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Recombination of germ-line TCR alpha and beta genes generates polypeptide receptors for MHC-peptide. Antigen exposure during long-term herpes simplex infections may shape the T-cell repertoire over time. We investigated the CD8 T-cell response to HSV-2 in chronically infected individuals by sequencing the hypervariable regions of T-cell receptor alpha and beta polypeptides from T-cell clones recognizing VP22 amino acids 49-57, an immunodominant epitope. The most commonly detected TCRBV gene segment, found in four of five subjects and in 12 of 50 independently derived T-cell clones, was TCRBV12-4. 19% to 72% of tetramer-binding cells in PBMC stained ex vivo with a TCRBV12 mAb. Three alpha chain and three beta chain public T-cell receptor sequences were shared between individuals. Public heterodimers were also detected. Promiscuous pairing of a specific TCRVA1-1 sequence with several different TCRB polypeptides was observed, implying a dominant structural role for the TCRA chain for these clonotypes. Functional avidity for cytotoxicity and interferon-gamma release was relatively invariant, except for one subject with both high avidity and unique TCR sequences and lower HSV-2 shedding. These data indicate that the CD8 response to a dominant alphaherpesvirus epitope converges on preferred T-cell receptor sequences with relatively constant functional avidity.
Virus-specific CD8 T-cells are an important component of the host response to herpes simplex virus (HSV) during several phases of this chronic infection. After epithelial inoculation, virus arrives in dorsal root ganglia (DRG) and replicates in neuronal cells. Infiltration of CD8 T-cells correlates with shut-down of lytic replication and the establishment of latency (1). HSV-specific CD8 T-cells are retained in murine DRG for several months (2), and in this species are associated with very tight clinical latency. If ganglia are explanted or transplanted, HSV reactivation is controlled by HSV-specific CD8 T-cells (3, 4). Specific CD8 T-cells also localize to HSV-infected human DRG (5). Periodic reactivations in the human DRG leads to peripheral lytic replication, with or without lesion formation. At sites of HSV symptomatic and asymptomatic reactivation in epithelial tissues, antigen-specific CD4 and CD8 T-cells are readily detectable (6, 7). The presence of HSV-2-specific CD8 CTL correlates temporally with viral clearance from symptomatic lesions (6-8). HSV-2-specific T-cells persist in the dermis and at the dermal-epidermal junction, even after viral clearance and epithelial healing (6, 8)
The specificity and clonotypic complexity of HSV-specific CD8 T-cells are relatively unexplored. Using genomic HSV-2 DNA libraries, we found HSV-2 epitopes recognized by prevalent HLA alleles (9-11). One epitope, amino acids 49-57 of virion protein 22 (VP22), encoded by gene UL49, is immunodominant in terms of cell number and population prevalence when referenced to the current integrated data set. Amongst persons with the restricting HLA B*0702 allele, about 50% have CD8 (+) lymphocytes in peripheral blood at a level high enough to detect with direct staining with tetramers of HLA B*0702 and the peptide (sequence RPRGEVRFL). Staining of direct PBMC with this tetramer (abbreviated B7-RPR) is the most consistently observed among HLA-appropriate persons compared to several others we have tested. Numerical abundances are up to 0.6% of CD8 cells in PBMC, higher than observed for other tetramers (10) and near the 0.5-1.0% range for integrated HSV-2-specific CD8 T-cells estimated using infected DC as APC or massive parallel peptide IFN-γ ELISPOT methods (12, 13).
In this report, we investigate the fine clonotypic structure of the CD8 response to B7-RPR. By sequencing the hypervariable CDR3 regions of both the alpha and beta chains of cellular and molecular clones, we now uncovered several examples of public T-cell receptor sequences and even heterodimeric pairs that are shared between individuals. Conserved alpha chains can pair with diverse beta chains, implying strong structural recognition of B7-bound RPR peptide by the TCR alpha chain, in contrast to some structural models. Previous, similar findings for CMV and EBV (14-16), chronic herpesviruses that reside in professional antigen presenting cells and that stimulate massive CD8 responses, can now be extended for first time to infection by a chronic alphaherpesvirus with tropism for epithelial cells and neurons and that stimulates a much more modest CD8 response.
Subjects with serologically (17) documented HSV-2 infection provided written informed consent and were enrolled in a protocol approved by the University of Washington Institutional Review Board. PBMC were enriched by Ficoll centrifugation and used fresh or after cryopreservation. Subjects were HLA typed at the Puget Sound Blood Center, Seattle, WA. Subjects 5491, 5, and 9 were in prior publications (9, 11). Subjects 12-13 are new. None of the subjects had symptomatic recurrences at the time of phlebotomy and each had a clinical history of symptomatic genital herpes. The times from last clinical recurrence were not recorded. Subject five was studied three times for greater than 30 sequential days using daily home-collected genital swabs for HSV DNA detection by PCR as described (18), with shedding rates of 11.2%, 0%, and 1.7%, while subjects 9 and 12 had single shedding studies with rates of 32.9% and 18.1%, respectively, and subjects 13 and 5491 have not had shedding studies.
T-cell clones and lines were derived by several pathways. RPR-specific T-cell clones 1B1 and 2F1 were derived from subject 5 PBMC by sorting CD8 cells expressing high surface cutaneous lymphocyte-associated antigen (CLA) (9, 11). For subjects 9, 12, and 13, PBMC were stained with anti-CD8α-FITC (Caltag, Burlingame, CA) and tetramer B7-RPR-PE. The peptide RPRGEVRFL is present in lab strain HG52 (19) and the majority of circulating HSV-2 strains cultured in Seattle, Washington, USA, although diversity is present (Koelle et al. unpublished). Cells in the tetramer (+), CD8α-high region were sorted (FacsAria, Becton Dickinson) into T-cell medium (TCM) (20) and rested overnight in a 96-well U-bottom plate in TCM and 50 U/ml recombinant human IL-2 (Novartis, New York, NY). Cells were cloned at 1-3 cells/well with allogeneic irradiated feeders, IL-2, and PHA as described (20). After two weeks, microcultures with visible growth were screened for specificity. In each case, the wells selected for screening, expansion and TCR analysis came from plates with <37% of cultures having visual growth, yielding a >95% chance of clonality per the Poisson distribution (21). Cultures with cytotoxicity in screening assays (below) were expanded with IL-2 and anti-CD3 mAb as described (9). Prior to harvesting RNA for TCR analysis, cultures were held at least two weeks to minimize persistent feeder cell-derived RNA. For subject 5491, PBMC were re-stimulated in bulk with peptide RPRGEVRFL, IL-2 and IL-7 as described (9). After 2 weeks, cells in the tetramer (+), CD8α region were sorted. Some of the cells were cloned by limiting dilution, and some were used for bulk RNA isolation.
To assess TCRBV expression, T-cell clones were stained with FITC-conjugated anti-TCR mAb clone 56C5.2 (Beckman Coulter, Miama, FL). This mAb is listed as anti-Vbeta8 in one system (22), corresponding to TCRBV 12 in standard International Immunogenetics nomenclature (23). For PBMC, single-step combined staining used mAb 56C5.2 (15 μl), anti-CD8α-PE-cyanin 5 (Caltag) (5 μl) and tetramer B7-RPR-PE (0.05 μl) per 4 × 106 PBMC. For PBMC, we analyzed 350 or more CD8α (+) tetramer-high cells per subject for TCRBV 12 expression.
T-cell clones and lines were tested for specificity in cytotoxicity assays (20). Target cells were 51Cr-labeled HLA B*0702 genotype LCL that were either pulsed with 1 μg/ml RPRGEVRFL (majority VP22 49-57 sequence), infected overnight with HSV-2 strain 333 at a multiplicity of infection of 10, or mock-infected, were washed and used at 2 × 103/well. Candidate clonal microcultures were screened in singlicate or duplicate using 12.5% to 25% of the microculture/well as effectors. The criteria for HSV-specificity was greater than 25% specific release for peptide-pulsed cells and less than 5% specific release for mock-treated cells. Clones meeting these criteria were expanded for further studies. After expansion, the specificity of each T-cell clones was confirmed as RPR-specific in assays with peptide-loaded and mock targets in triplicate at an effector to target (E:T) ratio of 20. The arithmetic mean of triplicate experimental, minimum (media only) and maximum (detergent lysis) counts per minute values were used to calculate percent specific release. Variance from duplicate wells was < 10%. We required that the clones meet the same criteria in the confirmatory assay for inclusion in this report. The same HLA B*0702 LCL line was used for all experiments. Some clones were also confirmed as recognizing HSV-2 in the context of viral protein synthesis by using HSV-2-infected target cells in cytotoxicity assays as reported (24).
For functional avidity determination, 51Cr-labeled HLA B*0702-positive LCL were pulsed for 90 minutes with 10-7 to 10-15 molar peptide in 1 × log10 steps and washed thrice prior to use in duplicate CTL assays an at E:T of 20. To estimate EC50, the concentration of peptide leading to 50% of maximal lysis for each T-cell clone, Prism version 5 software (GraphPad, San Diego, CA) was used with these settings: fit set to non-linear log(agonist) vs. response; data range corresponding to 10-7 to 10-13 molar peptide, least mean squares, and constraints set as bottom value = 0 with no constraint on top values. For selected points on specific T-cell clone-specific dose-response curves, values that were clear outliers from sigmoidal log10 (peptide) vs. specific release curves were omitted.
IFN-γ release was measured after co-incubating 6 × 104 cloned CD8 T-cells with 2.5 × 104 HLA B*0702-genoypte LCL in 200 μl T-cell medium for 24 hours. The LCL had previously been peptide-loaded and washed as described above for cytotoxicity assays. Frozen supernatants were thawed and measured by ELISA as described (9). EC50 values were estimated as described for CTL assays.
RNA was isolated using RNAeasy mini kits (Qiagen, Valencia, CA). cDNA synthesis used Superscript II (Invitrogen, Carlsbad, CA) with random hexamer primers. PCR used Taq DNA polymerase and 10X buffer, 1.5 mM MgCl2 and 200 μM each dNTP (all Invitrogen). Cycling conditions were 94 °C × 2 min, then 94 °C for 30 sec/55 °C for 30 sec/72 °C for 30 seconds × 35 cycles, and then 72 °C for 7 min. Primer sequences were as described (25). In initial rounds, tubes contained the constant region primer and a pool of 5 TCRBV primers (each primer at 0.4 μM). For TCRAV, the initial PCR used a C region primer and single variable region primers, each at 0.4 μM. PCR products were visualized on ethidium bromide or SYBR Safe (Invitrogen) 1.5% agarose/TBE gels. Positive pools (for TCRBV) were repeated with single constituent TCRBV primers. For single, strong bands in the expected molecular weight range, PCR product was gel-purified (Qiaquick, Qiagen) and sequenced using BigDye 3.0 (ABI, Foster City, CA). Separate reactions used the same CB or TCRBV primers used in PCR. Chromatograms were aligned and amibiguities resolved using Seqman (Lasergene, Madison, WI). Sequences were aligned (MegAlign, Lasergene), analyzed for TCR gene segment usage with a computer algorithm (26, 27), and named per conventions (23). Primer names corresponded to an older, alternate TCR nomenclature to the standard IMGT system (28, 29).
For putative T-cell clones that had more than one strong band in the expected molecular weight ranges at either the pool or single TCVBV amplification stage, each band was excised and sequenced. If more than one TCRBV band was predicted to encode a productive CDR3, the cells and sequence were excluded from analysis. If one band was predicted to encode a productive TCRBV CDR3 region and the other band(s) contained a stop codon or frameshift in the CDR3, the predicted productive sequence was used. T-cell clones with in-frame, productive TCRBV CDR3 sequences were had their TCRAV sequenced. If PCR products were obtained in the expected molecular weight range for greater than 1 TCRAV primer, each one was sequenced. If one or two sequences encoded in-frame TCRAV CDR3 sequences, data was included because T-cell clones can have two allelic TCRA chains (23). If more than 2 productive TCRAV sequences were obtained, data were excluded.
To examine the TCRBV repertoire in bulk tetramer (+) CD8 cells, RNA was purified and cDNA synthesis done as outlined above. PCR analysis used the C region primer and each individual V region primer in separate tubes. Bands in the expected molecular weight range (28) were excised and ligated in separate reactions into the pCR4-Topo vector (Invitrogen). Transformed E. coli were incubated on selective media. Insert DNA from plasmid preps (Invitrogen) of random colonies was sequenced with Invitrogen-recommended primers.
For TCRVA and TCRVB, CDR3 region DNA sequences were submitted to the IMGT/V-Quest algorithm (27) and the assigned V and J (TCRA chain) or V, D, and J (TCRB chain) genes compiled. In some cases due to nucleotide deletion and addition, unambiguous gene usage could not be defined, but if the predicted amino acid sequences were in-frame and contained framework CDR3 conserved residues, the sequences were retained for analysis. Genbank (http://www.ncbi.nlm.nih.gov/Genbank/) accessions are EF567065-EF567070, EF567072-EF567074, EF591993-EF592010, EF592012-EF592030 and GQ502862-GQ502902 (TCRBV), and GQ502787-GQ502861 (TCRAV).
Cellular or molecular clones for TCR sequencing were obtained from five persons (Table I). We used several pathways to obtain CDR3 data. The first two approaches obtained RPR-specific T-cell clones directly ex vivo from peripheral blood. For subject 5 (Table I), we sorted CD8 (+), CLA-high cells from PBMC. Circulating HSV-2-specific CD8 CTL preferentially express cutaneous lymphocyte antigen (CLA) (11). The second approach sorted CD8 (+) cells binding the B7-RPR tetramer from PBMC from subjects 9, 12, and 13 (Table I). Cells sorted by either criterion were immediately cloned with a non-specific mitogen. The third approach stimulated PBMC from subjects 5491 and 5 with RPR peptide, sort-purified CD8 (+) tetramer (+) cells, and then cloned them. Finally, for subject 5491 only, we made cDNA molecular clones for TCRBV CDR3 sequence analysis from a portion of the bulk-sorted cells.
Screening of clones derived by tetramer sorting (directly ex vivo or from bulk cultures) showed that more than 80% had specific lysis of peptide-loaded APC (not shown). Confirmatory cytotoxicity tests were positive on >95% of candidate clones (not shown). Screening CTL assays of CLA-sorted clones from subject 5 (first approach, above) showed that 5.9% were HSV-2-specific. All clones with HSV-2-specific lytic activity were also specific for the RPR peptide and restricted by HLA B*0702 (not shown), indicating immunodominance in this subject as reported (11). Two such clones (1B1 and 2F1) were available for TCR analysis. Overall, we obtained paired TCRAV and TCRBV data from 68 T-cell clones specific for the B7-RPR epitope (Table I). 50 of these were made by direct ex vivo methods (48 by tetramer sorting, 2 by CLA-based sorting), while 18 T-cell clones were derived after prior bulk stimulation with peptide. Additional TCRB data (but not TCRA) information was available from 19 molecular clones from subject 5491, yielding a total of 87 TCRB and 68 TCRA CDR3 sequences.
TCRBV preference for HSV-2 epitope-specific T-cells was initially investigated using bulk CD8 (+) tetramer (+) sorted cells from subject 5491. These were >96% pure by re-analysis of a fraction of sorted cells (not shown). When cDNA was amplified with TCRBV family-specific primers, we observed that only primers 6 and 8 (primers corresponding to TCRBV12 and TCVBV7 after nomenclature reconciliation (28, 29)) gave PCR products in the expected molecular weight ranges (Fig. 1). Bulk PCR products were ligated and random clones sequenced. Consistent with the PCR data, the TCRBV sequences contained either the TCRBV12-4 gene or the TCRBV7-3 gene (28). A small amount of heterogeneity was observed within the TCRBV families. For TCRBV12-4, 14 of 15 molecular clones had identical nucleotide sequences encoding CASRPQGRDNEQFF, while one clone (5491VB8.6) encoded the near-identical CDR3 region CASRRQGRDNEQFF. Within TCRBV7-3, three of the four CDR3 regions had the same nucleotides encoding CASRKTGGGTEAFF, and one (5491V6.12) encoded CASSLHLGGVTDTQYF.
We compared these molecular clones to single cell-origin T-cell clones from the same subject (5491), and detected both identity and subtle variation. Within the dominant TCRBV12-4, both variants mentioned above were present. Cell clones 5491-W19 and 5491-W21 had the TCRBV12 CDR3 CASRPQGRDNEQFF amino acid sequence (and identical nucleotide region) also seen in most of the TCRBV12-4 molecular clones from this subject (above). These two cellular clones also had a TCRAV19*01-using CDR3 sequence, CALSEANSWGKLQF, which exemplifies a TCRAV motif seen in four of five subjects (below). Cell clone 5491-W14 had an amino acid sequence identical to that of the TCRBV 12-4 minor variant CASRRQGRDNEQFF (above), but with a nucleotide sequence that differed at 3 positions (nucleotide-level data, including synonymous variants referred to throughout this report, are available in Genbank, with a focus retained herein on amino acid sequences). The subdominant TCRBV7-3 sequence (CASRKTGGGTEAFF) from the bulk molecular clones was present in cell clone 5491-W2, with the same nucleotide sequence. These data indicate that TCRBV12 dominated the RPR epitope-specific response in subject 5491, whether studied by molecular or cellular cloning methods.
We then measured TCRBV12 use by tetramer (+) cells in PBMC at the protein level. Three additional subjects were studied directly ex vivo using flow cytometry. From 0.084% to 0.22% of CD8α (+) cells bound the HSV-2 B7-RPR tetramer (Fig. 2). The proportion of CD8α (+), tetramer (+) cells expressing TCRBV12 ranged from 19.2% to 71.9% (Fig. 2). This was a 3 to 48-fold enrichment over the level of TCRBV12 usage observed in CD8α (+), tetramer (-) cells, and confirmed a general trend to TCRBV12 use by cells reacting with this epitope.
Amongst the 68 T-cell clones from 5 subjects, the most commonly used TCRBV gene was TCRBV12-4 (16 of 68, 24%). TCRBV12-4 also had the highest population prevalence, occurring in four of the five subjects studied. Amongst these four persons, each had identical or near-identical TCRBV12 CDR3 amino acid sequences (for example, clones 5.19, 9.43, 12.51, 5491.W14 (Table II). Clones 12.51 and 12.59 had the same CDR3 amino acid sequence, CASRRQGRDNEQFF, in cell (clone 5491.W14) and molecular clones from subject 5491, forming the first of three public TCRB CDR3 sequences. Within this public amino acid sequence, heterogeneity was again noted at the nucleotide level.
The TCRBV12-4 using receptors showed clear evidence of a common motif, with other subjects having near matches to CASRRQGRDNEQFF. Cell clones 5.1B1 and 5.2F1 had the sequence CASRVQGRDNEQFF, while cell clone 9.43 had sequence CASRMQGRDNEQFF. Together, these sequences define a CASRXQGRDNEQFF motif, with position 5 being either P, R, V, or M, represented in four of the five subjects. Other features of the motif included invariant (QG) amino acid residues at positions 6 and 7, arginine (positive) at position 8 in most clones, and one or more negatively charged residues at positions 9-11.
The second most prevalent TCRBV gene, used by 3 of the 5 subjects, was TCRBV9. Remarkably, all 9 T-cell clones from subjects 9, 12, and 13 that used TCRBV9 had the same TCRB CDR3 amino acid sequence (CASSVWGTDTQYF). This second public TCRB CDR3 amino acid sequence was encoded by 4 different nucleotide sequences, one of which was shared between subjects 9 and 13.
A third public TCRBV CDR3 example was noted within the group of clones from subjects 9 and 12 that used TCRBV5 family members. Each of 10 TCRBV5-using clones from subject 9, as well as clone 12.42, had the same sequence (CATRIGWGTDTQY) encoded by the same nucleotide sequence. We noted that 8 of the 10 used TCRBV5-1, while one each used TCRBV5-3 and one used TCRBV5-7. The nucleotide differences between these TCRBV5 family members lie outside of the CDR3 region (30).
The most frequently used TCRAV gene was TCRAV 19, used by 24 of 68 clones (35%) and four of the five subjects. TCRAV-19-based sequences occurred together with six different TCRBV genes, including TCRBV12-4, TCRBV5-1, TCRBV7, TCRBV11-2, TCRBV9, and TCRBV-24. Every cell clone using TCRBV12-4 (above) invariably expressed TCRAV19 mRNA. These TCRAV19 sequences frequently had identity, or similarity, within and between subjects. The CALSEAXSWGKLQF motif, with X being either N, D, or S, was present in each subject that had TCRBV12-4-using clones. The CALSEADSWGKLQF variant was public to 3 subjects (5, 9, and 12) and identical at the nucleotide level as well between selected clones from subjects 9 and 12 (details in Genbank).
This public TCRVA19 CALSEADSWGKLQF sequence was noted to occur with either the R, V, and M variants of the TCRBV12-4 CASRXQGRDNEQFF motif (above). The TCRBV12-4 motif variants (above) and the TCRAV19 motif variants, each had a single variable amino acid position. Because the TCRVA19 variants and TCRBV12-4 variants tolerated promiscuous rather than preferential binding to each other, no two persons shared a double-public TCR with amino acid identity for TCRBV12-4/TCRAV19 polypeptides. TCRAV19 almost invariably occurred with TCRBV12-4 and vice versa, although a few exceptions were noted in both directions (e.g. clones 5491.W5 and clone 12.5).
The TCRAV1-1-based sequence, CAVRDTNTNAGKSTF, was the second public TCRA sequence noted. In subject 9, this sequence occurred only with the TCRBV5-1 CATRIGWGTDTQYF sequence (itself also public). Clone 12.42 had the same amino acid and nucleotide sequences for both the TCRA and TCRB CDR3 regions as did the TCRAV1-1/TCRBV5 expanded clonotype in subject 9, thus defining a dually-public TCRA/B heterodimer. The TCRAV1-1 CAVRDTNTNAGKSTF sequence was also present in subject 13 (clone 13.18), and was thus seen in 3 of 5 subjects.
Remarkably, the TCRAV1-1 CAVRDTNTNAGKSTF sequence also occurred in a second dually-public TCR, paired with the TCRBV9-using CASSVWGTDTQYF in clones 12.14, 12.22, and also clone 13.18. This public TCRVA1-1 sequence was also found with TCRBV12-3 and TCRBV6-1 in various clones from subject 12. It was thus the most promiscuous specific TCRA CDR3 sequence, pairing with four different TCRB polypeptides within this one subject (subject 12). Overall, the TCRVA1-1 gene segment was found in 13 clones (20% of the total) and with six different TCRBV partners, namely TCRBV5-1, TCRBV6-1, TCRBV9, TCRBV12-3, TCRBV27, and TCRBV10-3. Together with TCRAV19, TCRVA1-1 appeared to be favored by T-cell clones specific for this epitope in HSV-2.
The third public TCRA CDR3 region noted was a TCRVA38-2-using sequence, CAFGRGAQKLVF. It was used by clones 9.30, 9.45, 9.47 and 9.46 (invariably paired with the dominant and public TCRBV5-1 CATRIGWGTDTQY, above), and also occurred in T-cell clone 12.48 with a TCRBV30-based CDR3 region with a very different TCRB CDR3 amino acid sequence.
It was not uncommon to detect several T-cell clones within-subject with both the same TCRA and TCRB CDR3 regions, likely representing clonal expansions. For example, of the ten clones from subject 9 with the same TCRBV5-1 nucleotide and amino acid sequences, five were paired with identical TCRVA1-1-based genes. Three clones (L4, H37, H40) from subject 5 had both TCRVA12-1 and TCRVA23-using genes and a TCRBV24-1 using gene that were each always identical to each other. A clonotypic expressing two coding TCRA mRNAs was likely present in subject 5, with some constituent cells (e.g. clone H32) possibly losing expression of one TCRA mRNA. Indeed, the sequences showing publicity, defined earlier in this report, and in-person immunodominance, defined as being present a high proportion of single cell-derived T-cells, were generally linked in our data set. In subject 9, 11 of 20 (55%) independently derive clones had public TCRA chains. 11 of 20 (55%) clones also had public TCRB genes, although these did not completely overlap with the clones with public alpha chains. Similarly, for subject 13, 9 clones each (out of 23, for 39%) had a public TCRA gene or a public TCRB gene. Within these subjects, the clones expressing public TCRA and/or TCRB frequently occurred in expanded clonotypes (Table II).
It has previously been reported that functional avidity, as measured by the concentration of peptide required to stimulate 50% of maximal cytotoxic activity, can correlate with CDR3 amino acid sequences or motifs (31). We therefore performed dose-response titrations for individual T-cell clones. EC50 values for cytolysis of peptide-loaded antigen presenting cells were obtained for 57 of the 68 clones (84%) with both TCRB and TVRA CDR3 sequences (above). Sample data (Fig. 3) show an inverse relationship between peptide concentration and cytolysis. Most EC50 values were in the 10-9 to 10-10 molar range for four of the five subjects (Fig. 4). Within-subject, we observed a range of about 1 log10 between the lowest and highest functional avidity.
Subject 5 is an individual with very low shedding rates of HSV-2 as measured by daily genital swab samples (11). Clone 1B1 and 2F1, derived directly from blood, had TCRs with public or prevalent motif sequences for both TCRA and TCRB chains. EC50 values were not obtained for these clones due to availability. We did note that a series of clones derived by tetramer-sorting cells after in vitro expansion tended to have very high avidity (low EC50) (Table II, Fig. 3 and Fig. 4). This correlated with TCR V regions and sequences largely unique to this series of clones. The highest avidity clones used diverse TCRA12-1, 19, 22, 23, and 26-2 sequences, and tended to use TCRB20-1 and 24-1, unique to this subject, and a variant (CASRIWGGADTQYF) of the public TCRBV5-1 CATRIGWGTDTQY sequence noted above.
We also determined the functional avidity for stimulation of IFN-γ release on a subset of the T-cell clones. Each clone showed peptide-specific IFNγ response that decreased with decreasing peptide concentration (Fig. 4). In contrast to cytotoxicity (Fig. 3), we did not observe a plateau at the higher concentrations of peptide, and it is possible that higher responses could have been observed at an optimal peptide concentration. EC50 values were generally obtained at higher concentrations of peptide, in the range of 10-7 to 10-8 molar, than were observed in CTL assays. Some clones had EC50 values as low as 3.75 × 10-11 (clone 20, Fig. 4). Overall, we did not observe a correlation between functional avidity, as measured by EC50 values, for cytotoxicity and IFN-γ effector functions (r2=0.109 using logarithmic regression) (not shown).
We sequenced the CDR3 hypervariable regions alpha and beta chains of HSV-2-reactive CD8 T-cells specific for the most immunodominant known HSV-2 epitope in humans, as defined by both population prevalence and percent tetramer positivity. Among the 68 clones studied, 50 were derived from single cells. This is the first in-depth examination of epitope-specific T-cell CDR3 repertoire to an alphaherpesvirus in it’s natural host. The CD8 response to HSV-2 strongly displays the public TCR phenomenon, as previously observed for CD8 responses to viruses in the betaherpesvirus (CMV) and gammaherpesvirus (EBV) families. Again similar to CMV and EBV,we observed that HSV-2-specific public TCRs are frequently also immunodominant within-subject. The host response to HSV-2 differs from responses to CMV and EBV, in that the peak and memory-phase overall CD8 magnitudes are much lower for HSV-2. In addition, the cell type HSV-2 uses for latent and lytic replication in vivo, neurons and epithelial cells, respectively, are not professional APC. In contrast, monocyte/macrophages and B-cells, cells that are chronically infected by CMV and EBV, respectively, are highly efficient APC. We therefore conclude that the chronicity of herpesvirus infection may be an important driver for selection towards public TCR sequences.
The CD8 response to HSV is functionally important both in humans and in animal models. Infiltration of HSV-2-specific cytotoxic T-cells into the skin is correlated temporally with clearance of infectious virus from recurrent genital HSV lesions (7). HSV-2-specific CD8 T-cell also persist at the dermal-epidermal junction, in proximity to the sensory nerve endings that are the conduit for virus delivery during recurrences, for several weeks after resolution of recurrent genital herpes in humans (6, 8). In mice and humans, HSV-specific CD8 T-cells persistently infiltrate sensory ganglia that harbor infected neurons (2, 5). Ablation of local CD8 cells or interferon-gamma contribute to ganglionic reactivation in mice (32, 33). In the case of SIV infection, it has been observed that public TCR-using CD8 wells are correlated with protection after vaccination (34). Vaccines delivering single HSV CD8 epitopes but neither CD4 nor antibody targets can protect mice from lethal HSV infection (35), and CD8-based HSV vaccine strategies are currently being studied in humans (36). The possibility that the targeting HSV-2 epitopes that can elicit public/immunodominant TCRs might be more effective than the targeting of epitopes that do not show these phenomena suggest that similar studies of other HSV-2 CD8 epitopes may be of interest.
In MHC H-2b haplotype mice, the CD8 response largely focuses on a single epitope in HSV glycoprotein B. This response uses two dominant TCRBV genes, but fine clonotypes have not been investigated (37). While murine neurons are chronically infected after HSV-1 infection, murine HSV-1 differ significantly from human HSV infection in that productive lytic recurrent infection capable of transmitting infection to the periphery is very rare (38). The human CD8 response during chronic infection may be shaped by chronic antigenic (re)-stimulation in both skin and ganglia to a larger extent than in mice. The subjects studied in this report each had long-established histories of HSV-2 infection. It is now established, based on PCR-based genital shedding studies, that essentially all HSV-2-infected persons periodically shed virus from the periphery (39). This implies re-exposure of the immune system to viral antigens on a chronic basis.
We observed a strong bias towards usage of specific TCRVA and TCRVB gene segments, the existence of several TCR single-chain and heterodimeric public amino acid sequences, and also TCR3 amino acid motifs in which distinct sequences shared conserved features. These findings are all interrelated. TCRVA19, TCRVA1-1, TCRVA4 together accounted for more than 50% of the observed TCRA chains. Similarly, TCRBV12-4, TCRBV9, and TCRBV5-1 comprised the majority of the TCRB chains. We detected three distinct public TCRA CDR3 sequences and three distinct public TCRB CDR3 sequences amongst a relatively small collection of subjects and T-cell clones.
Debate exists as to the relative contributions of TCRA or TCRB chains to peptide-MHC binding. This appears to vary for different TCR heterodimers and different MHC-peptide complexes, but in general, TCRB is thought to have more interactions with the C terminus of the peptide while TCRA may dominate interactions with the N terminus (40). There is no crystal structure for HLA B*0702 complexed with peptide/TCR, and important differences in TCR interactions have been noted between different human HLA alleles (41). Our data support a model in which the specific TCRVA1-1 CAVRDTNTNAGKSTF sequence form functional heterodimers that can bind B7-RPR in conjunction with quite diverse TCRB sequences using TCRBV 5-1, 6-1, 9, and 12-3. In this example, the TCRVA chain may thus be dominant and contribute more to the overall energetics of the interaction.
Occasional T-cell clones expressed two distinct mRNAs with protein-coding sequences without stop or frameshift mutations within the sequenced regions. Amongst the 50 clones directly derived from single cells in PBMC, 3 (6%) had this pattern. In addition, clones 5.L4, 5.H37, and 5.H48 each had the same two TCRA chains. These were derived after in vitro stimulation of PBMC with peptide, and likely represent progeny of a single cell present in PBMC which expressed both mRNAs. Other clones from this bulk expansion only seemed to express one of these two mRNAs. It is not know if expression of the other was lost, or was below the analytical threshold of detection.
We noted that the functional avidities of T-cell clones were generally quite similar, both within-subject and between subjects. The one exception was subject 5, whose T-cell clones had a functional avidity in cytotoxicity assays that was 2 to 3 orders of magnitude higher than for the other subjects. These clones had TCR sequences largely not seen in any other subject. As these clones were derived after in vitro enrichment of peptide-specific clones, it is not known how representative they are of epitope-specific T-cells from this subject. Possibly, their higher avidity is partially a consequence of selective outgrowth during in vitro expansion. Tetramer-based flow sorting was used for both direct PBMC- and culture-derived cloning, so this step is unlikely to have selected preferentially for high affinity clones. These exceptional high avidity clonotypes do, however, demonstrate in principle that highly avid HSV-2-specific T-cell clones can occur, and that comparisons of subjects with different phenotypes could disclose differences between groups. It is not known why the functional avidities we observed for IFN-γ release were generally ~100-fold lower than for cytotoxicity assays. In other T-cell systems, higher densities of TCR stimulation have been reported to be required to trigger interferon than cytotoxicity effector functions (42). It is not known why there was a slight decrease in percent cytotoxicity at high concentrations (Fig. 3). If this was due to slight peptide toxicity for the LCL target cells, then actual cytotoxicity EC50 values might be left-shifted to higher peptide concentrations, more similar to those seen for the IFN-γ assays. We did not, however, observe higher spontaneous 51Cr release for target cells incubated at 10(-6) or 10(-7) M peptide compared to target cells incubated with lower concentrations of peptide (data not shown). Our IFN-γ release assay also used a lower responder to stimulator ratio (2.4:1) than did the cytotoxicity assay format (20:1), possibly increasing the observed EC50. For these reasons, direct quantitative comparisons of EC50 values in the two effector assays must be performed with caution.
Our work-flow required clonal outgrowth prior to assay. Some virus-specific T-cells may not make IFN-γ or proliferate well directly ex vivo (43, 44). This may correlate with effector vs. central memory status as measured by a variety of markers. Of note, TCR clonotypes within CMV- and EBV-specific CD8 cells with varying effector vs. central memory phenotypes were recently examined directly ex vivo, and in general, every TCR clonotype was present within each phenotypic subset (16). B7-RPR-specific CD8 T-cells also display varied memory markers (9), and it is not yet known if TCR sharing is also true for HSV-2-specific memory CD8 T-cells with different memory phenotypes. In addition, our sample size was relatively limited and we studied a single HSV-2 epitope. T-cells were also made using three work-flows, and for these reasons our conclusions must be regarded as preliminary. Further research will be required to determine if our observations also hold true for population-prevalent responses such as HLA A*0201/HSV-2 UL47 551-559 (45). Studies of additional epitopes await validation of additional HSV tetramers that are solidly “on-screen” for direct PBMC staining.
In conclusion, the CD8 response to an immunodominant epitope in HSV-2, a chronic human pathogen to which CD8 cells respond at neuronal and epithelial sites of infection, displays within-person dominance and remarkable conservation of dominant TCR clonotypes between individuals. Within a small set of 5 subjects, three separate public TCRA amino acid sequences, three separate TCRB amino acid sequences, and two distinct public TCRA/B combinations were detected. Chronic exposure to an alphaherpesvirus thus appears to be similar to chronic infection by beta- and gamma-herpesviruses and retroviruses, agents with very different cell tropisms and viral loads, and thus appears to be a common pathway during long-term exposure to foreign antigens.
The staff of the University of Washington Virology Research Clinic recruited subjects and performed phlebotomy. Stacy Selke provided database management. Dr. Rhoda Morrow and the University of Washington diagnostic virology laboratory provided diagnostic serology. Dr. Jianhong Cao at the Fred Hutchinson Cancer Research Center Immune Monitoring lab provided peptide-HLA tetramers.
1Supported by NIH grants AI50132 and AI30731.