Abstract
High-throughput T cell receptor sequencing on sequentially banked blood samples from healthy individuals has shown that high-frequency clonotypes can remain relatively stable for up to 18 years, with minimal inflation, deflation, or turnover. These populations included T cell expansions specific for Epstein-Barr virus. Thus, in spite of exposure to a barrage of microorganisms over the course of life, the dominant clonotypes in the mature peripheral T cell repertoire can alter surprisingly little.
TEXT
The human immune system is thought to include approximately 1012 T cells expressing 1 million to 25 million unique T cell receptors (TCRs) (1–3). From the vast naive receptor pool, select clonotypes emerge to engage the series of infectious agents, aberrant cells, and occasional innocuous agents encountered over the course of life (4). Often these T cell expansions are further amplified by persistent pathogens that provide a constant stream of antigen (Ag) to the cellular compartment. In mice, chronic infection can lead to rapid memory inflation, T cell dysfunction, and “exhaustion” (5). To compensate, new naive T cells can be recruited into the Ag-specific repertoire (6). In humans, the effect of long-term persistent infection on the T cell repertoire is less well defined; however, the tracking of human memory clonotypes specific for viruses has shown long-term persistence and dominance in the peripheral circulation for many years (7–9). These studies, which employed conventional sequencing techniques and were limited to a small number of clonotypes specific for several viral epitopes, provide limited insight into the stability of the T cell repertoire in totum. To address this issue, we have utilized a sequential series of cryobanked peripheral blood mononuclear cell (PBMC) samples from healthy individuals with new high-throughput sequencing (HTS) technology to monitor global fluctuations in the T cell repertoire over many years.
Ex vivo cell sorting.
PBMCs were stained with antibodies to CD8, CD4, CD14, CD16, and CD19 (BioLegend) and Live/Dead fixable aqua dead cell stain (Molecular Probes). Approximately 106 CD8+ T cells were sorted using a FACSAria II cell sorter (BD Biosciences). For HTS, 3 μg of DNA was isolated using the QIAamp DNA blood minikit (Qiagen). Donor parameters are detailed in Table 1. Written consent was obtained from the blood donors, and the study was approved by an institutional review committee.
Table 1.
Donor parametersa
| Donor | Gender | HLA-A | HLA-B | EBV serostatus | CMV serostatus |
|---|---|---|---|---|---|
| H01 | Male | 23, 25 | 18, 49 | + | − |
| H02 | Male | 24, 29 | 4403, 4405 | + | − |
HLA, human leukocyte antigen.
High-throughput sequencing.
TCR sequencing was performed using the ImmunoSEQ platform, which combines template-switch anchored reverse transcription-PCR (RT-PCR) with the Illumina HiSeq system (3). Data filtering and T cell receptor beta (TRB) gene annotation were performed using a microassembler and standard algorithms as described previously (3). TRBV, TRBD, TRBJ, and CDR3 parameters were delineated according to definitions from International ImMunoGeneTics collaboration (10).
Epstein-Barr virus-specific T cell identification.
Short-term T cell cultures specific for Epstein-Barr virus (EBV) were raised as previously described (11). EBV-specific T cells were isolated using a FACSAria II cell sorter (BD Biosciences) through autologous lymphoblastoid cell line (LCL) stimulation followed by a surface tumor necrosis factor (TNF) capture assay (12). Rearranged, functional αβ TCRs were identified using TRBV gene-specific RT-PCR, bacterial subcloning, and Sanger sequencing as previously described (11).
To examine the stability of the human CD8+ T cell repertoire, the CD8+ T lymphocyte subsets were sorted to >99% purity from PBMC samples from a healthy Caucasian male (H01), collected at four time points between the ages of 26 and 44. The donor remained EBV seropositive and cytomegalovirus (CMV) seronegative during the sampling schedule and could recall no significant health problems during this period. High-throughput TCR sequencing of the four populations yielded an average of 4.7 million reads and 41,000 unique TCR sequences per sample. Full sequence statistics are shown in Table S1 in the supplemental material.
The 40 most frequent TCR β-chain sequences from the 1993 time point accounted for 16.7% of the total CD8+ repertoire and were tracked over the 18-year period. Surprisingly, 35 of these clonotypes could be identified across all time points, and all 40 β-chains could be observed at a significant frequency across at least three time points (Table 2; see also Table S2 in the supplemental material). The 40 most frequent TCR β-chain sequences from 2011, which accounted for 16.3% of the total CD8+ repertoire, were also tracked at the earlier three time points (see Table S3). All 40 of these 2011 β-chain sequences were observed in 2008, while 37 and 32 of the sequences were observed in 1997 and 1993, respectively. In total, 17 TCR β-chain sequences persisted in the top 40 most frequent in both 1993 and 2011. These data illustrate that the apex of the CD8+ T cell repertoire can remain surprisingly stable over many decades, with minimal turnover of highly expanded clonotypes.
Table 2.
Forty TCR β-chain sequences occurring at highest frequency in CD8+ T cells from donor H01 in 1993 and their frequencies in CD8+ T cells in 1997, 2008, and 2011
| Rank | Amino acid sequenceb | TRBV gene(s) | TRBJ gene | Frequency in CD8+ T cells by yr (%)a |
|||
|---|---|---|---|---|---|---|---|
| 1993 | 1997 | 2008 | 2011 | ||||
| 1 | CASSFPGNEQFF | 7-9 | 2-1 | 5.312 | 0.579 | 0.344 | 0.162 |
| 2 | CASSEPGTSQETQYF | 7-9 | 2-5 | 1.285 | 0.160 | 0.292 | 0.239 |
| 3 | CASSSTGSGETQYF | 7-9 | 2-5 | 0.923 | 0.214 | 0.655 | 0.278 |
| 4 | CASSFGTSSYNEQFF | 7-9 | 2-1 | 0.651 | 2.044 | 0.349 | 0.156 |
| 5 | CASSLGHAEAFF | 7-9 | 1-1 | 0.645 | 1.277 | 0.896 | 0.446 |
| 6 | CASSEQDGFNYGYTF | 7-9 | 1-2 | 0.598 | 0.576 | 0.772 | 0.602 |
| 7 | CASSLGDYRGYTF | 7-3 | 1-2 | 0.458 | 0.379 | 0.099 | 0.043 |
| 8 | CASSSLEGVYDEQFF | 7-9 | 2-1 | 0.423 | 0.935 | 0.224 | 0.167 |
| 9 | CASSQGVRGQHSYNEQFF | 4-2 | 2-1 | 0.416 | 0.844 | 0.389 | 0.315 |
| 10 | CASSLASNGYTF | 7-9 | 1-2 | 0.390 | 0.060 | 0.062 | 0.014 |
| 11 | CASSPRQGTHNEQFF | 19 | 2-1 | 0.333 | 0.040 | 0.040 | 0.009 |
| 12 | CASSPKLGGEQYF | 7-3 | 2-7 | 0.284 | 0.074 | 0.000 | 0.009 |
| 13 | CASSDQGHRDEKLFF | 6-1 | 1-4 | 0.275 | 0.184 | 0.136 | 0.126 |
| 14 | CASSLLPRHTDTQYF | 18 | 2-3 | 0.274 | 0.161 | 0.129 | 0.132 |
| 15 | CASAPPPGEGARELFF | 7-9/11-1 | 2-2 | 0.257 | 0.004 | 0.042 | 0.000 |
| 16 | CASFPDRGYTGELFF | 7-9 | 2-2 | 0.238 | 0.153 | 0.053 | 0.084 |
| 17 | CASRRVMSGTDTQYF | 7-8 | 2-3 | 0.226 | 0.017 | 0.002 | 0.006 |
| 18 | CASATWAGATDTQYF | 19 | 2-3 | 0.218 | 0.472 | 0.026 | 0.029 |
| 19 | CASSPQSLGGYTF | 18 | 1-2 | 0.211 | 0.501 | 0.438 | 0.381 |
| 20 | CASSFVPGQPQHF | 7-9 | 1-5 | 0.203 | 0.436 | 0.322 | 0.208 |
| 21 | CASIAGSFDEQFF | 7-9 | 2-1 | 0.196 | 0.056 | 0.028 | 0.000 |
| 22 | CASSPLPRRDSHSPLHF | 18 | 1-6 | 0.189 | 0.113 | 0.037 | 0.029 |
| 23 | CASSPTGGSYNSPLHF | 7-2/11-2/11-3 | 1-6 | 0.188 | 0.277 | 0.323 | 0.331 |
| 24 | CASSLAGGYSYEQYF | 7-6 | 2-7 | 0.174 | 0.154 | 0.019 | 0.073 |
| 25 | CASSHSRDLDYEQYF | 6-5/6-6 | 2-7 | 0.171 | 0.068 | 0.010 | 0.018 |
| 26 | CASSLVPWSETTGDTDTQYF | 7-6/7-7 | 2-3 | 0.169 | 0.095 | 0.082 | 0.098 |
| 27 | CASSRGGNNEQFF | 19 | 2-1 | 0.165 | 0.272 | 0.067 | 0.017 |
| 28 | CASSLRDASYEQYF | 7-9 | 2-7 | 0.160 | 0.086 | 0.016 | 0.000 |
| 29 | CASSLGAGGLEQFF | 7-6 | 2-1 | 0.157 | 0.003 | 0.040 | 0.001 |
| 30 | CASSYLTADGNQPQHF | 6-2/6-3 | 1-5 | 0.156 | 0.088 | 0.199 | 0.086 |
| 31 | CASSPIFRGLYTEAFF | 7-9/11-1 | 1-1 | 0.152 | 0.167 | 0.039 | 0.025 |
| 32 | CACNNSPLHF | 30 | 1-6 | 0.147 | 0.088 | 0.033 | 0.028 |
| 33 | CASVLEGFNQPQHF | 6-1/6-5/6-6 | 1-5 | 0.146 | 0.844 | 0.147 | 0.061 |
| 34 | CAGGTGSDTQYF | 5-4 | 2-3 | 0.146 | 0.268 | 0.163 | 0.253 |
| 35 | CASSLWGTTYEQYF | 7-9 | 2-7 | 0.143 | 0.082 | 0.039 | 0.002 |
| 36 | CASSPVPATYEQYF | 5-6 | 2-7 | 0.137 | 0.306 | 0.234 | 0.332 |
| 37 | CASSPSSGPYEQYF | 18 | 2-7 | 0.137 | 0.204 | 0.225 | 0.276 |
| 38 | CASSPETGILSGYTF | 7-6 | 1-2 | 0.128 | 0.121 | 0.037 | 0.045 |
| 39 | CASTARGNTGELFF | 6-1/6-5/6-6 | 2-2 | 0.119 | 0.129 | 0.330 | 0.370 |
| 40 | CASSLVGHYEQYF | 7-9 | 2-7 | 0.113 | 0.047 | 0.000 | 0.007 |
| Total (%) | 16.712 | 12.576 | 7.333 | 5.458 | |||
Frequency of sequence in each sample relative to the total no. of productive sequences per sample. Key: unshaded, <0.001%; shaded and regular font (not underlined), 0.001 to 0.1%; shaded and italic, 0.1 to 1%; shaded and underlined, 1 to 5%; shaded and bold, >5%.
Bold and italics indicate sequences corresponding to EBV-specific clonotypes identified by polychromatic flow cytometric sorting and TCR Sanger sequencing.
Interestingly, each of the prominent β-chain amino acid sequences observed at these time points was encoded by a single dominant nucleotide sequence, with alternative coding sequences observed at very low frequencies or not at all at any time point (see Tables S2 and S3 in the supplemental material). These data indicate that the selection pressures that drive these clonal expansions have acted upon individual clonotypes, which, once expanded to large numbers, remove any further pressure for the expansion of additional clonotypes with the same specificity from the naive T cell repertoire.
We also performed TCR HTS on the PBMCs of a second healthy Caucasian donor (H02) at the ages of 58 and 67. This donor also remained EBV seropositive and CMV seronegative during the sampling schedule and had no significant health problems. The 40 most frequent TCR β-chain sequences from this donor in 2002 occupied 10.3% of the total T cell repertoire, and 36 of these β-chains were also found in 2011, many at high frequencies, filling a total of 12.2% of the repertoire at this recent time point (Table 3; see also Table S4 in the supplemental material). These data support the conclusion that high-frequency T cell clonotypes can persist for many years in adults. As with donor H01, each of the prominent β-chain amino acid sequences was encoded by a single dominant nucleotide sequence, with alternative coding sequences observed at very low frequencies or not at all (see Table S4).
Table 3.
Forty TCR β-chain sequences occurring at highest frequency in PBMCs from donor H02 in 2002 and their frequencies in 2011
| Rank | Amino acid sequenceb | TRBV gene(s) | TRBJ gene | Frequency in PBMCs by yr (%)a |
|
|---|---|---|---|---|---|
| 2002 | 2011 | ||||
| 1 | CASRYRDDSYNEQFF | 7-9 | 2-1 | 5.019 | 5.664 |
| 2 | CASTPGRQSTRGNQPQHF | 2 | 1-5 | 0.981 | 2.707 |
| 3 | CASSLIGSGQSYNEQFF | 7-9/11-1 | 2-1 | 0.571 | 0.279 |
| 4 | CASSLAWGWKIDTQYF | 7-9/11-1 | 2-3 | 0.415 | 0.420 |
| 5 | CASSGGSGDADTQYF | 6-2/6-3 | 2-3 | 0.258 | 0.189 |
| 6 | CASSLMGGSETQYF | 7-2 | 2-5 | 0.253 | 0.388 |
| 7 | CASSSTLPGTTPHEQYF | 6-5/6-6 | 2-7 | 0.245 | 0.131 |
| 8 | CASSYGETQYF | 7-9 | 2-5 | 0.166 | 0.151 |
| 9 | CASSRQGANEQYF | 7-9 | 2-7 | 0.158 | 0.026 |
| 10 | CAWRGRGAAYEQYF | 30 | 2-7 | 0.147 | 0.557 |
| 11 | CASSLRLGGAHEQYF | 5-1 | 2-7 | 0.126 | 0.151 |
| 12 | CASHTGPGNSYEQYF | 6-1/6-5/6-6 | 2-7 | 0.124 | 0.262 |
| 13 | CASSPWDQETQYF | 7-2 | 2-5 | 0.115 | 0.063 |
| 14 | CASSNGPGQGASETQYF | 18 | 2-5 | 0.106 | 0.018 |
| 15 | CASSDSLPSLPAGGGNEQFF | Undefined | 2-1 | 0.104 | 0.007 |
| 16 | CASSLYGGTSYEQYF | 7-9 | 2-7 | 0.101 | 0.024 |
| 17 | CASSLGFTGELFF | 5-1 | 2-2 | 0.101 | 0.120 |
| 18 | CSAPDGTSGYNEQFF | 20-1 | 2-1 | 0.085 | 0.126 |
| 19 | CASSLPNIRNEQFF | 7-9 | 2-1 | 0.082 | 0.323 |
| 20 | CSAVGGRGYTF | 29-1 | 1-2 | 0.076 | 0.289 |
| 21 | CASSYSSGRVGYEQFF | 6-2/6-3 | 2-1 | 0.073 | 0.021 |
| 22 | CASSYRENTEAFF | 6-2/6-3 | 1-1 | 0.072 | 0.085 |
| 23 | CASSEGELSGAETQYF | 6-1 | 2-5 | 0.071 | 0.013 |
| 24 | CASSLNTGAPGELFF | 7-6 | 2-2 | 0.068 | 0.047 |
| 25 | CASSVGTGEQYF | 7-9 | 2-7 | 0.065 | 0.007 |
| 26 | CASSTGRSPDTQYF | 18 | 2-3 | 0.056 | 0.020 |
| 27 | CASSTPTSGRQTQYF | 19 | 2-5 | 0.053 | 0.007 |
| 28 | CASRSGLFSTDTQYF | 7-2 | 2-3 | 0.053 | 0.015 |
| 29 | CASSLAYQSETSYEQYF | 7-9/11-1 | 2-7 | 0.048 | 0.003 |
| 30 | CASSPRGYPEAYEQYF | 7-9/11-1 | 2-7 | 0.048 | 0.003 |
| 31 | CASSPRGPDTQYF | 7-9 | 2-3 | 0.046 | 0.019 |
| 32 | CASSPNGGATNEKLFF | 18 | 1-4 | 0.044 | 0.000 |
| 33 | CASLGPGRGLRGYTF | 7-9 | 1-2 | 0.044 | 0.002 |
| 34 | CASSPVAGDNEQFF | 18 | 2-1 | 0.044 | 0.008 |
| 35 | CASSFITDTQYF | 7-2 | 2-3 | 0.043 | 0.000 |
| 36 | CASSYSFSSVGYEQFF | 6-2/6-3 | 2-1 | 0.043 | 0.018 |
| 37 | CASSLLGQGKDTGELFF | 7-6 | 2-2 | 0.043 | 0.042 |
| 38 | CAWRYRGGNTEAFF | 30 | 1-1 | 0.042 | 0.000 |
| 39 | CASTDNTYEQYF | 2 | 2-7 | 0.041 | 0.028 |
| 40 | CASSVPTESSIQYF | 19 | 2-4 | 0.041 | 0.000 |
| Total (%) | 10.272 | 12.234 | |||
Frequency of sequence relative to the total no. of productive sequences. Key: unshaded, <0.001; shaded, in regular font and not underlined, 0.001 to 0.1; shaded and italic, 0.1% to 1%; shaded and underlined, 1% to 5%; shaded and bold, >5%.
Bold and italics indicate sequence corresponding to EBV-specific clonotype identified previously (11).
The most frequent and persistent TCR β-chain in donor H02, which was encoded by the TRBV7-9 and TRBJ2-1 gene segments, comprised more than 5% of the total repertoire in both 2002 and 2011. Interestingly, this β-chain corresponds to a T cell clonotype that recognizes the EBV nuclear antigen 3C and was previously identified for this donor using conventional sequencing techniques (8) and which has a high affinity for its target EBV epitope (13). It is well known that human herpesviruses trigger large CD8+ memory T cell expansions which inflate with age (14–18). To investigate which of the TCR β-chain sequences identified by HTS in donor H01 were herpesvirus specific, we used LCL stimulation and TNF capture to sort EBV-specific T cells from the 2011 sample, which were then utilized in cloning and TCR Sanger sequencing. We identified 13 EBV-specific β-chains that corresponded to sequences from the HTS analysis of the total CD8+ subset (Table 4). These included three highly prevalent β-chains that were within the 40 most frequently observed sequences from the 1993 or 2011 HTS data (Table 2; see also Tables S2 and S3 in the supplemental material). It is notable that the TNF capture technique may have underestimated the proportion of TCR β-chains that were EBV specific because only functionally “sound” CD8+ T cells are detected, with the exclusion of T cells that fail to secrete TNF.
Table 4.
TCR β-chain sequences and longitudinal analysis of EBV-specific clonotypes within the CD8+ compartment of donor H01
| CDR3 sequence | TRBV gene(s) | TRBJ gene | Frequency rank by yra |
|||
|---|---|---|---|---|---|---|
| 1993 | 1997 | 2008 | 2011 | |||
| CASSEQDGFNYGYTF | 7-9 | 1-2 | 6 | 7 | 6 | 6 |
| CASSYLTADGNQPQHF | 6-2/6-3 | 1-5 | 30 | 61 | 23 | 47 |
| CASTARGNTGELFF | 6-1/6-5/6-6 | 2-2 | 39 | 37 | 14 | 10 |
| CAISDPPGGVDEQYF | 10-3 | 2-7 | 47 | 102 | 68 | 67 |
| CASTSSAGLDTQYF | 5-4 | 2-3 | 55 | 125 | 28 | 19 |
| CSAYNRGDEAYEQYF | 20-1 | 2-7 | 70 | 151 | 145 | 113 |
| CASNQGGADTQYF | 19 | 2-3 | 75 | 109 | 551 | 175 |
| CSGGVPNTGELFF | 29-1 | 2-2 | 1,446 | 614 | ND | 29,800 |
| CASSLWETQYF | 28 | 2-5 | 44,058 | 33,700 | ND | ND |
| CASSPEGSFEPQHF | 12-3/12-4 | 1-5 | 15,106 | 17,427 | 12,318 | 10,528 |
| CSARSGTDRIEQYF | 20-1 | 2-7 | ND | ND | 11,049 | 1,383 |
| CASSYGEGAWNEQFF | 6-2/6-3 | 2-1 | ND | ND | ND | 4,011 |
| CSARDAGQEYEQYF | 20-1 | 2-7 | ND | ND | ND | 28,921 |
Frequency rank of β-chain sequence among all sequences identified within the CD8+ subset using HTS. ND, not determined.
In summary, this study has investigated the evolution of the human T cell repertoire in peripheral blood and revealed that the high-frequency clonotypes can remain surprisingly stable over long periods of time. Furthermore, these high-frequency, ever-present clones occupy a considerable amount of immunological space and include several Epstein-Barr virus-specific expansions. Although our archived PBMC numbers were too limited for further sorting into smaller subsets, future studies aimed at characterizing the phenotype of the long-lived T cell clonotypes will be worth pursuing. It will also be important to investigate the mechanisms that control T cell longevity due to its potential importance in influencing the efficacy of T cell-based vaccines and adoptive therapies.
Supplementary Material
ACKNOWLEDGMENTS
J.J.M. is an NHMRC Career Development Fellow (APP1031652), and S.R.B. is an NHMRC Principal Research Fellow (APP1021452). This work was supported by an NHMRC program grant (APP389830).
Footnotes
Published ahead of print 17 October 2012
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.02180-12.
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