Ellis L. Reinherz
Dana-Farber Cancer Institute
Department of Medicine
77 Ave. Louis Pasteur, HIM-419
Boston, MA 02115
Lab Members: 10 Postdoctoral Fellows, 3 Instructors
The αβ T cell receptor (TCR) is a multimeric complex consisting of eight polypeptides: the antigen binding αβ clonotypic heterodimer and the invariant CD3 subunit dimers CD3εγ, CD3εδ and CD3ζζ. Specific interaction between an antigenic peptide bound to an MHC molecule (pMHC) and the TCRαβ heterodimer triggers downstream signaling via the immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic tails of the CD3 subunits. In turn, interaction with intracellular adaptors and signaling molecules induce distinct patterns of tyrosine phosphorylation in mature T cells. TCR signaling is also critical for thymocyte development, being essential for selection of double positive (DP) thymocytes for maturation into single positive (SP) thymocytes and subsequent peripheral egress. In early thymic development, a surrogate α chain termed pTα, is expressed in double negative (DN) thymocytes along with the TCRβ chain to form the pre-TCR. The pre-TCR functions to terminate additional β chain rearrangements and fosters the transition from DN3 to DN4 developmental stages. As with the TCR, signal transduction by the pre-TCR is carried out by the non-covalently associated CD3 subunits.
Determination of exactly how clonotypic TCRαβ heterodimer interaction with a given pMHC evokes signaling via the associated CD3 subunits is a daunting task. A key feature of αβTCR recognition that emerged from the initial wave of TCR structures in complex with pMHC is that each TCR assumes a common diagonal docking mode on the pMHC with its Vα domain docking over the N-terminal portion of the antigenic peptide and the Vβ domain docking over the C-terminal part. The inherent twist of the β sheet platform that forms the floor of MHC molecule’s antigen-binding groove forces the two long α helices on the wall of the groove to each break into two segments, creating two peaks on the pMHC molecular surface. In order for the TCR to probe into the MHC groove to make requisite contact with an antigenic peptide, a diagonal binding geometry is required to slot between the two peaks. Within this framework, binding variations in twist, tilt and shift are observed. However, the general principles governing TCR-pMHC recognition and subsequent signal transduction remain essentially uncharted.
In this regard, the solution structure of a heterodimeric murine CD3εγ complex first revealed a unique side-to-side hydrophobic interface with conjoined β sheets between the two Ig-like ectodomains (C2-set folds). Rigidity of the parallel C-terminal G β-strands suggested the possibility that a piston-type or other mechanical displacement of CD3εγ upon TCR ligation might be involved in initiation of T cell signaling. The subsequent solution structure of the heterodimeric murine CD3εδ complex is also consistent with this view. The CD3ε subunit conformation of CD3εδ is virtually identical to that of CD3ε in CD3εγ, whereas the CD3δ ectodomain adopts a C1-set Ig fold with a narrower GFC front face β sheet more parallel to the ABED backface than those β sheets in CD3ε and γ. Nonetheless, the dimeric interface between CD3δ and CD3ε is highly conserved among species and similar in character to that of CD3εγ. The importance of the CD3γ ectodomain terminal β-strand and membrane proximal stalk motif (RxCxxCxE) in thymic development and receptor assembly are now well-documented, and by extension, those of CD3ε and δ as well. Recent crystal studies of a human CD3εγ ectodomain fragment complexed with the Fab fragment of OKT3 and that of human CD3εδ complexed with a UCHT1 single-chain antibody identify a similar architecture.
Having structures of αβ, CD3εγ and CD3εδ heterodimers permitted us to construct a plausible model for the topology of assembled subunit ectodomains of the TCR, incorporating several known TCR characteristics: transmembrane charge pairs involving TCR subunit chain associations, CD3ε-CD3δ-TCRα-CD3ζ-CD3ζ as one cluster and CD3ε-CD3γ-TCRβ as a second cluster; extracellular domain interactions involving other in vitro chain association data; TCR crosslinking results; and proximity of one CD3ε subunit to the TCR Cβ FG loop by quantitative T cell surface immunofluorescent antibody binding analysis. Structural insights from crystallographic data on the glycosylated N15 TCR in complex with H57 Fab and the likely position of glycans in both CD3εγ and CD3εδ were considered. Immediately evident was the central position of the TCR αβ heterodimer with a vertical dimension of 80Å projecting from the cell membrane, flanked on either side by the shorter (40Å) CD3 heterodimers, CD3εδ on the TCRα side and CD3εγ on the TCRβ side. The width of the CD3εδ and CD3εγ components, 50Å and 55Å, respectively, were comparable in size to that of the TCRαβ heterodimer (58Å), and together (excluding glycans) span ~160Å. These flanking CD3 components likely impede lateral movement of the TCRαβ heterodimer upon pMHC binding. As noted for CD3εγ, the intradomain disulfide bridge between Cys residues on the B and F strands at the center of each CD3εδ domain reinforces the domain structure. Further rigidity for potential signal transduction comes from the paired G β-strands in each CD3 heterodimer, coupled with the conserved RxCxxCxE cysteine-coordinated stalks. Crystallographic details on TCR, CD4 and CD8 interactions with pMHCI and pMHCII ligands reveal that the CD4 and CD8 co-receptors are located on the side adjacent to CD3εδ when binding to the same pMHC ligand as the TCR. Not surprisingly, CD3δ couples the TCR with lipid raft-associated CD8αβ required for effective activation and positive selection of CD8+ T cells. While the relatively flexible CD8 stalk region poses no steric constraints for concurrent TCR ligation, CD4 is a rigid concatamer with four Ig domains comprising its extracellular segment. Nevertheless, structural analysis shows that the membrane proximal ends of CD4 and TCRαβ are 100Å apart, providing ample space for CD3εδ in between.
Based on the CD3 structures, TCR signaling may require dynamic interaction rather than static on-and-off switching, such that the interfaces between the extracellular domains of the TCR αβ heterodimer and CD3 dimers may be quite small. We envisage the ectodomains of TCR αβ chains being supported by the CD3 heterodimers, while components of the TCR αβ dimer, such as the Cβ FG loop and the α-CP may serve as levers to help control vertical movements of CD3 subunits for signal transduction through the critical transmembrane (TM) segments. Given apparently weak ectodomain association between CD3 and TCRαβ, it is possible that this assembly undergoes dynamic quaternary change upon TCR ligation, thereby affecting cytoplasmic CD3 signaling. Developing an accurate view of conformational/quaternary alterations leading to outside-inside signaling is challenging but of interest to our group. How TCR signaling directs intrathymic development and modulates intrathymic trafficking is also under investigation.
Practical applications of immune recognition have resulted from basic principles uncovered during these studies. Bioinformatic approaches to define eptopes on tumor antigens and within proteomes of infectious agents will guide further vaccine design to elicit further protective effector and memory T cell responses. Likewise, targeted approaches to neutralizing antibody production are being investigated in HIV for stimulation of focused immune responses.
Kim ST, Takeuchi K, Sun Z-YJ, Touma, M, Castro CE, Fahmy A, Lang MJ, Wagner G, Reinherz EL. The αβ T cell receptor is an anisotropic mechanosensor. J Biol Chem. 2009; 284:31028-31037. PMC2781503
Kim MK, Sun Z-YJ, Rand KD, Shi X, Son L, Cheng Y, Fahmy AF, Majumdar S, Ofek G, Yang Y, Kwong PD, Wang J-H, Engen JR, Wagner G, Reinherz EL. Antibody-mediated mechanics on a membrane embedded HIV-1 envelope segment essential for gp41-targeted viral neutralization. Nat Struc Molec Biol. 2011; 18:1235-1243. PMC3253551
Keskin DB, Reinhold B, Lee SY, Zhang GL, Lank S, O'Connor DH, Berkowitz RS, Brusic V, Kim SJ, Reinherz EL. Direct identification of an HPV-16 tumor antigen from cervical cancer biopsy specimens. Front Immunol. 2011; 2:1-11. PMC3342284
Kim ST, Shin Y, Brazin K, Mallis R, Sun J, Wagner G, Lang M, Reinherz EL. TCR mechanobiology: Torques and tunable structures linked to early T cell signaling. Front Immunol. 2012; 3:article 76. PMC3342345
Wang JH, Reinherz EL. The structural basis of αβ T lineage immune recognition: TCR docking topologies, mechanotransduction and co-receptor function. Immunol Rev. 2012; 250:102-119. PMC3694212
Last Update: 1/6/2014