K. van der Merwe P.A. Nature. 2005; 436: 578-582Crossref PubMed Scopus (252) Google Scholar), using an introduced unique BamHI restriction site in the class I heavy chains. This enabled the use of the identical CD2, CD22, and CD4 inserts used previously, adding two, three, and four extra Ig-like domains, respectively. For the HLA-A2 SCT, only a CD4-extended version was made. Soluble versions of the HLA-A2 SCT proteins were made by removing the transmembrane and cytoplasmic regions. For the unextended soluble SCT, the HLA-A2 heavy chain terminates at residue Trp274. For the CD4-extended soluble SCT, Trp274 is followed by the sequence EDPPS before continuing with the CD4 insert. Both constructs then contain additional residues at the C-terminal end, comprising a Myc epitope tag, a biotinylation sequence, and a His6 tag to facilitate purification. The amino acid sequence of this region is TGEQKLISEEDLGLNDIFEAQKIEWHHHHHH. For expression, the constructs were cloned into the bicistronic retroviral expression vector pQCXIX (Clontech). The second multiple cloning site of the vector was used to express green fluorescent protein. Pantropic recombinant retrovirus was produced by transient transfection of the GP2–293 packaging cell line with the recombinant pQCXIX plasmids and pVSV-G vector (Clontech). Tissue culture supernatant containing retrovirus was used to infect 293T cells; multiple rounds of infection were carried out, and the level of infection was monitored by the level of green fluorescent protein expression. Once high levels of infection had been achieved (five or six rounds of infection), clones were made by limiting dilution. The clones that demonstrated very high levels of green fluorescent protein expression were tested by Western blotting for SCT protein expression in culture supernatant. The best expressing clones were expanded, and SCT protein was purified from culture supernatant using Ni2+-nitrilotriacetic acid-agarose (Qiagen). Protein was eluted with a gradient of imidazole, desalted into 10 mm Tris buffer, pH 7.5, and biotinylated using recombinant BirA enzyme according to the manufacturer's instructions (Avidity LLC). Biotinylated SCTs were purified by gel filtration (fast protein liquid chromatography) using Superdex 75 or 200 columns and stored in PBS. Biotinylation efficiency was checked by depletion assay in which 10 μl of streptavidin-coupled magnetic beads (Dynal) were incubated with 10 ml of SCTs (5 μg) for 20 min at room temperature, after which supernatant was separated by 10% SDS-PAGE and protein was detected by Coomassie staining. Human CD8αα was expressed and purified as described elsewhere (23Wyer J.R. Willcox B.E. Gao G.F. Gerth U.C. Davis S.J. Bell J.I. van der Merwe P.A. Jakobsen B.K. Immunity. 1999; 10: 219-225Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The concentration of CD8αα was determined using an extinction coefficient of 32,480 m−1 cm−1. Human G10 TCR was expressed and refolded in vitro as described (24Lee J.K. Stewart-Jones G. Dong T. Harlos K. Di Gleria K. Dorrell L. Douek D.C. van der Merwe P.A. Jones E.Y. McMichael A.J. J. Exp. Med. 2004; 200: 1455-1466Crossref PubMed Scopus (133) Google Scholar), and concentration was determined using an extinction coefficient of 63,995 m−1 cm−1. Splenocytes were harvested from F5 RAG−/− mice, primed with 10 μm NT60 peptide for 48 h, and purified by negative selection and Ficoll (Sigma) separation (>95% CD8+) before culturing for a further 6 days in complete medium containing 20 units/ml recombinant murine IL-2 (Sigma). The cells were rested in complete medium without IL-2 for 2 h prior to use in stimulation assays in which 104 T cells/well were incubated with varying numbers of SCT-expressing CHO cells (denoted as APC/F5 ratio). Culture supernatants were assayed for IL-2 by ELISA after incubation at 37 °C in 5% CO2 for 24 h. Alternatively, an F5 TCR-expressing CD8+ murine T cell clone (F5 CTL), propagated by several rounds of antigen-induced expansion, was incubated for 6 days with syngeneic B6 splenocytes loaded with 10 μm NT60 peptide in medium containing 100 units/ml recombinant murine IL-2. Viable lymphocytes were isolated by Ficoll gradient centrifugation. The cells were rested for 4 days in medium containing 20 units/ml IL-2 and used at day 10 post-priming. For human G10 CD8+ T cells, the well established G10 clone (24Lee J.K. Stewart-Jones G. Dong T. Harlos K. Di Gleria K. Dorrell L. Douek D.C. van der Merwe P.A. Jones E.Y. McMichael A.J. J. Exp. Med. 2004; 200: 1455-1466Crossref PubMed Scopus (133) Google Scholar) was expanded using irradiated heterologous peripheral blood mononuclear cells from three donors in the presence of IL-2. Viable cells were isolated on day 14 post-priming and used on the same day. The SLY10 T cell clone was cultured in human IL-15-containing medium for 24 h and used as above in stimulation assays. The B3Z murine T cell hybridoma (expressing the OT1 TCR) was used as a control for SCT accumulation by F5 RAG−/− cells. For imaging of G10 T cells, coverslips were cleaned by overnight incubation in 1 n HCl at 60 °C, followed by graded hydration by sonication in an ethanol series (95, 70, 50, 25, and 0%). Following brief coating with 0.01% poly-l-lysine, the coverslips were washed and coated with 0.05 mg/ml streptavidin in PBS (Sigma) for 2 h at 37 °C. Biotinylated SCTs (10 μg/ml in PBS) were immobilized onto coated coverslips by incubation for 1 h at room temperature. As a control, the coverslips were coated with 10 μg/ml biotinylated mouse anti-human HLA class I antibody (W6/32; Abcam). Following washing, G10 T cells in complete RPMI medium were incubated on coverslips for 1 min at 37 °C in 5% CO2. The medium was aspirated, and the cells were fixed in 2% paraformaldehyde for 10 min at 37 °C and permeabilized for 5 min in 0.05% Triton X-100. The samples were stained for CD3ϵ, Zap70, and CD45 by serial incubation of each antibody followed where appropriate by a species-specific and fluorophore-conjugated secondary antibody in the order mouse anti-CD3ϵ (UCHT1; AbD Serotec), anti-mouse Alexa 488 (Molecular Probes), rabbit anti-Zap70 (99F2; Cell Signaling Technology), anti-rabbit IgG Alexa 546 (Molecular Probes), and anti-human pan CD45 antibody directly conjugated with Alexa 647 (F10-89-4; AbD Serotec). The samples were washed extensively between all steps and sealed in anti-fade containing mounting agent (Molecular Probes). The images were acquired on a Zeiss LSM Pascal laser scanning confocal microscope equipped with 488/543/633 laser lines using the appropriate excitation and bandpass emission filters. The coverslip surface was identified by the second maxima of reflected light, and 1024 × 1024 pixel images were acquired using a Planapochromat ×63, NA 1.4 oil objective. A pinhole diameter corresponding to an optical slice thickness of 0.5 μm was chosen for imaging cell/glass interface-associated fluorescence. Multiple experiments were performed using identical labeling and imaging parameters. Cell conjugates for phoshotyrosine labeling were prepared by brief centrifugation at 4 °C of equal numbers of primed F5 RAG−/− T cells and SCT-expressing CHO cells labeled with a far-red emitting dye (DDAO; Molecular Probes). Following incubation at 37 °C for 2 min, the cells were fixed and permeabilized on glass coverslips. The samples were labeled with anti-phosphotyrosine antibody conjugated with Alexa 488 (G10; Santa Cruz Biotechnology) and prepared as above for imaging by confocal immunofluorescence microscopy. A similar method was used for imaging SCT accumulation. Conjugates of F5 RAG−/− or B3Z T cells, labeled with DDAO, and SCT-expressing CHO cells were labeled with a mouse anti-H2-Db monoclonal antibody (27-11-13S) followed by anti-mouse secondary conjugated with fluorescein isothiocyanate (BD Pharmingen). The Metamorph and ImageJ software packages were used for all image processing, and ImageJ was used for calculation of the Manders coefficient. All of the images were background subtracted prior to analysis. Interface enrichment was calculated as previously described using Metamorph (21Choudhuri K. Wiseman D. Brown M.H. Gould K. van der Merwe P.A. Nature. 2005; 436: 578-582Crossref PubMed Scopus (252) Google Scholar). Conjugates were fixed in a mixture of 2.5% glutaraldehyde and 2% formaldehyde in 100 mm cacodylate buffer, pH 7.4, with 2 mm Ca2+, post-fixed in buffered 1% osmium tetroxide and en bloc stained with 0.5% aqueous uranyl acetate. Ultrathin (∼60 nm thick) were cut and double stained with uranyl acetate and led citrate. All of the sections were examined in a Zeiss (LEO) Omega 912 electron microscope (Zeiss/LEO Electron Microscope Ltd., Oberkochen, Germany) equipped with a Proscan CCD camera (2048 by 2048 pixels). Digital images were captured with the integrated Soft Imaging Software image analysis package (Soft Imaging Software, GmbH, Münster, Germany), and absolute measurements were recorded directly from the images. Interfaces were imaged at ×35,500 magnification, and intermembrane distances were measured (at a 200% digital magnification) where apposed membranes were aligned (parallel) and where both membranes exhibited a trilaminar appearance, indicating that they were orthogonal to the image plane. Primed T cells (F5 RAG−/− or G10) in complete RPMI medium were placed in 96-well round-bottomed microtitre plates at 104 cells/well with varying numbers of CHO cells expressing native and elongated SCTs, represented in figures as T cell/APC ratio. The cells were incubated for 24 h at 37 °C in 5% CO2, and culture supernatants were collected for cytokine measurements. For PP2 inhibitor experiments, T cells were incubated with PP2 (Calbiochem) for 30 min, washed, and plated at 104 cells/well with CHO cells expressing native SCT at 5 × 104 cells/well and processed as described above. Equivalent amounts of PP2 corresponding to concentrations for T cells were added to CHO cells just before addition to microtitre wells to maintain overall PP2 concentrations. For IL-2 measurements, culture supernatants and mouse recombinant IL-2 standards (Sigma) were incubated in microtitre plates coated with a capture anti-mouse IL-2 antibody (BD Pharmingen) for 2 h at 37 °C, washed, and incubated for another 1 h with a biotinylated detection anti-mouse IL-2 antibody (BD Pharmingen). Following extensive washing, the plates were incubated with extravidin-horseradish peroxidase for 30 min and developed using 3,3′,5,5′-tetramethylbenzidine substrate (Sigma). Absorbance was measured at 450 nm in a spectrophotometric plate reader, and IL-2 concentration was interpolated from the absorbance of IL-2 standards. Commercially available ELISA kits employing a similar sandwich ELISA method were used for detection of human IFNγ (BD Pharmingen) and MIP1b (R & D Systems), according to the manufacturers' instructions. A standard 5- or 6-h 51Cr release assay was used to measure cytotoxicity. CHO target cells were detached using PBS containing 0.5 mm EDTA and labeled in RPMI medium with 51Cr Na2CrO4 (MP Biomedicals) for 1 h at 37 °C. After two washes, target cells were plated out in round-bottomed 96-well plates at 10,000 cells/well with F5 CTL effectors at the indicated effector to target ratios, in a total volume of 150 μl. The medium for the assay was complete RPMI + 10% fetal calf serum + 10 mm HEPES buffer, pH 7.4. After 5 or 6 h at 37 °C supernatant from each well was harvested, and 51Cr release was determined. The percentage specific lysis was calculated as follows: 100 × (release by CTL − medium release)/(3.3% Triton release − medium release). Each point was measured at least in duplicate against quadruplicate controls. Spontaneous release was less than 20% of Triton release in all experiments. For PP2 inhibition of CTL, target cells expressing a high level of H-2Db SCT were used with F5 CTL at an E:T target ratio of 10:1, and PP2 was included during the 5-h release period at concentrations from 0 to 10 μm. The CTL were preincubated with PP2 for 30 min before the target cells were added. PP3 was used as a control in the same experiment and gave no inhibition of lysis (data not shown). To ensure that proteins were monomeric, frozen aliquots of CD8αα and SCTs were purified by gel filtration on the day of experiments. Binding measurements were performed on a BIAcore 2000 instrument. Biotinylated SCTs were immobilized at ∼500 response units on streptavidin-coupled CM5 chips, and doubling dilutions of 154 μm CD8αα were injected over flow cells at 50 μl/min. G10 TCR binding to immobilized SCTs was measured similarly over a 0.1–7 μm concentration range. Binding curves were extracted and analyzed as described previously (23Wyer J.R. Willcox B.E. Gao G.F. Gerth U.C. Davis S.J. Bell J.I. van der Merwe P.A. Jakobsen B.K. Immunity. 1999; 10: 219-225Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Two-tailed T tests assuming unequal variance, one-way analysis of variance with Bonferroni's correction, and nonlinear curve fittings were performed using PRISM software. We first examined the effect of elongation of the mouse MHC class I molecule H-2Db on pMHC recognition by T cells expressing the F5 TCR, derived originally from influenza virus-infected mice (25Mamalaki C. Norton T. Tanaka Y. Townsend A.R. Chandler P. Simpson E. Kioussis D. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 11342-11346Crossref PubMed Scopus (143) Google Scholar). To avoid confounding the effects of elongation on peptide loading and/or assembly, the peptide epitope comprising residues 366–374 of influenza virus A/NT/60/68 nucleoprotein, β2-microglobulin, and H-2Db heavy chain were covalently joined by glycine/serine linkers and expressed as a single-chain trimer (DbSCT) (21Choudhuri K. Wiseman D. Brown M.H. Gould K. van der Merwe P.A. Nature. 2005; 436: 578-582Crossref PubMed Scopus (252) Google Scholar, 22Palmowski M.J. Parker M. Choudhuri K. Chiu C. Callan M.F. van der Merwe P.A. Cerundolo V. Gould K.G. J. Immunol. 2009; 182: 4565-4571Crossref PubMed Scopus (16) Google Scholar). Elongated forms of DbSCT were generated by the insertion of two, three, or four Ig domains from the ectodomains of CD2, CD22, and CD4 into the stalk region (Fig. 1A), and artificial APCs generated expressing comparable surface levels of these proteins on transporter associated with antigen processing (TAP)-deficient CHO cells (Fig. 1B). Incubation of T cells from F5 TCR transgenic mice with APCs expressing DbSCT resulted in IL-2 release, indicating specific recognition of the DbSCT by the F5 TCR (Fig. 1C). APCs expressing elongated forms of DbSCT stimulated considerably less IL-2 production, with the extent of IL-2 release correlated inversely with DbSCT size. To investigate early signaling events, we visualized phosphotyrosine accumulation at the contact interface in T cell/APC conjugates (supplemental Fig. S1A). An increase in interface phosphotyrosine was observed with DbSCT-expressing versus control CHO APCs, but no such increase was seen in conjugates with CHO cells expressing elongated DbSCT (Fig. 1D). We also measured DbSCT enrichment at the contact interface as a measure of TCR engagement (supplemental Fig. S1B). Elongated forms of DbSCT were enriched to the same extent as normal length DbSCT (Fig. 1D), indicating that elongation did not disrupt TCR binding. Finally, we used transmission electron microscopy to show that elongation of DbSCT increased the intermembrane distance at the contact interface between F5 T cells and DbSCT-expressing CHO cells (Table 1). It was notable, however, that the increase was significantly less than might be expected by the size of the inserts (∼7 nm for CD2 and ∼12 nm for CD4). This result, taken together with the finding that the intermembrane distance was more variable as the insert length increased (Table 1 and supplemental Fig. S2), suggests that the constructs were flexible and not fully extended at the interface.TABLE 1pMHC elongation increases the average intermembrane di
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