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Blood, 15 July 2005, Vol. 106, No. 2, pp. 601-608. Prepublished online as a Blood First Edition Paper on March 24, 2005; DOI 10.1182/blood-2004-12-4763.
IMMUNOBIOLOGY Ligand-induced conformational change in the T-cell receptor associated with productive immune synapsesFrom the Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, and Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Spain; and the Laboratory of Transplantation Immunology and Nephrology, University Hospital-Basel, Switzerland.
Triggering of the T-cell receptor (TCR) can produce very different responses, depending on the nature of the major histocompatibility complex/antigen peptide (MHCp) ligand. The molecular mechanisms that permit such fine discrimination are still unknown. We show here that an epitope in the cytoplasmic tail of the TCR CD3  subunit, recognized by antibody APA1/1, is only detected when the TCR is fully activated. Exposure of the APA1/1 epitope is shown to be fast and independent of tyrosine kinase activity and that it takes place even when T cells are stimulated at 0°C. These results suggest that APA1/1 detects a conformational change in the TCR. APA1/1 staining concentrates in a restricted area of the immunologic synapse. Most important, we show that full agonist, but not partial agonist, peptides induce exposure of the APA1/1 epitope, indicating a correlation between the induction of the conformational change in the TCR and full T-cell activation. Finally, the conformational change is shown to occur in T cells that are being stimulated by antigen in vivo. Therefore, these results demonstrate that the TCR undergoes a conformational change on MHCp binding in vitro and in vivo, and they establish a molecular correlate for productive TCR engagement. (Blood. 2005;106:601-608)
When T cells recognize antigen-presenting cells (APCs) that bear an appropriate antigen, large-scale rearrangement of the T-cell cytoskeleton occurs, coupled to a redistribution of plasma membrane and cytoplasmic molecules. This reorganization results in the formation of what is known as an immunologic synapse (IS).1,2 Antibody-staining experiments and time-lapse confocal microscopy have demonstrated that central and peripheral supramolecular activation clusters (cSMACs and pSMACs) form at the IS.3 Although the cSMAC contains the T-cell receptor (TCR) and signaling molecules such as protein kinase C  (PKC), the pSMAC is formed by a ring of integrins, such as leukocyte factor antigen 1 (LFA1) (CD11a/CD18), and accompanying cytoskeletal proteins, such as talin.3 It has been proposed that the formation of the IS serves to sustain TCR signaling, which is necessary for full T-cell activation.1,2 However, some studies indicate that TCR signaling takes place before the IS is formed,4 whereas others indicate that the IS is a site for TCR internalization and down-modulation of the activation signals.5
Much of the evidence supporting the need to form the IS for T-cell activation derives from the observation that antagonist and partial agonist peptides do not form bona fide ISs.2,6,7 Furthermore, TCR engagement with a partial agonist peptide results in a pattern of intracellular signaling distinct from that induced with a full-agonist peptide. Thus, stimulation with a partial agonist induces only partial phosphorylation of the CD3
The existence of conformational changes in the TCR was first proposed by Janeway and colleagues12 based on the TCR-induced cocapping of CD4.12 Although structural data generally preclude the existence of large rearrangements in the ectodomains of the TCR
We now show that antibody or major histocompatibility complex/antigen peptide (MHCp) engagement of the TCR promotes the exposure of an epitope recognized by a monoclonal antibody (mAb) (APA1/1) that binds to the cytoplasmic tail of CD3
Cells and mice
The human Jurkat T-cell line and the lymphoblastoid B-cell line Raji were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS; Sigma, St Louis, MO). CH7C17, a Jurkat cell transfectant expressing the TCR Antibodies and other reagents
Anti-CD3 T-cell stimulation and flow cytometry Spleen cells from 6-week-old OT-I RAG2-/- mice were stimulated with H-2Kb/Ig dimers loaded with the indicated peptide for 1 minute at 37°C. Anti-mouse immunoglobulin-PE (PharMingen) was added for 15 minutes at 37°C, and the cells were fixed and permeabilized using the Cytofix/Cytoperm kit (PharMingen) according to the manufacturer's instructions. Biotin-labeled APA1/1 was added at a concentration of 4 µg/mL for 30 minutes on ice. Cells were stained with specific fluorochrome-conjugated antibodies and analyzed by flow cytometry in a FACSCalibur flow cytometer using the CellQuest software (Becton Dickinson, San Jose, CA). For antibody stimulation, spleen or lymph node cells were incubated with 10 µg/mL 145-2C11 for 10 minutes at 37°C and were directly stained on ice with PE-labeled anti-CD4 or anti-CD8 antibodies. APA1/1 staining was analyzed after fixation and permeabilization, as for H-2Kb/Ig dimer stimulation. Pull-down assay
For the glutathione-S-transferase (GST)-Nck pull-down assay, cell lysates were first precleared with GST adsorbed to glutathione-Sepharose (Amersham Biosciences, Freiburg, Germany) before affinity chromatography with the amino-terminal SH3 domain of Nck Cell conjugation and immunofluorescence labeling Jurkat/Raji conjugates. To distinguish APCs from T cells, Raji cells were loaded with 10 µM chloromethyl aminocoumarin (CMAC) for 30 minutes at 37°C, washed, and resuspended in RPMI 1640 with 10% FBS. Cells were then incubated for 15 minutes in the presence of 1 µg/mL SEE. Jurkat cells were mixed with an equal number of Raji cells in a final volume of 50 µL and were incubated at 37°C for 15 minutes. Conjugates were plated on poly-L-lysine-coated slides, fixed for 10 minutes in 2% formaldehyde-Hanks balanced salt solution (HBSS) and were stained with the appropriate antibodies, using goat anti-mouse or rabbit Alexa488, Alexa594, or Alexa647 (Caltag, Burlingame, CA) as the secondary antibodies. Cells were visualized with an Axiovert 200 microscope (Carl Zeiss, Oberkochen, Germany) or with a Zeiss Radiance 2000 confocal microscope and were analyzed with the ImageJ 1.33v software (National Institutes of Health, Bethesda, MD). Images were acquired with a charge-coupled device (CCD) camera SPOT RT Slider (Diagnostic Instruments, Sterling Heights, MI). CH7C17/DAP-DR1-ICAM conjugates. The APCs DAP-DR1-ICAM were cultured on coverslips with 10 µM HA307-319 or HAK316A peptides for 24 hours at 37°C. Afterward, they were incubated with CH7C17 at a 1:1 ratio for 15 minutes at 37°C. Cells were fixed and stained as described in the previous paragraph for Jurkat/Raji conjugates. Spleen cell conjugates. Unfractionated spleen cells from OT-I C57BL/6 mice were incubated with 2 µM of the indicated peptide for 2 hours at 37°C. The cells were plated on poly-L-lysine-coated slides. Antibody staining was performed as described earlier in this section for Jurkat/Raji conjugates. Confocal microscopy T-cell/APC conjugates were identified based on cell morphology under differential interference contrast (DIC) microscopy with blue fluorescence for CMAC-labeled APCs (Jurkat/Raji conjugates) and green fluorescence for antibody marker-labeled APCs (spleen cell conjugates). The proportion of conjugates with APA1/1 or CD3 redistributed to the T-cell/APC contacts was calculated by randomly choosing 100 different conjugates. Confocal microscopy was carried out on a Zeiss Radiance 2000 using a 63 x 1.4 oil Plan-Apochomat objective lens. Image acquisition was with the Lasersharp 2000 5.2 software (Zeiss) and analysis with the Image J 1.33v software. Immunohistochemical studies Lymph nodes were fixed with 4% paraformaldehyde solution in phosphate-buffered saline (PBS), washed with PBS, and incubated overnight in a 30% sucrose solution in PBS at 4°C. Lymph nodes were embedded in OCT compound (Tissue-Tec; Miles Scientific, Naperville, IL), and 10-µm cryostat sections were obtained using a 2800 Frigocut N cryostat (Leica, Nussloch, Germany). Sections were fixed again with 4% paraformaldehyde in PBS, quenched with 20 mM NH4Cl, and stained for 1 hour with antibodies in a solution containing 1% FBS. Secondary antibodies were prepared as described in "Cell conjugation and immunofluorescence labeling," and the sections were mounted in fluorescent mounting medium (DAKO, Carpinteria, CA).
Conformational changes in the TCR revealed by exposure of the APA1/1 epitope
We have previously revealed the conformational change in the intracellular domain of the TCR using a pull-down assay with GST-Nck fusion protein.15 Consistent with these earlier results, stimulation of spleen T cells with anti-CD3 antibody induced the binding of Nck to the TCR (Figure 1A). Because the mAb APA1/1 binds in the proximity of the proline-rich sequence of CD3
Exposure of the APA1/1 epitope was not affected by inhibiting TCR-induced tyrosine kinase activity with the src-kinase inhibitor PP2 at a concentration that completely abrogated tyrosine phosphorylation (Figure 1C; data not shown). Moreover, the APA1/1 epitope was exposed very early after TCR engagement (1 minute) and also when the cells were stimulated on ice (Figure 1C). These results suggest that exposure of the APA1/1 epitope is independent of the activation of intracellular signaling pathways and independent of the cell's metabolic activity, corroborating the observations previously made in the GST-Nck pull-down assay15 and indicating that the APA1/1 antibody can be used to trace the conformational change in the TCR.
Conformational change of TCR induced by full, but not partial, agonist ligands
It was important to confirm that the conformational change in the TCR could be detected not only after stimulation with anti-CD3 antibodies but also with MHCp. To investigate this, we used spleen T cells from transgenic mice with the H-2Kb-restricted OT-I TCR. Stimulation of OT-I cells with H-2Kb/Ig dimers loaded with the OVA agonist peptide (OVAp, SIINFEKL) induced expression of the CD69 activation marker (Figure 2A). In contrast, stimulation of the cells with unloaded H-2Kb/Ig dimers or with dimers loaded with the partial agonist/antagonist E1 peptide (EIINFEDKL)16,21,22 failed to activate CD69 expression. Using the same set of peptide-loaded H-2Kb/Ig dimers, we found that the H-2Kb/Ig dimer loaded with the agonist OVAp, but not the empty dimer or the dimer loaded with E1, induced a shift in the mean intensity of APA1/1 fluorescence within 5 minutes (Figure 2B). The experiment was performed at room temperature to reduce down-modulation of the TCR promoted by the H-2Kb:Ig-OVAp agonist ligand. Although BIAcore experiments have shown that H-2Kb-E1 has a lower affinity for the OT-I TCR than H-2Kb-OVAp,11 the failure to detect the APA1/1 epitope after E1 engagement was not the result of poor binding. Indeed, a well-defined population of T cells bound to the E1-loaded H-2Kb/Ig dimer was detected (Figure 2C, left). However, cells that bound to H-2Kb-OVAp presented more intense APA1/1 immunofluorescence than cells bound to H-2Kb-E1 (Figure 2C, right). These results indicate that the conformational change revealed by exposure of the APA1/1 epitope in CD3
APA1/1 epitope expressed by a subset of TCRs located in the IS
To study the cellular distribution of TCR molecules that expose the APA1/1 epitope during antigen presentation, we chose Jurkat T cells and Raji lymphoblastoid B cells as APCs. In the presence of an appropriate superantigen (SEE), these cells have been shown to form a well-defined, mature IS.23 Interestingly, the APA1/1 epitope was clearly associated with the IS and colocalized with PKC Distinction of productive ISs by induced expression of APA1/1 epitope
Exposure of the APA1/1 epitope occurred on binding to the TCR of H-2Kb dimers complexed to an agonist peptide (OVAp) but not to a partial agonist (E1; Figure 2), Hence, we sought to determine whether both types of ligand induced a similar type of synapse in primary T cells. To this end, total spleen cells from OT-I TCR transgenic mice were cultured ex vivo with both peptides, and the formation of synapses between OT-I T cells and APCs was analyzed. The OT-I T cells formed conjugates with dendritic cells (DCs) identified with the CD11c marker (Figure 4A). In almost half the T-cell/DC conjugates, the TCR was localized in the IS (visualized with anti-CD3
Given these results, we have found a correlation between the formation of ISs containing APA1/1—indicative of a TCR conformational change—and the activation of T cells. To demonstrate a correlation between the formation of a productive IS and the conformational change in the TCR in a different T-cell/APC system, thus adding general validity to our findings, an MHC class 2-restricted agonist/antagonist system was used. CH7C17 cells, a Jurkat T-cell derivative expressing the HA1.7 TCR, were stimulated with APCs loaded with an antagonist (HAK316A) or an agonist peptide (HA307-319), and the formation of ISs was examined. As for the OT-I system, the APA1/1 antibody, more than anti-CD3  antibody, made a clear distinction between synapses formed by agonist and antagonist peptides (Figure 5A-B). In this HA1.7 TCR system, the HAK316A peptide did not induce CD69 expression, unlike the agonist peptide (Figure 5C), but it was clearly not a "null" agonist because it elicited TCR translocation to the IS (Figure 5A-B) and tyrosine phosphorylation (data not shown). The results shown in Figures 4 and 5 indicate that the conformational change in the TCR is induced by full but not partial agonists/antagonists, establishing a molecular correlate for productive TCR engagement in vivo. APA1/1 demonstrates the conformational change in TCR in vivo We finally studied whether the expression of the APA1/1 epitope could be detected in lymphoid tissue in vivo. Unlike a conventional anti-CD3 antibody, the APA1/1 antibody poorly stained lymph node sections of OT-I mice injected with vehicle alone (Figure 6A; PBS). Indeed, most T cells of nonimmunized TCR transgenic and nontransgenic mice were not recognized by the APA1/1 antibody (data not shown). However, when OT-I mice were subcutaneously injected with OVA, the draining lymph nodes showed a large increase in APA1/1 immunoreactivity as early as 6 hours after injection (Figure 6A; OVA). The total fluorescence signal within the lymph nodes (Figure 6A) was quantified in the green (conventional CD3) and red (APA1/1) channels using the ImageJ program. Although the fluorescence corresponding to the anti-CD3 antibody increased only 10% after OVA injection, APA1/1 fluorescence increased 300%. At higher magnifications, it was apparent that the APA1/1 antibody labeled almost two thirds (60%) of the OT-I T cells after OVA injection. Using CD11c as a marker for DCs, we found that OT-I T cells formed frequent APA1/1-positive synapses with these APC types (Figure 6B). The APA1/1 epitope was always distributed in a polarized manner (n = 199), clearly labeling the ISs formed with APCs bearing the H-2Kb-OVAp complex, as demonstrated by 25-D1.16 antibody staining (data not shown). Furthermore, most (81%) APA1/1-positive T cells formed synapses with APCs.
Hence, it appears that the APA1/1 epitope is exposed in CD8+ T cells in the lymph node when they encounter their nominal antigen (Figure 6), permitting the IS formation to be labeled in vivo. Furthermore, these results suggest that the APA1/1 antibody can be used to track and distinguish T cells whose TCRs are being engaged by fully stimulatory ligands in vivo, thus undergoing conformational change in vivo.
In this study, we show that an epitope in the tail of CD3 defined by APA1/1 is exposed when the TCR is engaged by stimulatory antibodies or MHCp. Furthermore, we show that the exposure of this epitope is rapid, takes place at 0°C, and is independent of PTK activity. Therefore, we conclude that exposure of the APA1/1 epitope is a hallmark of a conformational change in the TCR, similar to the Nck-binding assay (GST-Nck pull-down).15 Using CD8+ T cells from OT-I TCR transgenic mice as a model system, we show that the APA1/1 epitope is displayed when the TCR is engaged by a full agonist but not by a partial agonist/antagonist. This result adds an important control of specificity to the assay and suggests that the conformational change in the TCR is decisive for productive T-cell activation. In accordance with this idea, APA1/1 labels a restricted area of the cSMAC when formed with an APC bearing the full agonist MHCp and not the partial agonist. This restricted exposure of the APA1/1 epitope in the cSMAC suggests that only a fraction of the TCR in the IS undergoes the conformational change at any given time point. Finally, we demonstrate that in most resting T cells in the lymph node, the APA1/1 epitope is not accessible to the antibody; however, shortly after antigen exposure in vivo, it becomes exposed in most antigen-specific T cells. This result indicates that APA1/1 can be a useful tool to distinguish T cells whose TCR is being engaged by MHCp in vivo. Furthermore, the results demonstrate that the conformational change in the TCR occurs on antigen recognition in vivo.
Traditionally, it has been thought that the differential capacity of antibodies to detect specific epitopes reflects conformational changes. One of the most intensely studied examples of this is the increased affinity of integrins from inside-out signaling, promoting the exposure of neoepitopes in the alpha and beta subunit ectodomains.25,26 However, perhaps mechanistically, a more related example is that of the insulin receptor whose binding to insulin causes a conformational change in the associated G(i)
Our results show that the conformational change in the TCR is provoked by full agonists and not by partial agonists. Thus, it would be interesting to determine whether known molecular correlates of TCR signaling induced by antagonist peptides—partial phosphorylation of CD3 to the p21 form, recruitment but not phosphorylation of ZAP70, and, most important, poor CD3 phosphorylation8,9—are related to the failure of the TCR to modify its conformation. It is possible that TCR engagement induces a general rearrangement of the CD3 tails, exposing them to complete tyrosine phosphorylation by Lck. The failure to unmask the CD3 tails would impair Lck-mediated phosphorylation when T cells are stimulated with an antagonist peptide and might be reflected in a failure to recruit Lck to antagonist peptide-induced IS.7 Nevertheless, if the TCR conformational change affects only the tail of CD3, we could envision Nck recruitment, together with tyrosine phosphorylation of CD3 and CD3, as among the primary events impaired by antagonist peptide binding to the TCR.
Antagonist peptides have been shown to form abnormal ISs in which the TCR3 and MHCp2 are less densely accumulated than in ISs formed by full agonists. Furthermore, antagonist peptides seem to impair the recruitment of Lck to the synapse,7 which, on the other hand, correlates with an inhibition in the TCR-CD4 interaction in the IS.6 Interestingly, the conformational change in the TCR was initially proposed to explain cocapping of CD4 induced by anti-CD3 antibodies.28 Indeed the findings of Gascoigne and colleagues6 may indirectly show that antagonist peptides fail to induce the conformational change in the TCR and the TCR-CD4 interaction in the IS. Thus, antagonist peptides do not produce the 2 effects thus far described that are indicative of a conformational change in the TCR: CD4 cocapping and exposure of the proline-rich sequence in CD3
How recognition of a full agonist or an antagonist ligand is reflected in the differential exposure of the CD3 Recently, DCs were shown to form ISs with T cells in the absence of antigen, and it was proposed that this interaction could lead to the activation of signaling pathways involved in the maintenance of the naive T cell pool in vivo.24 In this work, the translocation of the TCR to the T-cell/DC synapse was indeed detected in the absence of antigen. In light of our ex vivo experiments with antagonist and agonist peptides, one might anticipate that APA1/1 would not be exposed in the T-cell/DC synapses in which an appropriate MHCp was not engaged and in which the TCR would not undergo a conformational change. This further supports the notion that the conformational change in the TCR is required for full activation. Finally, the results described here suggest that the APA1/1 antibody can be used as a tool to track productively engaged T cells in vivo. The antibody could, for example, be used in histochemical studies to identify a correlation between the induction of a conformational change in the TCR of infiltrating T cells and tumor or allograft rejection.
We thank C. Ardavín, B. Cubelos, M. Mittelbrunn, and E. Palmer for kindly providing reagents and protocols and A. Borroto, S. Ley, H. M. van Santen, and M. Sefton for critically reading the manuscript. We also thank T. Cerrato, M. A. Muñoz, and T. Villalba for their excellent technical support.
Submitted December 16, 2004; accepted March 16, 2005.
Prepublished online as Blood First Edition Paper, March 24, 2005; DOI 10.1182/blood-2004-12-4763.
Supported by grants from Comisión Interministerial de Ciencia y Tecnología (SAF2002-03589) (B.A.), Comunidad de Madrid 08.3/0030/2001 (B.A.), Formación de Profesorado Universitario Fellowship (R.M.R.), and Fundación Areces (to the CBMSO).
An Inside Blood analysis of this article appears in the front of the issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Balbino Alarcón, Centro de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco Madrid 28049, Spain; e-mail: balarcon{at}cbm.uam.es.
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Top
Abstract
Introduction
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heterodimer,
) and SD from data collected in triplicate. To quantify total TCR levels, permeabilized spleen T cells were stained either with anti-CD3
indicates stimulated samples; 
