Engagement of a T cell receptor by major histocompatibility complex irrespective of peptide

T cell receptors (TCR) identify target cells presenting a ligand consisting of a major histocompatibility complex molecule (MHC) and an antigenic peptide. A considerable amount of evidence indicates that the TCR contacts both the peptide and the MHC components of the ligand. In fully differentiated T cells the interaction between the peptide and the TCR makes the critical contribution to eliciting a cellular response. However, during the positive selection of thymocytes the contribution of peptide relative to MHC is less well established. Indeed it has been suggested that the critical interaction for positive selection is between the TCR and the MHC molecule and that peptides can be viewed as either allowing or obstructing this contact. This predicts that a given TCR is capable of engaging multiple MHC/peptide complexes. In this study a system is described which detects simply engagement of the TCR by MHC/peptide complexes rather than the functional outcome of such interactions. Using this approach the extent to which peptides can influence contacts between the TCR and the MHC molecule has been examined. The results show that the TCR does in fact engage a wide range of ligands in an MHC-restricted but largely peptide-independent manner, suggesting that only a few peptides are able to prevent the TCR from contacting the MHC molecule.     

Inhibition of thymocyte negative selection by T cell receptor antagonist peptides

The T cell receptor (TCR) recognizes antigenic peptide presented by major histocompatibility complex (MHC) molecules. Analogs of antigenic peptides have been shown to inhibit antigen-specific T cell responses, a phenomenon described as TCR antagonism. We have examined the effect of a natural variant of an antigenic peptide and a synthetic peptide analog, on the responses of mature T cells and immature thymocytes from an αβ TCR-transgenic mouse (F5), the TCR of which recognizes a nonamer peptide from the nucleoprotein (NP) of influenza virus in the context of the H-2D^b MHC molecule. Both peptides were shown to antagonize specifically the T cell cytolytic response without being able directly to stimulate mature T cells from these transgenic mice. Furthermore, a negative selection assay in vitro was used to demonstrate for the first time that antagonistic peptides are capable of antagonizing thymocyte deletion induced by antigenic peptides. These data suggest that the final selection of a T cell could be the result of a balance between the positive and negative influences of endogenous peptide ligands.     

Differential expression of alternative H2-M isoforms in B cells, dendritic cells and macrophages by proinflammatory cytokines

Major histocompatibility (MHC) class II heterodimers bind peptides generated by degradation of endocytosed antigens and display them on the surface of antigen presenting cells (APCs) for recognition by CD4+ T cells. Efficient loading of MHC class II molecules with peptides is catalyzed by the MHC class II-like molecule H2-M. The coordinate regulation of MHC class II and H2-M expression is a prerequisite for efficient MHC class II/peptide assembly in APCs determining both the generation of the T cell repertoire in the thymus and cellular immune responses in the periphery. Here we show that expression of H2-M and MHC class II genes is coordinately and cell type-specific regulated in splenic B cells, splenic dendritic cells (DCs) and peritoneal macrophages (Mphi) in response to proinflammatory and immunoregulatory cytokines, including GM-CSF, IFN-gamma, TGF-beta2, IL-4, IL-10 and viral IL-10. In addition, ratio-RT-PCR expression analysis of the duplicated H2-Mbeta-chain loci demonstrates for the first time that Mbl and Mb2 genes are differentially expressed in individual APC types. Mb2 is preferentially expressed in IL-4, GM-CSF, IL-10, vIL-10 and IFN-gamma stimulated splenic B cells, whereas splenic DCs express both Mb genes at almost equal levels. In contrast, peritoneal Mphi express predominantly Mb2 but stimulation with IFN-gamma induces a switch towards Mb1 expression. These data suggest a common mechanism that regulates coordinate expression of H2-M and MHC class II genes in professional APCs. Differential expression of Mb1 and Mb2, and by consequence alternative H2-M isoforms (Malphabeta1 or Malphabeta2), may influence the nature of the peptide repertoire presented by different APC types.     

Peptide presentation by class-I major histocompatibility complex molecules

MHC class-I molecules express distinct peptide-binding pockets within their antigen-binding groove. These are critically involved in the binding of antigenic peptides. The amino acid composition of a pocket dictates the structure of a peptide which can be bound in it. This is evident as a consensus amino acid motif which has to exist within a peptide in order for it to bind to a particular MHC allele. Perturbation of a MHC pocket by amino acid substitution can result in the abolition of peptide binding. Less drastic mutations of the peptide-binding groove, particularly the ones away from the critical pocket, can subtly alter the conformation of bound peptide. Both types of substitution exert an influence on the TCR recognition of antigenic peptide. Peptides are also critically involved in the positive selection of the class-I-restricted TCR repertoire in the thymus. These self peptides act by mimicking their foreign antigens. This mimicking involves the binding of self peptides and foreign antigenic peptides to the same pockets of the MHC class-I-antigen binding groove. Consequently, MHC class-I polymorphism in the antigen binding groove controls the intrathymic positive selection and peripheral antigen presentation by the same mechanisms. The majority of positively selecting self peptides could well originate from the extracellular processing of circulating self proteins. Using the diverse, extracellularly generated self peptides and the different determinant density requirements for positive versus negative selection, the immune system can ensure the repertoire diversity, avoiding both the massive clonal deletion of the selected repertoire and the autoreactivity of its T cells.     

Inefficient clustering of tyrosine-phosphorylated proteins at the immunological synapse in response to an antagonist peptide

Interactions of T cells with MHC plus peptide in the peripheral lymphoid system are important for their survival. In this study we investigated further the molecular consequences of such interactions using F5 TCR transgenic mice and peptides previously shown to induce either negative or positive selection in the thymus. Following TCR ligation with the negatively selecting agonist peptide, mature CD8(+) cells proliferated and up-regulated the activation marker CD69. Interestingly, ligation of this TCR with MHC molecules loaded with high concentrations of the positively selecting peptide also resulted in the aforementioned changes, but with slower kinetics. Analysis of the biochemical changes that occur following stimulation with these peptides showed that phosphorylation of key signaling molecules, such as ZAP-70, CD3zeta, Vav, SLP-76, LAT, and ERK-1 and 2, could be detected after exposure to agonist but not antagonist peptide. Confocal microscopy, however, revealed infrequent phosphorylation 'patches' at the site of contact between T cells and APC presenting the antagonist peptide. Our data suggest that peptides capable of inducing positive selection in the thymus can be recognized by mature T cells and cause proliferation, up-regulation of CD69 and accumulation of phosphorylated proteins at the immunological synapse with low efficiency; however no phosphorylation of signaling molecules can be detected using conventional biochemical assays.     

The multigenic structure of the MHC locus contributes to positive selection efficiency: a role for MHC class II gene-specific restriction

The study of T cell positive selection in the thymus has long been focused on the specificity of the MHC-TCR interactions, making use of genetically manipulated mice that display TCR specificities or selecting peptides of limited diversity. However, little is known on the role of the MHC molecules irrespective of the peptide specificity and the implications of MHC multigenic structure in thymic positive selection have not been addressed. Here, we investigated the effect of MHC class II genetic configuration on the positive selection efficiency of naturally generated pre-selection repertoires in the mouse thymus. Analysis of positively selected thymocyte populations in MHC-congenic and -transgenic mice revealed that expression of I-E molecule in the thymic cortex increases positive selection efficiency of CD4 cells by approximately 50%. We show that increments in positive selection attributable to either the I-A and I-E genes are not due to increased MHC class II expression in the thymic cortex and are not affected by the number of MHC alleles. Collectively, our findings imply that MHC class II gene-restricted TCR specificities significantly contribute to positive selection efficiency, introducing the notion that multigenic structure of the MHC locus serves to increase selection of non-overlapping TCR repertoires.     

Regulatory role of leukocyte common antigen‐related molecule (LAR) in thymocyte differentiation

The strength of interaction between the antigenic peptide‐loaded MHC (MHC/p) and the TCR determines T‐cell fate in the thymus. A high avidity interaction between the TCR and the MHC/p induces apoptosis of self‐reactive T cells (negative selection), whereas a moderate avidity interaction rescues thymocytes from apoptosis and permits further differentiation to mature T cells (positive selection). Leukocyte common antigen‐related molecule (LAR), a receptor‐like protein tyrosine phosphatase, is expressed on immature thymocytes, but its role in thymocyte differentiation has not yet been fully elucidated. We analyzed LAR‐deficient mice and demonstrated that LAR deficiency affected the differentiation and expansion of immature thymocytes as well as positive and negative selection. Furthermore, LAR deficiency resulted in a lower Ca²⁺ response. The results indicate that LAR is an important modulator of TCR signaling that controls thymocyte differentiation.     

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells

Summary: Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC), At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR). whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal I to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal I alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-l can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and [CAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.     

Conserved T-cell receptor class II major histocompatibility complex contact detected in a T-lymphocyte population.

T-cell receptor (TCR) interacts with an antigenic peptide deeply buried in the major histocompatibility complex (MHC) molecule. How class II MHC is contacted by TCR during antigen recognition remains largely elusive. Here we used a panel of I-Ek mutants to identify two I-Ek residues that were frequently contacted by TCR among a large pool of T cells specific for the same antigen. The restricted TCR interaction with I-Ek was independent of the antigen peptides. We also identified a dominant heteroclitic residue on I-Ek, beta81H, in which mutation led to increased recognition of antigens in individual T-cell clones. Moreover, both the conserved TCR-I-Ek interaction and the heteroclitic TCR-I-Ek recognition were detected in T lymphocytes freshly isolated from mice primed with the specific antigens. The identical TCR-I-Ek interaction in a heterogeneous T-cell population suggested the dominance of invariant TCR-class II MHC interaction.     

Altered major histocompatibility complex class II peptide loading in H2-O-deficient mice

The biosynthesis of MHC class II/peptide complexes involves classical, cell surface MHC products as well as the intracellular component H2-M, required for the removal of invariant chain-derived CLIP and for peptide loading. The function of another intracellular class II heterodimer, H2-O, is the matter of some controversy. The physical association of H2-O with H2-M and co-localization in class II+ vesicles suggest a related function in peptide exchange. Furthermore, the distinctive thymic distribution of H2-O raises the possibility of a specialized role in T cell thymic selection. To investigate the role of H2-O in vivo we generated mice carrying a targeted disruption in the H2-Oa gene. No evidence was obtained for a defect in removal of CLIP. However, the array of endogenous peptides bound by class II was altered and a defect in antigen presentation through H2-A to T cells was seen on the 129/Sv/ C57BL/6 mixed strain background but not in 129/Sv pure strain mice. Furthermore, H2-O-null mice showed enhanced selection of CD4+ single positive thymocytes. The findings indicate that H2-O interacts with H2-M in peptide editing but that the genetic background in which H2-O deficiency is manifest is also important. Overall, the experiments indicate that H2-O/HLA-DO should be regarded as neither up-regulating nor down-regulating the DM-dependent release of CLIP, but as a modulator of peptide editing, determining the presenting cell type specific peptide profile able to retain stability in the class II groove.     

Different affinity windows for virus and cancer‐specific T‐cell receptors: Implications for therapeutic strategies

T‐cell destiny during thymic selection depends on the affinity of the TCR for autologous peptide ligands presented in the context of MHC molecules. This is a delicately balanced process; robust binding leads to negative selection, yet some affinity for the antigen complex is required for positive selection. All TCRs of the resulting repertoire thus have some intrinsic affinity for an MHC type presenting an assortment of peptides. Generally, TCR affinities of peripheral T cells will be low toward self‐derived peptides, as these would have been presented during thymic selection, whereas, by serendipity, binding to pathogen‐derived peptides that are encountered de novo could be stronger. A crucial question in assessing immunotherapeutic strategies for cancer is whether natural TCR repertoires have the capacity for efficiently recognizing tumor‐associated peptide antigens. Here, we report a comprehensive comparison of TCR affinities to a range of HLA‐A2 presented antigens. TCRs that bind viral antigens fall within a strikingly higher affinity range than those that bind cancer‐related antigens. This difference may be one of the key explanations for tumor immune escape and for the deficiencies of T‐cell vaccines against cancer.     

The Diversity of Antigen-Specific TCR Repertoires Reflects the Relative Complexity of Epitopes Recognized

Antigen-selected T cell receptor (TCR) repertoires vary in complexity from very limited to extremely diverse. We have previously characterized two different CD8 T cell responses, which are restricted by the same mouse major histocompatibility complex (MHC) class I molecule, H-2 K^d. The TCR repertoire in the response against a determinant from Plasmodium berghei circumsporozoite protein (PbCS; region 252–260) is very diverse, whereas TCRs expressed by clones specific for a determinant in region 170–179 of HLA-CW3 (human) MHC class I molecule show relatively limited structural diversity. We had already demonstrated that cytolytic T lymphocyte (CTL) clones specific for the PbCS peptide display diverse patterns of antigen recognition when tested with a series of single Ala-substituted PbCS peptides or mutant H-2 K^d molecules. We now show that CW3-specific CTL clones display much less diverse patterns of recognition. Our earlier functional studies with synthetic peptide variants suggested that the optimal peptides recognized were 9 (or 8) residues long for PbCS and 10 residues long for CW3. We now present more direct evidence that the natural CW3 ligand is indeed a 10-mer. Our functional data together with molecular modeling suggest that the limited TCR repertoire selected during the CW3 response is not due to a paucity of available epitopes displayed at the surface of the CW3 peptide/K^d complex. We discuss other factors, such as the expression of similar self MHC peptide sequences, that might be involved in trimming this TCR repertoire.     

CTL responses to H2-M3-restricted Listeria epitopes

Summary: Cytotoxic T cells (CTL) play a critical role in the murine immune response to Listeria monocytogenes (Listeria), Bacterial antigens are presented lo Listeria-specific CTL by products of both conventional, polymorphic MHC class Ia and non-polymorphic MHC class Ib alleles. The H2-M3 class Ib gene product. M3, preferentially presents formylmethionine-initiating (fMet) peptides derived from the N termini of bacterial and mitochondrial proteins. Thus, M3 signals the presence of bacterial invaders to CTL effectors, Listeria-encoded fMet peptide epitopes for H2-M3-restricted CTL have recently been identified. These and other identified fMet peptides are predominantly comprised of hydrophobic residues and appear lo be cleaved from membrane-bound proteins. The subcellular location and membrane topology of such proteins may be significant factors in their selection as target antigens for H2-M3-restricied CTL. Such rules may prove useful for prediction of candidate fMet peptide epitopes from other bacterial proteins and species. Studies using synthetic fMet peptides to stimulate CTL ex vivo are also discussed, These latter studies indicate that Listeria infection boosts H2-M3-restricted CTL responses. However, in contrast to MHC class la-restricted CTL responses, fMet peptide-specific CTL are observed in a large proportion of cultures from non immunized, conventionally housed (non-SPF) mice. The CTL activity in these latter cultures may reflect priming in vivo on cross-reactive antigens, or may indicate that requirements for priming of H2-M3-restricted CTL are less stringent than for class Ia-restricted responses.     

Recognition of core and flanking amino acids of MHC class II-bound peptides by the T cell receptor

CD4 T cells recognize peptides bound to major histocompatibility complex (MHC) class II molecules. Most MHC class II molecules have four binding pockets occupied by amino acids 1, 4, 6, and 9 of the minimal peptide epitope, while the residues at positions 2, 3, 5, 7, and 8 are available to interact with the T cell receptor (TCR). In addition MHC class II bound peptides have flanking residues situated outside of this peptide core. Here we demonstrate that the flanking residues of the conalbumin peptide bound to I-A(k) have no effect on recognition by the D10 TCR. To study the role of peptide flanks for recognition by a second TCR, we determined the MHC and TCR contacting amino acids of the I-A(b) bound Ealpha peptide. The Ealpha peptide is shown to bind I-A(b) using four alanines as anchor residues. TCR recognition of Ealpha peptides with altered flanking residues again suggested that, in general, no specific interactions occurred with the peptide flanks. However, using an HLA-DM-mediated technique to measure peptide binding to MHC class II molecules, we found that the peptide flanking residues contribute substantially to MHC binding.     

Short peptides sensitize target cells to CTL specific for the MHC class Ib molecule,H2-M3

The MHC class Ib molecule H2-M3 presents N-formylated peptides from the N terminus of proteins encoded by the mitochondrial genome to CTL. A panel of CTL specific for a peptide derived from a mitochondrial protein, either COI or ND1, was used to determine the optimal peptide length for sensitizing antigen-deficient target cells. All long-term CTL lines and most CTL clones lysed target cells sensitized with either a COI hexamer or an ND1 heptamer. Only 3 out of 12 anti-ND1 clones preferred an octamer or nonamer peptide and no CTL required to longer peptides. The CTL preference for short peptides matches a shortened groove in M3. The CTL all lysed lymphoblasts encoding the appropriate mitochondrial antigen, suggesting that these target cells express naturally processed, endogenous, formylated peptides, ranging from six to nine amino acids in length.     

Structural analysis of two HLA-DR-presented autoantigenic epitopes: crucial role of peripheral but not central peptide residues for T-cell receptor recognition

Specific and major histocompatibility complex (MHC)-restricted T-cell recognition of antigenic peptides is based on interactions of the T-cell receptor (TCR) with the MHC alpha helices and solvent exposed peptide residues termed TCR contacts. In the case of MHC class II-presented peptides, the latter are located in the positions p2/3, p5 and p7/8 between MHC anchor residues. For numerous epitopes, peptide substitution studies have identified the central residue p5 as primary TCR contact characterized by very low permissiveness for peptide substitution, while the more peripheral positions generally represent auxiliary TCR contacts. In structural studies of TCR/peptide/MHC complexes, this has been shown to be due to intimate contact between the TCR complementarity determining region (CDR) three loops and the central peptide residue. We asked whether this model also applied to two HLA-DR presented epitopes derived from an antigen targeted in type 1 diabetes. Large panels of epitope variants with mainly conservative single substitutions were tested for human leukocyte antigen (HLA) class II binding affinity and T cell stimulation. Both epitopes bind with high affinity to the presenting HLA-DR molecules. However, in striking contrast to the standard distribution of TCR contacts, recognition of the central p5 residue displayed high permissiveness even for non-conservative substitutions, while the more peripheral p2 and p8 TCR contacts showed very low permissiveness for substitution. This suggests that intimate TCR interaction with the central peptide residue is not always required for specific antigen recognition and can be compensated by interactions with positions normally acting as auxiliary contacts.     

Structure of an autoimmune T cell receptor complexed with class II peptide-MHC: insights into MHC bias and antigen specificity

T cell receptor crossreactivity with different peptide ligands and biased recognition of MHC are coupled features of antigen recognition that are necessary for the T cell's diverse functional repertoire. In the crystal structure between an autoreactive, EAE T cell clone 172.10 and myelin basic protein (1-11) presented by class II MHC I-Au, recognition of the MHC is dominated by the Vbeta domain of the TCR, which interacts with the MHC alpha chain in a manner suggestive of a germline-encoded TCR/MHC "anchor point." Strikingly, there are few specific contacts between the TCR CDR3 loops and the MBP peptide. We also find that over 1,000,000 different peptides derived from combinatorial libraries can activate 172.10, yet the TCR strongly prefers the native MBP contact residues. We suggest that while TCR scanning of pMHC may be degenerate due to the TCR germline bias for MHC, recognition of structurally distinct agonist peptides is not indicative of TCR promiscuity, but rather highly specific alternative solutions to TCR engagement.     

Major histocompatibility complex class II-associated peptides determine the binding of the superantigen toxic shock syndrome toxin-1

Superantigens bind to major histocompatibility complex (MHC) class II proteins and interact with variable parts of the T cell antigen receptor (TCR) β-chain. Cross-linking the TCR with MHC class II molecules on the antigen-presenting cell by the superantigen leads to T cell activation that plays an essential role in pathogenesis. Recent crystallographic data have resolved the structure of the complexes between HLA-DR1 and staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1), respectively. For TSST-1, these studies have revealed possible contact sites between the superantigen and the HLA-DR1-bound peptide. Here, we show that TSST-1 binding is dependent on the MHC-II-associated peptides by employing variants of T2 mutant cells deficient in loading of peptides to MHC class II molecules as superantigen-presenting cells. On HLA-DR3-transfected T2 cells, presentation of TSST-1, but not SEB, was dependent on HLA-DR3-associated peptides. Thus, although these superantigens can be recognized in the context of multiple MHC class II alleles and isotypes, they clearly bind to specific subsets of MHC molecules displaying appropriate peptides.     

Allo- and self-restricted cytotoxic T lymphocytes against a peptide library: evidence for a functionally diverse allorestricted T cell repertoire

BALB/c-derived spleen cells were depleted of cytotoxic T lymphocytes (CTL) recognizing allogeneic (H2^b) and TAP-negative cells followed by stimulation with the same cells loaded with a synthetic library binding to H2-K^b. The resulting CTL lines were found to differ widely in peptide specificity and to exhibit an avidity towards the library as that demonstrated for syngeneic CTL. These results demonstrate that positive selection in the context of a certain MHC molecule does not seem to be required for generating high-avidity TCR that are restricted by the same molecule. However, positive selection increases the frequency of such CTL. By raising T cell lines from a repertoire which did not undergo negative selection by the restriction element in question, it becomes possible to produce effective self-peptide/MHC as well as nonself-peptide/MHC-specific CTL as tools for adoptive tumor immunotherapy.     

T cell stimulation in the absence of exogenous antigen: a T cell signal is induced by both MHC-dependent and -independent mechanisms

Mature T cells residing in peripheral lymphoid organs have frequent contact with antigen presenting cells (APC). Such contact may be required for T cell survival, but the degree to which signals in mature T cells are induced by TCR recognition of self peptide/MHC complexes is unclear. We have used induction of the early growth response gene 1 (Egr1) as an indicator of signal transduction in 3.L2 (I-Ek-restricted) T cells interacting with APC in the absence of exogenous antigen. The data show that Egr1 can be induced in 3.L2 T cells by TCR recognition of self peptides presented by I-Ek. However, a more transient induction of Egr1 can be induced in 3.L2 T cells interacting with dendritic cells derived from class II/beta2m double-deficient mice. Egr1 induction after T cell-APC contact was also observed in a freshly isolated polyclonal CD4 T cell population. The data suggest that self peptide/MHC recognition by the TCR induces a signal in T cells and that dendritic cells can also induce a more transient T cell signal by an MHC-independent mechanism.