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The Journal of Immunology, 2002, 168: 3894-3901.
Copyright © 2002 by The American Association of Immunologists

Complementarity-Determining Region 1 Sequence Requirements Drive Limited V{alpha} Usage in Response to Influenza Hemagglutinin 307–319 Peptide1

James V. Brawley and Patrick Concannon2

Molecular Genetics Program, Virginia Mason Research Center, Seattle, WA 98101; and Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 V{alpha} and V{beta} 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 CDR1{alpha} or CDR2{alpha}, were selected for response to the same peptide/MHC ligand. A wide variety of CDR2{alpha} 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 CDR1{alpha} 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 CDR1{alpha} are significantly more restricted than at CDR2{alpha}. This system for mutation and selection of TCRs in vitro may be of use in engineering T cells with defined specificities for therapeutic applications.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The TCR for Ag is a disulfide-linked heterodimer of {alpha}- and {beta}-chains that recognizes peptides presented by MHC molecules on the surface of APCs. Recognition of peptide-MHC complexes by the TCR is dominated by contacts among three complementarity-determining regions (CDRs)3 on each of the TCR {alpha}- and {beta}-chains and sites on both the peptide and MHC molecule. The TCR is encoded in a series of discontinuous gene segments that are recombined specifically within T cells to generate a functional receptor molecule. This rearrangement process creates a diverse TCR repertoire, but also imposes certain constraints on the sequence variation possible within the different CDRs. The V segments of the TCRA and TCRB loci encode both CDR1 and CDR2 as well as framework amino acids. Therefore, CDR 1 and 2 sequences are limited to the germline repertoire of available V segments and are coupled, i.e., there is no mechanism to vary the pairing of different CDR1 and CDR2 elements. In contrast, CDR3 sequences are much more heterogeneous due to their assembly from different D and J gene segments, the frequent loss of nucleotides at the junctions of these elements, and the addition of untemplated nucleotides during the joining process.

The two available MHC class II-peptide-TCR crystal structures (1, 2) show a common diagonal mode of binding between the TCR and peptide-MHC complex that was first proposed by Sun et al. (3) and that was observed earlier in class I-peptide-TCR structures (4, 5, 6, 7, 8, 9). In these structures, the {alpha}- and {beta}-chain CDR3 regions are centrally located and engaged in complex interactions both with each other and with peptide. The CDR2 regions are adjacent to, and have varying degrees of contact with, the MHC {alpha} helices. Besides contacting the MHC helices, CDR1{alpha} and CDR1{beta} interact with the amino terminus and the carboxy terminus of the peptide, respectively.

The relative contributions to T cell recognition of the different contacts involving CDRs observed in the available crystal structures have been difficult to assess. For some Ags, particular AV or BV gene segments dominate the repertoire of responding TCRs consistent with a strong requirement for particular CDR1 and/or CDR2 sequences in these responses (10, 11, 12, 13, 14). However, the peripheral T cell repertoire is highly dependent on thymic selection, making it difficult to draw direct conclusions about requirements for specific CDR sequences based on the usage of TCRs that contain them. In vitro mutagenesis studies can be used to bypass the effects of thymic selection, but it has not been possible to dissect the individual roles of CDRs 1 and 2 or to separate them from the effects of framework residues encoded in the V segments by these approaches because attempts to "graft" CDRs between TCRs have either failed (15) or incompletely transferred specificity (16). Indeed, most attempts to assess the roles of specific residues or sequences in CDRs with regard to Ag recognition by in vitro mutagenesis have yielded negative results (17, 18, 19, 20, 21, 22). Notable exceptions are in vivo studies in which antigenic challenge with an altered ligand has led to compensatory changes in the responding TCR. These experiments demonstrate specific CDR3 associations with peptide and suggest a dominant role for interactions involving CDR3 in determining TCR specificity (20, 23, 24, 25). However, other studies have reported effects of mutations in CDR3 more consistent with global structural alterations to the TCR rather than direct effects on the interaction of specific amino acid side chains (21, 26). In contrast to these studies that use T cell activation as an endpoint, Manning et al. (27) have examined the affinity of interactions involving CDRs by alanine-scanning mutagenesis and find that most of the binding energy of the TCR is encoded in CDRs 1 and 2.

In prior studies, we have probed the structural requirements for Ag recognition of two TCRs derived from clones, 3BC6.6 (3BC) and JS515.11 (JS), specific for the influenza hemagglutinin (HA) epitope 307–319, by transfection of cognate and mutant receptors into the receptor-deficient Jurkat-derived cell line JRT3 (21, 28). The human T cell response to HA 307–319 is dominated by T cells, like the 3BC and JS clones, that express receptors composed of the V{alpha}1 and V{beta}3 gene segments (2). An examination of the effects of in vitro mutations upon the specificity of these TCRs would potentially allow us to evaluate the relative contributions of CDRs 1 and 2 in this T cell response. However, consistent with results in other systems, most of the directed mutations that we have made within these TCRs resulted in either loss of recognition or, in a few cases, a general broadening of specificity for MHC, peptide, or both.

Given the unpredictable nature of the outcomes from directed mutagenesis studies, we sought to develop a random mutagenesis and selection approach that would allow us to broadly evaluate the sequence requirements for TCR recognition in vitro. In the current study, we describe such a system and use it to investigate the requirements for recognition at CDR1 and CDR2 of the TCR {alpha}-chain and specifically the prevalence of V{alpha}1 in the HA 307–319 response. We created libraries of TCRs with random sequences inserted at either CDR1{alpha} or CDR2{alpha}, allowing us to individually assess their contribution to the recognition of HA 307–319 presented by various HLA-DR alleles. Analysis of receptor sequences from cells selected based on TCR-mediated activation reveals that a wide variety of sequences at CDR2 of the TCR {alpha}-chain are compatible with recognition. In contrast, we could not isolate any CDR1{alpha} variants, beyond the parental TCR sequence, that allowed Ag recognition, suggesting that limitations imposed by the need for specific CDR1 interactions drive the limited V{alpha} diversity that characterizes this T cell response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

A derivative of the Jurkat T cell line, J.RT3-T3.5 (JRT3) (29), defective for endogenous TCR{beta} expression, was obtained from the American Type Culture Collection (Manassas, VA). Bare lymphocyte syndrome (BLS) 1 cells expressing exogenously introduced HLA class II genes (30, 31, 32) were used for Ag presentation. Cells were grown in RPMI 1640 supplemented with 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS. Phoenix A packaging cells were grown as described elsewhere (33).

Derivation of T cells with an activation-inducible reporter gene

The complete sequences of the {alpha}- and {beta}-chains of the 3BC TCR have been reported (28). The 527.1 cells expressing the rearranged TCRB gene from the 3BC T cell clone as well as an activation-inducible Kd gene were constructed as follows. A ClaI-HindIII DNA fragment containing a triple NFAT-binding element and minimal IL-2 promoter was transferred from NFATZ (34) to Bluescript (Stratagene, La Jolla, CA). The Kd gene was excised as a BamHI fragment from the plasmid pNA (35) and transferred to Bluescript so that the NFAT-binding element preceded Kd. The NFAT-Kd fragment from Bluescript was excised by HincII and partial digestion with SacI, yielding a 1.6-kb fragment that was cloned into the expression plasmid pMCFR (36), from which the promoter had been excised by PstI digestion, end-filling, and SacI digestion. This construct was inserted into JRT3 cells by electroporation after linearization with Asp700 I. Cells constitutively expressing Kd were removed by incubation with anti-Kd (hybridoma SF1-1.1) and sheep anti-mouse IgG M-450 Dynabeads (Dynal, Oslo, Norway) used according to the manufacturer’s instructions. The remaining cells were cloned by limiting dilution. A single clone, 435.8, was chosen for further work due to its high cell surface Kd expression in response to stimulation with ionomycin plus PMA but lack of expression in the absence of stimulation. The 3BC TCR {beta}-chain, cloned in the expression plasmid pHISACT, was linearized with EcoRI and electroporated into 435.8 cells. pHISACT is a derivative of pNA that is selectable with histidinol rather than G418. Histidinol-resistant transfectants were selected for strong V{beta}3 expression using anti-V{beta}3 (BD PharMingen, San Diego, CA) and Dynabeads. Some endogenous V{beta}8 expression was detected in this line and these cells were removed by using anti-V{beta}8 (BD PharMingen) and Dynabeads. The clone 527.1 was derived from this line and was checked for strong V{beta}3 expression and inducibility of Kd expression.

3BC {alpha} retrovirus

The TCRA gene from the 3BC T cell line was amplified by PCR using Pfu turbo (Stratagene) and primers containing a 5' SalI site (5'-TCTTCTCTAGTCGACGGTACCATAATGCTCCTGCTGC-3') and a 3' ClaI site (5'-TCTTCTCCAATCGATGGATCCAGTTGGTGGC-3'). After digestion, this product was cloned into a retroviral plasmid based on SAMEN' (37), in which a ClaI site had been introduced downstream of SalI in the multicloning site. This construct was introduced into Phoenix A packaging cells by calcium phosphate-mediated transfection (33). Supernatants were harvested and used for spin infection of 527.1 cells at 1800 rpm for 90 min. The 527.1 lines infected with virus were grown in 900 µg/ml active geneticin (Life Technologies, Gaithersburg, MD).

Construction of randomized CDR libraries

The random 3BC{alpha} CDR2 library was created by PCR amplification of two overlapping gene fragments, one extending from the 5' end of the gene to CDR2 and the other extending from CDR2 to the 3' end of the gene. Forward and reverse primers spanning CDR2 contained 12 bases of random nucleotides (coding for aa 50–53) flanked by 16 bases of cognate sequence on each side. The mutagenic CDR2 forward primer had the sequence 5'-GCTTCTCCTGAAGTACN12ACCCTGGTTAAAGGCA-3' and the CDR2 reverse primer’s sequence was 5'-TGCCTTTAACCAGGGTN12GTACTTCAGGAGAAGC-3'. The reverse CDR2 primer was paired with the 5' SalI-containing primer to create one fragment. The forward CDR2 primer was used with the 3' ClaI primer to make the other PCR fragment. Products of these PCRs were gel purified, digested with DpnI to remove residual plasmid template DNA, and used as substrate in a subsequent amplification reaction using the 5' SalI and 3' ClaI primers to produce full-length product. This product was then gel purified, cut with SalI and ClaI, and inserted into the SAMEN vector.

The CDR1 forward and reverse mutagenic primers also contained 12 random bases (coding for aa 27–30) flanked by 16 nucleotides at each end. Their sequences were 5'-GTGCAACTACTCATCGN12TATCTCTTCTGGTATG-3' (forward) and 5'-CATACCAGAAGAGATAN12CGATGAGTAGTTGCAC-3' (reverse). The PCR strategy and 5' and 3' primers used were the same as for the CDR2 library. Both CDR1- and CDR2-randomized DNAs were electroporated into XL-1 electrocompetent cells (Stratagene). Transfection of Phoenix A cells and infection of 527.1 cells was as described above.

Selection of cells from libraries

A total of 5 x 106 cells from a CDR-randomized library and 5 x 106 BLS cells as APCs were mixed with 10 µg/ml HA 307–319 peptide and 3 ng/ml PMA in 10 ml. At 12 h, cells were stained with phycoerythrin anti-Kd and CyChrome anti-CD19 (BD PharMingen). Cells were analyzed on a FACSort or sorted on a FACSVantage (BD Biosciences, Mountain View, CA). CD19-negative, Kd-positive cells were harvested in sorting experiments. Selected cells were either cloned by FACS at the time of sorting or grown in bulk culture before another round of stimulation or cloning by limiting dilution.

Stimulation of selected or random cells

Clones selected by FACS sorting were grown in 96-well plates. Clones were stimulated in triplicate when the average of all the wells in a plate was estimated to be ~50,000 cells. Plates were centrifuged and T cells were resuspended in medium such that there were 50,000 BLS cells in 150 µl of 3 ng/ml PMA. In standard experiments with peptide, HA 307–319 or variants were present at 10 µg/ml. In the experiments with altered ligands, 50,000 T cells were present in each well. Supernatants were harvested at 20–24 h and frozen. Thawed supernatants were applied to IL-2-dependent HT-2 cells. At 16 h, 1 µCi of [3H]thymidine (5 Ci/mmol) was added. HT-2 cells were harvested 8 h later, and incorporation of [3H]thymidine was measured by liquid scintillation counting.

DNA sequencing

DNA was extracted from cells using DNAzol (Life Technologies). TCRA genes were amplified by PCR using Taq DNA polymerase (Roche, Indianapolis, IN) and sequenced using a primer from the retroviral long-terminal repeat. Sequencing reactions were separated on a 377 sequencer (PE Applied Biosystems, Foster City, CA).

Determination of parental sequence representation in CDR1 library

Primers were created that amplified only the parental 3BC TCRA CDR1 sequence (5'-ACTCATCGTCTGTTCCACCA-3') or all constructs (5'-TCCAGCTTCTCCTGAAGTAC-3'). Individually, these primers were used with a C{alpha} region reverse primer (5'-AGAGTCTCTCAGCTGGTACA-3') to form a 314-bp parental or 257-bp common band. The parental specific 5' primer did not amplify the most closely homologous nonparental clone identified in the library, even at high template concentrations. The parental and common primer systems were used separately to amplify a series of input DNA concentrations from the library. Products of these PCRs were resolved on a DNASep (Transgenomic, Omaha, NE) high-performance liquid chromatography column under nondenaturing conditions where DNA fragments were separated by size. Pairs of one parental-specific product and one common product were coinjected. Peaks were measured by the manufacturer’s software and the abundance of parental sequences was determined from the relative peak sizes, when both products were in a linear range, and the amount of input library DNA. This number was adjusted to account for the differences in product size and relative priming efficiency determined by amplification of DNA from a clone that had the parental sequence.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A recipient cell line expresses the reporter gene product Kd on its surface after TCR-mediated activation by peptide presented by class II

To isolate functional CDR sequences without the bias inherent in directed mutation, we constructed a recipient T cell line that was TCR-negative but, upon reconstitution with genes encoding Ag-specific TCR, would respond to Ag stimulation by placing a unique reporter protein on the cell surface. We created a plasmid in which the mouse class I gene Kd was placed under the control of a triple NFAT-binding element and minimal IL-2 promoter (34). We selected against leaky transfectants expressing Kd constitutively and chose a clone that was completely negative for constitutive Kd expression yet responded well to ionomycin plus PMA.

The 3BC TCR uses V{beta}3 and V{alpha}1 to recognize HA 307–319 presented by the products of the DRA*0101 and DRB1*0401 genes. The 3BC V{beta}3 chain was introduced into JRT3 cells containing the NFAT-Kd construct, where it was expressed constitutively under the control of the human {beta}-actin promoter. A clone, 527.1, with high expression of V{beta}3 was selected. JRT3 expresses an endogenous {alpha}-chain (but not a {beta}-chain) allowing pairing with, and surface expression of, the exogenous {beta}-chain.

The parental 3BC V{alpha}1 chain was cloned into a retroviral vector, packaged, and used to infect 527.1 cells. Geneticin-resistant cells that grew out were stimulated with 10 µg/ml HA 307–319 presented by BLS cells into which DRA*0101 and DRB1*0401 (BLS.0401) had been introduced. Kd expression on T cells was analyzed (Fig. 1GoA) and it was found that 25% of T cells were Kd-positive, exhibiting a broad range of expression. As has been previously reported by other investigators using such inducible constructs (34), not all cells became Kd-positive after stimulation through the TCR. However, all cells could be activated by ionomycin plus PMA.



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  		FIGURE 1. Expression of the exogenous activation marker Kd on the surface of T cells after stimulation with HA 307–319 presented by DR4. Cells were stained with PE-anti-Kd and CyChrome-anti-CD19. Shown here are the CD 19-negative cells. A, 3BC receptor; B, CDR2 library; C, CDR2 library after one round of selection.

 
Responder clones can be selected from a CDR2 library by expression of the reporter gene

A library with random sequences at positions 50–53 of CDR2 of the 3BC {alpha}-chain was created as described in Materials and Methods. This randomized CDR2 library contained ~9.4 x 105 recombinants. When 527.1 cells containing the CDR2 library were stimulated by BLS.0401 presenting the HA 307–319 peptide and Kd expression was analyzed by flow cytometry, a small shoulder (5.6%) of Kd-positive cells was observed (Fig. 1GoB) that was selected and allowed to expand. A second round of stimulation with BLS.0401 plus HA 307–319 further enriched this population, resulting in 18.6% of the T cells becoming Kd-positive (Fig. 1GoC). These cells were sorted and cloned. As a negative control, CDR2 library cells were cloned by limiting dilution without selection. After expansion, control and selected CDR2 library clones were stimulated in triplicate by HA presented by BLS.0401 and assayed for IL-2 production in an HT-2 assay. The stimulation index (SI), defined as the ratio of incorporation of [3H]thymidine in cultures with HA to those without HA, was calculated for each set of clones. The selected cells exhibited a broad range of stimulation indices with the greatest density at high values and a median of 131 (Fig. 2GoA). In contrast, the median SI for the control population was 1.7. Although there was some overlap in the range, the distribution of SI values in the selected and control groups was significantly different (p < 0.0001), confirming that the selection of Kd-positive cells did result in enrichment for cells bearing TCRs that recognize the appropriate ligand.



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  		FIGURE 2. SIs for populations of T cells containing CDR2-modified TCRs. Stimulations were performed as in Materials and Methods. Each dot represents the average result for a single clone assayed in triplicate. Horizontal line denotes the median. A, Control and Kd-selected cells stimulated without knowledge of sequence (includes some duplicates and some cells that could not be sequenced). B, Control and selected cells from library that exhibited a single sequence.

 
Many random clones in the CDR2 library responded to ligand. Kd-selected clones with stimulation indices of 3.0 or greater were chosen for TCRA sequence analysis. Clones that yielded more than one sequence or from which the TCRA sequence did not amplify well were excluded from further analysis. Of 155 single-sequence clones, 74 were novel, 16 had the parental 3BC{alpha} sequence, and 65 were duplicates of novel sequences. The stimulation indices for these novel clones are shown in Fig. 2GoB. For comparison, TCRA sequences from control clones randomly chosen from the library were determined without knowledge of their SIs. Of 123 single-sequence control clones identified, 80 were novel and 30 had the parental sequence. Eleven additional sequences contained stop codons and there were two duplicates of novel sequences. A total of 26% of the novel sequences derived from control clones had an SI of >3.0, indicating that a significant number of the sequences in the CDR2 library allow recognition of the HA peptide presented in the context of DR4. All clones in both pools that had the parental amino acid sequence had the parental nucleotide sequence, suggesting that the most likely source for these clones was a small amount of parental DNA that had evaded removal by DpnI digestion during the PCR mutagenesis scheme.

A diverse repertoire of CDR2{alpha} sequences is compatible with Ag recognition

The set of novel CDR2{alpha} sequences selected from the CDR2 library for recognition of the HA 307–319 peptide presented by DR4 is shown in Fig. 3Go, ordered by SI With the exception of the contaminating parental clones, the sequences derived display little similarity to the parental CDR2{alpha} sequence TSAA, although the sequences TSPA and STLA each occur once. There were no sequences in common between this selected pool of CDR2 sequences and control clones derived from the library before selection. Overall, the usage of amino acids in the selected, compared with the control, CDR2{alpha} pools did not differ significantly. Indeed, the relative usage of individual amino acids was highly correlated between the two groups (r = 0.74; p = 0.0001). However, the distribution of amino acid usage by position within CDR2{alpha} (Fig. 4Go) was significantly biased for particular classes of amino acids in the selected compared with the control pools. At position 50, aliphatic hydrophobic residues were highly enriched in the selected population compared with controls (p < 0.0001). For example, 17 of the 26 valine residues and six of seven isoleucine residues observed in the selected CDR2{alpha} sequences occurred at position 50. Other specific amino acids such as proline, lysine, arginine, aspartic acid, and glutamic acid were completely absent at this position. At position 51, alanine and glycine were enriched, whereas aliphatic hydrophobic amino acids were scarce. The charged amino acids lysine, arginine, aspartic acid, and glutamic acid were present 29 times at positions 52 and 53, but only once at positions 50 and 51. This distribution was significantly different from that observed in the control sequences (p = 0.0002). Although no single motif could capture all of the observed variation at each CDR2 amino acid position, amino acid preferences observed at specific positions appeared to be correlated. For example, sequences with aliphatic hydrophobic residues at position 50 were more likely to have negatively charged amino acids (D or E) at 52 or 53 (p = 0.042), whereas sequences without these amino acids at position 50 had an increased frequency of positive charges (K or R) at 52 or 53 (p = 0.0365)



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  		FIGURE 3. Novel sequences for aa 50–53 of the TCRA gene expressed in clones from CDR2 library that respond to Ag. The parental sequence is TSAA. Sequences are ordered such that 1 is the sequence with the highest SI.

 


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  		FIGURE 4. Representation of chemically similar amino acids by position in CDR2. Graphs indicate the percentage of CDR2 sequences among selected ({square}) or control ({blacksquare}) that contain a member of the indicated group of amino acids. Each graph corresponds to a specific position in CDR2{alpha} (positions 50–53) as indicated.

 
Isolation of responsive cells from a randomized CDR1 library requires additional rounds of selection

A TCR {alpha}-chain CDR1 retroviral expression library of 5.1 x 105 recombinants, with random sequences encoding residues 27–30, was made by the same strategy as was used for the CDR2 library. When 527.1 cells infected with this library were challenged with 10 µg/ml HA presented by BLS.0401, the resultant Kd expression profiles (Fig. 5GoA) showed no apparent difference between the results obtained with and without added peptide. To enrich for potentially rare responding clones, we sorted cells from the leading edge of the peak. These cells were allowed to expand and were subjected to two more rounds of Ag challenge and selection. By the third round of selection, the profile showed clear evidence of enrichment. When cells selected in that third round were allowed to expand and then were stimulated again, they gave the profile shown in Fig. 5GoB (31% positive).



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  		FIGURE 5. Expression of the exogenous activation marker Kd on the surface of T cells after stimulation with HA 307–319 presented by DR4. Cells were stained with PE-anti-Kd and CyChrome-anti-CD19. Shown here are the CD 19-negative cells. A, CDR1 library; B, CDR1 library after three rounds of selection.

 
Examination of CDR1 sequences from control and selected clones shows few sequences allow recognition

T cell clones were derived by limiting dilution from the initial CDR1 library before Ag challenge and also from the three-time-selected cells. A plot of stimulation indices obtained after secondary proliferation assays for these clones is shown in Fig. 6GoA. None of the nonselected control clones had an SI >3 and the median SI was 1.0. In contrast, nearly all of the selected clones had SI values above 100 and the median SI was 416. TCRA genes from selected and control clones were sequenced as before, and those with single sequences were further analyzed. The distributions of SI values among the 54 control clones and 92 selected clones for which single sequences were obtained were comparable to those for the overall populations (Fig. 6Go).



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  		FIGURE 6. SIs for populations of T cells containing CDR1-modified TCRs. Stimulations were performed as in Materials and Methods. Each dot represents the average result for a single clone assayed in triplicate. Horizontal line denotes the median. A, Control and Kd-selected cells stimulated without knowledge of sequence (includes some duplicates and some cells that could not be sequenced). B, Control and selected cells from library that exhibited a single sequence.

 
The 92 selected CDR1 clones all yielded the parental sequence SVPP. In contrast, the 54 control CDR1 library clones contained no parental sequences. As in the case of the CDR2 library, all of the CDR1 clones with the sequence SVPP were also identical at the nucleotide level, suggesting that they derived from parental sequences. However, unlike the CDR2 library, it required three rounds of selection to obtain these clones, suggesting that they are quite rare in the original library.

To estimate the frequency of clones containing the parental sequence in the CDR1 library, we performed PCR on dilutions of DNA from the library using a common 3' primer and either a parental-specific 5' primer or a 5' primer from the framework region, which should amplify all library sequences. Taking into account the relative amplification efficiencies of the two systems using a single clone with the parental sequence, we estimate that one in 500 sequences in the library is parental in origin. Among nonselected CDR1 clones, there were 54 novel sequences, no duplicate sequences were observed, and great diversity was seen with respect to amino acid sequence (data not shown). Although we can’t be certain this library contains all possible sequences for CDR1, we note that sequences SRYP, SAPV, SSPA, and TGPD, which are similar to the parental SVPP, were present in our set of 54 random clones, and all were negative in stimulation assays. These observations in conjunction with the failure to obtain any novel sequences among the selected clones suggest that there may be few such sequences that are permissible for recognition.

Sensitivity and specificity of selected clones

Our previous site-directed mutagenesis studies of CDR2{alpha} in TCRs responding to the HA 307–319 peptide presented in the context of DR4 revealed that some substitutions in this CDR resulted in altered specificity. Given the diversity of CDR2{alpha} sequences recovered from the library screen, we tested whether some of these clones might have broader specificities for either HLA-DR or peptide or higher sensitivity. Five clones were selected from each of three pools: control positives (i.e., clones chosen at random that subsequently proved to be HA peptide responsive), selected clones with novel sequences and high stimulation indices, and clones with parental sequences. These clones were stimulated with HA peptide concentrations ranging from 0.1 ng/ml to 50 µg/ml presented by BLS.0401. No significant increase in sensitivity was noted, although clones did vary in their responses at different concentrations of peptide. We have previously demonstrated that the parental 3BC TCR does not respond to the following HA variants with substitutions at TCR-directed residues: K308A, K308E, V310K, and V310L. None of the clones studied above were found to have acquired responsiveness to these peptides.

The HA 307–319 peptide is known to bind a variety of DR gene products, including DR1, with relatively similar affinities to DR4 (38). The three sets of clones were challenged with the HA peptide in the context of HLA-DR1. The DRB1*0101 allele, although it differs from the 0401 allele at 10 positions, does share a common sequence in the {alpha}-helix region of the molecule likely to contact TCR, often referred to as the "shared epitope" because of its occurrence in DRB1 alleles associated with susceptibility to rheumatoid arthritis. None of the clones responded to the HA peptide when presented by DR1. Because clones responding to the HA peptide in the context of DR1 might be rare in the CDR2{alpha} randomized library or not cross-reactive with the peptide when presented on the DR4 allele, we attempted to isolate such clones directly by challenging the library with the HA peptide presented by BLS.0101 cells. Although there was no initial evidence of a responding population, the leading edge of the distribution of cells was sorted and rechallenged, as was previously done successfully to isolate rare responder clones from the CDR1{alpha} library. However, after two rounds of such selection, there was no apparent enrichment for Kd-positive cells upon additional restimulation.

The DRB1*0401 and 0101 alleles may differ at too many positions to be accommodated frequently by changes in CDR2{alpha} alone. Therefore, we investigated whether 16 additional clones isolated for reactivity to the HA peptide presented by DR4w4 (DRB1*0401) might cross-react (as the 3BC receptor does) with the peptide presented by DR4w14 (DRB1*0404). The DRB1*0401 and 0404 alleles differ at only two amino acid positions, one of which, 86, affects peptide binding, whereas the other, 71, is potentially TCR directed. Because of the known effect of the difference at position 86 on peptide binding, a higher peptide concentration (50 µg/ml) was used to compensate for the reduced binding to this class II allele. All of the clones responded to the HA peptide presented by either DR4w4 or DR4w14. In addition, one clone responded to the variant peptides 308A and 310L when presented by the DRB1*0401 gene product.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study, we describe a system that allows the selection of T cells expressing in vitro mutagenized TCRs based on Ag-specific cellular activation. Using this system, in conjunction with libraries of cloned TCR genes in which random sequences have been inserted in place of CDR{alpha}s 1 and 2, we have enriched for cells bearing TCRs of a desired specificity and successfully cloned these cells. Because this system employs activation-based rather than affinity-based selection, it allows us to more closely mimic biological T cell recognition, but without the constraints on sequence imposed by the germline V segment repertoire and the effects of thymic selection. We have applied this system to examine the relative roles of CDR1{alpha} and CDR2{alpha} in the T cell response to the influenza HA peptide 307–319. This response is dominated by TCRs using V{alpha}1 in conjunction with V{beta}3 (2), suggesting a particularly important role for the V{alpha}1 segment-encoded CDRs 1 and 2 in this system.

Given the strong preference for V{alpha}1 in the HA 307–319 response, it was surprising that when the randomized CDR2{alpha} library-infected cells were stimulated with the peptide presented by BLS.0401 cells, 26% of the clones allowed recognition. Only one additional round of selection was necessary to obtain cells that, when cloned and restimulated, were 84% positive by HT-2 assay. Consistent with this observation that a high percentage of random clones were stimulated in an Ag-specific manner is the fact that a diverse collection of CDR2{alpha} sequences was found expressed in the selected clones.

Sample sequencing from the library before activation-dependent selection revealed that the distribution of amino acids in CDR2{alpha} was dependent on codon number, as expected. Amino acids such as methionine and tryptophan, which are each represented by only a single codon, were less common, but nevertheless present in the library sequences. There was a slightly increased GC content in the library, possibly reflecting some thermodynamic selection due to the strength of base pairing during the annealing of the oligonucleotides used in the mutagenesis.

Amino acid representation in CDR2{alpha} sequences derived from clones selected for recognition of the HA peptide was similar, overall, to that in the library before selection. However, there were position-specific biases in the usage of particular amino acids. Aliphatic hydrophobic residues were enriched at position 50, whereas charged residues, which were quite frequent, were confined almost entirely to positions 52 and 53. The nature of the charge present at these positions, either positive or negative, was dependent in part on the presence or absence of an aliphatic hydrophobic residue at position 50.

Our CDR2{alpha} results, showing that diverse sequences are permissive for HA 307–319 recognition in the context of DR4, do not provide an explanation for the preferential use of V{alpha}1 in this T cell response, although the position-specific differences in amino acid usage observed argue against the trivial possibility that CDR2{alpha} is not involved. The results are somewhat surprising in light of the published structure of a complex of a TCR using V{alpha}1 and V{beta}3 interacting with the HA peptide complex presented by DR1 (rather than DR4) (2). In this structure, the CDR2{alpha} residues S51 and A52 contact the DRB1*0101 molecule at residues T77 and E69, respectively. These residues (T77 and E69) are conserved in the DRB1*0401 allele and would presumably be available for interaction with TCR residues. However, we observed no statistically significant preference for S at position 51 or for A at position 52 in sequences from TCRs selected for recognition of the HA peptide presented by DR4. Ding et al. (6) studied two TCRs that each recognize a peptide derived from the HIV Tax protein when presented by HLA-A2. They observed that the same TCR positions could be used to contact the identical ligand even when the positions on the TCR chain were occupied with different amino acids. Thus, some of the diversity in the CDR2{alpha} sequence we observed could reflect redundancy. However, even using DR1 APCs and the HA peptide in our system, we were unable to select TCRs from our randomized CDR2{alpha} library, suggesting that interactions involving CDR2{alpha} may be broadly permissive for recognition of the HA peptide in the context of DR4, whereas there are more specific sequence requirements for recognition in the context of DR1. This could be because of constraints imposed by the binding of the 3BC CDR3. Although we had difficulty finding a DR1 responsive clone, it is noteworthy that all 16 clones tested for recognition of HA presented by DRB1*0404 responded positively. This suggests that any specific contact does not involve position 71 of the MHC {beta}-chain or that fixed components of the TCR, such as CDR3, mandate its recognition of *0404 along with *0401.

In contrast to our results for CDR2{alpha}, we found that it was difficult to modify positions 27–30 of CDR1{alpha} and retain recognition. In our library screen, multiple extra rounds of selection were required, relative to CDR2{alpha}, to obtain a population of cells in which a high proportion responded to peptide. Moreover, when the V{alpha} sequences from these clones were determined, all were of parental origin. By PCR, we estimate that clones with the parental sequence are no more frequent than one in 500 in our CDR1{alpha} randomized library. Presumably, novel sequences that can accommodate recognition are even less common, suggesting significant constraints on the allowable CDR1{alpha} sequence compared with CDR2{alpha}.

The natural sequence for residues 27–30 of the human V{alpha}1 CDR1{alpha} is SVPP. In the structure described by Hennecke et al. (2), V28 interacts with H81 of the DRB1 chain of the DR1 molecule. This position, H81, is conserved in the DRB1 subunit of the DR4 molecule used to present Ag in our system. In addition to its interaction with H81 of the DR molecule, V28 also makes van der Waals contacts with K308 and V310 of the peptide. The requirement for these multiple contacts with both peptide and HLA restricting element may greatly constrain the possible choices for amino acids at this position that would still allow recognition.

It is possible that the predominant usage of V{alpha}1 in the response to the HA 307–319 peptide is predicated on the need for interactions involving V28 of the TCR {alpha}-chain. This residue is only present in two TCRA gene segments, the AV1 gene segment used here and AV10S1 (39). TCR recognition in the structure described by Hennecke et al. (2) is dominated by electrostatic interactions between the three lysines of the peptide and acidic residues in CDR3{alpha} and CDR1{beta}. The CDR3{alpha} residues involved, at positions 94 and 102, are conserved in the 3BC TCR studied here, as is the usage of V{beta}3. Thus, it may be that V28 is the only solution to the recognition problem once the constraints imposed by the requirements for appropriate CDR3 sequences and pairing with V{beta}3 are applied.

As part of this study, we have developed an activation-based system that allows the selection of TCRs from CDR randomized libraries. In designing this system, we considered several alternative approaches. Rather than relying on an activation-induced marker, is should be possible to directly select TCRs of desired specificity using class II tetramers loaded with the appropriate peptide. However, we found that DR4 tetramers loaded with the HA peptide could not stain JRT3 cells expressing the 3BC TCR (E. Novak, J. Brawley, P. Concannon, and G. Nepom, unpublished observations). We believe that this lack of tetramer staining results from interference by the noncognate TCR {alpha}-chain expressed by JRT3 cells. Instead of using an activation-induced reporter gene construct, we considered the use of endogenous markers such as CD25 and CD69, but found that both were up-regulated in JRT3 cells even in the absence of activation. Given these apparent limitations on the system imposed by the use of JRT3 cells, it might seem reasonable to reconstruct T cell recognition in a heterologous system that might be more easily manipulated. For example, Kranz and coworkers (40) were able to use random mutagenesis of CDR3{alpha} in a yeast-based system to select a mutant TCR with 100-fold improved affinity for peptide-MHC. However, the relationship between the affinity of isolated MHC-peptide-TCR complexes and biological activation in the context of accessory molecules and cellular signaling pathways is not well defined. Despite the demonstrated ability of the yeast system to enrich for high-affinity interactions, we opted for a system based on cellular activation that might more closely mimic T cell recognition in vivo. The ability to generate TCRs of known specificity but improved sensitivity using a system of this type has potential application in the implementation of T cell-mediated therapies (37, 41) as well as contributing to the better understanding of the relative roles of CDR sequences in T cell recognition.


   Acknowledgments
 
We thank A. Morrison, S. Cei, and E. Olson for DNA sequencing gels and K. Allen for flow cytometry assistance. We are grateful to G. Nepom and W. Kwok for BLS cells, M. Nishimura for the SAMEN vector, and M. Bevan for a Kd plasmid and a hybridoma secreting anti-Kd. We thank J. Gebe, G. Nepom, and D. Ostrov for review of this manuscript and M. West for assistance in preparation of this manuscript.


   Footnotes
 
1 This work was supported by Grant AI39636 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Patrick Concannon, Molecular Genetics Program, Virginia Mason Research Center, 1201 Ninth Avenue, Seattle, WA 98101. E-mail address: patcon{at}vmresearch.org Back

3 Abbreviations used in this paper: CDR, complementarity-determining region; HA, hemagglutinin; BLS, bare lymphocyte syndrome; SI, stimulation index. Back

Received for publication November 26, 2001. Accepted for publication February 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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