A conserved energetic footprint underpins recognition of human leukocyte antigen-E by two distinct αß T cell receptors.

αß T cell receptors (TCRs) interact with peptides bound to the polymorphic major histocompatibility complex class Ia (MHC-Ia) and class II (MHC-II) molecules as well as the essentially monomorphic MHC class Ib (MHC-Ib) molecules. Although there is a large amount of information on how TCRs engage with MHC-Ia and MHC-II, our understanding of TCR/MHC-Ib interactions is very limited. Infection with cytomegalovirus (CMV) can elicit a CD8+ T cell response restricted by the human MHC-Ib molecule human leukocyte antigen (HLA)-E and specific for an epitope from UL40 (VMAPRTLIL), which is characterized by biased TRBV14 gene usage. Here we describe an HLA-E-restricted CD8+ T cell able to recognize an allotypic variant of the UL40 peptide with a modification at position 8 (P8) of the peptide (VMAPRTLVL) that uses the TRBV9 gene segment. We report the structures of a TRBV9+ TCR in complex with the HLA-E molecule presenting the two peptides. Our data revealed that the TRBV9+ TCR adopts a different docking mode and molecular footprint atop HLA-E when compared with the TRBV14+ TCR-HLA-E ternary complex. Additionally, despite their differing V gene segment usage and different docking mechanisms, mutational analyses showed that the TCRs shared a conserved energetic footprint on the HLA-E molecule, focused around the peptide-binding groove. Hence, we provide new insights into how monomorphic MHC molecules interact with T cells.

Subtle changes in TCRα CDR1 profoundly increase the sensitivity of CD4 T cells.

Changes in the peptide and MHC molecules have been extensively examined for how they alter T cell activation, but many fewer studies have examined the TCR. Structural studies of how TCR differences alter T cell specificity have focused on broad variation in the CDR3 loops. However, changes in the CDR1 and 2 loops can also alter TCR recognition of pMHC. In this study we focus on two mutations in the CDR1α loop of the TCR that increased the affinity of a TCR for agonist Hb(64-76)/I-E(k) by increasing the on-rate of the reaction. These same mutations also conferred broader recognition of altered peptide ligands. TCR transgenic mice expressing the CDR1α mutations had altered thymic selection, as most of the T cells were negatively selected compared to T cells expressing the wildtype TCR. The few T cells that escaped negative selection and were found in the periphery were rendered anergic, thereby avoiding autoimmunity. T cells with the CDR1α mutations were completely deleted in the presence of Hb(64-76) as an endogenous peptide. Interestingly, the wildtype T cells were not eliminated, identifying a threshold affinity for negative selection where a 3-fold increase in affinity is the difference between incomplete and complete deletion. Overall, these studies highlight how small changes in the TCR can increase the affinity of TCR:pMHC but with the consequences of skewing selection and producing an unresponsive T cell.

Localization of NK1.1(+) invariant Valpha19 TCR(+) cells in the liver with potential to promptly respond to TCR stimulation.

Previously, we found that more than a half of the NK1.1(+) T cell lines prepared from CD1(-/-) livers expressed invariant Valpha19-Jalpha33 TCR alpha chains. Over-expression of the invariant Valpha19-Jalpha33 TCR alpha transgene (Tg) with a natural TCR alpha promoter and an enhancer in mice induced the development of NK1.1(+) T cells (Valpha19 NKT cells) in the lymphoid organs, especially in the liver. Preferential usage of the Valpha19 Tg by NKT cells in the transgenic mouse livers was indirectly indicated by the observation that few NK1.1(+) TCRalphabeta(+) cells of the Valpha19 Tg livers were stained with a cocktail of anti-TCR Valpha antibodies in the FACS analysis. Upon invariant TCR engagement in vivo following injection of mice with anti-CD3 antibody, NKT cells of the Tg mouse livers as well as spleens promptly produced immunoregulatory cytokines such as IL-4 and IFN-gamma and altered surface receptor expression. Collectively, localization of Valpha19 NKT cells in the liver is suggested that are ready to immediately response against antigen stimulation.

Modulation of CD4 co-receptor limits spontaneous autoimmunity when high-affinity transgenic TCR specific for self-antigen is expressed on a genetically resistant background.

Myelin proteolipid protein (PLP) 139-151 is an immunodominant peptide that induces experimental autoimmune encephalomyelitis (EAE) in H-2(s) SJL/J mice. While PLP 139-151-specific TCR transgenic (tg) 4E3 mice develop fulminant spontaneous disease on the susceptible SJL/J background, spontaneous EAE is dramatically reduced on the H-2(s) congenic B10.S background. On this resistant background, we observed a high frequency of positively selected tg CD4-CD8- (DN) thymocytes and peripheral DN tg T cells. Splenic DN tg T cells responded to anti-CD3 stimulation similarly to CD4+ cells, but proliferative and cytokine responses to PLP 139-151 were blunted, implying that CD4 co-receptor down-regulation modulated T cell responses to the self-antigen in vitro. Adoptive transfer of tg DN CD3hi cells into RAG-deficient wild-type (WT) recipients induced EAE less efficiently than transfer of CD4+ T tg cells indicating the blunted responses of DN tg T cells to self-antigen in vivo. The frequency of tg DN T cells was irrespective of thymic expression of the autoantigen. These data implicate that down-regulation of CD4 co-receptor in the thymus, which is independent from the expression of thymic autoantigen, results in a blunted response to the autoantigen in the periphery and limits the incidence of spontaneous autoimmunity in genetically resistant mice bearing a large autoreactive tg T cell repertoire.

TCRgamma silencing during alphabeta T cell development depends upon pre-TCR-induced proliferation.

During thymus development, immature T cells become committed to two distinct lineages based upon expression of alphabeta or gammadelta TCR. In the alphabeta lineage, developing thymocytes progressively extinguish transcription of the TCRgamma genes by a poorly understood process known as gamma silencing. We show that alphabeta lineage thymocytes in mice lacking a functional pre-TCR undergo limited proliferation and fail to silence TCRgamma genes during development. Stimulation of pre-TCR-deficient immature thymocytes with anti-CD3 Abs does not directly down-regulate TCRgamma transcription but restores TCRgamma silencing following proliferation. Collectively our data reveal an important role for pre-TCR induced proliferation in activating the TCRgamma silencer in alphabeta lineage thymocytes, a process that may reinforce alphabeta or gammadelta lineage commitment.

Binding of natural variants of staphylococcal superantigens SEG and SEI to TCR and MHC class II molecule.

SEG and SEI are staphylococcal superantigens (SAgs) identified recently that belong to the egc operon and whose genes are in tandem orientation. Only a few allelic variants of SEG and SEI have been reported. Here we analyzed four Staphylococcus aureus strains with genotypic variation in both SAgs. However, both SAgs retain key residues in their putative TCR and MHC binding sites and, accordingly, their superantigenic properties. Thus, SEI significantly stimulates mouse T-cells bearing Vbeta3, 5 and 13, while SEG stimulates Vbeta7 and 9 in the draining node when inoculated in the footpad. As another member of the SEB subfamily, SEG also stimulates mouse Vbeta8.1+2. However, the increase in Vbeta8.1+2 T-cells observed at day 2 after inoculation reverts to normal values at day 4, whereas it remains high at day 4 following inoculation with SEC3 or SSA. T-cell stimulation assays in the mouse and analysis of the putative Vbeta8.2 binding site on SEG, which includes three non-conserved residues, suggest a possibly unique interaction between Vbeta8.2 and SEG. We also analyzed biochemical and biophysical characteristics of SEI and SEG binding to their cognate human beta chains by surface plasmon resonance, and binding to the HLA-DR1 MHC class II molecule by gel filtration. SEI binds human Vbeta5.2 and Vbeta1 with apparent K(D)'s of 23 and 118 microM, respectively; SEG binds Vbeta13.6 with a K(D) of 5 microM. As suggested by sequence homology, SEI requires Zn2+ for strong binding to DR1, which goes undetected in the presence of EDTA. SEG and SEI have characteristics such as co-expression, different interaction with MHC class II and stimulation of completely different subsets of human and mouse T-cells, which indicate complementary superantigenic activity and suggest an important advantage to staphylococcal strains in producing them both.

The fourth way? Harnessing aggressive tendencies in the thymus.

During late stages of thymic development, T cells must chose between different fates, dictated by their TCR specificity. Typically, this is thought of as a choice between three alternatives (being positive selection for useful T cells vs negative selection or neglect for harmful or useless T cells). However, there is growing evidence for a fourth alternative, in which T cells are positively selected by agonist ligands, which would normally be expected to induce T cell deletion. In this review, we will discuss where and when agonist selection is induced and whether this should be considered as a novel form of thymic selection or as an alternative differentiation state for Ag-exposed T cells.

Conversion of a T cell antagonist into an agonist by repairing a defect in the TCR/peptide/MHC interface: implications for TCR signaling.

The structure of the A6 alphabetaTCR/HTLV-1 Tax-peptide/MHC I complex with proline 6 of Tax substituted with alanine (P6A), an antagonist, is nearly identical to the structure with wild-type Tax agonist. Neither the proline in the agonist nor the alanine in the antagonist is contacted by the alphabetaTCR. Here, we demonstrate that antagonist activity of P6A is associated with low affinity of the A6 alphabetaTCR for Tax-P6A/HLA-A2. We show that stepwise repair of a packing defect in the TCR/MHC interface using N-alkylated amino acids results in stepwise increases in TCR affinity and activity. Kinetic and thermodynamic measurements suggest that for some ligands the range of T cell outcomes does not correlate with either their alphabetaTCR affinity or the half-life of the alphabetaTCR/peptide/MHC complex.

Cross-antagonism of a T cell clone expressing two distinct T cell receptors.

Inhibition of T cell activation can be mediated by analogs of the original antigenic peptide (TCR antagonists). Here, a T cell clone expressing two distinct TCR was used to investigate whether such inhibition involves an active mechanism by examining whether an antagonist for one TCR could influence responses stimulated by the other TCR engaging its agonist. Our results demonstrate functional cross-inhibition under these conditions involving the ability of antagonist: TCR interactions to diminish Lck enzymatic activity associated with the agonist-recognizing second TCR, apparently through enhancement of SHP-1 association with these receptors. Our findings reveal that inhibition of cellular responses by antagonists arises at least in part from active negative regulation of proximal TCR signaling and identify elements of the biochemical process.

Four A6-TCR/peptide/HLA-A2 structures that generate very different T cell signals are nearly identical.

The interactions of three singly substituted peptide variants of the HTLV-1 Tax peptide bound to HLA-A2 with the A6 T cell receptor have been studied using T cell assays, kinetic and thermodynamic measurements, and X-ray crystallography. The three peptide/MHC ligands include weak agonists and antagonists with different affinities for TCR. The three-dimensional structures of the three A6-TCR/peptide/HLA-A2 complexes are remarkably similar to each other and to the wild-type agonist complex, with minor adjustments at the interface to accommodate the peptide substitutions (P6A, V7R, and Y8A). The lack of correlation between structural changes and the type of T cell signals induced provides direct evidence that different signals are not generated by different ligand-induced conformational changes in the alphabeta TCR.

Positive selection of low responsive, potentially autoreactive T cells induced by high avidity, non-deleting interactions.

Using a novel cell suspension model we investigated the relative abilities of nominal peptide and variants thereof to modulate de novo positive selection of lymphocytic choriomeningitis virus (LCMV)-specific TCR transgenic T cells. Confirming our earlier findings intermediate concentrations (10(-7) to 10(-5) M) of the nominal agonist peptide, p33, induced CD8 co-receptor down-modulation at the level of the entire receptor and the CD8beta chain as a consequence of high but non-deleting signal interactions. Agonist peptide variants caused down-modulation of the CD8beta chain but to a lesser degree. An antagonist peptide capable of inducing positive selection did not cause such modifications of the co-receptor. The positively selected TCRhiCD8alpha alpha and TCRhiCD8- cells were functional but not as efficient as TCRhiCD8alphabeta cells, presumably due to lower avidity interactions in the absence of the CD8beta chain or entire co-receptor. CD8beta mRNA was absent in these cells and was not up-regulated when further stimulated with fresh antigen-presenting cells pulsed with 10(-5) M p33. Effectively our data suggest that it is not the agonist or antagonist nature of a peptide per se but the overall strength of signalling that determines whether a cell will be positively or negatively selected, or die by neglect. Furthermore the agonist/antagonist properties of peptides defined at the level of mature T cell function do not unequivocally predict their effect on positive/negative selection. The ability of the T cell to down-modulate its CD8 co-receptor in response to high but non-deleting peptide interactions during positive selection allows the survival of T cells with a broader range of affinities and represents a possible mechanism by which low responsive but potentially autoreactive cells may escape into the periphery.

Partial signaling by CD8+ T cells in response to antagonist ligands.

Structural variants of an agonist peptide-major histocompatibility complex (MHC) molecule ligand can show partial agonist and/or antagonist properties. A number of such altered ligands appear to act as pure antagonists. They lack any detectable ability to induce T cell effector function and have been described as unable to induce calcium transients and turnover of inositol phosphates. This has been interpreted as an inability of these ligands to initiate any T cell receptor (TCR)-dependent signal transduction, with their antagonist properties ascribed to competition with offered agonist for TCR occupancy. Yet antagonists for mature CD8+ T cells can induce positive selection of thymocytes, implying active induction of T cell differentiation events, and partial agonists or agonist/antagonist combinations elicit a distinctive pattern of early TCR-associated tyrosine phosphorylation events in CD4+ T cells. We have therefore directly examined proximal TCR signaling in a CD8+ T cell line in response to various related ligands. TCR engagement with natural peptide-MHC class I agonist resulted in the same pattern of early TCR-associated tyrosine phosphorylation events as seen with CD4+ cells, including accumulation of both the p21 and p23 forms of phosphorylated zeta, phosphorylation of CD3 epsilon, and association of phosphorylated ZAP-70 with the TCR. Two antagonists that lacked the ability to induce any detectable CTL effector response (cytolysis, esterase release, gamma interferon secretion, interleukin-2 receptor alpha upregulation) were nevertheless found to also induce TCR-dependent phosphorylation events. In these cases, there was preferential accumulation of the p21 form of phospho-zeta without net phosphorylation of CD3 epsilon, as well as the association of nonphosphorylated ZAP-70 kinase with the receptor. These data show that variant ligands induce similar TCR-dependent phosphorylation events in CD8+ T cells as first observed in CD4+ cells. More importantly, they demonstrate that some putatively pure antagonists are actually a subset of partial agonists able to induce intracellular biochemical changes through the TCR. This delivery of a partial signal by antagonists raises the possibility that antagonism in some cases may result from active interference with stimulation of effector activity by agonist in mature T cells, while the same variant signal could selectively trigger intracellular events that allow positive without negative selection in thymocytes.