Immunity to T cell receptor peptides in multiple sclerosis. III. Preferential immunogenicity of complementarity-determining region 2 peptides from disease-associated T cell receptor BV genes

Vaccination with synthetic TCR peptides from the BV5S2 complementarity-determining region 2 (CDR2) can boost significantly the frequency of circulating CD4+ peptide-specific Th2 cells in multiple sclerosis (MS) patients, with an associated decrease in the frequency of myelin basic protein (MBP)-reactive Th1 cells and possible clinical benefit. To evaluate the immunogenicity of CDR2 vs other regions of the TCR, we vaccinated seven MS patients with overlapping BV5S2 peptides spanning amino acids 1-94. Six patients responded to at least one of three overlapping or substituted CDR2 peptides possessing a core epitope of residues 44-52, and one patient also responded to a CDR1 peptide. Of the CDR2 peptides, the substituted (Y49T)BV5S2-38-58 peptide was the most immunogenic but cross-reacted with the native sequence and had the strongest binding affinity for MS-associated HLA-DR2 alleles, suggesting that position 49 is an MHC rather than a TCR contact residue. Two MS patients who did not respond to BV5S2 peptides were immunized successfully with CDR2 peptides from different BV gene families overexpressed by their MBP-specific T cells. Taken together, these results suggest that a widely active vaccine for MS might well involve a limited set of slightly modified CDR2 peptides from BV genes involved in T cell recognition of MBP.     

T cell receptor recognition of MHC class II-bound peptide flanking residues enhances immunogenicity and results in altered TCR V region usage

Naturally processed MHC class II-bound peptides possess ragged NH2 and COOH termini. It is not known whether these peptide flanking residues (PFRs), which lie outside the MHC anchor residues, are recognized by the TCR or influence immunogenicity. Here we analyzed T cell responses to the COOH-terminal PFR of the H-2A(k) immunodominant epitope of hen egg lysozyme (HEL) 52-61. Surprisingly, the majority of T cells were completely dependent on, and specific for, the COOH-terminal PFR of the immunogen. In addition, there were striking correlations between TCR V beta usage and PFR dependence. We hypothesize that the V alpha CDR1 region recognizes NH2-terminal PFRs, while the V beta CDR1 region recognizes COOH-terminal PFRs. Last, peptides containing PFRs were considerably more immunogenic and mediated a greater recall response to the HEL protein. These results demonstrate that PFRs, which are a unique characteristic of peptides bound to MHC class II molecules, can have a profound effect on TCR recognition and T cell function. These data may have important implications for peptide-based immunotherapy and vaccine development.     

T cell recognition of hapten. Anatomy of T cell receptor binding of a H-2kd-associated photoreactive peptide derivative

To elucidate the structural basis of T cell recognition of hapten-modified antigenic peptides, we studied the interaction of the T1 T cell antigen receptor (TCR) with its ligand, the H-2Kd-bound Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI) containing photoreactive 4-azidobenzoic acid (ABA) on P. berghei circumsporozoite Lys259. The photoaffinity-labeled TCR residue(s) were mapped as Tyr48 and/or Tyr50 of complementary determining region 2beta (CDR2beta). Other TCR-ligand contacts were identified by mutational analysis. Molecular modeling, based on crystallographic coordinates of closely related TCR and major histocompatibility complex I molecules, indicated that ABA binds strongly and specifically in a cavity between CDR3alpha and CDR2beta. We conclude that TCR expressing selective Vbeta and CDR3alpha sequences form a binding domain between CDR3alpha and CDR2beta that can accommodate nonpeptidic moieties conjugated at the C-terminal portion of peptides binding to major histocompatibility complex (MHC) encoded proteins.     

The orientation of a T cell receptor to its MHC class II:peptide ligands

The TCR found on CD4 T cells recognizes peptides bound to self MHC class II molecules as well as non-self MHC class II molecules. We have used the receptor on a cloned T cell line called D10.G4.1 (D10) to perform a structure-function analysis of this interaction. The D10 T cell clone recognizes not only a peptide from conalbumin (CA-wt) bound to syngeneic I-Ak against which it was raised, but also the allogeneic MHC molecules I-A(b,v,p,q,d). In the present study, we show that residue 30 in complementarity-determining region 1 (CDR1) of the TCR alpha-chain interacts with the I-A alpha-chain at hvr2 (residues 52, 53, and 55). We also show that residue 51 in CDR2 of the TCR alpha-chain interacts with the peptide at peptide residue 2. Finally, we show that residue 29 in CDR1 of the TCR beta-chain affects recognition of the glutamic acid at residue 66 in the I-A beta-chain. These data suggest an orientation of TCR relative to its peptide:MHC class II ligands. We argue that this orientation will be shared by all CD4 TCRs, and that it is only subtly different from the common orientation proposed for receptors binding to MHC class I.     

Topology of T cell receptor-peptide/class I MHC interaction defined by charge reversal complementation and functional analysis

The molecular interactions between the CD8 co-receptor dependent N15 and N26 T cell receptors (TCRs) and their common ligand, the vesicular stomatitis virus octapeptide (VSV8) bound to H-2Kb, were studied to define the docking orientation(s) of MHC class I restricted TCRs during immune recognition. Guided by the molecular surfaces of the crystallographically defined peptide/MHC and modeled TCRs, a series of mutations in exposed residues likely contacting the TCR ligand were analyzed for their ability to alter peptide-triggered IL-2 production in T cell transfectants. Critical residues which diminished antigen recognition by 1000 to 10,000-fold in molar terms were identified in both N15 Valpha (alphaE94A or alphaE94R, Y98A and K99) and Vbeta (betaR96A, betaW97A and betaD99A) CDR3 loops. Mutational analysis indicated that the Rp1 residue of VSV8 is critical for antigen recognition of N15 TCR, but R62 of H-2Kb is less critical. More importantly, the alphaE94R mutant could be fully complemented by a reciprocal charge reversal at Kb R62 (R62E). This result suggests a direct interaction between N15 TCR Valpha E94R and Kb R62E residues. As Rp1 of VSV8 is adjacent to R62 in the VSV8/Kb complex and essential for T cell activation, this orientation implies that the N15 Valpha CDR3 loop interacts with the N-terminal residues of VSV8 with the Valpha domain docking to the Kb alpha2 helix while the N15 Vbeta CDR3 loop interacts with the more C-terminal peptide residues and the Vbeta domain overlies the Kb alpha1 helix. An equivalent orientation is suggested for N26, a second VSV8/Kb specific TCR. Given that genetic analysis of two different class II MHC-restricted TCRs and two crystallographic studies of class I restricted TCRs offers a similar overall orientation of V domains relative to alpha-helices, these data raise the possibility of a common docking mode between TCRs and their ligands regardless of MHC restriction.     

Point mutations in the beta chain CDR3 can alter the T cell receptor recognition pattern on an MHC class I/peptide complex over a broad interface area

To study how the T cell receptor interacts with its cognate ligand, the MHC/peptide complex, we used site directed mutagenesis to generate single point mutants that alter amino acids in the CDR3beta loop of a H-2Kb restricted TCR (N30.7) specific for an immunodominant peptide N52-N59 (VSV8) derived from the vesicular stomatitis virus nucleocapsid. The effect of each mutation on antigen recognition was analyzed using wild type H-2Kb and VSV8 peptide, as well as H-2Kb and VSV8 variants carrying single replacements at residues known to be exposed to the TCR. These analyses revealed that point mutations at some positions in the CDR3beta loop abrogated recognition entirely, while mutations at other CDR3beta positions caused an altered pattern of antigen recognition over a broad area on the MHC/peptide surface. This area included the N-terminus of the peptide, as well as residues of the MHC alpha1 and alpha2 helices flanking this region. Assuming that the N30 TCR docks on the MHC/peptide with an orientation similar to that recently observed in two different TCR-MHC/peptide crystal structures, our findings would suggest that single amino acid alterations within CDR3beta can affect the interaction of the TCR with an MHC surface region distal from the predicted CDR3beta-Kb/VSV8 interface. Such unique recognition capabilities are generated with minimal alterations in the CDR3 loops of the TCR. These observations suggest the hypothesis that extensive changes in the recognition pattern due to small perturbations in the CDR3 structure appears to be a structural strategy for generating a highly diversified TCR repertoire with specificity for a wide variety of antigens.     

Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen

The T cell receptor (TCR) inherently has dual specificity. T cells must recognize self-antigens in the thymus during maturation and then discriminate between foreign pathogens in the periphery. A molecular basis for this cross-reactivity is elucidated by the crystal structure of the alloreactive 2C TCR bound to self peptide-major histocompatibility complex (pMHC) antigen H-2Kb-dEV8 refined against anisotropic 3.0 angstrom resolution x-ray data. The interface between peptide and TCR exhibits extremely poor shape complementarity, and the TCR beta chain complementarity-determining region 3 (CDR3) has minimal interaction with the dEV8 peptide. Large conformational changes in three of the TCR CDR loops are induced upon binding, providing a mechanism of structural plasticity to accommodate a variety of different peptide antigens. Extensive TCR interaction with the pMHC alpha helices suggests a generalized orientation that is mediated by the Valpha domain of the TCR and rationalizes how TCRs can effectively "scan" different peptides bound within a large, low-affinity MHC structural framework for those that provide the slight additional kinetic stabilization required for signaling.     

Conserved sequence motifs of CDR3 loops of TCR specific for two major epitopes of the grass pollen allergen PhI p 1

We analyzed the cDNA sequence data of complementarity determining regions (CDR3) of epitope mapped alphabeta TCR of T cell clones (TCC). The TCC were specific for the major timothy grass (Phleum pratense) pollen allergen Phl p 1 and were derived from the peripheral blood of seven unrelated grass pollen-allergic individuals. Each TCR recognized one of two immunodominant T cell epitopes, PP73 or PP103, of Phl p 1. Although a diversity of recombined V and J segments was observed, amino acid motifs as long as five residues were conserved among CDR3 loops of TCR from TCC of different atopic individuals specific for the same peptide. The conserved sequences could comprise as much as 60% of the CDR3. All amino acid residues of the motifs of the CDR3beta and most of the CDR3alpha of all TCR used in this study were encoded by randomly added nucleotides. This indicates that they were specifically selected for by the peptide bound to the MHC class II molecule. For one selected patient, a larger number of TCC, specific for PP103, was analyzed. The TCR repertoire was limited to three different TCR. The same MHC class II molecule, DRB1*1301, was identified to present PP103 to each of the three TCR.     

Use of T cell receptor/HLA-DRB1*04 molecular modeling to predict site-specific interactions for the DR shared epitope associated with rheumatoid arthritis

OBJECTIVE: To use molecular modeling tools to analyze the potential structural basis for the genetic association of rheumatoid arthritis (RA) with the major histocompatibility complex (MHC) "shared epitope," a set of conserved amino acid residues in the third hypervariable region of the DRbeta chain. METHODS: Homology model building techniques were used to construct molecular models of the arthritis-associated DRB1*0404 molecule and a T cell receptor (TCR) from T cell clone EM025, which is specific for DR4 molecules containing the shared epitope sequence. Interactive graphics techniques were used to orient the TCR on the DR molecule, guided by surface complementarity analysis. RESULTS: The predicted TCR-MHC-peptide complex involved multiple interactions and specificity for the shared epitope. TCR residues CDR1beta D30, CDR2beta N51, and CDR3beta Q97 were positioned to potentially participate in hydrogen bond interactions with the shared epitope DRbeta residues Q70 and R71. CONCLUSION: These results suggest a structural mechanism in which specific TCR recognition and possibly Vbeta selection are directly influenced by the disease-associated MHC polymorphisms.     

A case of congenital mumps infection complicated with persistent pulmonary hypertension

Whether T-cell receptors (TCRs) recognize antigenic peptides bound to major histocompatability complex (MHC) molecules through common or distinct docking modes is currently uncertain. We report the crystal structure of a complex between the murine N15 TCR [1-4] and its peptide-MHC ligand, an octapeptide fragment representing amino acids 52-59 of the vesicular stomatitis virus nuclear capsid protein (VSV8) bound to the murine H-2Kb class I MHC molecule. Comparison of the structure of the N15 TCR-VSV8-H-2Kb complex with the murine 2C TCR-dEV8-H-2Kb [5] and the human A6 TCR-Tax-HLA-A2 [6] complexes revealed a common docking mode, regardless of TCR specificity or species origin, in which the TCR variable Valpha domain overlies the MHC alpha2 helix and the Vbeta domain overlies the MHC alpha1 helix. As a consequence, the complementary determining regions CDR1 and CDR3 of the TCR Valpha and Vbeta domains make the major contacts with the peptide, while the CDR2 loops interact primarily with the MHC. Nonetheless, in terms of the details of the relative orientation and disposition of binding, there is substantial variation in TCR parameters, which we term twist, tilt and shift, and which define the variation of the V module of the TCR relative to the MHC antigen-binding groove.     

Isolation and characterization of murine clonogenic osteoclast progenitors by cell surface phenotype analysis

MHC class I molecules bind short peptides for presentation to CD8+ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class Ia molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1c, which carries unusual key residues at both ends of the groove, and RT1.Aa which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7-15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-Kb, 8-13-mer and H2-Db, 9-15-mer peptides). The rat MHC class Ia molecule, RT1-Aa, revealed a preference for 9-15-mer peptides, whereas RT1-A1c showed a more stringent preference for 9-12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.     

Sleep apnea in end stage renal disease

Antibodies directed against the beta chain of the T-cell receptor (TCR) have been detected in animals and in humans in a number of distinct immune states that do not involve direct immunization with either T cells or TCR epitopes. When C57B1/6 mice are infected experimentally with the LP-BM5 retrovirus mixture they produce increased titres of autoantibodies directed against TCR V beta complementarity determining region 1 (CDR1) epitopes. Here, the authors utilized hybridoma technology to isolate monoclonal immunoglobulin (Ig)M antibodies (MoAbs) that arose at the peak of infection. The authors characterized the binding specificity tested using synthetic peptides modelling the CDR1 segments of 24 distinct V beta gene products and determined the VH gene usage by two such monoclonals. One binds to a restricted set of TCR V beta CDR1 peptides, and the second reacts with approximately half of the CDR1 peptide homologues. These MoAbs are specific for T-cell receptor beta chains and do not bind to immunoglobulin light chains or to unrelated protein molecules. Both MoAbs bind to intact T cells expressing the V beta domain (human V beta 8 and mouse V beta 11) from which selection peptides were derived, and costimulate a V beta specific in vitro T cell proliferative response induced by the staphylcoccal enterotoxin E (SEE) superantigen.     

Phosphorylation of phospholamban correlates with relaxation of coronary artery induced by nitric oxide, adenosine, and prostacyclin in the pig

A silicon-based biosensor microphysiometer measures real time cell response by monitoring an increase in extracellular acidification rate in response to ligands for specific membrane receptors. We used the microphysiometer to identify the minimal structure and critical residues of an antigenic peptide for its interaction with T cell receptor (TCR) using a synthetic peptide analog of human myelin basic protein (MBP) corresponding to residues 84-102 [MBP(83-102)Y83]. MBP(83-102)Y83 peptide analogs were allowed to interact with TCRs on a DRB5*0101-restricted Herpes virus saimiri (HVS) transformed human T cell clone (SS8T) which also contains major histocompatibility complexes (MHC) class II (DR2) molecules. Cultured SS8T cells were exposed to 11 N-terminus and 11 C-terminus truncated peptides separately in the microphysiometer chambers to determine the minimal amino acid residues required for the T cell response. In parallel, 13 analogs of the MBP(83-102)Y83 peptide with single alanine substitutions were tested in this assay to identify critical amino acid residues involved in TCR interactions. A minimal core length of MBP(91-100) peptide and residues F-91, K-93, N-94, I-95 and V-96 were essential for TCR interaction. Acidification rate measurements correlated well with enhanced levels of gamma-IFN (interferon gamma) and TNF-beta cytokine production and suggested that the increase in the extracellular acidification rate is a direct result of early T cell signaling events.     

A molecular basis for how a single TCR interfaces multiple ligands

CD8+ T cells respond to Ags when their clonotypic receptor, the TCR, recognizes nonself peptides displayed by MHC class I molecules. The TCR/ligand interactions are degenerate because, in its life time, the TCR interacts with self MHC class I-self peptide complexes during ontogeny and with self class I complexed with nonself peptides to initiate Ag-specific responses. Additionally, the same TCR has the potential to interact with nonself class I complexed with nonself peptides. How a single TCR interfaces multiple ligands remains unclear. Combinatorial synthetic peptide libraries provide a powerful tool to elucidate the rules that dictate how a single TCR engages multiple ligands. Such libraries were used to probe the requirements for TCR recognition by cloned CD8+ T cells directed against Ags presented by H-2Kb class I molecules. When H-2Kb contact residues were examined, position 3 of the peptides proved more critical than the dominant carboxyl-terminal anchor residue. Thus, secondary anchor residues can play a dominant role in determining the antigenicity of the epitope presented by class I molecules. When the four solvent-exposed potential TCR contact residues were examined, only one or two of these positions required structurally similar residues. Considerable structural variability was tolerated at the remaining two or three solvent-exposed residues of the Kb-binding peptides. The TCR, therefore, requires close physico-chemical complementarity with only a few amino acid residues, thus explaining why TCR/MHC interactions are of low affinity and degenerate.     

Direct binding of the MHC class I molecule H-2Ld to CD8: interaction with the amino terminus of a mature cell surface protein

MHC class I molecules (MHC-I) display peptides from the intracellular pool at the cell surface for recognition by T lymphocytes bearing alphabeta TCR. Although the activation of T cells is controlled by the interaction of the TCR with MHC/peptide complexes, the degree and extent of the activation is influenced by the binding in parallel of the CD8 coreceptor with MHC-I. In the course of quantitative evaluation of the binding of purified MHC-I to engineered CD8, we observed that peptide-deficient H-2Ld (MHC-I) molecules bound with moderate affinity (Kd = 7.96 x 10(-7) M), but in the presence of H-2Ld-binding peptides, no interaction was observed. Examination of the amino terminal sequences of CD8alpha and beta chains suggested that H-2Ld might bind these protein termini via its peptide binding cleft. Using both competition and real-time direct assays based on surface plasmon resonance, we detected binding of empty H-2Ld to synthetic peptides representing these termini. These results suggest that some MHC molecules are capable of binding the amino termini of intact cell surface proteins through their binding groove and provide alternative explanations for the observed binding of MHC molecules to a variety of cell surface receptors and coreceptors.     

On defining the rules for interactions between the T cell receptor and its ligand: a critical role for a specific amino acid residue of the T cell receptor beta chain

The specificity of T cell-mediated immune responses is primarily determined by the interaction between the T cell receptor (TCR) and the antigenic peptide presented by the major histocompatibility complex (MHC) molecules. To refine our understanding of interactions between the TCR and the antigenic peptide of vesicular stomatitis virus (VSV) presented by the class I MHC molecule H-2Kb, we constructed a TCR alpha chain transgenic mouse in a TCR alpha-deficient background to define specific structural features in the TCR beta chain that are important for the recognition of the VSV/H-2Kb complex. We found that for a given peptide, a peptide-specific, highly conserved amino acid could always be identified at position 98 of the complementarity-determining region 3 (CDR3) loop of TCR beta chains. Further, we demonstrated that substitutions at position 6, but not position 1, of the VSV peptide induced compensatory changes in the TCR in both the amino acid residue at position 98 and the length of the CDR3beta loop. We conclude that the amino acid residue at position 98 of the CDR3beta loop is a key residue that plays a critical role in determining the specificity of TCR-VSV/H-2Kb interactions and that a specific length of the CDR3beta loop is required to facilitate such interactions. Further, these findings suggest that the alpha and beta chains of TCRs interact with amino acid residue(s) toward the N and C termini of the VSV peptide, respectively, providing functional evidence for the orientation of a TCR with its peptide/MHC ligand as observed in the crystal structures of TCR/peptide/MHC complexes.     

Importance of a conserved TCR J alpha-encoded tyrosine for T cell recognition of an HLA B27/peptide complex

Human HLA B27-restricted cytotoxic T lymphocytes (CTL) specific for the influenza A epitope NP383-391 use similar TCR alpha and beta chains, with two closely related J alpha segments used by six of nine CTL clones from three unrelated donors (Bowness et al., Eur J. Immunol. 1993. 23: 1417-1421). The role of TCR complementarity-determining region (CDR)3alpha residues 93 and 100-102 was examined by site-directed mutagenesis, following expression of the TCR alpha and beta extracellular domains from one clone as a TCR zeta fusion heterodimer in rat basophil leukemia (RBL) cells. For the first time we have measured direct binding of tetrameric HLA B*2705/NP383-391 complexes to transfected TCR. Independently peptide-pulsed antigen-presenting cells (APC) were used to induce TCR-mediated degranulation of RBL transfectants. Our results show a key role for the conserved TCRalpha CDR3 J alpha-encoded residue Y102 in recognition of HLA B27/NP383-391. Thus the Y102D mutation abolished both tetramer binding and degranulation in the presence of peptide-pulsed APC. Even the Y102F mutation, differing only by a single hydroxyl group from the native TCR, abolished detectable degranulation. Further mutations F93A and S100R also abolished recognition. Interestingly, the N101A mutation recognized HLA B27/NP in functional assays despite having significantly reduced tetramer binding, a finding consistent with "kinetic editing" models of T cell activation. Modeling of the GRb TCR CDR3alpha loop suggests that residue Y102 contacts the HLA B*2705 alpha1 helix. It is thus possible that selection of germ-line TCRAJ-encoded residues at position 102 may be MHC driven.     

Induction of acute inflammation in vivo by staphylococcal superantigens. II. Critical role for chemokines, ICAM-1, and TNF-alpha

Antibody-staining experiments have shown that closely related members of the TCRAV3 family are reciprocally selected into the CD4 or CD8 peripheral T cell subsets. This has been attributed to the individual AV3 members interacting preferentially with either MHC class I or MHC class II molecules. Single amino acid residues present in the complementarity-determining regions (CDR) CDR1alpha and CDR2alpha are important in determining MHC class specificity. We have now extended these observations to survey the expressed repertoire of the AV3 family in C57BL/6 mice. Three of the four expressed AV3 members are preferentially selected into the CD4+ subset of T cells. These share the same amino acid residue in both CDR1alpha and CDR2alpha that differ from the only CD8-skewed member. Preferential expression of an individual AV3 is not caused by other endogenous alpha- or beta-chains, by any conserved CDR3 sequence, or by the usage of TCRAJ regions. This study shows that residues in the CDR1 and CDR2 regions are primary determinants for MHC class discrimination and suggests that polymorphism found within a TCRAV family has an important effect on the overall shaping of the T cell repertoire.     

Fine specificity of the myelin-reactive T cell repertoire: implications for TCR antagonism in autoimmunity

The use of altered peptide ligands (APL) to modulate T cell responses has been suggested as a means of treating T cell-mediated autoimmune disorders. We have assessed the therapeutic potential of TCR antagonist peptides in autoimmunity using murine experimental autoimmune encephalomyelitis (EAE) as a model. The Tg4 transgenic mouse expresses an MHC class II-restricted TCR specific for the immunodominant encephalitogenic epitope of myelin basic protein, Ac1-9 (acetylated N-terminal nonamer). We have used T cell lines derived from Tg4 mice to define the TCR contact residues within Ac1-9. APL with appropriate substitutions at the primary TCR contact residue were effective antagonists of Tg4 T cells. These antagonist APL, however, were found to induce EAE in susceptible, nontransgenic strains of mice. Underlying this, the Ac1-9-specific T cell repertoire of normal mice, rather than reflecting the Tg4 phenotype, showed considerable diversity in fine specificity and was able to respond to the Tg4 antagonist APL. Defining antagonist APL in vitro using T cell clones, therefore, was not a reliable approach for the identification of APL with EAE-suppressing potential in vivo. Our findings highlight the complexities of the autoreactive T cell repertoire and have major implications for the use of APL in autoimmune diseases.