Structural basis for enabling T-cell receptor diversity within biased virus-specific CD8+ T-cell responses E. Bridie Daya,1, Carole Guillonneaua,1, Stephanie Grasb,1, Nicole L. La Grutaa, Dario A. A. Vignalic, Peter C. Dohertya,c,2, Anthony W. Purcelld, Jamie Rossjohnb, and Stephen J. Turnera,2 a
Department of Microbiology and Immunology, dBio21, Department of Biochemistry, University of Melbourne, Parkville, VIC 3010, Australia; Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia; and cDepartment of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105 b
Contributed by Peter C. Doherty, May 2, 2011 (sent for review March 7, 2011)
Pathogen-specific responses are characterized by preferred profiles of peptide+class I MHC (pMHCI) glycoprotein-specific T-cell receptor (TCR) Variable (V)-region use. How TCRV-region bias impacts TCRαβ heterodimer selection and resultant diversity is unclear. The DbPA224–specific TCR repertoire in influenza A virusinfected C57BL/6J (B6) mice exhibits a preferred TCRV-region bias toward the TRBV29 gene segment and an optimal complementarity determining region (CDR3) β-length of 6 aa. Despite these restrictions, DbPA224-specific BV29+ T cells use a wide array of unique CDR3β sequences. Structural characterization of a single, TRBV29+DbPA224-specific TCRαβ-pMHCI complex demonstrated that CDR3α amino acid side chains made specific peptide interactions, but the CDR3β main chain exclusively contacted peptides. Thus, length but not amino acid sequence was key for recognition and flexibility in Vβ-region use. In support of this hypothesis, retrovirus expression of the DbPA224-specific TCRVα-chain was used to constrain pairing within a naive/immune epitope-specific repertoire. The retrogenic TCRVα paired with a diversity of CDR3βs in the context of a preferred TCRVβ spectrum. Overall, these data provide an explanation for the combination of TCRV region bias and diversity within selected repertoires, even as they maintain exquisite pMHCI specificity. T cell repertoire
| T-cell receptor bias | crystal structure
T
he adaptive T-cell response to viruses is mediated via T-cell receptor (TCR)-αβ glycoprotein heterodimer recognition of pathogen-derived peptides (p) complexed to cell-surface MHC glycoproteins. During T-cell development, somatic recombination of variable (V) and joining (J) or V, diversity (D), and J gene segments (1) leads to the emergence of a diverse spectrum of TCRα- and TCRβ-chains. Segments of hypervariability within the Vα and Vβ domains, termed complementarity determining regions (CDRs), form the TCRαβ binding site and mediate contact with a given pMHC complex. Although the CDR1 and -2 regions are determined by germ-line sequences of V-genes, the CDR3 region is encoded at the junction of spliced VJ (TCRα) and VDJ (TCRβ) gene segments. The murine TCRβ (TRB) locus consists of 35 V, 2 D, and 12 J gene segments and the TCRα (TRA) locus has 71 V and 60 J gene segments (2). Beyond the random splicing of different TCRα and TCRβ genes, the overall spectrum of TCR diversity largely reflects the inefficient splicing of DNA encoding the CDR3α and CDR3β loops (3) plus the addition of nontemplated encoded nucleotides at the V(D)J junctions (4). Finally, different pairings of rearranged TCRα- and TCRβ-chains are selected during thymic differentiation to give the naive TCRαβ repertoire. The diversity of a particular pMHC-specific immune repertoire is defined by the spectrum of different T-cell clones that is fully recruited from the available, naive pool of antigen-specific T cells (5, 6). At this stage, there are no general rules that predict the overall character of any given response. Some pMHCspecific repertoires consist of many different TCRαβ-defined clonotypes (7, 8) but others are much more limited in extent (9). 9536–9541 | PNAS | June 7, 2011 | vol. 108 | no. 23
However, accessing a breadth of TCRs within a pathogen-specific response can be an important predictor of efficient immune control (7, 10–13). As such, understanding the factors that shape pathogen-specific TCR repertoire diversity clearly has important implications for promoting effective immunity. Pathogen-specific T-cell responses are often characterized by the biased use of particular TCR V-regions that present with varying degrees of diversity (14). The nature of a given TCR bias can reflect the selection of a spectrum of sequences within a single TCR V-region, or be limited to a few (or even unique) conserved CDR3 amino acid sequences within specific TCRVβ- or Vα-chains. These individual profiles in turn define the extent of TCR sequence diversity within the host population. For example, the minimal sharing of essentially “private” TCR repertoires unique to particular individuals (8, 15) translates to extensive diversity at the population level. Alternatively, TCR sequences that are found again and again for different individuals give “public” repertoires and more limited diversity across the population (9). However, it should be noted that these two concepts are not necessarily mutually exclusive, with the possibility that highly diverse TCR repertoires could also exhibit a degree of sharing of particular TCR sequences between individuals. The fact that many “shared” antigen-specific TCR repertoires are represented by near germ-line DNA sequences has led to the suggestion that the ease of generating particular TCR sequences during TCR gene recombination can account for repeated selection of particular TCR sequences between individuals (16, 17). Other studies have established that biased TCR use may also reflect the selection of TCRs with optimal structural characteristics that impart exquisite specificity for a given pMHC molecular topography (18–25). Such antigen-driven selection will, of course, be shaped by other factors, such as the availability of naive T-cell precursors and the level and duration of antigenpresentation during infection. Although preferred TCRV-region use has been suggested to reflect a dominant role for either the TCRα or TCRβ, with there being little (or no) role for the other chain (26), recent analysis of biased antigen-specific TCR repertoires has suggested that the pairing of complementary TCRα- and TCRβ-chains is key to optimal TCR/pMHC binding (27, 28). Thus, a specific sequence
Author contributions: E.B.D., C.G., S.G., N.L.L.G., A.W.P., J.R., and S.J.T. designed research; E.B.D., C.G., S.G., N.L.L.G., and S.J.T. performed research; D.A.A.V. contributed new reagents/analytic tools; E.B.D., C.G., S.G., N.L.L.G., P.C.D., A.W.P., J.R., and S.J.T. analyzed data; and E.B.D., C.G., S.G., N.L.L.G., D.A.A.V., P.C.D., A.W.P., J.R., and S.J.T. wrote the paper. The authors declare no conflict of interest. Data deposition: Crystallography, atomic coordinates, and structure factors have been deposited (accession no. 3PQY). 1
E.B.D., C.G., and S.G. contributed equally to this work.
2
To whom correspondence may be addressed. E-mail:
[email protected] or sjturn@ unimelb.edu.au.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1106851108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1106851108
Results Structural Basis for Maintenance of CDR3β Diversity Within TCRBiased Repertoires. Structural overview. Prior studies showing that
biased TCRs exhibit exquisite structural specificity for their pMHC (19–21) raise the question: What is the molecular basis for maintenance of diverse antigen-specific TCR repertoires in the face of preferred Vα and Vβ usage? To address this question, we cloned and expressed the DbPA224-specific TCR from a T-cell hybridoma (named 6218) that is known to demonstrate a high degree of specificity for its cognate DbPA224 pMHCI epitope (29). The 6218 TCR (Fig. S1) consists of a TRAV21-AJ53 (CDR3α amino acid sequence, SGGSNYKL) and TRBV29-BJ27 combination (CDR3β amino acid sequence, SFGREQ). Importantly, the 6218 chain exhibits the canonical features of the diverse TRBV29+ DbPA224-specific repertoire, namely a CDR3β length of 6 aa and TRBJ2-7 use (8). We solved the structure of the 6218-TCR-H2-Db-SSLENFRAYV complex at a resolution of 3.20 Å to an Rfac and Rfree of 24.6% and 31.6%, respectively (Table S1). The bound 6218 TCR, comprised of the TRAV21TRAJ53 and TRBV29-TRBJ2-7, aligned at ∼60° across the long axis of the H2Db-SSLENFRAYV binding cleft (Fig. 1 A and B), a docking mode that falls within the range of TCR-pMHC complexes determined to date (reviewed in ref. 30). The 6218 TCR located over the C terminus of the H2Db-SSLENFRAYV complex, with a total buried surface area (BSA) of 1,730 Å2 at the interface between the PA-TCR and the DbPA224 complex and both the Vα and Vβ contributing equally to the pMHC interface (∼ 430 and 440 Å2 BSA, respectively) (Fig. 1B). Although the CDR2α and CDR1β made little contact with the pMHC (6.3% and 7.2%, respectively), roughly equal and significant contributions were made by the CDR3α (23.5%), CDR3β (18.8%), CDR2β (18.8%), and CDR1α (17.5%) loops. Day et al.
Fig. 1. Structural determination of the 6218 PA-DbPA224 ternary complex. (A) A ribbon representation of the PA TCR/DbPA224 complex, showing the TCRα-chain in pale pink, the TCRβ-chain in pale blue, H2Db in white, and the PA224 peptide in dark purple. The CDR regions of the PA TCR are colored purple (CDR1α), green (CDR2α), yellow (CDR3α), red (CDR1β), blue (CDR2β), and orange (CDR3β). This color system is used throughout. (B) The footprint of the PA TCR on the surface of the DbPA224 complex. Regions of the DbPA224 complex that are contacted by the CDR loops of the PA TCR are shaded using the color system described in A and the orientation of the Vα and Vβ regions are indicated. (C–E) Contacts made by the CDR2β and MHCα1 helix (C), CDR3α (D), and CDR3β (E) loops with the PA224 peptide are shown. The residues involved in the interaction are represented in stick and the red dashed lines indicate hydrogen bonds. The coloring system is the same as described in A.
TRBV29 makes unique TCR–MHC interactions. Although the complex was determined to moderate resolution, the quality of the electron density at the interface was clear (Fig. S2), permitting a detailed description of the contacts (Table S2). The TCRβchain interacts more extensively with H2-Db than the α-chain and, but the CDR1β loop makes a relatively small contribution to the overall BSA at the interface, Glu30β contacts residues from both α1 (Asn80) and α2 helices (Lys-146) of H2-Db. The CDR2β loop contacts a large region of the α1-helix, spanning residues Gln72 to Arg79. This process involves a network of hydrogen bonds and hydrophobic interactions, principally Ser60β H-bonding with Gln72 and Arg75, and Asp58β salt-bridging to Arg-75 and Arg-79 (Fig. 1C). In addition, the TRBV29 framework residue, Arg84β, interacts with Arg79 and Asn80. Notably, TRBV29 is the only Vβ gene to contain this constellation of amino acids, namely Glu30β in CDR1β the framework Arg84 as well as the D58xxS60 motif of the CDR2β loop. As such, the TRBV29 chain is uniquely suited to making appropriate MHC contacts when recognizing the DbPA224 complex and provides molecular insight into its preferred use within DbPA224-specific CTL repertoires. Strikingly, the CDR3β contacts H2-Db via its two nongerm-line–encoded residues, Phe108β and the Arg110β. The side chain of Phe108β sits atop Lys146 of H2-Db, forming a large number of hydrophobic contacts, but the long side chain PNAS | June 7, 2011 | vol. 108 | no. 23 | 9537
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requirement for one TCR chain is likely to limit the extent of acceptable diversity in the other. This constraint, together with the fact that that TCR/pMHC binding is ultimately determined at the level of the structural interface (19, 20), raises an important question, namely: How can a specific T-cell repertoire reconcile the need to maintain exquisite specificity, preferred TCRVregion usage and CDR3 diversity at the same time? The present analysis addresses these issues for the H-2Dbrestricted acid polymerase peptide 224–233 (SSLENFRAYV, DbPA224)-specific cytotoxic T lymphocyte (CTL) response. The DbPA224-specific TCR repertoire in influenza A virus-infected WT C57BL/6J (B6) mice exhibits an optimal CDR3β length of 6 aa and a preferred TCRV-region bias toward the TRBV29 gene segment (8). Despite these restrictions, DbPA224-specific BV29+ CTLs use a wide array of unique CDR3β sequences (8). As such, this is an ideal model to examine how diversity can be maintained in the face of preferred TCRV-region use. We solved the ternary structure of a canonical DbPA224-specific TCR complexed to its cognate pMHC complex and found that the CDR3 loops differed in the type of contacts made with the pMHC. Although the CDR3α loop made peptide contact via the amino acid side chains, the CDR3β loop made main-chain interactions. To test the hypothesis that main-chain interactions have the capacity to enable the selection of a diverse TCR repertoire as they maintain structural characteristics for optimal pMHC recognition, we used retroviral transduction to generate mice with fixed expression of a single DbPA224-specific TCRα-chain (termed retrogenic, or Rg mice). Importantly, although fixing the DbPA224specific TRAV resulted in a preferred TRBV29 bias in the virusspecific T-cell repertoire, extensive TCR diversity was maintained. This comprehensive analysis illustrates how topographical constraints determine the spectrum of CDR3α and CDR3β sequences in a given antigen-specific response, establishing the key role of the TCR/pMHC structural interface in determining both TCR bias and repertoire diversity.
of Arg110β packs against Gln149 and Ala150 of H2-Db. As such, these contacts argue against the CDR3 being solely responsible for interactions with peptide (31). Peptide–TCR Interaction. The 6218 TCR is positioned directly over the prominent P7-Arg of the SSLENFRAYV peptide (Fig. 1 A and B), previously reported as a key functional TCR contact residue (32). In the binary H2-Db-SSLENFRAYV complex, the P7-Arg side chain exhibited a degree of flexibility and, following ligation by the 6218 TCR, was reorientated to avoid steric clashes with the CDR3β loop. The P7-Arg side-chain (peg) inserts into a pocket (notch) formed by the CDR1α, CDR3α, CDR1β, CDR2β, and CDR3β loops, although peptide contact is almost exclusively via the CDR3 loops (Fig. 1 A and B). The CDR3α loop interacts with residues P4-Glu to P9-Tyr, mostly via the 107SGGSNY112 motif (Fig. 1D), and dominates peptide contact (66% BSA). The “SGGS” motif within the CDR3α loop results in an H-bonded turn that enables the CDR3α loop to come into close proximity with the backbone of the SSLENFRAYV peptide (Fig. 1D). This feature, coupled with the lack of bulky side chains within the “SGGS” motif, results in extensive main-chain interactions with residues P5-Asn and P6-Phe (Fig. 1D). Furthermore, CDR3α side chain-mediated interactions are observed between Ser107α, Ser110α, Asn111α, and Tyr112α, and positions P4-Glu and P7-Arg (Fig. 1D). Tyr112α also interacts with the C-terminal residues, P8-Ala and P9-Tyr (Fig. 1D), suggesting that this residue is key for pMHC recognition All together, the combination of the SGGS motif and the Tyr112α within the 6218 PA TCR CDR3α imparts a high degree of specificity toward H2-Db-SSLENFRAYV. In contrast to the amino acid side-chain contacts made by the CDR3α, the CDR3β loop engages P7-Arg and P8-Ala via the main chain of Phe108β, Gly109β, and Arg110β (Fig. 1E). A hydrogen bond between the conserved Ser107β and Gly109 help maintains the conformation of the CDR3β loop. Furthermore, the lack of bulky side chains at positions 107 and 109 permit the main chain of the CDR3β loop to be in close contact with the peptide. The nongerm-line–encoded side-chains Phe108β and Arg110β are pushed away from the peptide and interact with H-2Db (Fig. 1E). Phe-108 is mostly solvent-exposed, and its location and mode of packing against Lys146 provides a basis for understanding how an aromatic residue preferably occupies this position within DbPA224immune repertoires (8). Similarly, Arg110β is largely solventexposed, such that its substitution by another residue could easily be accommodated without changing the overall structure of the complex. As the CDR3β contacts the peptide exclusively via it’s main chain, with no contribution from the amino acid side chains within the CDR3β loop, it appears that it is the conformation of the CDR3β loop (rather than amino acid composition per se) that is the dominant structural characteristic enabling optimal recognition of the SSLENFRAYV peptide. Accordingly our data provide a molecular explanation for how sequence diversity within the CDR3β loop could be tolerated in the face of preferred TRBV use for virus-specific TCR repertoires.
(Fig. 2A). Because of the lack of a specific mAb to detect the TRAV21 gene segment by flow cytometry, we made the assumption that (in the absence of a functional TCRα constant gene segment because of transduction of TRAC-region–deficient bone marrow) GFP staining of CD8+ T cells was a measure of efficient retroviral transduction and expression of the TRAV21 TCRα in the PAα-Rg mice (33), although this need not, of course, lead to the expression of a TCR specific for the DbPA224 epitope. To determine whether the TRAV21 (6218 TCRα) DbPA224specific CD8+ CTLs can indeed respond in vivo, the PAα-Rg mice were infected intranasally with the HKx31 virus and DbPA224specific CTL responses were measured by DbPA224 tetramer binding (Fig. 2B) and intracellular cytokine staining (Fig. 2C). Tetramer+ GFP+CD8+DbPA224+ cells were prominent in the bronchoalveolar lavage (BAL) (Fig. 2D) and spleen (Fig. 2E) populations from infected PAα-Rg mice, but the normally equivalent (in WT mice) DbNP366-specific CTL response from both the nontransduced (GFP−) and Rg (GFP+) TCR repertoires was minimal. Clearly, even when free to pair with any available TCRβ, the 6218 TCRα-chain cannot establish a significant DbNP366 specific TCR repertoire (Fig. 2 D and E). The PAα-Rg GFP+DbPA224-specific CTLs, on the other hand, showed patterns of IFN-γ, TNF-α, and IL-2 polyfunctionality (Fig. 2B and Fig. S3) comparable to those observed for endogenous DbPA224-specific T cells (35). Ectopic expression of a single DbPA224-specific TCRα thus provides an immune CTL repertoire that, at least in the functional sense, mimics the WT response. Preferred TCRαβ Pairing Determines DbPA224 Specificity. As an initial probe to screen how the Rg TRAV21 shapes TRBV use within the DbPA224-specific CTL repertoire, we used a panel of TCRVβspecific mAbs to scan both the broad GFP+CD8+ (nonspecific) and GFP+CD8+DbPA224-specific populations from PAα-Rg mice (Fig. 3). Although there was little overall impact of forced TRAV21-AJ53 expression on TRBV use within the breadth of naive GFP+CD8+ T cells (Fig. 3, white bars), the TRBV29 TCRβchain was used exclusively in the immune GFP+DbPA224-specific CTL response (Fig. 3, black bars). This finding represented a significant increase in the TRBV29 bias compared with the endog-
Generation of DbPA224-Specific Single TCRα-Chain Retrogenic Mice.
The fact that the 6218 TRBV29+CDR3β loop contacts the DbPA224 complex via main-chain interactions led to the view that, should the DbPA224-specific repertoire be limited by enforced expression of the TRAV21 chain, the spectrum of potential CDR3β interactions would still enable TRBV29+ TCR repertoire diversity to be maintained in response to infection. To test this hypothesis, we used a retroviral transduction system (33, 34) to fix expression of the DbPA224-specific TCRα (analyzed above) in retrogenic PAα-Rg mice. The 6218 TRAV21-AJ53 TCR chain was cloned into the bicistronic pMIG retroviral construct, enabling cell transduction to be detected by GFP expression. Significant numbers of T cells were GFP+ at 8 wk after reconstitution 9538 | www.pnas.org/cgi/doi/10.1073/pnas.1106851108
Fig. 2. Retroviral-mediated expression of DbPA224-specific TCRα-chain in mice. (A) PAα retrogenic mice were bled 5 to 6 wk after reconstitution and GFP expression was analyzed for lymphocytes, CD8+ and CD4+ T cells by flow cytometry. (B) PAα-Rg mice were infected with 104 pfu of the A/HKx31 influenza A virus and transduced (GFP+) CD8+ lymphocytes populations were stained with the DbPA224 tetramer 10 d after infection. (C) GFP+CD8+ T cells from immune PAα-Rg mice T cells isolated from BAL or spleen were analyzed for expression of IFN-γ and TNF-α production by intracellular cytokine staining after 5-h stimulation with or without PA224 peptide in the presence of golgi plug and hIL-2. (D and E) DbNP366- and DbPA224-specific CD8+ T cells were enumerated from the BAL (D) and spleen (E) of PAα-Rg mice 10 d after infection. Data show mean ± SD for three to four mice. *P < 0.05; **P < 0.01.
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b
enous D PA224-specific CTL repertoire from infected B6 mice (Fig. 3). Apparently the TRAV21-AJ53/TRBV29 TCRαβ combination imparts optimal specificity for the DbPA224 complex demonstrating, in turn, that preferred TCRαβ pairing dictates biased TCRV-region use for the DbPA224-specific TRBV29 repertoire in PAα-Rg mice. Diversity in This TCRVα-Constrained TCRVβ Repertoire. Although exclusive selection of TRBV29 chain and pairing with the TRAV21 PA TCRα-chain suggests that preferred TCRαβ pairing is a key driver for biased TCR chain use, the question remains: Can TCR diversity be maintained in the face of such bias? To address this question, single GFP+TRBV29+DbPA224+ CTLs were sorted for subsequent TRBV29 amplification and CDR3β sequencing (Table 1 and Table S3). The exclusive TRBV29 gene-segment use within these individually-analyzed (and diverse) PAα GFP+DbPA224-specific CTLs was characterized by the repeated selection of a fixed 6-aa CDR3β length (Table 1), a canonical feature of the WT DbPA224-specific repertoires induced by infection (8, 32). Strikingly, despite these strict limitations on TRBV use and CDR3β length, the spectrum of selected clonotypes expressed a diverse array of CDR3β sequences (Table 1 and Table S3), comparable to that observed for endogenous DbPA224-specific responses (8). Interestingly, as found previously for the WT DbPA224-specific repertoire (8, 32), there was a clear preference for the selection of an aromatic amino acid (Phe, Tyr, Trp) at position 108 of the CDR3β loop (Table S3). This finding likely reflects the key CDR3β mediated MHC contacts identified in the structural analysis (Fig. 1). Furthermore, fixed expression of the 6218 PA TCRα-chain did not result in dominant selection of the parent TRBV29 CDR3β (SFGREQ) that we used for our structural analysis (Fig. 1). In
Discussion A hallmark of many pathogen-specific T-cell immune repertoires is preferred use of specific TRAV or TRBV gene segments (14). Structural studies have demonstrated that such TCR bias reflects optimal TCR binding for any given pMHC complex. For example, both the LC13 TCR, specific for EBV EBNA3/HLA-B0801, and JM22 TCR, specific for Influenza Matrix 1/HLA-A0201, provide examples of biased TCR use with defined viral determinants (36, 37). Both TCRs use a “peg-notch” type of interaction to recognize their cognate pMHC complexes and rely on a number of amino acid side-chain–mediated contacts to achieve a high degree of specificity (19, 20, 38). Our data demonstrate that although preferred TCR V-region use reflects a requirement for specific TCRαβ clonotype pairing to impart optimal pMHC specificity, the combinatorial nature of the TCRαβ heterodimer in mediating contact has a major impact on the extent of diversity within a given, selected, antigen-specific TCR repertoire. Some earlier studies have suggested that increased diversity within a particular TCRα- or TCRβ-chain is reflective of a diminished contribution to pMHC recognition (26, 28). In contrast, we demonstrate here that forced expression of a TCRα-chain resulted in preferred TCRβV-region use and a fixed CDR3β length within a virus-specific repertoire. This finding suggests that both chains make significant contributions to specific pMHC recognition. This result supports an earlier finding where enforced expression of an irrelevant TRAV2 TCRα-chain resulted in diminished, suboptimal TCR repertoire diversity within the TRBV29+ DbPA224-specific CTL response (27) induced by infection. Importantly, our data provide a structural explanation for the paradox of how TCR diversity can be maintained where restriction in one TCR chain limits pairing in the other. The exclusive selection of the TRBV29 gene segment by DbPA224-specific CTL in PAα Rg mice can be thought of as a function of key MHC contacts made by both the germ-line
Table 1. Summary of TRBV29+ CDR3β sequences from total GFP+CD8+ T cells and GFP+CD8+DbPA224-specific CD8+ T cells from PAα-Rg mice
Mice analyzed TCRs sequenced Modal CDR3β loop length‡ TRBJ preference‡ No. of clonotypes No. of clonotypes per mouse Percentage of shared clonotypes¶
CD8+*
DbPA224+CD8+†
2 94 9–10 aa (72.2%) BJ2-1 (24.1%), BJ2-3 (14.1%), BJ2-7 (16.0%) 69 34.5 0
4 267 6 aa (99.6%) BJ2-1 (41.8%), BJ2-7 (49.9%) 63 22.5 ± 5.7§ 69.7 ± 20.5
*Single TRBV29+GFP+CD8+ cells sorted from the spleens of naive PAα-Rg mice. † Single DbPA224+TRBV29+GFP+CD8+CD44hi cells sorted from the spleens of PAα-Rg mice 10 d after infection with HKx31 virus. ‡ More than 10% of sequences. Values in parenthesis indicate the proportion of sequences with this characteristic. § Mean ± SD for four mice. ¶ Sequences found in more than one mouse. Mean ± SD for four mice.
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Fig. 3. Preferred TCRVβ pairing within DbPA224-specific CTL from PAα-Rg mice. TRBV expression was examined by isolating splenocytes from either WT B6 or PAα mice infected 10 d earlier with A/HKx31 virus and staining with a panel of PE-conjugated mAbs specific for a range of different TRBV gene segments. Data show TRBV use for CD8+DbPA224-specific CTL from infected B6 mice (white bars), GFP+CD8+ T cells tetramer-negative (gray bars), or GFP+CD8+DbPA224-specific (black bars). Mean ± SD values are shown for three to four mice per group. *P < 0.001; **P < 0.01 for TRBV29 use.
fact, this specific CDR3β sequence was detected only at low frequency (4/65 and 2/66 sequences) in two of four mice analyzed (Table S3). In addition, although there was an increase in the degree of TCR sharing between individual mice (Table 1) compared with the WT DbPA224-specific repertoire (8), no one clonotype consistently dominated the response. This diversity of CDR3β use between individuals replicates the essentially “private” characteristic of the endogenous TRBV29+DbPA224+ repertoire (8, 32). Thus, although fixed expression of the DbPA224-specific TRAV21-AJ53 TCRα-chain resulted in preferred TRBV29 use and a consistent CDR3β length, CDR3β diversity was maintained within this “forced immune repertoire.” Collectively, these data confirm the hypothesis developed from our structural analysis: main-chain contacts ensure the maintenance of TCR diversity in the face of limited TCRV-region use.
and nongerm-line TRBV29-BJ2-7 TCR CDR loops. In particular, the TRBV29 Glu30β, a residue found only at this position within the TRBV29 germ-line sequence, contacts residues from both the α1 (Asn80) and α2 helices (Lys146) of H2-Db. As such, the selection of TRBV29 likely reflects the capacity to make optimal MHC contacts when paired with the TRAV21-AJ53 TCRα-chain. Such contacts ensure that the expressed TCR is able to dock selectively on a particular MHC, providing a structural basis for MHC restriction (31). Surprisingly, we also found that the TRAV21-AJ53/BV29-2–7 TCR CDR3 loops, particularly the CDR3β residues, Phe108β and the Arg110β, make a number of contacts with the MHCα helices. The spectrum of TRBV29 chains used by DbPA224-specific TCRs from the PAα Rg mice showed repeated selection of an aromatic residue at position 108 of the CDR3β loop. Moreover, on reflection, this preference for aromatic residues at position 108 is also characteristic of WT TRBV29+ DbPA224-specific repertoires induced after infection (8, 32). The codon sequence for aromatic residues at this position require N-region addition, indicating a potentially important role for nongerm-line–encoded CDR loops to ensure MHC (rather than bound peptide) recognition. Given previous suggestions that conserved, germ-line TRBV CDR residues alone are important for ensuring efficient MHC docking and positive selection (31, 39, 40), it seems likely from the analysis reported here that CDR3β MHC contacts may also function in this way during thymic differentiation for at least some TCRs. This finding is consistent with a recent TCR-pMHC structural analysis that shows that CDR3 regions from several virus-specific TCRs contact the MHC (41). To probe this issue further, we are currently exploiting the retrogenic strategy used here to determine how specific point mutations with the CDR regions influence pMHC recognition and T-cell development. Despite the fact that forced TRAV21-AJ53 TCR expression led to exclusive pairing with the TRBV29 gene segment, there was still evidence of extensive CDR3β sequence diversity within the TRBV29 DbPA224-specific repertoire. This speaks to the question: How are diverse antigen-specific TCR repertoires maintained in the face of biased Vα and Vβ usage? The answer looks to be in the type of interactions made between the CDR3 loops and the pMHC complex. Structural analysis of the TRAV21-AJ35/ TRBV29-BJ2-7/DbPA224 ternary complex demonstrated that the CDR3β loop backbone, and not the specific CDR3β loop amino acid side-chains, makes specific contact with the bound PA224 peptide. Importantly, despite the maintenance of diversity, this selection was limited to TCRs with a CDR3β loop length of 6 aa. Therefore, we suggest that main-chain interactions have the capacity to enable the selection of a TCR repertoire that has the necessary structural characteristics for optimal pMHC recognition, but is not dependent on specific amino acid composition. In this way TCR sequence diversity can be maintained within the CDR3 loop despite preferred Vα and Vβ use in virus-specific TCR repertoires. Taken together, these data demonstrate how topographical constraints shape both TCRV-region bias and how molecular interactions at the TCR/pMHC structural interface determine TCR repertoire diversity. It should be noted that up to half of the WT DbPA224-specific CTL response consists of non-TRBV29 populations. As such, other TCRαβ combinations are capable of binding to the DbPA224
complex. This raises, of course, the question of whether these non-TRBV29 TCR repertoires use a similar mode of docking for DbPA224 complex recognition. A recent report established that two different TCRs specific for the EBNA3/HLA-B0801 complex used completely different docking modes (18). Thus, a diverse antigen-specific TCR repertoire is likely to use a full range of docking modes, ensuring efficient pMHC recognition. Current efforts are focused on solving further DbPA224-TCR complexes to address this key issue in T-cell recognition.
1. Davis MM, Chien YH (1999) T cell antigen receptors. Fundamental Immunology, ed Paul WE (Lippincott-Raven, Philadeliphia), pp 341–366. 2. Lefranc MP (2001) IMGT, the international ImMunoGeneTics database. Nucleic Acids Res 29:207–209. 3. Pannetier C, et al. (1993) The sizes of the CDR3 hypervariable regions of the murine Tcell receptor beta chains vary as a function of the recombined germ-line segments. Proc Natl Acad Sci USA 90:4319–4323. 4. Cabaniols JP, Fazilleau N, Casrouge A, Kourilsky P, Kanellopoulos JM (2001) Most alpha/beta T cell receptor diversity is due to terminal deoxynucleotidyl transferase. J Exp Med 194:1385–1390.
5. La Gruta NL, et al. (2010) Primary CTL response magnitude in mice is determined by the extent of naive T cell recruitment and subsequent clonal expansion. J Clin Invest 120:1885–1894. 6. Malherbe L, Hausl C, Teyton L, McHeyzer-Williams MG (2004) Clonal selection of helper T cells is determined by an affinity threshold with no further skewing of TCR binding properties. Immunity 21:669–679. 7. Price DA, et al. (2004) T cell receptor recognition motifs govern immune escape patterns in acute SIV infection. Immunity 21:793–803. 8. Turner SJ, Diaz G, Cross R, Doherty PC (2003) Analysis of clonotype distribution and persistence for an influenza virus-specific CD8+ T cell response. Immunity 18:549–559.
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Materials and Methods Mice and Viruses. Female C57BL/6J mice (B6, H2KbDb) were bred at the University of Melbourne (Parkville, Australia). Retrogenic (Rg) mice expressing the 6218 DbPA224-specific α-(TRAV21-AJ53) chain (PAα) were generated by retroviral-mediated transduction of bone marrow (33, 34). For analysis of influenza-specific CD8+ T-cell responses, mice were anesthetized with isofluorane and infected intranasally with 104 pfu of the A/HKx31 (H3N2) influenza virus in 30 μL PBS. Ethics approval for the animal experiments was obtained from the University of Melbourne Animal Ethics Committee. Flow Cytometry, Tetramer, and Antibody Staining. Lymphocytes from PAα-Rg mice were stained on ice for 20 min with anti–CD8α-PerCPCy5.5, anti–CD4APC, and anti–Vβ7-PE (binds TRBV29) (BD Pharmigen) in FACS buffer (1% BSA with 0.02% sodium azide in PBS). For analysis of virus-specific CD8+ T cells, lymphocytes were stained with optimal dilutions of the DbNP366 or DbPA224 tetramer conjugated to PE or APC (Molecular Probes) for 1 h at room temperature. The lymphocytes were then washed in FACS buffer and stained with combinations of anti–CD8α-PerCPCy5.5, anti–Vβ7-PE and biotinylated anti-CD44 (BD Pharmingen) for 20 min on ice, with the latter being detected by incubation with streptavidin-APC-Cy7 (Molecular Probes). Cell staining profiles were determined by flow cytometry using a BD LSRII flow cytometer and data were analyzed using FloJo software (Tree Star). Isolation of CD8+ T Cells, RT-PCR, and Sequencing. Splenocytes were isolated from either naive mice or from immune mice at 10 d after infection and stained in sort buffer (0.1% BSA in PBS) with various combinations of DbPA224tetramer, anti–CD8α-PerCPCy5.5, anti–Vβ7-PE, and biotinylated anti-CD44 (detected following incubation with Streptavidin APC-Cy7) as above. Singlecell sorting was performed using a BD FACS Aria. For transcription of cDNA from single, sorted cells, 5 μL of cDNA mix containing 0.25 μL of Sensiscript reverse transcriptase, 1× cDNA buffer, 0.5 mM dNTPs (Qiagen), 0.125 μg of oligo(dT) (Promega), 100 μg/mL gelatin (Roche), 100 μg/mL tRNA (Roche), 20 U RNase OUT (Invitrogen Life Technologies), and 0.1% Triton X-100 (SigmaAldrich) was added to wells and plates incubated at 37 °C for 90 min. Plates were stored at −80 °C and the TRAV21 and TRBV29 transcripts were later amplified by nested PCR and sequenced (8). Expression, Refolding, Crystalization, and Structural Determination of the 6218 TCR-Db-PA224 Complex. Details of the conditions utlised for expression, refolding and crystalization of the 6218 TCRαβ-H2Db-PA224 complex can be found in the SI Materials and Methods. ACKNOWLEDGMENTS. We thank the staff at the General Medical Sciences and National Cancer Institute Collaborative Access Team beamline at the Argonne Photon Source for assistance. This work was supported by Australian National Health and Medical Research Council (NHMRC) program Grants 5671222 (to P.C.D., S.J.T., and J.R.), NHMRC Project Grant 508929 (to A.W.P. and S.J.T.), National Institute Health Grant AI70251 (to P.C.D.), a NHMRC Dora Lush Postgraduate Award (to E.B.D.), a Marie Curie Postdoctoral Fellowship (to C.G.), an NHMRC RD Wright Career Development Award (to N.L.L.G.), an NHMRC Senior Research Fellowship (to A.W.P.), an Australian Research Council Federation Fellowship (to J.R.), a Pfizer Senior Research Fellowship (to S.J.T.), and the Juvenile Diabetes Research Foundation International and the American Lebanese Syrian Associated Charities.
Day et al.
Day et al.
27. Valkenburg SA, et al. (2010) Fixing an irrelevant TCR alpha chain reveals the importance of TCR beta diversity for optimal TCR alpha beta pairing and function of virus-specific CD8+ T cells. Eur J Immunol 40:2470–2481. 28. Zhong W, et al. (2007) CTL recognition of a protective immunodominant influenza A virus nucleoprotein epitope utilizes a highly restricted Vbeta but diverse Valpha repertoire: Functional and structural implications. J Mol Biol 372:535–548. 29. Crowe SR, et al. (2003) Differential antigen presentation regulates the changing patterns of CD8+ T cell immunodominance in primary and secondary influenza virus infections. J Exp Med 198:399–410. 30. Rudolph MG, Stanfield RL, Wilson IA (2006) How TCRs bind MHCs, peptides, and coreceptors. Annu Rev Immunol 24:419–466. 31. Garcia KC, Adams JJ, Feng D, Ely LK (2009) The molecular basis of TCR germline bias for MHC is surprisingly simple. Nat Immunol 10:143–147. 32. Turner SJ, et al. (2005) Lack of prominent peptide-major histocompatibility complex features limits repertoire diversity in virus-specific CD8+ T cell populations. Nat Immunol 6:382–389. 33. Holst J, et al. (2006) Generation of T-cell receptor retrogenic mice. Nat Protoc 1: 406–417. 34. Szymczak AL, et al. (2004) Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol 22:589–594. 35. La Gruta NL, Turner SJ, Doherty PC (2004) Hierarchies in cytokine expression profiles for acute and resolving influenza virus-specific CD8+ T cell responses: correlation of cytokine profile and TCR avidity. J Immunol 172:5553–5560. 36. Argaet VP, et al. (1994) Dominant selection of an invariant T cell antigen receptor in response to persistent infection by Epstein-Barr virus. J Exp Med 180:2335–2340. 37. Lehner PJ, et al. (1995) Human HLA-A0201-restricted cytotoxic T lymphocyte recognition of influenza A is dominated by T cells bearing the V beta 17 gene segment. J Exp Med 181:79–91. 38. Ishizuka J, et al. (2008) The structural dynamics and energetics of an immunodominant T cell receptor are programmed by its Vbeta domain. Immunity 28: 171–182. 39. Feng D, Bond CJ, Ely LK, Maynard J, Garcia KC (2007) Structural evidence for a germline-encoded T cell receptor-major histocompatibility complex interaction ‘codon’. Nat Immunol 8:975–983. 40. Scott-Browne JP, White J, Kappler JW, Gapin L, Marrack P (2009) Germline-encoded amino acids in the alphabeta T-cell receptor control thymic selection. Nature 458: 1043–1046. 41. Burrows SR, et al. (2010) Hard wiring of T cell receptor specificity for the major histocompatibility complex is underpinned by TCR adaptability. Proc Natl Acad Sci USA 107:10608–10613.
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9. Kedzierska K, Turner SJ, Doherty PC (2004) Conserved T cell receptor usage in primary and recall responses to an immunodominant influenza virus nucleoprotein epitope. Proc Natl Acad Sci USA 101:4942–4947. 10. Kim SK, et al. (2005) Private specificities of CD8 T cell responses control patterns of heterologous immunity. J Exp Med 201:523–533. 11. Messaoudi I, Guevara Patiño JA, Dyall R, LeMaoult J, Nikolich-Zugich J (2002) Direct link between mhc polymorphism, T cell avidity, and diversity in immune defense. Science 298:1797–1800. 12. Price DA, et al. (2009) Public clonotype usage identifies protective Gag-specific CD8+ T cell responses in SIV infection. J Exp Med 206:923–936. 13. Menezes JS, et al. (2007) A public T cell clonotype within a heterogeneous autoreactive repertoire is dominant in driving EAE. J Clin Invest 117:2176–2185. 14. Turner SJ, Doherty PC, McCluskey J, Rossjohn J (2006) Structural determinants of T-cell receptor bias in immunity. Nat Rev Immunol 6:883–894. 15. Cibotti R, et al. (1994) Public and private V beta T cell receptor repertoires against hen egg white lysozyme (HEL) in nontransgenic versus HEL transgenic mice. J Exp Med 180:861–872. 16. Quigley MF, et al. (2010) Convergent recombination shapes the clonotypic landscape of the naive T-cell repertoire. Proc Natl Acad Sci USA 107:19414–19419. 17. Venturi V, et al. (2006) Sharing of T cell receptors in antigen-specific responses is driven by convergent recombination. Proc Natl Acad Sci USA 103:18691–18696. 18. Gras S, et al. (2009) The shaping of T cell receptor recognition by self-tolerance. Immunity 30:193–203. 19. Kjer-Nielsen L, et al. (2003) A structural basis for the selection of dominant alphabeta T cell receptors in antiviral immunity. Immunity 18:53–64. 20. Stewart-Jones GB, McMichael AJ, Bell JI, Stuart DI, Jones EY (2003) A structural basis for immunodominant human T cell receptor recognition. Nat Immunol 4:657–663. 21. Tynan FE, et al. (2005) T cell receptor recognition of a ‘super-bulged’ major histocompatibility complex class I-bound peptide. Nat Immunol 6:1114–1122. 22. Tynan FE, et al. (2007) A T cell receptor flattens a bulged antigenic peptide presented by a major histocompatibility complex class I molecule. Nat Immunol 8:268–276. 23. Gras S, et al. (2009) Structural bases for the affinity-driven selection of a public TCR against a dominant human cytomegalovirus epitope. J Immunol 183:430–437. 24. Cole DK, et al. (2009) Germ line-governed recognition of a cancer epitope by an immunodominant human T-cell receptor. J Biol Chem 284:27281–27289. 25. Miles JJ, et al. (2010) Genetic and structural basis for selection of a ubiquitous T cell receptor deployed in Epstein-Barr virus infection. PLoS Pathog 6:e1001198. 26. Yokosuka T, et al. (2002) Predominant role of T cell receptor (TCR)-alpha chain in forming preimmune TCR repertoire revealed by clonal TCR reconstitution system. J Exp Med 195:991–1001.
