Multivalent structure of an αβT cell receptor

Abstract

Whether there is one or multiple ␣␤T cell antigen receptor (TCR) recognition modules in a given TCR͞ CD3 complex is a long-standing controversy in immunology. We show that T cells from transgenic mice that coexpress comparable amounts of two distinct TCR␤ chains incorporate at least two ␣␤TCRs in a single TCR͞CD3 complex. Evidence for bispecific ␣␤TCRs was obtained by immunoprecipitation and immunoblotting and confirmed on the surface of living cells both by f luorescence resonance energy transfer and comodulation assays by using antibodies specific for TCR␤-variable regions. Such (␣␤) 2 TCR͞CD3 or higher-order complexes were evident in T cells studied either ex vivo or after expansion in vitro. T cell activation is thought by many, but not all, to require TCR cross-linking by its antigen͞major histocompatibility complex ligand. The implications of a multivalent (␣␤) 2 TCR͞CD3 complex stoichiometry for the ordered docking of specific antigen͞major histocompatibility complex, CD4, or CD8 coreceptors and additional TCRs are discussed.The T cell receptor complex (TCR͞CD3) consists of disulfidelinked ␣␤ heterodimers noncovalently associated with invariant proteins of the CD3 (CD3␥, ␦, and ) and the (, , and FcRI␥) families (1-3). The TCR␣ and TCR␤ components contain variable (V) and constant (C) domains homologous to those of antibodies (Ig), but they are not secreted and recognize antigen fragments embedded in molecules of the major histocompatibility complex (MHC) (4-6). The CD3͞ subunits have both signaling and structural functions (7,8). Whether one or multiple ␣␤TCR heterodimers occur in a TCR͞CD3 complex currently is unresolved. A reassessment of TCR stoichiometry began after the finding that two CD3 chains occur per TCR͞CD3 complex (9, 10), because early studies indicated a 1:1 ratio for TCR͞CD3 chains using mAbs to TCR and CD3 for estimation of the number of binding sites (1). On the one hand, several groups have suggested that TCR, like Ig, is multivalent, [i.e., (␣␤) 2 TCR and (HϩL) 2 Ig]. It was argued that such an arrangement: i) maximizes the interactions between charged transmembrane residues in ␣␤TCR (3 ϩ ) and CD3͞ (6 Ϫ ) chains, rendering a more stable complex, and ii) fits with the hydrodynamic measurements of TCR͞CD3 complex size (11-13). On the other hand, recent biochemical analyses argue against a multivalent TCR model because immunoprecipitations with mAb to V ␣ or V ␤ failed to reveal the predicted association of two TCR␣s or two TCR␤s in lysates of T cells bearing two distinct ␣␤TCRs (14, 15).Previous studies of the TCR͞CD3 subunit stoichiometry (9, 10, 15) have relied on the ability to coprecipitate from surface-labeled cells two homologous forms of a given subunit that were distinguishable after gel electrophoresis and autoradiography. Although such an experimental design allowed to show that two CD3 subunits, of human and mouse origin, occur per TCR͞CD3 complex (9, 10), it may be not of universal application. We and others have noticed that the similarity between two distinct TCR␣...

Stoichiometry of Signalling Complexes in Immune Cells: Regulation by the Numbers

“…Thus, the overall stoichiometry of the TCR complex was found to be 1:1:1:1:2:2 (for TCR, TCR, CD3 , CD3 , CD3 , and CD3 , respectively) (Rudolph et al, 2006;Wucherpfennig et al, 2010) These findings corroborated the stoichiometry proposed in a previous study (Blumberg et al, 1990), and are supported by other results, as well (Call et al, 2004). However, other works suggested that a second heterodimer was incorporated into the complex, suggesting an ( ) 2 stoichiometry (Exley et al, 1995;Fernandez-Miguel et al, 1999;San Jose et al, 1998). Finally, an ( ) 2 configuration was also suggested (Rubin et al, 2002).…”

Evidence for a functional sidedness to the αβTCR

“…Our model of the αβTCR-CD3 complex, with CD3δε and CD3γε clustered on one side of the TCR (7), stands in contrast to those in which the CD3 heterodimers dock on opposite sides of the TCR (21) or in which two TCRs are associated with one set of signaling dimers (16). To discriminate between these models, we used a dimerization reporter system based on the knowledge that erythropoietin receptor (EPOR) signaling, via the Jak-Stat pathway when two signaling domains are in juxtaposition (22), can drive BaF3 cell proliferation (23).…”

“…Residues that stabilize TCR-CD3 interactions have been implicated in signaling (6,7,9) and subtle conformational changes have been proposed (10). In addition, dimerization of the complex is thought to be important because forcing TCRs into juxtaposition can induce signaling (11,12); however, the evidence for dimerization has been controversial (13)(14)(15)(16), and the structural features that would mediate such interactions have not been identified. A more complete understanding of how the subunits fit together within and between complexes is needed to advance our understanding of how this molecular machinery works.…”

TCRs Genetically Linked to CD28 and CD3ε Do Not Mispair with Endogenous TCR Chains and Mediate Enhanced T Cell Persistence and Anti-Melanoma Activity

“…Indeed, TCR:28ε-mediated, but not wt TCR-mediated, immune synapses were readily detectable in primary human T cells, showed enhanced accumulation of TCR, CD3, and CD8 (Figs. 5, 6), and are expected to facilitate Ag-dependent T cell activation (44,46,47). Notably, TCR:z mediates immune synapses in which densities of TCR, CD3, and CD8 are diluted over enlarged contact areas (23), whereas TCR:28ε mediates immune synapses in which densities of the aforementioned molecules are enriched in normal-sized immune synapses.…”