MR angiography of patients with peripheral arterial disease before and after transluminal angioplasty

The strength of the cytotoxic T lymphocyte (CTL) response is believed to influence the final outcome of hepatitis B virus (HBV) infection. Among the different CTL epitopes so far identified, the sequence 18-27 of the HBV nucleocapsid antigen is widely recognized by CTL of HLA-A2-positive patients with acute self-limited HBV infection, and represents the main component of a peptide-based therapeutic vaccine aimed at stimulating the antiviral CTL response in patients with chronic hepatitis B. In the present study, we further analyzed the features of this important HBV region by the following: 1) defining the contribution of individual residues of the epitope to the interaction with the T-cell receptor (TCR) and with the HLA-A0201 molecule; 2) assessing the antigenicity of this viral region in the context of the different HLA-A2 subtypes; and 3) testing whether this sequence can stimulate not only HLA-class I but also HLA class II restricted T-cell responses. A clear hierarchy was observed in the ability of individual residues to act as TCR or HLA binding sites. Furthermore, the sequence HBc18-27 was able to be recognized by specific CTL when presented in the context of different HLA-A2 subtypes. Finally, this HBV region was also found to stimulate HLA class II restricted T-cell responses. These data further increase the potential coverage and efficacy of therapeutic vaccines based on the HBc18-27 sequence.     

Evaluation of a corporate-sponsored health care program for retired employees

Five HLA-B27 subtypes, B*2701, B*2703, B*2704, B*2705, and B*2706, were tested for direct binding with twenty-six synthetic nonapeptides carrying the primary anchor residue motifs (combination of amino residues at positions 2 and 9) relevant to B*2705. The peptide sequences were derived from human HSP89 alpha, P53 and MBP. The alpha chains were immunospecifically isolated from LH (B*2701), CH (B*2703), WE1 (B*2704), BTB (B*2705), and LIE (B*2706) cells and their peptide binding was measured by the HLA class I alpha chain refolding assay. The data obtained indicated that the B27 subtypes tested can bind a common set of peptides carrying several different anchor residue motifs. The motifs, R-K and R-R, reported for B*2705 and a new motif H-R were accepted by B*2703, B*2704, and B*2706, but not by B*2701. However, other motifs, including known B*2702 and/or B*2705 motifs, R-H, R-L, R-A, and R-F, and a new motif found here, R-G, were apparently accepted by all B27 subtypes tested. The observed cross-peptide binding in the B27 subgroup is compatible with the so-called arthritogenic peptide hypothesis in the pathogenesis of ankylosing spondylitis.     

The same natural ligand is involved in allorecognition of multiple HLA-B27 subtypes by a single T cell clone: role of peptide and the MHC molecule in alloreactivity

The human alloreactive CTL clone 27S69, raised against B*2705, cross-reacts with B*2702 and B*2703, but not with B*2701, B*2704, B*2706, or B*2710. Its natural epitope was identified by electrospray/ion trap mass spectrometry, as the proteasome-derived RRFFPYYV octamer. This is the first HLA-B27 ligand shown to be immunogenic in alloreactivity. The RRFFPYYVY nonamer, also found in the B*2705-bound peptide pool, was recognized much less efficiently, demonstrating that an alloreactive CTL distinguishes between very similar natural ligands. Molecular modeling suggested that this was due to the different conformation of each peptide in complex with B*2705. B*2702- and B*2703-RMA-S cells were lysed by CTL 27S69 when sensitized with the octamer, demonstrating that cross-reaction with these subtypes is through recognition of the same peptide as in B*2705. B*2704-, B*2706-, and B*2710-RMA-S cells were not sensitized for lysis, in spite of efficient binding of the octamer, indicating that polymorphism in these subtypes directly impairs allorecognition. B*2701-RMA-S and -C1R cells were sensitized for lysis by the octamer, suggesting lack of the endogenous peptide epitope on this subtype. Absence of the octamer in the B*2701-bound peptide pool further suggested that B*2701 polymorphism impairs the generation of this peptide.     

Proteasomes generate in vitro a natural peptide of influenza-A nucleoprotein functional in HLA-B27 antigen assembly

We have studied the degradation of a set of long peptides (9-30 amino acids) from the nucleoprotein of influenza A. In common for all these peptides is the core sequence NH2-Ser-Arg-Tyr-Trp-Ala-Ile-Arg-Thr-Arg-COOH, NP383-391, known as an antigenic peptide specific for the HLA-B27 class I antigen. We show that this peptide is generated by enriched cytosolic proteasomes of two sizes, 20S and 12S. The 12S proteasome is the precursor, the preproteasome, to the 20S mature proteasome as shown by pulse-chase experiment and is most likely responsible for the proteolytic activity in the 12S region. Cleavage at the N-terminus is distinct and restricted to residue 383, independent of the N-terminal extension of the peptide. The C-terminus is generated via cleavage at three sites. Intermediate and final peptide products were identified by mass spectrometry. Finally, we show that the NP383-391 peptide generated by proteasomes in vitro is functional inasmuch as it possesses the ability to stimulate assembly of in vitro translated HLA-B27 antigens.     

Antagonist HIV-1 Gag peptides induce structural changes in HLA B8

In the cellular immune response, recognition by CTL-TCRs of viral antigens presented as peptides by HLA class I molecules, triggers destruction of the virally infected cell (Townsend, A.R.M., J. Rothbard, F.M. Gotch, G. Bahadur, D. Wraith, and A.J. McMichael. 1986. Cell. 44:959-968). Altered peptide ligands (APLs) which antagonise CTL recognition of infected cells have been reported (Jameson, S.C., F.R. Carbone, and M.J. Bevan. 1993. J. Exp. Med. 177:1541-1550). In one example, lysis of antigen presenting cells by CTLs in response to recognition of an HLA B8-restricted HIV-1 P17 (aa 24-31) epitope can be inhibited by naturally occurring variants of this peptide, which act as TCR antagonists (Klenerman, P., S. Rowland Jones, S. McAdam, J. Edwards, S. Daenke, D. Lalloo, B. Koppe, W. Rosenberg, D. Boyd, A. Edwards, P. Giangrande, R.E. Phillips, and A. McMichael. 1994. Nature (Lond.). 369:403-407). We have characterised two CTL clones and a CTL line whose interactions with these variants of P17 (aa 24-31) exhibit a variety of responses. We have examined the high resolution crystal structures of four of these APLs in complex with HLA B8 to determine alterations in the shape, chemistry, and local flexibility of the TCR binding surface. The variant peptides cause changes in the recognition surface by three mechanisms: changes contributed directly by the peptide, effects transmitted to the exposed peptide surface, and induced effects on the exposed framework of the peptide binding groove. While the first two mechanisms frequently lead to antagonism, the third has more profound effects on TCR recognition.     

Evidence for cooperation between TCR V region and junctional sequences in determining a dominant cytotoxic T lymphocyte response to herpes simplex virus glycoprotein B

TCR repertoire availability has the potential to influence the immune response to foreign antigens. Here we have analysed how changes in V region availability influence the H-2b-restricted cytotoxic T lymphocyte (CTL) response to a dominant peptide determinant derived from the herpes simplex virus glycoprotein B (gB). We have previously shown that C57BL/6 mice mount a gB-specific, Kb-restricted CTL response which is dominated by a TCRBV10+ population and a TCRBV8S1+ subpopulation, both containing highly conserved CDR3 elements. We find that this dominant gB-specific CTL pool is lost in C57/L mice which have a different TCRBV haplotype. A population of CTL with diverse TCRBV and junctional sequence usage, which otherwise represents a minor subset in the gB-specific response, appears to emerge as a consequence of this TCRBV gene variation. The loss of preferential V region-encoded complementarity determining regions (CDR) 1- and/or CDR2-ligand interactions in this emerging population also results in a change in CDR3 sequence usage and a corresponding focusing of an otherwise promiscuous pattern of cross-reactivity with a panel of gB498-505 substitution analogues. This suggests that the difference between the two distinct TCR populations is the relative contributions of the CDR towards ligand recognition. Therefore, preferential V region-ligand interaction, at the expense of CDR3 peptide recognition, appears to control the dominant TCR selection in the C57BL/6 response to this peptide determinant.     

A functionally significant allelic polymorphism in a T cell receptor V beta gene segment

The effect of an allelic polymorphism in the BV1S1 gene segment on recognition of major histocompatibility complex (MHC)-peptide complexes by a specific T cell receptor (TCR) was studied using RBL 2H3 cells transfected with TCR-CD3 zeta chimeric receptors. An HLA-A2-restricted human immunodeficiency virus (HIV) pol-specific cytotoxic T lymphocyte (CTL) clone utilizing the BV1S1A2 gene in combination with AV2S1A2 was identified and the extracellular domains of the TCR were fused to CD3 zeta. In degranulation assays RBL 2H3 transfectants expressing this receptor maintained the specificity of the parental CTL clone. The allelic variant BV1S1A1N1 containing a glutamine for histidine substitution at position 48 in the loop of the second complementarity-determining region was generated by site-directed mutagenesis. Transfection of this molecule as a CD3 zeta chimera together with the original AV2S1A2 CD3 zeta molecule resulted in cell surface expression of both chains but a loss of recognition of HLA-A2 HIV pol peptide-pulsed targets. The effect of this polymorphism on MHC-peptide recognition supports current models of TCR MHC-peptide interaction and provides evidence for a functional role for polymorphism in the TCRV genes.     

Glu227-->Lys substitution in the acidic loop of major histocompatibility complex class I alpha 3 domain distinguishes low avidity CD8 coreceptor and avidity-enhanced CD8 accessory functions

Cytotoxic T lymphocyte (CTL) activation requires specific T cell receptor (TCR)-class I major histocompatibility complex (MHC) antigen complex interactions as well as the participation of coreceptor or accessory molecules on the surface of CTL. CD8 can serve as a coreceptor in that it binds to the same MHC class I molecules as the TCR to facilitate efficient TCR signaling. In addition, CD8 can be "activated" by TCR stimulation to bind to class I molecules with high avidity, including class I not recognized by the TCR as antigenic complexes (non-antigen [Ag] class I), to augment CTL responses and thus serve an accessory molecule function. A Glu/Asp227-->Lys substitution in the class I alpha 3 domain acidic loop abrogates lysis of target cells expressing these mutant molecules by alloreactive CD8-dependent CTL. Lack of response is attributed to the destruction of the CD8 binding site in the alpha 3 domain which is likely to disrupt CD8 coreceptor function. The relative importance of the class I alpha 3 domain acidic loop Glu227 in coreceptor as opposed to accessory functions of CD8 is unclear. To address this issue, we examined CTL adhesion and degranulation in response to immobilized class I-peptide complexes formed in vitro from antigenic peptides and purified class I molecules containing wild-type or Glu227-->Lys substituted alpha 3 domains. The alpha 3 domain mutant class I-peptide complexes were bound by CTL and triggered degranulation, however to much lower levels than wild-type class I-peptide complexes. In further experiments, it is directly demonstrated that the alpha 3 domain mutant class I molecules, which lack the Glu227 CD8 binding site, still serve as TCR-activated, avidity-enhanced CD8 accessory ligands. However, mutant class I peptide Ag complexes failed to effectively serve as CD8 coreceptor ligands to initiate TCR-dependent signals required to induce avidity-enhanced CD8 binding to coimmobilized non-Ag class I molecules. Thus the Glu227-->Lys mutation effectively distinguishes CD8 coreceptor and avidity-enhanced CD8 accessory functions.     

Cross-reactive memory T cells for Epstein-Barr virus augment the alloresponse to common human leukocyte antigens: degenerate recognition of major histocompatibility complex-bound peptide by T cells and its role in alloreactivity

In the present report, cytotoxic T lymphocyte (CTL) clones are described that display dual specificity for one of two common human leukocyte antigens (HLA B14 or B35) as alloantigens, and an immunodominant epitope (FLRGRAYGL) from Epstein-Barr virus (EBV) that binds to HLA B8. These T cell clonotypes were isolated from several unrelated HLA B8+, EBV-exposed individuals, and each distinct cross-reactivity pattern was associated with a common, public T cell receptor (TCR). In some individuals, CTL cross-reactive with these alloantigens completely dominated the memory response to this EBV epitope. Moreover, these memory T cells to EBV could be reactivated as a significant component of the repertoire of CTL responding to allogeneic stimulator cells expressing either HLA B14 or B35. These data illustrate how a history of infection with an immunogenic virus such as EBV can augment responsiveness to particular alloantigens; such influences may underlie the observed clinical association between herpesvirus infection and both allograft rejection and graft-versus-host disease. We have also explored the molecular basis for T cell cross-reactivity with alloantigens using the HLA B35 allo-reactive CTL clonotype. To elucidate the structural features of peptides that may be cross-recognized by these T cells, mono-substituted analogs of the viral epitope were screened for recognition, revealing broad specificity for major histocompatibility complex (MHC)-bound peptide. Based on the particular amino acid changes tolerated by the CTL at each peptide position, the human protein sequence database was searched for possible sequences that were recognized in association with HLA B35. Four peptides were identified (MPEATVYGL, IPIAPVYGM, KPSPPYFGL, and KPIVVLHGY) that were powerful activating ligands for the CTL when presented on HLA B35 but not B8. Thus, equivalent epitopes, capable of fully activating a single TCR, were formed by peptides with minimal obvious sequence homology bound to either HLA B8 or B35. These data indicate that degenerate peptide recognition by TCR may play an important role in the vigorous response of self-MHC-restricted T cells to alloantigens.     

The J558 VH CDR3 region contributes little to antibody avidity; however, it is the recognition element for cognate T cell control of the alpha(1-->3) dextran-specific antibody response

Analysis of the humoral immune response of BALB/c mice to alpha(1-->3) dextran (Dex) reveals novel aspects of T cell-mediated control of 'type 2 thymus-independent' responses against polysaccharide antigens. The IgM and IgG antibody response, dominated by the J558 idiotype (Id), is controlled by Id-specific T cells. These regulatory T cells, for which the T cell clone 178-4 Ts with characterized TCR alpha and beta chain sequences is the prototype, expand in all BALB/c mice upon immunization with Dex. They suppress in a cognate interaction the expansion of J558 Id-bearing B cells, committed for production of IgG antibodies. Furthermore they provide a gate which precludes variability in the VH CDR3 region of IgG antibodies appearing occasionally in the periphery. The VH CDR3 region is the recognition element of 178-4 Ts analogous T cells but contributes little to affinity for the antigen. For recognition by 178-4 Ts cells not even minimal sequence deviations of the J558 Id peptide are allowed. The tight germline programmed complementarity between J558 Id-bearing Dex-specific B and J558 Id-specific 178-4 Ts analogous T cells leaves little room on both sides for ontogenetic variability.     

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.     

A MAGE-1-encoded HLA-A24-binding synthetic peptide induces specific anti-tumor cytotoxic T lymphocytes

Although several MAGE-1 peptides have already been identified, the MAGE-1-encoded peptide presented by HLA-A24, which is the most common allele in Japanese population and is also frequently present in Caucasians, might have a wide applicability for immunotherapy using these peptides. To identify this potential peptide, we examined the induction of specific cytotoxic T lymphocytes (CTL) from the peripheral-blood mononuclear cells (PBMC) in HLA-A24 healthy donors by in vitro stimulation with MAGE-1-encoded synthetic peptides with a binding affinity for HLA-A24, by a simplified method. Of the 5 peptides tested, the highest HLA binder (NYKHCFPEI) was able to elicit CTL from unseparated PBMC by stimulation with freshly isolated, peptide-pulsed PMBC as antigen-presenting cells (APC) and by also using interleukin 7 and keyhole-limpet hemocyanin for a primary culture. The induced CTL could thus lyse HLA-A24 tumor cells expressing MAGE-1, as well as the peptide-pulsed target cells, in an HLA-class-I-restricted manner. By using the MAGE-1/HLA-A24 peptide, NYKHCFPEI, we found it possible to immunize many more patients, especially Japanese patients, by means of such peptide-based immunotherapeutic approaches to MAGE-1-positive malignant tumors.     

Pores formed by influenza hemagglutinin

The mouse H4 minor histocompatibility antigen (HA) includes a single H2Kb-bound peptide that stimulates rejection of skin allografts and generation of cytolytic T lymphocytes (CTL). We evaluated the diversity of the CTL response to this single minor HA peptide by sequencing alpha and beta chains of T-cell receptors (Tcr) from H4-specific CTLs as a first step toward understanding the diversity of Tcrs specific for single minor HA. We selected H4-specific CTL clones from short-term lines that were restricted by Kb (19 clones), Kbm5 (7 clones), and Kbm11 (10 clones). Whereas multiple V alpha and V beta family members were identified in the panel of Kb-restricted CTLs, five VB genes and one VA subfamily were predominant in CTLs derived from multiple individuals. Similar distributions were observed with Kbm5- and Kbm11-restricted CTLs, suggesting that these two mutants did not alter Tcr gene usage observable at the VA and/or VB gene levels. Negatively charged residues were present in the CDR3 regions of 12/13 unique Kb-restricted beta chains. A comparable observation was made with Kbm5-restricted CTLs, and the distributions of these residues among CDR3 positions were similar in the two CTL panels. However, the Kbm11 mutation dramatically altered the distribution of these residues resulting in their presence in positions 10-12 of CDR3 regions in all CTLs. These results indicate that the Tcrs expressed by CTLs specific for this single minor HA peptide are oligoclonal and characterized by the presence of negatively charged residues in beta CDR3 regions, the distribution of which is profoundly altered by the Kbm11 mutation.     

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 clonal composition of a peptide-specific oligoclonal CTL repertoire selected in response to persistent EBV infection is stable over time

The TCR repertoire of a peptide-specific HLA A11-restricted CTL response to persistent infection with EBV was followed for a period of 57 mo. Sequencing of TCR V alpha and V beta chains and alanine scanning mutagenesis analysis of 83 CTL clones isolated in five reactivation experiments demonstrated that this repertoire is composed of at least four distinct CTL clonotypes that are constantly reactivated from donor's blood and express structurally heterogeneous TCRs. Target cell recognition and CD8 blocking experiments indicate that the four clonotypes possess different avidity and TCR affinity for the specific Ag. This demonstrates that at least in some individuals a heterogeneous peptide-specific memory CTL repertoire selected by a persistent Ag can be remarkably stable in time and accommodate a range of TCR affinities and T cell avidities. Our results suggest that competition for the specific Ag may be not the major force driving the maintenance of memory CTLs and that the nature of the first antigenic challenge may largely determine the clonal composition of memory.     

TCRB junctional regions from HLA-B27-restricted T cells and HLA-B27 binding peptides display conserved hydropathy profiles in the absence of primary sequence homology

Analysis of formal amino acid sequence identity between different TCRB chain (TCRB) hypervariable regions (CDR3) is commonly used to localize relevant sites of TCR antigen interaction or to yield indirect information on unknown corresponding antigens. However, this analysis sometimes fails to demonstrate expected concordances, e.g. between CDR3 from T cell clones of identical reactivity. Since this may be due to ignorance of physico-chemical parameters, we have now use hydropathy profile analysis as an additional method to examine TCRB-CDR3 and putative peptide antigens. Superimposed hydropathy plots (SHOP) of 20 TCRB-CDR3 from HLA-B27-restricted autoreactive and Yersinia enterocolitica-specific synovial cytotoxic T lymphocytes (CTL) isolated from patients with reactive arthritis (ReA) revealed restricted distribution of polar amino acids resulting in characteristically different SHOP profiles between the two CTL groups. Similarly, Yersinia-derived and self nonapeptides known to bind HLA-B27 differed in SHOP profiles. To validate the method we have extended SHOP analysis to published TCRB sequence data from additional TCRB-CDR3 from peptide-specific CTL but not in TCRB from HLA-B27-alloreactive CTL or non-HLA-B27-restricted control CTL. We here demonstrate that SHOP may improve TCR-CDR3 sequence analysis by detection of structural constraints which remain cryptic by conventional sequence analysis. Our data suggest that electrostatic properties rather than rigid sequence motifs determine T cell specificities.     

HLA-B27 (B*2701) specificity for peptides lacking Arg2 is determined by polymorphism outside the B pocket

B*2701 differs from B*2705-by three amino acid changes: D-->Y74, D-->N77, L-->A81, and from B*2702 only by two: D-->Y74 and T-->I80. Tyr74 is located in the C/F cavity of the peptide-binding site, and is unique to B*2701 among HLA-B27 subtypes. Binding of natural B*2705 and B*2702 ligands to B*2701, and to mutants mimicking subtype changes, was analyzed. In addition, sequencing of the peptides bound in vivo by B*2701 and the Y74 mutant was carried out. The main distinctive feature of B*2701 was its presentation of peptides with Gln2. Synthetic analogs bound in vitro similarly as the corresponding ligands with Arg2. Moreover, both Gln2 and Arg2 were dominant upon pool sequencing of B*2701-bound peptides, and 2 of 8 natural ligands contained Gln2. Suitability of Gln2 was largely determined by the Y74 change, as indicated by: 1) binding of Gln2 analogs to this mutant, and 2) detection of Gln2 by pool sequencing of Y74-bound peptides. B*2701 bound peptides with C-terminal aromatic or Leu residues, and interacted with these motifs more strongly than B*2702. The Y74 mutation alone was not responsible for poor binding of peptides with C-terminal basic residues to B*2701, since they bound efficiently and at least one was presented in vivo by this mutant. Most peptides bound to the A81 mutant worse than to B*2705, but frequently better than to B*2701 or B*2702, suggesting that other subtype changes were compensatory. The peptide specificity of B*2701 suggests that this subtype may determine susceptibility to spondyloarthropathy.     

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.     

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.