T-cell Receptor Contact Research Articles

Complementary mutations in an antigenic peptide allow for crossreactivity of autoreactive T-cell clones.

T cells recognize antigen by formation of a trimolecular complex in which the T-cell receptor (TCR) recognizes a specific peptide antigen within the groove of a major histocompatibility complex (MHC) molecule. It has generally been assumed that T-cell recognition of two distinct MHC-antigen complexes is due to similarities in the three-dimensional structure of the complexes. Here we report results of experiments examining the crossreactivity of TCRs recognizing the myelin basic protein peptide MBPp85-99 and several of its analogs in the context of MHC. We demonstrate that single conservative amino acid substitutions of the antigenic peptide at the predominant TCR contact residues at positions 91 and 93 totally abrogate reactivity of specific T-cell clones. Yet, when a conservative substitution is made at position 91 concomitant with a substitution at position 93, the T-cell clones regain reactivity equivalent with that of the original stimulating peptide. Thus, the exact nature of the amino acid side chains engaging one TCR functional pocket may change the apparent selectivity of the other predominant TCR functional pocket, thus suggesting a remarkable degree of receptor plasticity. This ability of the TCR-MHC-peptide complex to undergo conformational changes provides a conceptual framework for reconciling the apparent paradox of the extreme selectivity of the TCR and its remarkable crossreactivity with different MHC-peptide complexes.

Differential activation of human autoreactive T cell clones by altered peptide ligands derived from myelin basic protein peptide (87–99)

We have examined the functional consequences induced by interaction of DR2a-restricted myelin basic protein (MBP) (87-99)-specific T cell clones (TCC) with altered peptide ligands (APL) derived from MBP peptide (87-99). The immunodominant MBP peptide (87-99) has been implicated as a candidate antigen in multiple sclerosis (MS) by several lines of evidence. In the present study, we have defined the T cell receptor (TCR) contact residues for DR2a-restricted, (87-99)-specific T helper type 1 T cells to design APL suitable to modify the functions of such T cells potentially relevant for the pathogenesis of MS. We show that neutral (L-alanine substitutions) or conservative exchanges of the primary and secondary TCR contact residues lead to various alterations of T cell function, ranging from differences in interleukin-2 receptor up-regulation to anergy induction and TCR antagonism. The potential usefulness of APL as an immunomodulating therapy for DR2+ MS patients is discussed.

Specificity of the H-2 L(d)-restricted cytotoxic T-lymphocyte response to the mouse hepatitis virus nucleocapsid protein.

Cytotoxic T lymphocytes provide protection against persistent infection of the central nervous system by the JHM strain of mouse hepatitis virus. In BALB/c (H-2d) mice, the dominant response is directed against an Ld-restricted peptide in the nucleocapsid protein (APTAGAFFF). Characterization of the fine specificity of this response revealed that the predicted anchor residues at positions 2 and 9 were the most critical for class I binding. Amino acids at positions 7 and 8 were identified as T-cell receptor contact residues. Virus-induced cytotoxic T lymphocytes to other Ld motif-containing nucleocapsid peptides were not detected, despite the identification of two epitopes with reduced Ld affinity. These data suggest that mutations within four residues of the dominant epitope could contribute to the persistence of the JHM strain of mouse hepatitis virus.

A Structural Model for TCR Recognition of the HLA Class II Shared Epitope Sequence Implicated in Susceptibility to Rheumatoid Arthritis

HLA molecules associated with rheumatoid arthritis (RA) contain a discrete structural element known as the shared epitope, a set of conserved amino acid residues located on the alpha helical portion of the class II beta chain. Each of the different HLA molecules associated with RA contain the same shared epitope sequence, although they may vary markedly in other regions of the class II structure, which also determine peptide-class II interactions. Previous mutagenesis studies and structural modelling indicate that key polymorphic amino acid side chains within the shared epitope sequence are in locations likely to contact the T cell receptor (TCR) during the trimolecular activation reaction between the HLA-peptide complex and TCR. We have evaluated the potential structural basis for such shared epitope recognition by analysing detailed molecular models of the arthritis-associated DRB1*0404 molecule and a T cell receptor from T cell clone EM025, specific for HLA-DR4 molecules which carry the shared epitope. A likely orientation for the trimolecular complex was deduced in which the EM025 alpha chain interacts with the DR alpha chain and the EM025 beta chain interacts with the DR beta chain; residues Q70 and R71 within the DR beta chain shared epitope region are positioned for hydrogen bond interactions directly with Q97 of the TCR beta CDR3 region, D30 of the TCR beta CDR1 region, and possibly N51 of the TCR beta CDR2 region, indicating a degree of specific selection and interaction which encompasses multiple TCR contacts. These findings suggest a structural basis for the genetic associations with the HLA shared epitope and the potential contribution of this region to oligoclonal T cell selection and expansion in RA.

T cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen.

Two unusual characteristics of the memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, have provided an unique opportunity to investigate self tolerance and T cell receptor (TCR) plasticity in humans. First, the response is exceptionally restricted, dominated by cytotoxic T lymphocytes (CTL) with identical TCR protein sequences (Argaet, V. P., C. W. Schmidt, S. R. Burrows, S. L. Silins, M. G. Kurilla, D. L. Doolan, A. Suhrbier, D. J. Moss, E. Kieff, T. B. Sculley, and I. S. Misko. 1994. J. Exp. Med. 180:2335-2340). Second, CTL expressing this receptor are cross-reactive with the alloantigen HLA B* 4402 on uninfected cells (Burrows, S. R., R. Khanna, J. M. Burrows, and D. J. Moss. 1994. J. Exp. Med. 179:1155-1161). No CTL using this conserved public TCR could be reactivated from the peripheral blood of EBV exposed individuals expressing both HLA B8 and B*4402, demonstrating the clonal inactivation of potentially self-reactive T cells in humans. A significant FLRGRAYGL-specific response was still apparent, however, and TCR sequence analysis of multiple CTL clones revealed an oligoclonal TCR repertoire for this determinant within these individuals, using diverse V and J gene segments and CDR3 regions. In addition, a significant public TCR component was identified in which several distinct alpha/beta rearrangements are shared by CTL clones from a number of unrelated HLA B8+, B*4402+ donors. The striking dominance of public TCR in the response to this EBV epitope suggests a strong genetic bias in TCR gene recombination. Fine specificity analysis using peptide analogues showed that, of six different antigen receptors for FLRGRAYGL/HLA B8, none associate closely with the peptide's full array of potential TCR contact residues. Whereas the HLA B*4402-cross-reactive receptor binds amino acids toward the COOH terminus of the peptide, others preferentially favor an NH2-terminal determinant, presumably evading an area that mimics a structure presented on HLA B*4402. Thus, tolerance to a background major histocompatibility antigen can effectively diversify the TCR repertoire for a foreign epitope by deflecting the response away from an immunodominant combination of TCR-binding residues.

Mapping T cell recognition: the identification of a T cell receptor residue critical to the specific interaction with an influenza hemagglutinin peptide

The fine specificity of T cell receptor (TCR) interaction with the influenza hemagglutinin peptide HA 307-319 in the context of the DR1 (DRA, DRB1 0101) and DR4 (DRA, DRB1 0404) was studied in two human T cell clones (HA1.7 and Cl-1) derived from different individuals. Sequencing of amplified TCR transcripts revealed that these two clones express highly related TCR alpha chains, with a conserved junctional motif, but very different TCR beta chains. Modeling studies led to the prediction that the conserved glutamic acid residue in the TCR alpha chain could interact with the lysine at position 316 in the peptide, a known TCR contact residue. HA1.7 TCR-CD3 zeta chimeric constructs were expressed in the rat basophil line (RBL) and shown to confer specific antigen recognition. In two TCR alpha chain mutants, with the conserved glutamic acid residue altered to alanine and lysine, respectively, peptide recognition was lost. Specific recognition was not rescued by altered peptide ligands. Furthermore, Jurkat derivatives expressing the related Jurkat TCR alpha chain paired with the HA 1.7 TCR beta chain did not recognize the HA 307-319/DR1 complex. These data provide evidence for the critical interaction of a TCR residue with antigenic peptide.

Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substituted in their T-cell receptor contact residues defined by x-ray crystallographic data of the Tax(11-19) peptide in the groove of HLA-A2. CD8 T-cell stimulation with the wild-type peptide antigen led to activation of p56lck kinase activity, interleukin 2 secretion, cytotoxicity, and clonal expansion. A Tax analog peptide with an alanine substitution of the T-cell receptor contact residue tyrosine-15 induced T-cell-mediated cytolysis without activation of interleukin 2 secretion or proliferation. Induction of p56lck kinase activity correlated with T-cell-mediated cytotoxicity, whereas interleukin 2 secretion correlated with [3H]thymidine incorporation and proliferation. Moreover, Tax peptide analogs that activated the tyrosine kinase activity of p56lck could induce unresponsiveness to secondary stimulation with the wild-type peptide. These observations show that a single amino acid substitution in a T-cell receptor contact residue of Tax can differentially signal CD8 T cells and further demonstrate that primary activation has functional consequences for the secondary response of at least some Tax-specific CD8 T cells to HTLV-I-infected target cells.

T cell receptor-major histocompatibility complex class II interaction is required for the T cell response to bacterial superantigens.

Bacterial and retroviral superantigens (SAGs) stimulate a high proportion of T cells expressing specific variable regions of the T cell receptor (TCR) beta chain. Although most alleles and isotypes bind SAGs, polymorphisms of major histocompatibility complex (MHC) class II molecules affect their presentation to T cells. This observation has raised the possibility that a TCR-MHC class II interaction can occur during this recognition process. To address the importance of such interactions during SAG presentation, we have used a panel of murine T cell hybridomas that respond to the bacterial SAG Staphylococcal enterotoxin B (SEB) and to the retroviral SAG Mtv-7 when presented by antigen-presenting cells (APCs) expressing HLA-DR1. Amino acid substitutions of the putative TCR contact residues 59, 64, 66, 77, and 81 on the DR1 beta chain showed that these amino acids are critical for recognition of the SAG SEB by T cells. TCR-MHC class II interactions are thus required for T cell recognition of SAG. Moreover, Mtv-7 SAG recognition by the same T cell hybridomas was not affected by these mutations, suggesting that the topology of the TCR-MHC class II-SAG trimolecular complex could be different from one TCR to another and from one SAG to another.

Peptide antagonists that promote positive selection are inefficient at T cell activation and thymocyte deletion

We set out to determine whether thymocytes from T cell receptor (TCR) transgenic animals specific for a class I-restricted determinant from ovalbumin (OVA) showed the same fine specificity for antigen-driven deletion in single-cell suspension culture as required for mature T cell activation. The transgenic TCR is specific for the Kb-restricted peptide OVA257-264 (SIINFEKL) which is known to have four TCR contact residues at position 1, 4, 6, and 7 from the crystal structure of this fragment in complex with Kb. OVA257-264 analogs systematically substituted at each of these positions were assayed for their ability to promote immature double-positive thymocyte deletion or mature T cell activation of a cytotoxic T lymphocyte line derived from this transgenic mouse. In the absence of additional antigen-presenting cells, single-cell thymocyte suspensions showed that the specificity for double-positive thymocyte deletion and mature T cell activation was virtually identical, demonstrating a limited cross-reactivity with a number of variants having conservative substitutions at these exposed residues. These peptides were considerably more efficient at both thymic deletion and mature T cell activation than a number of non-conservative substitution analogs known to act as antagonists of OVA257-264 and capable of selecting transgenic T cells in thymic organ culture. Therefore, both peripheral T cell activation and thymic deletion have an overall similar pattern of peptide specificity which differs from that required for positive selection. This suggests that a subset of major histocompatibility complex-presented peptides could promote positive selection without causing either thymic deletion or peripheral activation of those selected T cells.

Resting and activated T cells display different requirements for CD8 molecules.

Clonotype-positive (1B2+) T cells from 2C T cell receptor (TCR) transgenic mice were used to define the role of CD8 molecules in the induction phase vs. the effector phase of the primary response to class I alloantigens. Three main findings are reported. First, in the presence of exogenous lymphokines, resting CD8+ 2C cells gave strong proliferative responses to two alloantigens, Ld and Kbm11. In the absence of added lymphokines, however, CD8+ 2C cells responded only to Ld and not to Kbm11; Ld stimulated both interleukin 2 (IL-2) and IL-2 receptor (R) synthesis, whereas Kbm11 elicited only IL-2R synthesis. The primary response of CD8+ 2C cells was thus helper-independent (HI) to Ld but helper-dependent (HD) to Kbm11, presumably reflecting that Ld is a stronger antigen than Kbm11. Second, in contrast to CD8+ cells, CD8- 2C cells mounted only an HD and not an HI response to the strong Ld antigen; conversely, selecting for a minor subset of CD8hi cells enabled 2C cells to mount an HI response to the weak Kbm11 antigen. These findings, together with experiments with heterozygous vs. homozygous stimulator cells, suggest that HI and HD responses reflect differences in the overall avidity of T antigen presenting cell (APC) interaction: high-avidity interaction leads to strong intracellular signaling and an HI response, whereas low-avidity interaction causes weak signaling and an HD response; high-avidity T/APC interaction is heavily dependent on CD8 expression. Third, CD8 expression was found to be less important for CTL activity than for primary proliferative responses. Thus, in contrast to HI proliferative responses, CTL responses of 2C cells to Ld were CD8 independent. However, 2C lysis of Ld targets became strongly CD8 dependent in the presence of limiting doses of anti-TCR (1B2) antibody. Collectively, the data suggest that, both for T cell induction and the expression of effector function, CD8 molecules play a decisive role in augmenting TCR-mediated signaling, either by promoting TCR contact with antigen or delivering kinases (p56lck) to the TCR/CD3 complex, or both.

T cell receptor interaction with peptide/major histocompatibility complex (MHC) and superantigen/MHC ligands is dominated by antigen.

While recent evidence strongly suggests that the third complementarity determining regions (CDR3s) of T cell receptors (TCRs) directly contact antigenic peptides bound to major histocompatibility complex (MHC) molecules, the nature of other TCR contact(s) is less clear. Here we probe the extent to which different antigens can affect this interaction by comparing the responses of T cells bearing structurally related TCRs to cytochrome c peptides and staphylococcal enterotoxin A (SEA) presented by 13 mutant antigen-presenting cell (APC) lines. Each APC expresses a class II MHC molecule (I-Ek) with a single substitution of an amino acid residue predicted to be located on the MHC alpha helices and to point "up" towards the TCR. We find that very limited changes (even a single amino acid) in either a CDR3 loop of the TCR or in a contact residue of the antigenic peptide can have a profound effect on relatively distant TCR/MHC interactions. The extent of these effects can be as great as that observed between T cells bearing entirely different TCRs and recognizing different peptides. We also find that superantigen presentation entails a distinct mode of TCR/MHC interaction compared with peptide presentation. These data suggest that TCR/MHC contacts can be made in a variety of ways between the same TCR and MHC, with the final configuration apparently dominated by the antigen. These observations suggest a molecular basis for recent reports in which either peptide analogues or superantigens trigger distinct pathways of T cell activation.

Clone-specific T cell receptor antagonists of major histocompatibility complex class I-restricted cytotoxic T cells.

A previous report showed that the proliferative response of helper T cells to class II major histocompatibility complex (MHC)-restricted antigens can be inhibited by analogues of the antigen, which act as T cell receptor (TCR) antagonists. Here we define and analyze peptide variants that antagonize various functions of class I MHC-restricted cytotoxic T lymphocyte (CTL) clones. Of 64 variants at individual TCR contact sites of the Kb-restricted octamer peptide ovalbumin257-264 (OVAp), a very high proportion (40%) antagonized lysis by three OVAp-specific CTL clones. This effect was highly clone specific, since many antagonists for one T cell clone have differential effects on another. We show that this inhibition of CTL function is not a result of T cell-T cell interaction, precluding veto-like phenomena as a mechanism for antagonism. Moreover, we present evidence for direct interaction between the TCR and antagonist-MHC complexes. In further analysis of the T cell response, we found that serine esterase release and cytokine production are susceptible to TCR antagonism similarly to lysis. Ca2+ flux, an early event in signaling, is also inhibited by antagonists but may be more resistant to the antagonist effect than downstream responses.

Differential T cell receptor photoaffinity labeling among H-2Kd restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. Labeling of the alpha-chain correlates with J alpha segment usage.

Using a direct binding assay based on photoaffinity labeling, we studied the interaction of T cell receptor (TCR) with a Kd-bound photoreactive peptide derivative on living cells. The Kd-restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was reacted NH2-terminally with biotin and at the TCR contact residue Lys259 with photoreactive iodo, 4-azido salicylic acid (IASA) to make biotin-YIPSAEK(IASA)I. Cytotoxic T lymphocyte (CTL) clones derived from mice immunized with this derivative recognized this conjugate, but not a related one lacking the IASA group nor the parental PbCS peptide. The clones were Kd restricted. Recognition experiments with variant conjugates, lacking substituents from IASA, revealed a diverse fine specificity pattern and indicated that this group interacted directly with the TCR. The TCR of four clones could be photoaffinity labeled by biotin-YIPSAEK(125IASA)I. This labeling was dependent on the conjugates binding to the Kd molecule and was selective for the TCR alpha (2 clones) or beta chain (1 clone), or was common for both chains (1 clone). TCR sequence analysis showed a preferential usage of J alpha TA28 containing alpha chains that were paired with V beta 1 expressing beta chains. The TCR that were photoaffinity labeled at the alpha chain expressed these J alpha and V beta segments. The tryptophan encoded by the J alpha TA28 segment is rarely found in other J alpha segments. Moreover, we show that the IASA group interacts preferentially with tryptophan in aqueous solution. We thus propose that for these CTL clones, labeling of the alpha chain occurs via the J alpha-encoded tryptophan residue.

Microheterogeneity in the recognition of a HLA-DR2-restricted T cell epitope from a meningococcal outer membrane protein.

The trimolecular interaction of T cell receptor (TcR), antigen and major histocompatibility complex (MHC) class II was analyzed using a panel of HLA-DR2-restricted T cell clones recognizing the 49-61 region of a meningococcal class I outer membrane protein (OMP). The clones, all CD3+CD4+CD8-TcR alpha/beta+, were selected by restimulation with the synthetic peptide OMP(49-61), which contains an immunodominant T helper determinant. Using a series of peptides that were sequentially truncated from the N or C terminus, four different epitope fine-specificity patterns were identified. Furthermore, each clone was found to exhibit a distinct recognition pattern for a panel of 20 single-residue substitution analogues of the minimal epitope OMP(50-58). Most substitutions that were not tolerated in the nonamer were allowed when the analogues were prepared departing from the native peptide OMP(49-61). Obviously, the residues outside the minimal epitope contribute to stabilization of the trimolecular complex. These findings suggest that defining the minimal size of T cell determinants may be of limited value. By performing proliferation competition assays putative MHC and TcR contact residues were identified in the peptide. Most likely, Ile 51 and Phe 54 act as MHC-anchoring residues, whereas Asp 53 represents a critical TcR contact residue for all of the clones. MHC anchoring may be provided by other residues as well, since Ile 51 and Phe 54 can be substituted by conservative residues [as OMP(50-58) and OMP(49-61) analogues] and with Ala [as OMP(49-61) analogues only]. Some evidence was found for interaction of particular side chains at other positions with TcR molecules, but this contribution was not equally important for all clones. Apparently, the clonotypic TcR can see a single epitope in different ways in the context of the same MHC restriction element. Since most clones use different V alpha and V beta genes (which encompass the putative MHC-binding regions first and second complementarity-determining regions, CDR1 and CDR2) different modes of interaction with the HLA-DR2 molecule indeed are likely to occur.

Crystal structure of the major histocompatibility complex class I H-2Kb molecule containing a single viral peptide: implications for peptide binding and T-cell receptor recognition.

To study the structure of a homogenous major histocompatibility complex (MHC) class I molecule containing a single bound peptide, a complex of recombinant mouse H-2Kb, beta 2-microglobulin (beta 2m), and a fragment of the vesicular stomatitis virus (VSV) nuclear capsid protein, VSV-(N52-59) octapeptide (Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu), was prepared by exploiting a high-yield bacterial expression system and in vitro cocomplex formation. The structure of mouse H-2Kb revealed its similarity to three human class I HLA molecules, consistent with the high primary sequence homology and common function of these peptide-presenting molecules. Electron density was located in the peptide-binding groove, to which a single peptide in a unique conformation was unambiguously fit. The peptide extends the length of the groove, parallel to the alpha-helices, and assumes an extended, mostly beta-strand conformation. The peptide is constrained within the groove by hydrogen bonding of its main-chain atoms and by contacts of its side chains with the H-2Kb molecule. The amino-terminal nitrogen atom of the peptide forms a hydrogen bond with the hydroxyl group of Tyr-171 of H-2Kb at one end of the groove, while the carboxyl-terminal oxygen forms a hydrogen bond with the hydroxyl group of Tyr-84 at the other end. Since the amino acids at both ends are conserved among human and mouse MHC molecules, this anchoring of each end of the peptide appears to be a general feature of peptide-MHC class I molecule binding and imposes restrictions on its length. The side chains of residues Tyr-3, Tyr-5, and Leu-8 of the VSV octapeptide fit into the interior of the H-2Kb molecule with no appreciable surface exposure, a finding in support of previous biological studies that showed the importance of these residues for binding. Thus, the basis for binding of specific peptide sequences to the MHC class I molecule is the steric restriction imposed on the peptide side chains by the architecture of the floor and sides of the groove. The side chains of Arg-1, Val-4, and Gln-6 and the main-chain of Gly-7 of the octapeptide are exposed on the surface of the complex, thus confirming their availability for T-cell receptor contact, as previously demonstrated by T-cell recognition experiments.

Functional analysis of the antigen binding site on the T cell receptor alpha chain.

We have identified residues on a T cell receptor (TCR) alpha chain that are important for interaction with antigen/major histocompatibility complex (MHC). Using site-directed mutagenesis, we modified DNA encoding the postulated antigen/MHC binding loops on the TCR alpha chain expressed by the T cell clone D5, which recognizes p-azobenzenearsonate-conjugated antigens presented by cells bearing I-Ad. These variant TCR alpha chains were expressed in conjunction with the wild-type D5 TCR beta chain on the surface of hybridoma cells, and were tested for the ability to recognize hapten-conjugated antigens presented by I-Ad. Individual amino acid substitutions in each of the three antigen binding loops (alpha 1, alpha 2, alpha 3) of the D5 TCR alpha chain affected antigen recognition, demonstrating that all three loops are important in recognition of antigen/MHC. A subset of the single amino acid substitutions completely eliminated antigen recognition, thus identifying the residues that are particularly important in the recognition of antigenic peptide/MHC by the D5 TCR. Because the wild-type D5 TCR recognizes arsonate and certain structural analogues of arsonate conjugated to a variety of protein antigens, we were able to test whether the TCR substitutions affected the specificity of the D5 TCR for hapten or carrier antigen. One substitution introduced into antigen binding loop alpha 3 markedly altered the pattern of carrier recognition. Together, these results verify the Ig model for the TCR and are consistent with the proposition that residues forming the first and second antigen binding loops of the TCR contact the MHC, while those forming the third loop contact mainly antigenic peptides.

Molecular components of T-cell recognition.

We review recent data that increase our understanding of the ternary complex of the T cell receptor (TCR), antigenic peptides, and molecules of the major histocompatibility complex (MHC). Studies using synthetic peptide analogs for T-cell antigens have identified peptide residues that appear to interact with the MHC molecule and/or the TCR. The logical extension of these studies, using a complete replacement set of peptide analogues for a model peptide antigen, has more precisely defined the biochemical character of putative MHC and TCR contact residues, and indicated that the TCR is highly sensitive to subtle changes in peptide conformation. Insight into the binding site for peptide on the TCR has recently come from variant peptide immunization of TCR single-chain transgenic mice. These experiments indicate that residues encoded by the V(D)J junctions of both TCR chains contact peptide directly. TCR-MHC contacts have also been studied, using in vitro-mutagenized MHC molecules, particularly those altered at residues predicted to point "up," toward the TCR. These studies reveal that TCR-MHC contacts appear to be quite flexible, and vary between even closely related TCRs. A measure of the affinity of TCR for peptide/MHC complexes has come from competition experiments using soluble MHC complexed with specific peptides. This affinity, with a KD of 5 x 10(-5) M, is several orders of magnitude lower than that of most antibodies for their protein antigens and suggests that the sequence of events leading to T-cell activation begins with antigen-independent adhesion.

Interaction of the pertussis toxin peptide containing residues 30-42 with DR1 and the T-cell receptors of 12 human T-cell clones.

The interaction of the immunodominant pertussis toxin peptide containing residues 30-42 (p30-42) with soluble DR1 molecules and the T-cell receptor (TCR) of 12 DR1-restricted human T-cell clones has been analyzed. Peptide analogues of p30-42 containing single alanine substitutions were used in DR1-binding and T-cell proliferation assays to identify the major histocompatibility complex and TCR contact residues. Each T-cell clone was found to recognize p30-42 with a different fine specificity. However, a common core comprising amino acids 33-39 was found to be important for stimulation of all T-cell clones. Within this core two residues, Leu33 and Leu36, interact with the DR1 molecule, whereas Asp34, His35, Thr37, and Arg39 are important for TCR recognition in most of the clones. Computer modeling of the structure of p30-42 showed that an alpha-helical conformation is compatible with the experimental data. The analysis of TCR rearrangement revealed that the peptide was recognized by T-cell clones expressing different variable region alpha (V alpha) and variable region beta (V beta) chains, although a preferential use of V alpha 8-V beta 13 and V alpha 11-V beta 18 combinations was found in clones from the same donor. Understanding the details of the interaction of antigenic peptides with the major histocompatibility complex and TCR molecules should provide the theoretical basis to design T-cell epitopes and obtain more immunogenic vaccines.

Vesicular stomatitis virus antigenic octapeptide N52-59 is anchored into the groove of the H-2Kb molecule by the side chains of three amino acids and the main-chain atoms of the amino terminus.

This study describes an analysis of the interaction of individual amino acid residues of the vesicular stomatitis virus (VSV) nucleocapsid antigenic octapeptide (N52-59; Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu) with the H-2Kb molecule and T-cell receptors (TCRs). Tyr-3, Tyr-5, and Leu-8 were the positions in the peptide found to be H-2Kb contact residues by analyzing single alanine-substituted peptides in a competition assay with a Kb-restricted antigenic nonapeptide of Sendai virus. Arg-1, Gly-2, Val-4, Gln-6, and Gly-7 of the peptide were identified as putative TCR contact residues by testing the peptide analogs for their capacity to sensitize targets for VSV-specific cytolytic T-lymphocyte clones. The octamer N52-59 was the optimal length of the peptide required for binding to Kb. This peptide length requirement and the finding of an irregular interspersing of major histocompatibility complex and TCR contact residues are most consistent with the conclusion that the peptide is in an extended conformation in the antigen binding groove. Furthermore, data on binding of truncated peptides show that, although the Arg-1 side chain has been assigned as a TCR contact residue, the main-chain atoms of the N-terminal amino group are most likely involved in interacting with the major histocompatibility complex molecule. A panel of H-2Kb point mutants was constructed to explore the effect of altered amino acid residues on the binding of N52-59. Mutants with amino acid substitutions along the floor of the groove all bound the VSV peptide but modulated its interaction with Kb, apparently causing subtle changes in the spatial arrangement of some specific TCR contact residues in the peptide.

A possible basis for major histocompatibility complex-restricted T-cell recognition

Four distinct T-cell antigen-receptor gene loci have now been identified and partly characterized: alpha, beta, gamma and delta. All of these loci can rearrange in an immunoglobulin-like fashion and express polypeptides that contribute to either alpha:beta or gamma:delta T-cell receptor-CD3 complexes. Surprisingly, the T-cell receptor (TCR) delta coding regions are located entirely, or almost entirely, within the TCR alpha locus and share at least some of the V region gene segments, thus at least partly linking the two different types of receptor heterodimers. Analysis of potential T-cell receptor diversity, particularly that of the delta chain, indicates a striking concentration of somatic polymorphism in the V-J junctional region of the two heterodimers, four to six orders of magnitude higher than similar calculations for immunoglobulin light- and heavy-chain combinations. In contrast, the number of possible V region combinations in T-cell receptors is one hundredth to one thousandth that of immunoglobulins. TCR alpha: beta heterodimers are known to recognize many possible fragments of antigens embedded in the peptide-binding clefts of a relatively small number of major histocompatibility complex (MHC) molecules. Thus it is attractive to speculate that the V-J junctional portions of both types of T-cell receptor contact peptide antigens, whereas the remaining diversity regions contact the MHC. This contention is supported by molecular modelling studies and has interesting implications for the evolution of antigen-receptor genes.