The X-ray crystal structure of a Valpha2.6Jalpha38 mouse T cell receptor domain at 2.5 A resolution: alternate modes of dimerization and crystal packing.

We describe here the structure of a murine T cell receptor (TCR) Valpha2.6Jalpha38 (TCRAV2S6J38) domain, derived from a T cell hybridoma with specificity for the H-2Ddmajor histocompatibility complex class I molecule bound to a decamer peptide, P18-I10, from the HIV envelope glycoprotein gp120, determined by X-ray crystallography at 2.5 A resolution. Unlike other TCR Valpha domains that have been studied in isolation, this one does not dimerize in solution at concentrations below 1 mM, and the crystal fails to show dimer contacts that are likely to be physiological. In comparison to other Valpha domains, this Valpha2.6 shows great similarity in the packing of its core residues, and exhibits the same immunoglobulin-like fold characteristic of other TCR Valpha domains. There is good electron density in all three complementarity-determining regions (CDRs), where the differences between this Valpha domain and others are most pronounced, in particular in CDR3. Examination of crystal contacts reveals an association of Valpha domains distinct from those previously seen. Comparison with other Valpha domain structures reveals variability in all loop regions, as well as in the first beta strand where placement and configuration of a proline residue at position 6, 7, 8, or 9 affects the backbone structure. The great variation in CDR3 conformations among TCR structures is consistent with an evolving view that CDR3 of TCR plays a plastic role in the interaction of the TCR with the MHC/peptide complex as well as with CDR3 of the paired TCR chain.     

Conserved and heterogeneous lipid antigen specificities of CD1d-restricted NKT cell receptors

CD1d-restricted NKT cells use structurally conserved TCRs and recognize both self and foreign glycolipids, but the TCR features that determine these Ag specificities remain unclear. We investigated the TCR structures and lipid Ag recognition properties of five novel Valpha24-negative and 13 canonical Valpha24-positive/Vbeta11-positive human NKT cell clones generated using alpha-galactosylceramide (alpha-GalCer)-loaded CD1d tetramers. The Valpha24-negative clones expressed Vbeta11 paired with Valpha10, Valpha2, or Valpha3. Strikingly, their Valpha-chains had highly conserved rearrangements to Jalpha18, resulting in CDR3alpha loop sequences that are nearly identical to those of canonical TCRs. Valpha24-positive and Valpha24-negative clones responded similarly to alpha-GalCer and a closely related bacterial analog, suggesting that conservation of the CDR3alpha loop is sufficient for recognition of alpha-GalCer despite CDR1alpha and CDR2alpha sequence variation. Unlike Valpha24-positive clones, the Valpha24-negative clones responded poorly to a glucose-linked glycolipid (alpha-glucosylceramide), which correlated with their lack of a conserved CDR1alpha amino acid motif, suggesting that fine specificity for alpha-linked glycosphingolipids is influenced by Valpha-encoded TCR regions. Valpha24-negative clones showed no response to isoglobotrihexosylceramide, indicating that recognition of this mammalian lipid is not required for selection of Jalpha18-positive TCRs that can recognize alpha-GalCer. One alpha-GalCer-reactive, Valpha24-positive clone differed from the others in responding specifically to mammalian phospholipids, demonstrating that semi-invariant NKT TCRs have a capacity for private Ag specificities that are likely conferred by individual TCR beta-chain rearrangements. These results highlight the variation in Ag recognition among CD1d-restricted TCRs and suggest that TCR alpha-chain elements contribute to alpha-linked glycosphingolipid specificity, whereas TCR beta-chains can confer heterogeneous additional reactivities.     

Reciprocal expression in CD4 or CD8 subsets of different members of the V alpha 11 gene family correlates with sequence polymorphism

Previous staining studies with TCR V alpha 11-specific mAbs showed that V alpha 11.1/11.2 (AV11S1 and S2) expression was selectively favored in the CD4+ peripheral T cell population. As this phenomenon was essentially independent of the MHC haplotype, it was suggested that AV11S1 and S2 TCRs exert a preference for recognition of class II MHC molecules. The V alpha segment of the TCR alpha-chain is suggested to have a primary role in shaping the T cell repertoire due to selection for class I or II molecules acting through the complementarity determining regions (CDR) 1 alpha and CDR2 alpha residues. We have analyzed the repertoire of V alpha 11 family members expressed in C57BL/6 mice and have identified a new member of this family; AV11S8. We show that, whereas AV11S1 and S2 are more frequent in CD4+ cells, AV11S3 and S8 are more frequent in CD8+ cells. The sequences in the CDR1 alpha and CDR2 alpha correlate with differential expression in CD4+ or CD8+ cells, a phenomenon that is also observed in BALB/c mice. With no apparent restriction in TCR J alpha usage or CDR3 alpha length in C57BL/6, these findings support the idea of V alpha-dependent T cell repertoire selection through preferential recognition of MHC class I or class II molecules.     

High-affinity, peptide-specific T cell receptors can be generated by mutations in CDR1, CDR2 or CDR3

The third complementarity-determining regions (CDR3s) of antibodies and T cell receptors (TCRs) have been shown to play a major role in antigen binding and specificity. Consistent with this notion, we demonstrated previously that high-affinity, peptide-specific TCRs could be generated in vitro by mutations in the CDR3alpha region of the 2C TCR. In contrast, it has been argued that CDR1 and CDR2 are involved to a greater extent than CDR3s in the process of MHC restriction, due to their engagement of MHC helices. Based on this premise, we initiated the present study to explore whether higher affinity TCRs generated through mutations in these CDRs or other regions would lead to significant reductions in peptide specificity (i.e. the result of greater binding energy gained through interactions with major histocompatibility complex (MHC) helices). Yeast-display technology and flow sorting were used to select high-affinity TCRs from libraries of CDR mutants or random mutants. High-affinity TCRs with mutations in the first residue of the Valpha, CDR1, CDR2, or CDR3 were isolated. Unexpectedly, every TCR mutant, including those in CDR1 and CDR2, retained remarkable peptide specificity. Molecular modeling of various mutants suggested that such exquisite specificity may be due to: (1) enhanced electrostatic interactions with key peptide or MHC residues; or (2) stabilization of CDRs in specific conformations. The results indicate that the TCR is positioned so that virtually every CDR can contribute to the antigen-specificity of a T cell. The conserved diagonal docking of TCRs could thus orient each CDR loop to sense the peptide directly or indirectly through peptide-induced effects on the MHC.     

The diversity of H3 loops determines the antigen‐binding tendencies of antibody CDR loops

Of the complementarity‐determining regions (CDRs) of antibodies, H3 loops, with varying amino acid sequences and loop lengths, adopt particularly diverse loop conformations. The diversity of H3 conformations produces an array of antigen recognition patterns involving all the CDRs, in which the residue positions actually in contact with the antigen vary considerably. Therefore, for a deeper understanding of antigen recognition, it is necessary to relate the sequence and structural properties of each residue position in each CDR loop to its ability to bind antigens. In this study, we proposed a new method for characterizing the structural features of the CDR loops and obtained the antigen‐binding ability of each residue position in each CDR loop. This analysis led to a simple set of rules for identifying probable antigen‐binding residues. We also found that the diversity of H3 loop lengths and conformations affects the antigen‐binding tendencies of all the CDR loops.     

Amino acids specifying MHC class preference in TCR V alpha 2 regions.

Some TCR variable regions are preferentially expressed in CD4+ or CD8+ T cells, reflecting a predilection for interacting with MHC class II or class I molecules. The molecular basis for MHC class bias has been studied previously, in particular for V alpha 3 family members, pointing to a dominant role for two amino acid positions in complementary-determining regions (CDRs) 1 and 2. We have evaluated the generality of these findings by examining the MHC class bias of V alpha 2 family members, an attractive system because it shows more variability within the CDR1 and -2, exhibits variation in the framework regions, and includes a member for which the crystal structure has been determined. We find that preferential recognition of MHC class I or II molecules does not always depend on residues at the same positions of CDR1 and -2; rules for one family may be reversed in another. Instead, there are multiple influences exerted by various CDR1/2 positions as well as the CDR3s of both the TCR alpha- and TCR beta-chains.     

Predicted complementarity determining regions of the T cell antigen receptor determine antigen specificity.

The antigen receptor on T cells (TCR) has been predicted to have a structure similar to a membrane-anchored form of an immunoglobulin F(ab) fragment. Virtually all of the conserved amino acids that are important for inter- and intramolecular interactions in the VH-VL pair are also conserved in the TCR V alpha and V beta chains. A molecular model of the TCR has been constructed by homology and we have used the information from this, as well as the earlier structural predictions of others, to study the basis for specificity. Specifically, regions of a TCR cloned from an antigen-specific T cell were stitched into the corresponding framework of a second TCR. Results indicate that the substitution of amino acid sequences corresponding to the complementarity determining regions (CDRs) of immunoglobulin can convey the specificity for antigen and major histocompatibility complex molecules. These data are consistent with a role, but not an exclusive role, for CDR3 in antigen peptide recognition.     

Structure, specificity and CDR mobility of a class II restricted single-chain T-cell receptor.

Using NMR spectroscopy, we determined the solution structure of a single-chain T-cell receptor (scTCR) derived from the major histocompatibility complex (MHC) class II-restricted D10 TCR. The conformations of complementarity-determining regions (CDRs) 3beta and 1alpha and surface properties of 2alpha are different from those of related class I-restricted TCRs. We infer a conserved orientation for TCR V(alpha) domains in complexes with both class I and II MHC-peptide ligands, which implies that small structural variations in V(alpha) confer MHC class preference. High mobility of CDR3 residues relative to other CDR or framework residues (picosecond time scale) provides insight into immune recognition and selection mechanisms.     

从抗TNF抗体的CDR肽库中获得拮抗TNF的短肽

为设计来自抗体的短肽 ,以抗肿瘤坏死因子 (TNF)嵌合抗体 (cA2 )CDRs为模板 ,在其两侧各加 3个随机氨基酸残基 ( X3 CDR X3 ) ,构建了 6个以CDR为基础的肽库 .经过 3轮亲和选择 ,挑取单克隆 ,进一步经ELISA检测TNF阳性噬菌体克隆 ,分离得到 7个ELISA阳性较好的噬菌体肽克隆 ,分别命名为CDR2L1、CDR2L2、CDR2L3、CDR1L1、CDR2H1、CDR3H1、CDR3H 2 .应用MTT方法 ,检测 7个克隆对TNF生物学活性的拮抗作用 .结果显示 :来自CDR2L ,CDR3H肽库中的CDR2L2、CDR2L3,CDR3H2噬菌体肽具有明显的拮抗TNF诱导L92 9细胞的细胞毒作用 ,其中以CDR2L2噬菌体肽的拮抗活性最强 .而来源于CDR1L ,CDR2H肽库的CDR1L1和CDR2H1噬菌体肽和来自CDR2L ,CDR3H肽库中的CDR2L1和CDR3H1噬菌体肽没有明显的拮抗TNF作用 .研究结果初步表明 :从cA2抗体CDR肽库中筛选得到的噬菌体CDR模拟肽具有亲本抗体相似的结合活性和生物学效应 ,从而为开发已知抗体 (特别是治疗用抗体 )CDR为基础的肽药物创建一个技术平台奠定基础     

Modeling of the TCR-MHC-peptide complex

The methodology for generating a homology model of the T1 TCR-PbCS-K(d) class I major histocompatibility complex (MHC) class I complex is presented. The resulting model provides a qualitative explanation of the effect of over 50 different mutations in the region of the complementarity determining region (CDR) loops of the T cell receptor (TCR), the peptide and the MHC's alpha(1)/alpha(2) helices. The peptide is modified by an azido benzoic acid photoreactive group, which is part of the epitope recognized by the TCR. The construction of the model makes use of closely related homologs (the A6 TCR-Tax-HLA A2 complex, the 2C TCR, the 14.3.d TCR Vbeta chain, the 1934.4 TCR Valpha chain, and the H-2 K(b)-ovalbumine peptide), ab initio sampling of CDR loops conformations and experimental data to select from the set of possibilities. The model shows a complex arrangement of the CDR3alpha, CDR1beta, CDR2beta and CDR3beta loops that leads to the highly specific recognition of the photoreactive group. The protocol can be applied systematically to a series of related sequences, permitting the analysis at the structural level of the large TCR repertoire specific for a given peptide-MHC complex.     

Fine specificity of TCR complementarity-determining region residues and lipid antigen hydrophilic moieties in the recognition of a CD1-lipid complex

alphabeta TCR can recognize peptides presented by MHC molecules or lipids and glycolipids presented by CD1 proteins. Whereas the structural basis for peptide/MHC recognition is now clearly understood, it is not known how the TCR can interact with such disparate molecules as lipids. Recently, we demonstrated that the alphabeta TCR confers specificity for both the lipid Ag and CD1 isoform restriction, indicating that the TCR is likely to recognize a lipid/CD1 complex. We hypothesized that lipids may bind to CD1 via their hydrophobic alkyl and acyl chains, exposing the hydrophilic sugar, phosphate, and other polar functions for interaction with the TCR complementarity-determining regions (CDRs). To test this model, we mutated the residues in the CDR3 region of the DN1 TCR beta-chain that were predicted to project between the CD1b alpha helices in a model of the TCR/CD1 complex. In addition, we tested the requirement for the negatively charged and polar functions of mycolic acid for Ag recognition. Our findings indicate that the CDR loops of the TCR form the Ag recognition domain of CD1-restricted TCRs and suggest that the hydrophilic domains of a lipid Ag can form a combinatorial epitope recognized by the TCR.     

Immunobiological analysis of TCR single-chain transgenic mice reveals new possibilities for interaction between CDR3alpha and an antigenic peptide bound to MHC class I

The interaction between TCRs and peptides presented by MHC molecules determines the specificity of the T cell-mediated immune response. To elucidate the biologically important structural features of this interaction, we generated TCR beta-chain transgenic mice using a TCR derived from a T cell clone specific for the immunodominant peptide of vesicular stomatitis virus (RGYVYQGL, VSV8) presented by H-2K(b). We immunized these mice with VSV8 or analogs substituted at TCR contact residues (positions 1, 4, and 6) and analyzed the CDR3alpha sequences of the elicited T cells. In VSV8-specific CTLs, we observed a highly conserved residue at position 93 of CDR3alpha and preferred Jalpha usage, indicating that multiple residues of CDR3alpha are critical for recognition of the peptide. Certain substitutions at peptide position 4 induced changes at position 93 and in Jalpha usage, suggesting a potential interaction between CDR3alpha and position 4. Cross-reactivity data revealed the foremost importance of the Jalpha region in determining Ag specificity. Surprisingly, substitution at position 6 of VSV8 to a negatively charged residue induced a change at position 93 of CDR3alpha to a positively charged residue, suggesting that CDR3alpha may interact with position 6 in certain circumstances. Analogous interactions between the TCR alpha-chain and residues in the C-terminal half of the peptide have not yet been revealed by the limited number of TCR/peptide-MHC crystal structures reported to date. The transgenic mouse approach allows hundreds of TCR/peptide-MHC interactions to be examined comparatively easily, thus permitting a wide-ranging analysis of the possibilities for Ag recognition in vivo.     

Conserved CDR3 regions in T-cell receptor (TCR) CD8(+) T cells that recognize the Tax11-19/HLA-A*0201 complex in a subject infected with human T-cell leukemia virus type 1: relationship of T-cell fine specificity and major histocompatibility complex/peptide/TCR crystal structure

We investigated the T-cell receptor (TCR) repertoire of CD8(+) T cells that recognize the Tax11-19 immunodominant epitope of Tax protein expressed by human T-cell leukemia virus (HTLV-1) that is implicated in the disease HTLV-1-associated myelopathy (HAM/TSP). A panel of Tax11-19-reactive CD8(+) T-cell clones was generated by single-cell cloning of Tax11-19/HLA-A*0201 tetramer-positive peripheral blood lymphocytes from an HTLV-1-infected individual. The analyses of TCR usage revealed that the combination of diverse TCR alpha and beta chains could be used for the recognition of Tax11-19 but the major population of T-cell clones (15 of 24 clones) expressed the TCR V beta 13S1 and V alpha 17 chain. We found striking similarities in CDR3 regions of TCR alpha and beta chains between our major group of CD8(+) T-cell clones and those originating from different subjects as previously reported, including TCRs with resolved crystal structures. A 3-amino-acid sequence (PG-G) in the CDR3 region of the V beta chain was conserved among all the Tax11-19-reactive T-cell clones expressing V beta 13S1 and V alpha 17 chains. Conserved amino acids in the CDR3 region do not directly contact the Tax11-19 peptide, as corroborated by the crystal structure of B7-TCR, a TCR that is almost identical to VB13S1 clones isolated in this study. Analysis of fine peptide specificity using altered peptide ligands (APL) of Tax11-19 revealed a similar recognition pattern among this panel of T-cell clones. These data suggest that the PG-G amino acids in the CDR3 beta loop provide a structural framework necessary for the maintenance of the tertiary TCR structure.     

Complementarity-determining region 1 sequence requirements drive limited V alpha usage in response to influenza hemagglutinin 307-319 peptide

We have developed a T cell activation-based system that allows for the selection of TCRs with defined peptide/MHC specificities from libraries in which complementarity-determining region (CDR) sequences have been randomized by in vitro mutagenesis. Using this system, we have explored the sequence requirements for CDR1 and CDR2 of the TCR alpha-chain in a human T cell response characterized by restricted Valpha and Vbeta usage. Libraries of T cells expressing receptors built on the framework of a TCR specific for the influenza virus peptide hemagglutinin 307-319 presented by HLA-DR4, but with random sequences inserted at CDR1alpha or CDR2alpha, were selected for response to the same peptide/MHC ligand. A wide variety of CDR2alpha sequences were found to be permissive for recognition. Indeed, >25% of T cell clones chosen at random displayed a significant response. In contrast, a similar challenge of a randomized CDR1alpha library yielded only the parental sequence, and then only after multiple rounds of selection. T cell clones cross-reactive on closely related HLA alleles (subtypes of DR4) could be isolated from randomized libraries, but not clones restricted by more distantly related alleles such as HLA-DR1. These results indicate that, in the context of this T cell response, the structural requirements for recognition at CDR1alpha are significantly more restricted than at CDR2alpha. This system for mutation and selection of TCRs in vitro may be of use in engineering T cells with defined specificities for therapeutic applications.     

Systematic mutagenesis of TCR complementarity-determining region 3 residues: a single conservative substitution dramatically improves response to both multiple HLA-DR alleles and peptide variants.

To define the relative contributions of HLA and peptide contacts with TCR complementarity-determining region (CDR) 3 residues in T cell recognition, systematic mutagenesis and domain swapping was conducted on two highly similar TCRs that both respond to the influenza hemagglutinin (HA) peptide, HA307-319, but with different HLA restrictions. Despite the primary sequence similarity of these TCRs, exchange of as little as two CDR3 residues between them completely abrogated responsiveness. At position 95 within CDR3alpha, various substitutions still allowed for some degree of recognition. One modest substitution, alanine for glycine (essentially the addition of a methyl group), significantly broadened the specificity of the TCR. Transfectants expressing this mutant TCR responded strongly in the context of multiple HLA-DR alleles and to HA peptide variants with substitutions at each TCR contact residue. These results suggest that the conformations of CDR3 loops are crucial to TCR specificity and that it may not be reliable to extrapolate from primary sequence similarities in TCRs to similarities in specificity. The ease with which a broad specificity is induced in this mutant TCR has implications for the mechanisms and frequency of alloreactivity and promiscuity in T cell responses.     

Antigen-driven selection of TCR In vivo: related TCR alpha-chains pair with diverse TCR beta-chains

Ag-driven selection mediates effective T cell help and the development of Th cell memory in vivo. To analyze the dynamics of interclonal competition during the selection process in vivo, we use the I-Ek-restricted murine response to pigeon cytochrome c (PCC). The dominant PCC-specific clonotype expresses Valpha11Vbeta3 V regions with preferred sequence features in the third hypervariable regions (CDR3). In the current study we define and quantitatively monitor four subdominant PCC-specific clonotypes that express Valpha11 paired with non-Vbeta3 TCR beta-chains (Vbeta6, Vbeta8.1/8. 2, Vbeta8.3, and Vbeta14). The subdominant clonotypes emerge with similar dynamics to the dominant clonotype and together amount to similar numbers as the dominant clonotype in vivo. These subdominant clonotypes do not efficiently enter germinal centers, although they enter the memory compartment and rapidly re-emerge upon secondary challenge. Analysis of CDR3 diversity in the TCR alpha-chains identifies many preferred sequence features expressed by the dominant clonotype. These studies quantitatively demonstrate selection for diverse Th cells in vivo and highlight TCR alpha-chain dominance in Ag-driven selection for best fit.     

The T cell receptor V alpha 11 gene family. Analysis of allelic sequence polymorphism and demonstration of J alpha region-dependent recognition by allele-specific antibodies.

Allelic polymorphism in TCR loci may play an important role in shaping the T cell repertoire and in disease susceptibility. We have used a combination of antibody and sequence analysis to investigate polymorphism in the murine V alpha 11 family. Two different antibodies have been analyzed that recognize particular V alpha 11 family members of the V alpha b and V alpha d haplotypes. One antibody shows J alpha dependency, suggesting a conformational element to the epitope. Investigation of the anti-V alpha 11 staining pattern on different mouse strains indicates that there is a marked influence of MHC haplotype on V alpha 11 selection and that V alpha 11 is preferentially expressed on CD4+ cells. Sequence analysis of V alpha 11 genes from the V alpha a, V alpha b, and V alpha d haplotypes shows two potential regions for the haplotype-specific epitopes. The relatedness of the different V alpha 11 family members from different haplotypes suggests that the V alpha 11.1/11.2 gene duplication is relatively recent, but that V alpha 11.3 separated much earlier. Differences between V alpha 11.3 and V alpha 11.1/11.2 are concentrated in the putative complementarity determining regions (CDR), whereas differences between alleles are not clearly clustered. However, the V alpha 11.1a and V alpha 11.1d alleles differ from V alpha 11.1b and V alpha 11.2b in CDR1. A V alpha 11.2-expressing anti-cytochrome c T cell has the same V-J junction as a V alpha 11.1-bearing cell with a similar fine specificity, indicating that V alpha 11.1b and V alpha 11.2b do not contribute different Ag specificities.     

Maturation of shark single-domain (IgNAR) antibodies: evidence for induced-fit binding

Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.     

A single cell analysis of TCR AV24AJ18+ DN T cells.

The T-cell receptor (TCR) BV gene of human TCR AV24+ double-negative (DN) T cells, a novel subset of natural killer (NK) T cells, was investigated by single-cell sorting and single-cell polymerase chain reaction (PCR) methods. Seven of eleven TCR AV24+ DN T-cell clones utilized TCR BV8, three BV9, and one BV6. Six of seven TCR AV24/BV8+ DN T-cell clones had identical TCR beta and alpha chains, indicating that they were the same clone. All three TCR AV24/BV9+ DN T-cell clones also demonstrated the same amino acids in the CDR3 region. These findings strongly suggest that the usage of TCR beta and alpha chains on TCR AV24+ DN T cells is extremely restricted, supporting the notion that these cells recognize highly limited T-cell epitopes on antigens. All TCR AV24+ clones expressed the NKR-P1A mRNA, and so were true NK T cells. IL-2 and IL-4 mRNAs were detected in all clones, suggesting that the majority of these cells were Th0-type T cells. Six clones overexpressed Fas-ligand (Fas-L) mRNA and Fas antigen was detected on all clones at the mRNA level. In conclusion, TCR AV24+ DN T cells might recognize restricted T-cell epitopes on antigens and function as Th0-type T cells, inducer cells to Th1- or Th2-type T cells (regulatory T cells), and as Fas-L-positive cytolytic T cells.     

CTL recognition of a protective immunodominant influenza A virus nucleoprotein epitope utilizes a highly restricted Vbeta but diverse Valpha repertoire: functional and structural implications

To investigate protective immunity conferred by CTL against viral pathogens, we have analyzed CD8(+) T cell responses to the immunodominant nucleoprotein epitope (NP(366-374)) of influenza A virus in B6 mice during primary and secondary infections in vivo. Unlike the highly biased TCR Vbeta repertoire, the associated Valpha repertoire specific for the NP(366-374)/D(b) ligand is quite diverse. Nonetheless, certain public and conserved CDR3alpha clonotypes with distinct molecular signatures were identified. Pairing of public Valpha and Vbeta domains creates an alphabeta TCR heterodimer that binds efficiently to the NP(366-374)/D(b) ligand and stimulates T cell activation. In contrast, private TCRs, each comprising a distinct alpha chain paired with the same public beta chain, interact very differently. Molecular dynamics simulation reveals that the conformation and mobility of the shared Vbeta CDR loops are governed largely by the associated Valpha domains. These results provide insight into molecular principles regarding public versus private TCRs linked to immune surveillance after infection with influenza A virus.