T cell receptor recognition via cooperative conformational plasticity

Although T cell receptor cross-reactivity is a fundamental property of the immune system and is implicated in numerous autoimmune pathologies, the molecular mechanisms by which T cell receptors can recognize and respond to diverse ligands are incompletely understood. In the current study we examined the response of the human T cell lymphotropic virus-1 (HTLV-1) Tax-specific T cell receptor (TCR) A6 to a panel of structurally distinct haptens coupled to the Tax 11-19 peptide with a lysine substitution at position 5 (Tax5K, LLFG[K-hapten]PVYV). The A6 TCR could cross-reactively recognize one of these haptenated peptides, Tax-5K-4-(3-Indolyl)-butyric acid (IBA), presented by HLA-A*0201. The crystal structures of Tax5K-IBA/HLA-A2 free and in complex with A6 reveal that binding is mediated by a mechanism of cooperative conformational plasticity involving conformational changes on both sides of the protein-protein interface, including the TCR complementarity determining region (CDR) loops, Valpha/Vbeta domain orientation, and the hapten-modified peptide. Our findings illustrate the complex role that protein dynamics can play in TCR cross-reactivity and highlight that T cell receptor recognition of ligand can be achieved through diverse and complex molecular mechanisms that can occur simultaneously in the interface, not limited to molecular mimicry and CDR loop shifts.     

Identification of T cell recognition sites in S-antigen: dissociation of proliferative and pathogenic sites

Experimental autoimmune uveoretinitis (EAU) is a predominantly CD4+ T cell-mediated autoimmune inflammatory disease of the retina and uveal tract of the eye and the pineal gland. S-antigen, a protein found in retinal photoreceptor cells and pinealocytes, is a potent agent for the induction of EAU in susceptible species and strains. In order to identify the T cell recognition sites of S-antigen responsible for its uveitogenicity and proliferative responses, cyanogen bromide (CB) fragments as well as synthetic peptides were used to test the proliferative responses of two uveitogenic T cell lines, R9 and R17, prepared against native bovine and human S-antigen, respectively. Two nonoverlapping synthetic peptides which are known to actively induce EAU, amino acid residues 286-297 and 303-314 of the bovine sequence, were unable to induce proliferative responses in either S-antigen-specific T cell line. However, both of these sites were adjacent to synthetic peptides, residues 273-292 and 317-328, respectively, which were unable to actively induce EAU, but elicited strong proliferative responses from T cell lines raised to bovine and human S-antigen. Repeated in vitro selection of the R9 T cell line with a synthetic peptide containing one of these proliferative sites, residues 317-328, gave rise to a transiently uveitogenic T cell line. Several species-specific T cell epitopes were identified, but none of these were found to be involved in a uveitogenic response. Our results indicate that spatially separated and distinct T cell epitopes are present in S-antigen which are responsible for the active induction of EAU, lymphocyte proliferation, and the ability to adoptively transfer EAU.     

Cross-reactive antigen recognition by an encephalitogenic T cell receptor. Implications for T cell biology and autoimmunity.

The dominant immune response to rat myelin basic protein in H-2u mice is directed against the acetylated, N-terminal peptide Ac1-11 (AcASQKR-PSQRHG). This peptide causes encephalomyelitis on injection into mice of the H-2u haplotype. Only two residues of the peptide are required for ligation of the TCR from an Ac1-11-specific T cell hybridoma. Proline at position 6 could not be substituted by any other L-amino acid, whereas glutamine at position 3 could be replaced by phenylalanine, histidine, methionine, or tyrosine. Cross-reactive recognition of these residues appears to be specific, because increasing the affinity of each analogue for its MHC restriction element, by replacing lysine with tyrosine at position 4, did not alter the pattern of cross-reactivity. For the majority of substitutions at this position, a lack of stimulation could not be explained by failure to bind to I-Au. However, competition binding studies showed that introduction of proline at position 3 reduced the efficacy of binding to I-Au. Cross-reactive analogues of Ac1-11 were injected into H-2u mice to test the extent to which cross-reactive T cell activation might lead to autoimmune disease in this model. An analogue containing methionine at position 3 caused clinical experimental autoimmune encephalomyelitis in a small percentage of H-2u mice.     

Human T cell subpopulations defined by a monoclonal antibody. II. Evidence for cell cooperation in the response to alloantigens and generation of cytolytic cells

It has been shown previously that the 5/9 monoclonal antibody defines a small T cell subpopulation in human peripheral blood that includes all the cells responsible for proliferation to tetanus toxoid and to alloantigens as well as the helper cells for B cell differentiation. In the present study, human peripheral blood T cells were fractionated according to their reactivity with the 5/9 monoclonal antibody and stimulated in mixed lymphocyte culture (MLC). In spite of a strong proliferative response in MLC, 5/9+ cells generated no cytolytic activity against PHA-activated lymphocytes bearing the stimulating alloantigens (CTL activity) or against the K562 human cell line (NK activity). The precursors of these cytolytic effector cells were present in the 5/9- fraction. However, 5/9+ cells or soluble factors derived from 5/9+ cells were needed to induce 5/9- cells to respond in MLC and develop cytolytic activity. Both 5/9+ and 5/9- cell populations, upon MLC stimulation, were able to lyse L1210 mouse lymphoma cells in the presence of specific antibodies (ADCC).     

Structural bases of T cell antigen receptor recognition in celiac disease

Celiac disease (CeD) is a human leukocyte antigen (HLA)-linked autoimmune-like disorder that is triggered by the ingestion of gluten or related storage proteins. The majority of CeD patients are HLA-DQ2.5⁺, with the remainder being either HLA-DQ8⁺ or HLA-DQ2.2⁺. Structural studies have shown how deamidation of gluten epitopes engenders binding to HLA-DQ2.5/8, which then triggers an aberrant CD4⁺ T cell response. HLA tetramer studies, combined with structural investigations, have demonstrated that repeated patterns of TCR usage underpins the immune response to some HLADQ2.5/8 restricted gluten epitopes, with distinct TCR motifs representing common landing pads atop the HLA–gluten complexes. Structural studies have provided insight into TCR specificity and cross-reactivity towards gluten epitopes, as well as cross-reactivity to bacterial homologues of gluten epitopes, suggesting that environmental factors may directly play a role in CeD pathogenesis. Collectively, structural immunology-based studies in the CeD axis may lead to new therapeutics/diagnostics to treat CeD, and also serve as an exemplar for other T cell mediated autoimmune diseases.     

The mouse T cell receptor: structural heterogeneity of molecules of normal T cells defined by xenoantiserum

We have previously demonstrated that T lymphomas may express clonally specific epitopes that are carried by a T-cell-restricted, disulfide-bonded heterodimeric glycoprotein. We have used a monoclonal antibody, 124-40, to isolate the lymphoma-specific antigen and raise a xenoantiserum to the molecule. This antiserum immunoprecipitates a family of disulfide-bonded dimers from normal thymocytes and T cells, but is unreactive with B cells. Peptide maps prepared after limited proteolytic digestion indicate that the molecules from the different cell populations have homologous primary structures. Comparison of two-dimensional tryptic peptide maps indicate that, in addition to several common peptides, the molecules exhibit considerable structural heterogeneity. Taken together, these data indicate that the T-cell-specific heteroduplex has regions of constant and variable structure consistent with the properties expected for the T cell antigen receptor.     

CD8-independent tumor cell recognition is a property of the T cell receptor and not the T cell

The CD8 coreceptor enhances T cell function by stabilizing the TCR/peptide/MHC complex and/or increasing T cell avidity via interactions with the intracellular kinases Lck and LAT. We previously reported a CD4(+) T cell (TIL 1383I), which recognizes the tumor-associated Ag tyrosinase in the context of HLA-A2. To determine whether CD8 independent tumor cell recognition is a property of the TCR, we used retroviral transduction to express the TIL 1383I TCR in the CD8(-) murine lymphoma, 58 alpha(-)/beta(-). Immunofluorescent staining of TCR-transduced cells with human TCR V beta subfamily-specific and mouse CD3-specific Abs confirmed surface expression of the transferred TCR and coexpression of mouse CD3. Transduced effector cells secreted significant amounts of IL-2 following Ag presentation by tyrosinase peptide-pulsed T2 cells as well as stimulation with HLA-A2(+) melanoma lines compared with T2 cells alone or HLA-A2(-) melanoma cells. Further analysis of TCR-transduced clones demonstrated a correlation between T cell avidity and cell surface expression of the TCR. Therefore, the TIL 1383I TCR has sufficient affinity to mediate recognition of the physiologic levels of Ag expressed by tumor cells in the absence of CD8 expression.     

Multisite mutagenesis of interleukin 5 differentiates sites for receptor recognition and receptor activation

Multisite mutagenesis of single-chain and monomeric forms of human interleukin 5 (IL-5) was performed to investigate mechanistic features of receptor activation and the possibility of differentiating sites of activation from those for receptor interaction. The normally dimeric human IL-5 contains two domains, each containing a four-helix bundle. IL-5 has previously been re-engineered into the monomeric, one-domain GM1 form by introducing an eight-residue linker between the third and fourth helices. In this study, we tested a combination of mutations in a single-chain IL-5 (scIL-5) construct, [(89)SLRGG(92),W(110)/(89)AAAAA(92), A(110)]scIL-5. This mutein was found to retain substantial IL-5 receptor alpha-chain binding but with selectively suppressed proliferation of the IL-5-dependent cell line TF-1.28. This result confirms recent findings that IL-5 receptor alpha-chain recognition can be supported by the (89)SLRGG(92) epitope and that, in contrast, Glu110 is important in receptor activation. On the basis of this result, two mutants of GM1 were constructed with the intent to retain receptor alpha-chain binding while modifying receptor activation epitopes. In the first, [(88)SLRGG(92),W(110)]GM1, the wild-type CD-loop sequence (89)EERRR(92) was converted to the mimotope (89)SLRGG(92), and Glu110 to Trp. In the second, [A(13), A(110)]GM1, wild-type Glu13, and Glu110 were both mutated to Ala. GM1 and mutants were expressed in high yield in Escherichia coli, purified under denaturing conditions from inclusion bodies, and refolded. Monomers were screened for binding to shIL-5Ralpha-Fc using optical biosensor and ELISA and for bioactivity by proliferation of TF-1.28 cells. Both [(88)SLRGG(92),W(110)]GM1 and [A(13),A(110)]GM1 were found to interact with the shIL-5Ralpha-Fc, with affinities of 69-585 nM, 2-15-fold weaker than that of the original GM1. The mutants also were able to compete with IL-5 for binding to shIL-5Ralpha in an ELISA. In contrast, both mutants exhibited a disproportionately decreased capacity to stimulate TF-1. 28 cell proliferation. [A(13),A(110)]GM1 bioactivity was 160-fold lower than that of GM1, while that for the [(88)SLRGG(92),W(110)]GM1 mutant was 2600-fold lower. The largely retained IL-5 receptor alpha-chain binding affinities versus relatively suppressed bioactivities of [A(13),A(110)]GM1 and [(88)SLRGG(92),W(110)]GM1 variants, in particular the latter, point to the existence of separable IL-5 epitopes for receptor binding and activation and establish the potential to design smaller IL-5 mimetic antagonists.     

T cell receptor binding transition states and recognition of peptide/MHC

T cell receptor recognition of peptide/MHC has been described as proceeding through a "two-step" process in which the TCR first contacts the MHC molecule prior to formation of the binding transition state using the germline-encoded CDR1 and CDR2 loops. The receptor then contacts the peptide using the hypervariable CDR3 loops as the transition state decays to the bound state. The model subdivides TCR binding into peptide-independent and peptide-dependent steps, demarcated at the binding transition state. Investigating the two-step model, here we show that two TCRs that recognize the same peptide/MHC bury very similar amounts of solvent-accessible surface area in their transition states. However, 1300-1500 A2 of surface area is buried in each, a significant amount suggestive of participation of peptide and associated CDR3 surface. Consistent with this interpretation, analysis of peptide and TCR variants indicates that stabilizing contacts to the peptide are formed within both transition states. These data are incompatible with the original two-step model, as are transition state models built using the principle of minimal frustration commonly employed in the investigation of protein folding and binding transition states. These findings will be useful in further explorations of the nature of TCR binding transition states, as well as ongoing efforts to understand the mechanisms by which T cell receptors recognize the composite peptide/MHC surface.     

Protein-DNA interactions at recognition sites for the dioxin-Ah receptor complex

Gel retardation analyses reveal a cluster of six binding sites for the liganded Ah receptor within a 700-base pair DNA domain upstream of the mouse CYP1A1 gene. The nucleotide sequences of the binding sites define a consensus recognition motif for the liganded receptor. The consensus motif is not symmetric. Alteration of the consensus motif produces a decrease in the receptor-DNA interaction. The ligand receptor binds as a monomer to its recognition motif and preferentially binds to double-stranded DNA. These observations reveal apparent differences between 2,3,7,8-tetrachlorodibenzo-p-dioxin and steroid hormones in their respective mechanisms of action.     

Suppressor T cell recognition of major and minor histocompatibility alloantigens: selected suppression of MHC-directed responses by minor alloantigen Ts

The induction of suppression by i.v. administered alloantigens in the murine host was analyzed as a model of the possible effects of blood transfusion on transplant survival. The results indicated that suppressor T cells (Ts) specific for minor histocompatibility alloantigens could be readily induced by the i.v. presentation of minor alloantigen-disparate spleen cells. In contrast, similar priming with cells differing solely at the H-2 major histocompatibility complex stimulated only positive T cell immunity, with no evidence of suppression. The induction of H-2 directed Ts activity could be accomplished only by i.v. priming with major plus minor incompatible donor cells, suggesting that suppressor cell recognition of minor alloantigens may have facilitated the generation of Ts against H-2-encoded major transplantation antigens. A role for minor histocompatibility antigens in the regulation of H-2-specific immunity at the effector level was also indicated. Ts induced by i.v. pretreatment with minor antigen-disparate donor cells not only suppressed the delayed-type hypersensitivity (DTH) response to the relevant minor alloantigens, but also inhibited DTH against unrelated H-2 alloantigens introduced during subsequent intradermal immunization. Suppression of H-2-directed T cell reactivity was specific in that the presence of the Ts-inducing minor alloantigens was also required and occurred only when the minor and unrelated major alloantigens were presented within the same inoculum, if not on the same cell surface. The capacity of Lyt-2+Ts or Ts-derived suppressive factors specific for one set of cell surface molecules to modulate responses to an unrelated group of surface antigens does not appear to represent a general phenomenon, because similar suppression of immunity to unrelated tumor-specific transplantation antigens by minor-specific Ts was not observed. These results are discussed with respect to the possible mechanism of H-2-directed suppression and the role of the I region in Ts recognition of antigen.     

Mechanisms for T cell receptor triggering

There is considerable controversy about the mechanism of T cell receptor (TCR) triggering, the process by which the TCR tranduces signals across the plasma membrane after binding to its ligand (an agonist peptide complexed with an MHC molecule). Three main types of mechanism have been proposed, which involve aggregation, conformational change and segregation. Here, we review recently published evidence for each type of mechanism and conclude that all three may be involved. This complexity may reflect the uniquely demanding nature of TCR-mediated antigen recognition, which requires the detection of a very weak 'signal' (very rare foreign peptide-MHC ligands) in the presence of considerable 'noise' (abundant self peptide-MHC molecules).     

T cell receptor junctional regions and the MHC molecule affect the recognition of antigenic peptides by T cell clones.

Nine independent pigeon cytochrome c-specific T cell clones were analyzed by using a panel of antigenic peptide analogs presented in association with three allelic IE-encoded MHC glycoproteins. Eight of the T cell clones expressed a TCR composed of a unique alpha- and beta-chain amino acid sequence, and concordantly, each of these T cell clones exhibited a unique Ag specificity. This was true for several clones which differed only in TCR V-J junctional regions. Interestingly, for a given clone, the response to some of the peptide analogs depended to a large extent on the allelic form of the presenting MHC molecule. A simple interpretation of these data would suggest that certain positions of the peptide Ag are most important for Ag-MHC molecule interactions, and that these specific interactions can influence the antigenic epitope recognized by the TCR. We suggest that an antigenic peptide binds to an MHC glycoprotein in a distinct way, but may retain a measure of flexibility.     

Glucocorticoid receptor binds cooperatively to adjacent recognition sites.

In order to define the mechanism of synergistic induction mediated by multiple glucocorticoid response elements (GRE), the affinity of the glucocorticoid receptor to a single or duplicated GRE was analyzed by gel retardation, nitrocellulose filter binding and by footprinting experiments. Direct measurement of the relative affinity and indirect determination by competition showed greater than 10-fold higher affinity of the glucocorticoid receptor to a duplicated GRE when compared to a single element. Maximal stability of the GRE-receptor complex was obtained using two closely spaced GREs positioned on the same side of the DNA helix. Increasing the distance or changing the helical position of the GREs considerably increased the off rate of the receptor. DNase I footprinting shows in addition to the protection of the GRE region, an altered pattern in the nonprotected intervening DNA indicating structural alteration of the DNA helix by the receptor bound to adjacent GREs.     

Modeling T cell receptor recognition of CD1-lipid and MR1-metabolite complexes

Background

T cell receptors (TCRs) can recognize diverse lipid and metabolite antigens presented by MHC-like molecules CD1 and MR1, and the molecular basis of many of these interactions has not been determined. Here we applied our protein docking algorithm TCRFlexDock, previously developed to perform docking of TCRs to peptide-MHC (pMHC) molecules, to predict the binding of αβ and γδ TCRs to CD1 and MR1, starting with the structures of the unbound molecules.

Results

Evaluating against TCR-CD1d complexes with crystal structures, we achieved near-native structures in the top 20 models for two out of four cases, and an acceptable-rated prediction for a third case. We also predicted the structure of an interaction between a MAIT TCR and MR1-antigen that has not been structurally characterized, yielding a top-ranked model that agreed remarkably with a characterized TCR-MR1-antigen structure that has a nearly identical TCR α chain but a different β chain, highlighting the likely dominance of the conserved α chain in MR1-antigen recognition. Docking performance was improved by re-training our scoring function with a set of TCR-pMHC complexes, and for a case with an outlier binding mode, we found that alternative docking start positions improved predictive accuracy. We then performed unbound docking with two mycolyl-lipid specific TCRs that recognize lipid-bound CD1b, which represent a class of interactions that is not structurally characterized. Highly-ranked models of these complexes showed remarkable agreement between their binding topologies, as expected based on their shared germline sequences, while differences in residue-level interactions with their respective antigens point to possible mechanisms underlying their distinct specificities.

Conclusions

Together these results indicate that flexible docking simulations can provide accurate models and atomic-level insights into TCR recognition of MHC-like molecules presenting lipid and other small molecule antigens.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-319) contains supplementary material, which is available to authorized users.     

Representation of heavy but not light chain Ig idiotypes on T cell receptors for alloantigens.

We have analyzed idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants by the use of different anti-idiotypic antibodies. Such antisera were produced in (Lewis X DA) F1 rats against Lewis anti-DA alloantibodies (= B cell product) and Lewis T lymphocyte receptors with the same specificity. We found that B lymphocytes bear unique idiotypic determinants which are not present on the corresponding T lymphocytes. T cell unique (not shared by B lymphocytes) idiotypes were so far not detected. T cells idiotypic determinants which are present on heavy but not light chains of the corresponding alloantibodies.     

A T cell clone expresses two T cell receptor alpha genes but uses one alpha beta heterodimer for allorecognition and self MHC-restricted antigen recognition

All of the T cell receptor alpha- and beta-chain rearrangements present in a dual reactive T cell clone were characterized. This clone exhibits allelic exclusion of its beta-chain genes in that only one of the two alleles is productively rearranged. Unexpectedly, it displays two productive V alpha-gene rearrangements, which are both transcribed into 1.5 kb mRNA. The contribution of each of the two productive alpha genes to the dual recognition was analyzed by gene transfer. To this end, each of the two alpha genes was separately transfected with the single productively rearranged beta gene. Transfer of only one of the two alpha beta combinations restored both allogeneic MHC recognition and self MHC-restricted antigen recognition. Thus, T cell dual recognition results from the cross-reactive recognition of an allo-MHC product by a single antigen-specific and MHC-restricted alpha beta T cell receptor. Furthermore, the presence of two productively rearranged alpha-chain genes in a T cell clone raises questions concerning the level at which allelic exclusion operates in T cells.