A response calculus for immobilized T cell receptor ligands

To address the molecular mechanism of T cell receptor (TCR) signaling, we have formulated a model for T cell activation, termed the 2D-affinity model, in which the density of TCR on the T cell surface, the density of ligand on the presenting surface, and their corresponding two-dimensional affinity determine the level of T cell activation. When fitted to T cell responses against purified ligands immobilized on plastic surfaces, the 2D-affinity model adequately simulated changes in cellular activation as a result of varying ligand affinity and ligand density. These observations further demonstrated the importance of receptor cross-linking density in determining TCR signaling. Moreover, it was found that the functional two-dimensional affinity of TCR ligands was affected by the chemical composition of the ligand-presenting surface. This makes it possible that cell-bound TCR ligands, despite their low affinity in solution, are of optimal two-dimensional affinity thereby allowing effective TCR binding under physiological conditions, i.e. at low ligand densities in cellular interfaces.     

High affinity T cell receptors from yeast display libraries block T cell activation by superantigens

The alphabeta T cell receptor (TCR) can be triggered by a class of ligands called superantigens. Enterotoxins secreted by bacteria act as superantigens by simultaneously binding to an MHC class II molecule on an antigen- presenting cell and to a TCR beta-chain, thereby causing activation of the T cell. The cross-reactivity of enterotoxins with different Vbeta regions can lead to stimulation of a large fraction of T cells. To understand the molecular details of TCR-enterotoxin interactions and to generate potential antagonists of these serious hyperimmune reactions, we engineered soluble TCR mutants with improved affinity for staphylococcal enterotoxin C3 (SEC3). A library of randomly mutated, single-chain TCRs (Vbeta-linker-Valpha) were expressed as fusions to the Aga2p protein on the surface of yeast cells. Mutants were selected by flow cytometric cell sorting with a fluorescent-labeled SEC3. Various mutations were identified, primarily in Vbeta residues that are located at the TCR:SEC3 interface. The combined mutations created a remodeled SEC3-binding surface and yielded a Vbeta domain with an affinity that was increased by 1000-fold (K(D)=7 nM). A soluble form of this Vbeta mutant was a potent inhibitor of SEC3-mediated T cell activity, suggesting that these engineered proteins may be useful as antagonists.     

Induction of T cell anergy by low numbers of agonist ligands.

Engagement of TCR by its ligand, the MHC/peptide complex, causes T cell activation. T cells respond positively to stimulation with agonists, and are inhibited by antagonist MHC/peptide ligands. Failure to induce proper conformational changes in the TCR or fast TCR/MHC dissociation are the leading models proposed to explain anergy induction by antagonist ligands. In this study, we demonstrate that presentation of between 1 and 10 complexes of agonist/MHC II by unfixed APC induces T cell anergy that persists up to 7 days and has characteristics similar to anergy induced by antagonist ligand or TCR occupancy without costimulation. Furthermore, anergy-inducing doses of hemagglutinin 306-318 peptide led to the engagement of less than 1000 TCR/CD3 complexes. Thus, engagement of a subthreshold number of TCR by either a low density of agonist/MHC or a 2-3 orders of magnitude higher density of antagonist/MHC causes anergy. Moreover, we show that anergy induced by low agonist concentrations is inhibited in the presence of IL-2 or cyclosporin A, suggesting involvement of the calcineurin signaling pathway.     

Production of a soluble gammadelta T-cell receptor to identify ligands for the murine intestinal intraepithelial gammadelta T cell population

Although the functions and antigen recognition requirements of alphabeta T cells are well characterised, the antigens recognised by gammadelta T cells and the consequences of this recognition are unclear. gammadelta T cells are enriched within epithelia, where they eradicate transformed epithelial cells and regulate inflammation. To understand how this occurs, we need to understand the cellular ligands recognised by the gammadelta cell through the gammadelta T-cell receptor (TCR). We have therefore generated a soluble TCR (sTCR) to identify ligands for the murine gammadelta intestinal intraepithelial lymphocyte (IEL) population. sTCR was produced in the baculovirus expression system and purified by affinity chromatography on an anti-TCRdelta affinity column. sTCR was recognised by a panel of conformation-specific anti-TCRgammadelta antibodies. We will now use our sTCR to directly test the binding of putative ligands to the TCR using surface plasmon resonance, and to isolate the ligand biochemically.     

The T cell receptor's alpha-chain connecting peptide motif promotes close approximation of the CD8 coreceptor allowing efficient signal initiation

The CD8 coreceptor contributes to the recognition of peptide-MHC (pMHC) ligands by stabilizing the TCR-pMHC interaction and enabling efficient signaling initiation. It is unclear though, which structural elements of the TCR ensure a productive association of the coreceptor. The alpha-chain connecting peptide motif (alpha-CPM) is a highly conserved sequence of eight amino acids in the membrane proximal region of the TCR alpha-chain. TCRs lacking the alpha-CPM respond poorly to low-affinity pMHC ligands and are unable to induce positive thymic selection. In this study we show that CD8 participation in ligand binding is compromised in T lineage cells expressing mutant alpha-CPM TCRs, leading to a slight reduction in apparent affinity; however, this by itself does not explain the thymic selection defect. By fluorescence resonance energy transfer microscopy, we found that TCR-CD8 association was compromised for TCRs lacking the alpha-CPM. Although high-affinity (negative-selecting) pMHC ligands showed reduced TCR-CD8 interaction, low-affinity (positive-selecting) ligands completely failed to induce molecular approximation of the TCR and its coreceptor. Therefore, the alpha-CPM of a TCR is an important element in mediating CD8 approximation and signal initiation.     

T cell receptors are structures capable of initiating signaling in the absence of large conformational rearrangements

Native and non-native ligands of the T cell receptor (TCR), including antibodies, have been proposed to induce signaling in T cells via intra- or intersubunit conformational rearrangements within the extracellular regions of TCR complexes. We have investigated whether any signatures can be found for such postulated structural changes during TCR triggering induced by antibodies, using crystallographic and mutagenesis-based approaches. The crystal structure of murine CD3ε complexed with the mitogenic anti-CD3ε antibody 2C11 enabled the first direct structural comparisons of antibody-liganded and unliganded forms of CD3ε from a single species, which revealed that antibody binding does not induce any substantial rearrangements within CD3ε. Saturation mutagenesis of surface-exposed CD3ε residues, coupled with assays of antibody-induced signaling by the mutated complexes, suggests a new configuration for the complex within which CD3ε is highly exposed and reveals that no large new CD3ε interfaces are required to form during antibody-induced signaling. The TCR complex therefore appears to be a structure that is capable of initiating intracellular signaling in T cells without substantial structural rearrangements within or between the component subunits. Our findings raise the possibility that signaling by native ligands might also be initiated in the absence of large structural rearrangements in the receptor.     

Coreceptor CD8-driven modulation of T cell antigen receptor specificity

The CD8 coreceptor modulates the interaction between the T cell antigen receptor (TCR) and peptide-major histocompatibility class I (pMHCI). We present evidence that CD8 not only modifies the affinity of cognate TCR/pMHCI binding by altering both the association rate and the dissociation rate of the TCR/pMHCI interaction, but modulates the sensitivity (triggering threshold) of the TCR as well, by recruiting TCR/pMHCI complexes to membrane microdomains at a rate which depends on the affinity of MHCI/CD8 binding. Mathematical analysis of these modulatory effects indicates that a T cell can alter its functional avidity for its agonists by regulating CD8 expression, and can rearrange the relative potencies of each of its potential agonists. Thus we propose that a T cell can specifically increase its functional avidity for one agonist, while decreasing its functional avidity for other potential ligands. This focussing mechanism means that TCR degeneracy is inherently dynamic, allowing each TCR clonotype to have a wide range of agonists while avoiding autorecognition. The functional diversity of the TCR repertoire would therefore be greatly augmented by coreceptor-mediated ligand focussing.     

Critical relationship between TCR signaling potential and TCR affinity during thymocyte selection

Whether a developing thymocyte becomes positively or negatively selected is thought to be determined by the affinity/avidity of its TCR for MHC/peptide ligands expressed in the thymus. Presumably, differences in affinity translate into differences in the potency of the ensuing TCR-mediated signals, and these differences in signal strength determine the outcome of thymocyte selection. However, there is little direct evidence establishing a relationship between TCR-ligand affinity and signal strength during positive and negative selection. The TCR complex contains multiple signaling motifs, known as immunoreceptor tyrosine-based activation motifs (ITAMs) that are required for T cell activation. To examine the effects of TCR signal strength on selection, the signaling potential of the TCR was modified by substituting transgenic TCR zeta-chains containing either three, one, or zero ITAMs for endogenous (3-ITAM) zeta-chain. These zeta-chain variants were then bred into different alphabetaTCR transgenic backgrounds. We report that reductions in TCR signaling potential have distinct effects on the selection of thymocytes expressing different TCRs, and that the requirement for zeta-chain ITAMs critically depends upon the specificity and apparently, affinity, of the TCR for its selecting ligand(s).     

T cell tolerance induced by cross-reactive TCR ligands can be broken by superagonist resulting in anti-inflammatory T cell cytokine production

Cross-reactive activation of potentially autoreactive T cells by high-affinity nonself ligands may be important in breaking self-tolerance in autoimmunity. In a mouse transgenic for a cross-reactive TCR, we have previously shown that a hyper-stimulating altered peptide ligand, L144, induced unresponsiveness to the self peptide, proteolipid protein 139-151. In this study, we demonstrate that a superagonist ligand can break T cell tolerance induced by the lower affinity cognate Ag. T cells tolerant to the cognate ligand, Q144, responded to superagonist, L144, by proliferation and the production of mainly IL-4 and IL-10 in vitro. In contrast, T cells that were tolerized to the superagonist were unable to respond to any peptide that cross-reacted with the transgenic TCR. Low-dose immunization with the superagonist L144 was able to break tolerance to the cognate ligand in vivo and resulted in a blunted proliferative response with production of Th2 cytokines.     

Height,but not binding epitope,affects the potency of synthetic TCR agonists

Under physiological conditions, peptide-major histocompatibility complex (pMHC) molecules can trigger T cell receptors (TCRs) as monovalent ligands that are sparsely distributed on the plasma membrane of an antigen-presenting cell. TCRs can also be triggered by artificial clustering, such as with pMHC tetramers or antibodies; however, these strategies circumvent many of the natural ligand discrimination mechanisms of the T cell and can elicit nonphysiological signaling activity. We have recently introduced a synthetic TCR agonist composed of an anti-TCRβ Fab′ antibody fragment covalently bound to a DNA oligonucleotide, which serves as a membrane anchor. This Fab′-DNA ligand efficiently triggers TCR as a monomer when membrane associated and exhibits a potency and activation profile resembling agonist pMHC. In this report, we explore the geometric requirements for efficient TCR triggering and cellular activation by Fab′-DNA ligands. We find that T cells are insensitive to the ligand binding epitope on the TCR complex but that length of the DNA tether is important. Increasing, the intermembrane distance spanned by Fab′-DNA:TCR complexes decreases TCR triggering efficiency and T cell activation potency, consistent with the kinetic-segregation model of TCR triggering. These results establish design parameters for constructing synthetic TCR agonists that are able to activate polyclonal T cell populations, such as T cells from a human patient, in a similar manner as the native pMHC ligand.     

Non-Fc receptor-binding humanized anti-CD3 antibodies induce apoptosis of activated human T cells

Human trials in organ allografts have demonstrated that murine anti-CD3 mAbs are immunosuppressive. By mimicking Ag, anti-CD3 can produce T cell activation, anergy, or death. Activation of resting T cells in vivo results in dose-limiting cytokine release and is caused by Ab-mediated cross-linking of T cells and Fcgamma receptor (FcR)-bearing cells. With the goal of minimizing cytokine-induced toxicity, anti-CD3 have been engineered to lower Fc binding avidity. Preclinical murine studies have indicated that non-FcR-binding anti-CD3 can induce apoptosis of Ag-activated T cells. Since induction of T cell apoptosis may be an important mechanism of immunosuppression by anti-CD3, we tested whether Fc mutations affect the ability of anti-human CD3 to induce apoptosis of activated T cells. We compared wild-type murine anti-CD3, M291, and OKT3 and their humanized, FcR- and non-FcR-binding structural variants in quantitative assays of T cell apoptosis. Non-FcR-binding variants produced more sustainable phosphorylation of extracellular signal-regulated kinase-2, greater release of IFN-gamma, and more effectively caused activation-dependent T cell apoptosis. Non-FcR-binding variants dissociated more quickly from the T cell surface and caused less internalization of the TCR, which then remained available in greater abundance on the cell surface for signaling. Cross-linking of non-FcR-binding variants by antiglobulin enhanced TCR internalization and minimized induction of T cell apoptosis. We conclude that non-FcR-binding, humanized anti-CD3 have improved ability to induce apoptosis of activated T cells, presumably by allowing durable expression of the TCR and sustained signaling.     

Evidence that structural rearrangements and/or flexibility during TCR binding can contribute to T cell activation

While in many cases the half-life of T cell receptor (TCR) binding to a particular ligand is a good predictor of activation potential, numerous exceptions suggest that other physical parameter(s) must also play a role. Accordingly, we analyzed the thermodynamics of TCR binding to a series of peptide-MHC ligands, three of which are more stimulatory than their stability of binding would predict. Strikingly, we find that during TCR binding these outliers show anomalously large changes in heat capacity, an indicator of conformational change or flexibility in a binding interaction. By combining the values for heat capacity (DeltaCp) and the half-life of TCR binding (t(1/2)), we find that we can accurately predict the degree of T cell stimulation. Structural analysis shows significant changes in the central TCR contact residue of the peptide-MHC, indicating that structural rearrangements within the TCR-peptide-MHC interface can contribute to T cell activation.     

Partial T cell activation with an altered superantigenic ligand

T cells have the capacity to respond to ligands as full, weak, partial or null agonists, or indeed as antagonists. In the present paper, it is reported that staphylococcal enterotoxin B (SEB) mutated in a T cell receptor (TCR) contact site (SEBDelta61Y) behaves as an altered ligand for a T cell clone (AC20) that expresses the Vbeta17 TCR. The T cells were partially activated by SEBDelta61Y, as shown by TCR down-modulation and up-regulation of the IL-2 receptor. However, these cells did not secrete IL-2, IL-3, IL-4 or IFN-gamma, nor did they proliferate. Analysis of intracellular protein tyrosine phosphorylation after cellular activation provided further evidence that SEBDelta61Y could transduce a signal via the Vbeta17 TCR. The events following receptor ligation were clearly different when the T cells were stimulated with SEB or SEBDelta61Y, manifested as both quantitatively and qualitatively different patterns of phosphorylation of intracellular substrates. In contrast, only quantitative differences were apparent when a transfectant expressing the same alpha/beta TCR was stimulated with the different superantigens. Together, these results provide the first demonstration that altered TCR ligands are not restricted to peptides substituted at secondary TCR contact residues. Rather, an altered superantigenic ligand mutated in the TCR binding site can behave as a partial agonist.     

TCR affinity promotes CD8+ T cell expansion by regulating survival

Ligation with high affinity ligands are known to induce T lymphocytes to become fully activated effector cells while ligation with low affinity ligands (or partial agonists) may result in a delayed or incomplete response. We have examined the quantitative features of CD8(+) T cell proliferation induced by peptides of different TCR affinities at a range of concentrations in the mouse OT-I model. Both the frequency of cells responding and the average time taken for cells to reach their first division are affected by peptide concentration and affinity. Consecutive division times, however, remained largely unaffected by these variables. Importantly, we identified affinity to be the sole regulator of cell death in subsequent division. These results suggest a mechanism whereby TCR affinity detection can modulate the subsequent rate of T cell growth and ensure the dominance of higher affinity clones over time.     

The study of high-affinity TCRs reveals duality in T cell recognition of antigen: specificity and degeneracy

TCRs exhibit a high degree of Ag specificity, even though their affinity for the peptide/MHC ligand is in the micromolar range. To explore how Ag specificity is achieved, we studied murine T cells expressing high-affinity TCRs engineered by in vitro evolution for binding to hemoglobin peptide/class II complex (Hb/I-Ek). These TCRs were shown previously to maintain Ag specificity, despite having up to 800-fold higher affinity. We compared the response of the high-affinity TCRs and the low-affinity 3.L2 TCR toward a comprehensive set of peptides containing single substitutions at each TCR contact residue. This specificity analysis revealed that the increase in affinity resulted in a dramatic increase in the number of stimulatory peptides. The apparent discrepancy between observed degeneracy in the recognition of single amino acid-substituted Hb peptides and overall Ag specificity of the high-affinity TCRs was examined by generating chimeric peptides between the stimulatory Hb and nonstimulatory moth cytochrome c peptides. These experiments showed that MHC anchor residues significantly affected TCR recognition of peptide. The high-affinity TCRs allowed us to estimate the affinity, in the millimolar range, of immunologically relevant interactions of the TCR with peptide/MHC ligands that were previously unmeasurable because of their weak nature. Thus, through the study of high-affinity TCRs, we demonstrated that a TCR is more tolerant of single TCR contact residue substitutions than other peptide changes, revealing that recognition of Ag by T cells can exhibit both specificity and degeneracy.     

Kinetics of MHC-CD8 interaction at the T cell membrane

CD8 plays an important role in facilitating TCR-MHC interaction, promoting Ag recognition, and initiating T cell activation. MHC-CD8 binding kinetics have been measured in three dimensions by surface plasmon resonance technique using purified molecules. However, CD8 is a membrane-anchored, signaling kinase-linked, and TCR-associated molecule whose function depends on the cell membrane environment. Purified molecules lack their linkage to the membrane, which precludes interactions with other structures of the cell as well as signaling. Furthermore, three-dimensional binding in the fluid phase is biologically and physically distinct from two-dimensional binding across apposing cell membranes. As a first step toward characterizing the molecular interactions between T cells and APCs, we used a micropipette adhesion frequency assay to measure the adhesion kinetics of single mouse T cells interacting with single human RBCs coated with MHC. Using anti-TCR mAb we isolated and characterized the specific two-dimensional MHC-CD8 binding from the trimolecular TCR-MHC-CD8 interaction. The TCR-independent MHC-CD8 interaction has a very low affinity that depends on the MHC alleles, but not on the peptide complexed to the MHC and whether CD8 is an alphaalpha homodimer or an alphabeta heterodimer. Surprisingly, MHC-CD8 binding affinity varies with T cells from different TCR transgenic mice and these affinity differences were abolished by treatment with cholesterol oxidase to disrupt membrane rafts. These data highlight the relevance and importance of two-dimensional analysis of T cells and APCs and indicate that membrane rafts play an important role in modulating the affinity of cell-cell interactions.     

Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness

Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ～1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ～1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies.