T-cell receptor repertoire of cytomegalovirus-specific cytotoxic T-cells after allogeneic stem cell transplantation

Cytomegalovirus (CMV) infection is a major complication during allogeneic stem cell transplantation (allo-SCT). However, mechanisms of adaptive immunity that drive this remain unclear. To define early immunological responses to CMV after transplantation, we using next-generation sequencing to examine the repertoire of T-cell receptors in CD8+/CMV pp65 tetramer+ cells (CMV-CTLs) in peripheral blood samples obtained from 16 allo-SCT recipients with HLA-A*24:02 at the time of CMV reactivation. In most patients, TCR beta repertoire of CMV-CTLs was highly skewed (median Inverse Simpson’s index: 1.595) and, 15 of 16 patients shared at least one TCR-beta clonotype with ≥ 2 patients. The shared TCRs were dominant in 12 patients and, two clonotypes were shared by about half of the patients. Similarity analysis showed that CDR3 sequences of shared TCRs were more similar than unshared TCRs. TCR beta repertoires of CMV-CTLs in 12 patients were also analyzed after 2–4 weeks to characterize the short-term dynamics of TCR repertoires. In ten patients, we observed persistence of prevailing clones. In the other two patients, TCR repertoires became more diverse, major clones declined, and new private clones subsequently emerged. These results provided the substantive clue to understand the immunological behavior against CMV reactivation after allo-SCT.


Results
T-cell receptor repertoire analysis of CMV-CTLs. The median number of sorted CD8 + /CMV pp65 tetramer + cells used for TCR repertoire analysis was 6666 cells/sample (range 1826-119,706 cells/sample, Fig. 1a) and the median number of assigned reads was 127,998 reads/sample (range 67,181-460,219, Supplementary Table S1). The diversity of TCR repertoire of CMV-CTLs at the time of CMV reactivation was quite low in most patients, and this was represented by a low median Inverse Simpson's index score of 1.595 (95% confidence interval 0.990-3.688) when compared to scores of previously published TCR diversity of bulk T-cells in peripheral blood (PB) of healthy individuals (729.7 ± 493.9, Fig. 1b) 37 .
Both TCR-alpha and TCR-beta were analyzed in 10 patients and their diversity showed good correlation (P < 0.001, Fig. 1c). No obvious association between the number of sorted cells and TCR diversity was observed (P = 0.86, Fig. 1d). The TCR-beta repertoire of the entire T-cell population was analyzed in seven patients (median Inverse Simpson's index: 24.7) and this diversity was significantly higher (P < 0.001) than that of CMV-CTLs in each patient (median Inverse Simpson's index: 1.43, Fig. 1e-g). After allo-SCT, the TCR-beta repertoire diversity of the whole T-cell population was lower than what has previously been reported for normal populations 38 .
As stated above, we analyzed the TCR repertoire of CMV-CTLs as well as of the entire T-cell population at CMV reactivation in 7 patients. In 3 patients among them, the TCR repertoire of CMV-CTLs and the entire T-cell population 2-4 weeks after CMV reactivation was also evaluated. In these 10 samples, we identified 30 clones which comprised more than 1% of the CMV-CTLs in each sample and, among them, 24 clones were also identified in the entire TCR repertoire in each sample. When we calculated the frequency of each CMV-specific clone in the entire T-cell population by multiplying the frequency of CMV-CTLs in entire T-cells and frequency of each clonotype in CMV-CTLs, calculated frequencies were generally comparable to the actual frequency of CMV-CTLs. Nevertheless, in some cases, the calculated frequency was much lower than the actual frequency (Supplementary Table S2).
We performed analyses to evaluate possible relationships, but found no significant associations between clinical parameters with the TCR repertoire diversity of CMV-CTLs (Fig. 2, Supplementary Table S1). There were no apparent differences in diversity and dominant clones between CMV-IgG positive and negative donors.
We also evaluated the combination of V and J family usages in CMV-CTLs (Fig. 3c) and entire T-cells (Fig. 3d). Although there seemed to be some discrepancy in V-J usage between CMV-CTLs and entire T-cells, no statistically significant difference was observed in the frequency of each V-J combination between CMV-CTLs and entire T-cells.
Shared TCRs and similarity analysis. We next focused on the frequency, V and J usage, and complementarity determining region 3 (CDR3) sequences of shared TCR-beta in CMV-CTLs, and 37 types of shared TCRs were identified (1.2% of the 2992 type unique reads). Interestingly, 15 of the 16 patients had at least one shared TCR (Supplementary Table S3), and shared clonotypes were dominant in 12 patients (Fig. 4a). With regard to TRBV and TRBJ usage, there was a substantial bias of usage, with 20 of the 37 shared TCR being TRBV7-3/ Scientific Reports | (2020) 10:22218 | https://doi.org/10.1038/s41598-020-79363-2 www.nature.com/scientificreports/ TRBJ1-1, and 6 of them being TRBV7-9/TRBJ2-3 (Fig. 4b). However, most of these TCRs were shared by only a few patients. Of note, we also identified two clonotypes, namely TRBV2/TRBJ2-6/CASNADASSGANVLTF and TRBV11-2/TRBJ2-5/CASSLVTSGPGETQYF, which were shared by about half of the 16 patients (detected in 8 and 9 patients respectively, Fig. 4c; Supplementary Table S1). Similarity analysis using MUSCLE algorithm and Jalview software showed that CDR3 sequences of shared TCRs had a higher degree of similarity compared to those of non-shared TCRs ( Fig. 4d- Clonal dynamics of TCR repertoire. When we sequentially analyzed TCR beta repertoires in 12 patients to evaluate clonal transition, shared clonotypes were consistently detected in 10 of 11 patients with shared TCR. Additionally, two patterns of subsequent clonal behavior were detected in the TCR repertoire of CMV-CTLs. In 10 patients, variants of clones persisted and TCR repertoires of CMV-CTLs remained oligoclonal ( Fig. 5a-c, Supplementary Fig. S1). Frequency of V-J pairs at each time point and transition of CMV-CTL clonotypes in case 15 are shown as an example of such cases (Fig. 5d,e). However, in the two other patients, TCR repertoires of CMV-CTLs became more diverse. The Inverse Simpson's index increased from the time of reactivation to 2-4 weeks after (1.129-10.90 in one patient and 2.583-23.69 in the other), major clones markedly decreased, and new private clones subsequently appeared (Fig. 5a-c, Supplementary Figure S1). V-J usage at each time point and transition of CMV-CTL clones in case 8 are shown as an example (Fig. 5f,g). A polyclonal pattern, or a diverse TCR repertoire in CMV-CTLs at the time of reactivation [n = 1] or after 2-4 weeks [n = 2] was detected in 3 of 16 patients. This pattern was observed exclusively in patients who were administered corticosteroid (prednisolone, 20-30 mg/day) upon CMV reactivation (42.9% vs. 0.0%, P = 0.063). The 3D figure of the TRBV and TRBJ combination also indicated that some CMV-specific TCRs identified at CMV reactivation disappeared   www.nature.com/scientificreports/

Discussion
In this study, we used unbiased NGS to characterize the TCR repertoire of CMV-CTLs in 16 patients of type HLA-A*24:02 who underwent allo-SCT and suffered from CMV infection. Just as Miyama et al. demonstrated that CMV-CTLs in healthy individuals were oligoclonal even after in vitro stimulation 8 , this study also showed similar results in vivo in 12 patients who had undergone allo-SCT. These results are also consistent with a previous report by Link et al. that evaluated the TCR repertoire of CMV-CTLs in four patients of type HLA-A*02:01 after allo-SCT 23 . In this study, a larger number of patients was analyzed and results indicated that shared TCRs were common within allo-SCT recipients with CMV reactivation. These data were consistent with data from the previous study which reported that repertoire overlap was significantly driven by CMV but otherwise remained markedly limited 39 . Although frequency of shared TCRs and the number that is shared can depend on cohort size and sampling depth 40 , the higher frequency of shared clonotypes and markedly similar CDR3 sequences observed suggested that they may play important roles. In this study, we constructed the first TCR repertoire database from CMV-CTLs that are specific for HLA-A*24:02. In VDJdb 41 , only 38 sequences of 4 patients from 2 studies 22,42 were registered as TCRs of HLA-A*24:02-specific anti-CMV pp65 tetramer-positive CTLs. Although our shared TCR sequences were not included in this database, it might be due to a lack of cases, and more data should be accumulated to clarify the characteristics and distribution of these sequences. Our results showed that several specific TCRs were consistently and exclusively detected after CMV reactivation, suggesting that these TCRs had a crucial role in controlling CMV infection. We compared the TCR repertoire of CMV-CTLs and entire T-cells, thereby demonstrating that the frequency of CMV-specific TCRs in entire T-cells were comparable to the frequency which was calculated from frequency of CMV-CTLs in entire T-cells and frequency of specific TCRs in CMV-CTLs in many cases. Additionally, in sequentially analyzed cases, various clones were consistently identified. While these results suggested the robustness of such TCRs and our analytical methods, there were discrepancies. The exact reason was unclear, www.nature.com/scientificreports/ but perhaps such clones were not activated well, and most of the clones with deviation had exceedingly low frequency. Therefore, coverage might be insufficient to detect such minor populations. The affinity of each TCR and CMV pp65 tetramer could also affect the results, as it was previously reported that affinity could influence the sorting efficiency 9,24 .
In this study, we observed an interesting change in TCR repertoire diversity and clonal shift of CMV-CTLs in some patients. However, the exact mechanisms and clinical significance of this phenomenon are unclear. Although the TCR repertoire generally remains stable 43,44 , a change of TCR repertoire diversity and clonal dynamics of antigen-specific T-cells have also been reported in some contexts, including allo-SCT settings 22,24 . For example, Poiret et al. analyzed the behavior of CMV-CTLs after allo-SCT and showed that the population of CMV-CTLs could change over time 24 ; similar results were also reported by Nakasone et al. 22 . Ramien et al. analyzed the TCR repertoire of patients with multiple sclerosis and reported that TCR clones became significantly more diverse with the amelioration of disease symptoms during pregnancy, with possible induction of immunotolerance in the expectant mothers 45 . Costa et al. reported that in HIV-1 patients, the diversity of the HIV-specific TCR repertoire could change and that changes in diversity of the antigen-specific repertoire and magnitude of CD8 + T-cell responses were inversely correlated 26 . Other reports have also suggested that dominant and prominent TCRs, rather than a diverse TCR repertoire, appeared to be clinically important in specific immunity 22,46 . Persistence of dominant TCRs might also be important as we observed that increases in TCR diversity were accompanied by decreases in dominant clones. Considering that all three patients in our study who had diverse TCR repertoires were systemically administered corticosteroid therapy, it is likely that severe immunosuppression may have hampered the stable proliferation of CMV-CTLs and may have thus facilitated clonal shift. Additionally, while previous studies have often focused on clonal dynamics over a longer period, this study showed that the composition of specific T-cell clones could undergo change in a short period of time 22 .
Some reports indicated clinical efficacy of cellular therapy for CMV diseases after allo-SCT [30][31][32] . However, although infused T-cells derived from stem cell donors were generally persistent at least for some years, in many cases, T-cells from TPD disappeared soon after infusion probably because they were rejected by the allograft derived from the stem cell donor 31,47 . The longevity of infused T-cells should be important for the control of CMV because late and/or recurrent CMV infection is common among allo-SCT recipients 48 . Considering that late CMV infection often occurs after allo-SCT 49,50 , administration of persistent antiviral T-cells may be superior to antiviral drugs as patients may be spared the side effects and costs. The exact reason underlying TCR longevity remains unclear, but it is reported that antigen exposure has a crucial role for T-cell expansion and persistence 51 ; therefore the reactivity of each TCR against the relevant epitope may be important, although other strategies such as repeated infusion, use of allo-SCT donor T-cells, post-transfer vaccination 52 , and application www.nature.com/scientificreports/ of chimeric antigen receptor-modified T-cells 53 may also lead to longevity of infused T-cells. Our results suggest that characterization of "elite" TCR clones is warranted for more sophisticated adoptive T-cell therapy. It is presumed that public TCRs detected in many individuals may have higher compatibility of variable patients, may exhibit lower immunogenicity, and can be a good candidate for adoptive T-cell therapy from TPD. We believe that these results provide a substantive clue to understand the immunological behavior against CMV reactivation after allo-SCT and that the selection of appropriate TCRs could be important for the development of more sophisticated cellular therapy against CMV. Our study has several limitations. Firstly, the functions of the TCRs were not evaluated in this study. As TCR repertoire analyses are purely quantitative analyses, identification of TCR alpha/beta pairs and a comprehensive qualitative evaluation is necessary for functional assessment. In light of previous reports that have indicated higher avidity and activity of shared TCRs in several contexts 8,9,25 , the shared clonotypes that were identified could have prominent immunological functions. Additionally, as indicated by Poiret et al. through the analysis of three different HLA-A*02:01-specific CMV tetramers, the affinity of CMV-CTLs may be a promising biomarker for evaluation of the function of identified TCRs 24 . Secondly, we could not evaluate whether CMV-specific TCR repertoire behavior after HLA-mismatched transplantation was different from HLA-matched transplantation because all patients in our study underwent HLA-A matched allo-SCT, as with previous studies 8,9,11,12 . Considering recent increase of haploidentical transplantation 54 , characterization of immune reconstitution after HLA-mismatched transplantation is warranted. Thirdly, as we only analyzed patients who suffered from CMV reactivation, the difference of TCR repertoire between those and patients who did not experience CMV reactivation was unclear. Nakasone et al. reported that an oligoclonal CMV-specific TCR repertoire was not a hallmark of reactivation 55 , and it seemed less important to identify clinically functional TCRs against CMV. We speculate that the absence of relevant TCRs in patients who suffer from repeated and protracted CMV infection/diseases, rather than patients who do not experience CMV reactivation, may be better circumstantial evidence for the clinical importance of such clones. However, unfortunately most of such patients were administered a high dose of corticosteroid and marked lymphopenia prevented us from reliable analyses because not enough RNA could be obtained for repertoire analysis after tetramer sorting. Perhaps this technical barrier may be overcome through single-cell or ultra-low-input sequencing. Finally, although CMV reactivation was reported to be associated with lower relapse risk especially in patients with AML 3,56,57 , and Yew et al. suggested the association between TCR repertoire after allo-SCT and relapse/graft-versus-host disease (GVHD) 18 , as only one patient with ALL suffered from relapse in this patient cohort, it was not possible to identify any associations between CMV reactivation, TCR repertoire, and relapse. Additionally, GVHD was not apparently associated with CMV-specific TCR repertoire diversity or the existence of specific clones in this study. This was probably due to the small sample size or timing of sample collection considering the fact that Buhler et al. reported that clonality one year after allo-SCT was not associated with risk of clinical events 39 . Future studies with more cases and/or sequential evaluation are therefore warranted.
In conclusion, the TCR repertoire of CMV-CTLs after allo-SCT is oligoclonal in most cases at the time of CMV reactivation, but may undergo short-term dynamic changes in a minority of patients. Shared TCRs were frequently detected in allo-SCT recipients and exhibited high sequence similarity. Functional tests are warranted to identify key TCRs that may guard against CMV.

Methods
Patients and transplant procedures. Sixteen patients with type HLA-A*24:02 who underwent allo-SCT at our hospital and experienced CMV reactivation were included in this study. One patient underwent a second allo-SCT. For all other patients, this was their first allo-SCT. Intensity of each conditioning regimen was classified, as previously defined 58 . Donors that had 8/8 allele matches for HLA A, B, Cw and DRB1 were considered to be HLA-matched. Calcineurin inhibitor and methotrexate (short-term) were used for prophylaxis of GVHD. Surveillance of CMV pp65 antigenemia was started from time of neutrophil engraftment, and was monitored weekly until ≥ 100 days after allo-SCT. CMV reactivation was defined as being positive for CMV antigenemia by C7-HRP testing. No patient received prophylactic therapy for CMV. Peripheral blood samples were collected either weekly or fortnightly from time of neutrophil engraftment until approximately 100 days after allo-SCT, and the samples collected immediately after CMV reactivation and 2-4 weeks later were subsequently analyzed.
Written informed consent was obtained from every patient. This study was performed in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Tokyo Metropolitan Cancer and Infectious Diseases Center, Komagome Hospital, Tokyo, Japan (number 1966).

Patient characteristics. Sixteen transplant recipients were included in this study conducted from March
to October 2018. Their clinical characteristics are summarized in Table 1 and Supplementary Table S1. The median age of the recipients was 50 years (range 20-71). Patients were being treated for acute myeloid leukemia (n = 7), acute lymphoblastic leukemia (n = 7), or myelodysplastic syndromes (n = 2). Eleven patients received bone marrow, three patients received PB stem cells, and two patients received unrelated cord blood transplantation. Two donors were related to recipients and 14 were unrelated. Eleven patients underwent a myeloablative conditioning regimen and five underwent reduced intensity conditioning. Of the 16 patients, 9 received grafts from a HLA-matched donor. Only one patient who received a bone marrow transplant from an unrelated, HLA-DRB1 mismatched donor, was prophylatically administered anti-thymocyte globulin (Thymoglobulin, 2.5 mg/ kg) for GVHD. All recipients were CMV IgG-positive and 11 donors were CMV-seropositive. There was no CMV reactivation/disease observed in any patient prior to allo-SCT. The first clinically significant CMV infection was observed within a median of 35 days (range 16-55) post-transplantation. The median percentage of sorted CD8 + /CMV pp65 tetramer + cells upon CMV reactivation was 0.63% (range 0.05-6.56). All patients were Scientific Reports | (2020) 10:22218 | https://doi.org/10.1038/s41598-020-79363-2 www.nature.com/scientificreports/ preemptively treated with ganciclovir, valganciclovir, or foscarnet, and no patient developed CMV disease. Nine patients suffered from acute GVHD (grade 1 in seven and grade 2 in two patients) and eleven developed chronic GVHD (mild in seven and moderate in four patients) after allo-SCT.
Flow cytometry analysis and cell sorting. Mononuclear cells were purified from whole PB (7 mL) by density gradient sedimentation using Ficoll-Paque PLUS (GE Healthcare Life Science, Marlborough, MA). Tetramer and anti-CD8 antibody staining was performed as previously described 8 . Briefly, peripheral blood mononuclear cells (PBMC) were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD8 (BD Bioscience, San Jose, CA) and phycoerythrin (PE)-conjugated HLA-A*24:02-specific anti-CMV pp65 tetramer (MBL, Nagoya, Japan) in accordance with manufacturer's instructions. FITC + /PE + PBMCs were considered CMV-CTLs, and were sorted using the FACSMelody (BD Biosciences, San Jose, CA, USA) cell sorter (Fig. 1a). Samples that were not stained for tetramer were used as negative controls for non-specific tetramer staining. We mention that 7-AAD or other staining solution to remove dead/damaged cells was not used because marked lymphocytopenia and a low FITC + /PE + fraction incentivized us to use a minimal amount of antibodies. Our preliminary experiments suggested that after gating lymphocytes and eliminating doublet cells using FSC-A/ FSC-H and SSC-A/SSC-H, CD8 + /7-AAD + cells were infrequent (data not shown).
TCR repertoire analysis. Semi-quantitative analysis of the TCR repertoire was performed using highthroughput NGS, as previously described 8  Mismatched donor 7

GVHD prophylaxis
Tacrolimus and short-term methotrexate 14 Cyclosporine and short-term methotrexate 2 www.nature.com/scientificreports/ restriction site. Double-stranded cDNA was synthesized with SuperScript III Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and blunt ends were created with T4 DNA polymerase (Invitrogen). P10EA/P20EA adaptors were ligated to the 5′ end of the cDNA, and cDNA was digested with NotI. After removal of adaptors and primers, a second PCR was performed using TRA-or TRB-constant region-specific and P20EA primers. Following the second PCR, a third PCR was performed using the same conditions with Illumina adaptors for constant region-specific and P20EA primers, and the products were analyzed by high-throughput sequencing using an MiSEQ platform (Illumina, San Diego, CA, USA). Sequences with low quality scores were discarded and only TCR clonotypes whose reads were more than 9 in each patient were analyzed. TCR repertoire was analyzed using bioinformatics software from Repertoire Genesis Incorporation (Ibaraki, Japan). As previously described, TCR clonotypes with the same TRBV/TRBJ gene segments and CDR3 amino acid sequences that were observed in ≥ 2 patients were considered to be shared 8 . Diversity of TCR repertoire was estimated using the Inverse Simpson's index (1/λ), which was calculated with the formula below:

T-cell depletion
where N represents the total number of sequence reads, n i is the number of the ith unique sequence read, and S is the species number of unique sequence reads 37 . A greater score indicates higher diversity. We randomly selected 30,000 reads per sample to standardize the number of TCRs in every sample, and random sampling was repeated 100 times 59 . Median values of the index were used for comparing the diversity of TCR repertoires. A TCR-beta repertoire was considered as polyclonal when 1/λ was higher than the 95% confidence interval of 1/λ of CMV-CTL TCR-beta at CMV reactivation. Multiple alignments of CDR3 sequences were performed using MUSCLE (multiple sequence comparison by log-expectation) 60 , and mutual similarity was analyzed with Jalview software 61 . Illustrations of consensus sequences were created with WebLogo 62 .

Statistical methods. Differences in numerical and categorical variables were compared using a t test and
Fisher's exact test, respectively. The level of statistical significance was set at P value < 0.05. Statistical analyses were performed using R (version 3.5.0; R Foundation for Statistical Computing, Vienna, Austria).

Code availability
Accession codes Sequence data for TCR repertoire analyses have been deposited in the GenBank/EMBL/DDBJ sequence read archive (SRA) under the accession code DRA010029.