Ligand-induced conformational change in the T-cell receptor associated with productive immune synapses

Abstract

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. IntroductionWhen 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 downmodulation 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 fullagonist peptide. Thus, stimulation with a partial agonist induces only partial phosphorylation of the CD3 and CD3⑀ chains and the recruitment, but not the phosphorylation, of ZAP70 (zetaassociated protein of 70 kDa), 8,9 resulting in the activation of a subset of the downstream signaling pathways that are activated by an agonist peptide. 10 Anta...

Lipid-dependent conformational dynamics underlie the functional versatility of T-cell receptor

“…The FRET efficiency of E1-stimulated T cells was high, indicating low opening level of CD3ε CD . The FRET result agreed well with a previous study showing that the N4 can induce the exposure of the proline-rich sequence of CD3ε CD whereas E1 cannot [24]. The antigen dose (5 µg/ml) that we used in Figure 7B was at the saturating level to trigger all TCR (C-E) The signals of three representative residues, T42 in the N-terminal half of the cytoplasmic domain, Y67 and Y78 in the C-terminal half of the cytoplasmic domain, were enlarged and shown.…”

“…The allosteric regulation has been found in many receptors such as G-protein-coupled receptors [74]. The relevance of allosteric regulation in TCR triggering is supported by the findings that antigen-induced conformational changes have been observed in both extracellular and cytoplasmic domains [23,24,34,75]. Moreover, mutating CD3ε stalk region to prevent outside-in conformational transition leads to impaired TCR signaling [18,19,37].…”

“…In vivo experiments reveal that CD3ε-Nck interaction not only enhances TCR sensitivity to weak ligands but also contributes to the downregulation of TCR expression on the cell surface [20,21]. PRS thus may play positive or negative roles in TCR signaling in different scenarios [23,24]. These studies together show that the functional roles of ITAM, PRS and BRS are complicated and inter-regulated.…”

“…Second, antigen engagement induces the exposure of CD3ε PRS to recruit the adaptor protein Nck [23]. Intriguingly, only strong antigen but not weak antigen can induce the intracellular PRS exposure [24]. Third, the conserved cysteine motif in the CD3 stalk regions maintains the conformations of CD3 transmembrane and cytoplasmic domains [36].…”

“…Earlier biochemical studies show that different antigens can trigger distinct ITAM phosphorylation programs [16]. Besides the ITAM, other motifs in CD3 cytoplasmic domains also play important roles in TCR signaling, including the lipid-interacting Basic residue Rich Sequence (BRS) in CD3ε and CD3ζ chains and the Nck-interacting Proline-Rich Sequence (PRS) in CD3ε chain [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32]. The physiological importance of BRS and PRS has been demonstrated by in vivo experiments [25,28,33].…”