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. Author manuscript; available in PMC: 2020 Oct 1.
Published in final edited form as: Br J Haematol. 2019 Jun 20;187(2):206–218. doi: 10.1111/bjh.16053

T-cell receptor sequencing demonstrates persistence of virus-specific T cells after antiviral immunotherapy

Michael D Keller 1,2,†, Sam Darko 3,†, Haili Lang 2, Amy Ransier 3, Christopher A Lazarski 2, Yunfei Wang 4, Patrick J Hanley 2,6, Blachy J Davila 6, Jennifer R Heimall 7, Richard F Ambinder 8, A John Barrett 9, Cliona M Rooney 5, Helen E Heslop 5, Daniel C Douek 3, Catherine M Bollard 2,6
PMCID: PMC6786907  NIHMSID: NIHMS1033531  PMID: 31219185

Abstract

Viral infections are a serious cause of morbidity and mortality following haematopoietic stem cell transplantation (HSCT). Adoptive cellular therapy with virus-specific T cells (VSTs) has been successful in preventing or treating targeted viruses in prior studies, but the composition of ex vivo expanded VST and the critical cell populations that mediate antiviral activity in vivo are not well defined. We utilized deep sequencing of the T-cell receptor beta chain (TCRB) in order to classify and track VST populations in 12 patients who received VSTs following HSCT to prevent or treat viral infections. TCRB sequencing was performed on sorted VST products and patient peripheral blood mononuclear cells samples. TCRB diversity was gauged using the Shannon entropy index, and repertoire similarity determined using the Morisita-Horn index. Similarity indices reflected an early change in TCRB diversity in eight patients, and TCRB clonotypes corresponding to targeted viral epitopes expanded in eight patients. TCRB repertoire diversity increased in nine patients, and correlated with cytomegalovirus (CMV) viral load following VST infusion (p=0.0071). These findings demonstrate that allogeneic VSTs can be tracked via TCRB sequencing, and suggests that T-cell receptor repertoire diversity may be critical for the control of CMV reactivation after HSCT.

Keywords: Haematopoietic stem cell transplantation, viral infection, adoptive immunotherapy, T-lymphocyte

INTRODUCTION

Viral infections are common and potentially life-threatening in patients with compromised T-cell immunity, such as those undergoing haematopoietic stem cell transplantation (HSCT) and individuals with moderate to severe primary immunodeficiency disorders.(Bollard and Heslop 2016, Heimall, et al 2017) Among HSCT recipients, reactivation infections with herpesviruses, such as cytomegalovirus (CMV) and Epstein–Barr virus (EBV), are leading causes of morbidity and account for up to a third of post-transplant mortality.(Cohen 2015, El Chaer, et al 2016) Antiviral medications are available for many of these infections, but are often limited by toxicities, such as marrow suppression and nephrotoxicity, and are also compromised by emerging viral resistance.(Sellar and Peggs 2012, Smith 2003) As the underlying cause of viral susceptibility is largely related to poor or absent T-cell immunity, adoptive immunotherapy with virus-specific T cells (VSTs) from healthy donors has been a logical treatment approach that has been successfully used in many prior phase I-II studies.(Blyth, et al 2013, Creidy, et al 2016, Gerdemann, et al 2013, Heslop, et al 2010, Koehne, et al 2015, Leen, et al 2013, Leen, et al 2009, Leen, et al 2006)

Clinical grade VSTs are generated either by cell selection or by ex vivo expansion, and have been utilized from haematopoietic stem cell donors or partially human leucocyte antigen (HLA)-matched, third party donors.(Gerdemann, et al 2012, Leen, et al 2013, Peggs, et al 2011, Uhlin, et al 2012) With either strategy, the incidence of graft-versus-host disease (GVHD) following VST infusion is dramatically lower than that observed following unmanipulated donor lymphocyte infusion.(Doubrovina, et al 2012) Up to 5 viruses have been simultaneously targeted with VSTs, with most studies targeting CMV, EBV and adenovirus.(Papadopoulou, et al 2014, Tzannou, et al 2017) In an early study using gene-marked VST from HSC donors, the infused cells were detectable by polymerase chain reaction (PCR) and persisted for up to 9 years following infusion.(Heslop, et al 2010)

Although VST have proven efficacy, the exact cell types within the VST product that persist in vivo and mediate antiviral activity are not known. How VST products contribute to immune reconstitution in recipients is also unknown.(Busch, et al 2016) Previous reports do not resolve these issues because they have only addressed overall specificity to an epitope by the entire cell population. In recent years, deep sequencing of the CDR3 region of the T-cell receptor beta chain (TCRB) has been utilized to track T-cell populations over time.(Chapuis, et al 2017, Chen, et al 2012, Hanley, et al 2015, Suessmuth, et al 2015, Yu, et al 2014) Given that the CDR3 region of TCRB is unique to each clonal T-cell population, it can be used to track T-cells of a given specificity as well as to measure T-cell population diversity over time.(Rempala and Seweryn 2013)

In this study, we report longitudinal TCRB sequencing of 12 HSCT recipients who received VST infusions post-transplant on two protocols. TCRB sequencing showed that adoptively transferred VST persist for at least 48 months post-infusion and clonotypes derived from the VST product contribute to the peripheral TCRB repertoire. Furthermore, we discovered that TCRB diversity inversely correlated with CMV viral load, suggesting that VST administration contributes to the qualitative recovery of the TCRB repertoire necessary for control of CMV reactivation.

METHODS

Patient Demographics:

We studied 12 patients from two studies which infused trivirus-specific T cells to patients at Texas Children’s Hospital and Houston Methodist Hospital between 2004 and 2008 (Leen, et al 2006) and at Children’s National Medical Center between 2014 and 2017 (Table I). Ten of these patients were described in previously published reports.(Leen, et al 2006, Naik, et al 2016) Subjects with available VSTs as well as pre/post-infusions samples for analysis were included. The infused VST product was available for study in 11 of 12 patients. The studies were approved by the US Food and Drug Administration and local Institutional Review Boards.

Table I:

Patient Clinical Descriptions and Outcomes

Patient Age (years)/ Gender Dx HSCT Donor GVHD Prophylaxis Viral Infections VST
Dose		 Response AE Outcome
1 56 / M AML MRD Alemtuzumab, FK506, MTX CMV 2×107/ m2 CMV-CR,EBV-PR None DLI (Day +247) due to decreasing chimerism.
2 7 / F ALL MUD Alemtuzumab, FK506, MTX EBV, Adv 2×107/ m2 Adv-CR,EBV-CR Grade I skin, mild LFT elevation A&W
3 3 / F Thalassemia MUD Alemtuzumab, FK506, MTX EBV, Adv 5×107/ m2 Adv-CR,EBV-CR None A&W
4 47 / F AML MUD Alemtuzumab, FK506, MTX none 1×108/m2 Free of CMV/EBV/Adv None Re-transplanted, A&W
5 15 / M AML MMRD/ CD34+ selected Alemtuzumab CMV 1×108/m2 CMV-NR None Died, CMV (Day +39)
6 9 / M SAA MMRD/ CD34+ selected Alemtuzumab CMV and EBV 1×108/m2 CMV-CR, EBV-PR None A&W
7 8 /F MDS MMRD Alemtuzumab, FK506, MTX EBV 2×107/ m2 EBV-CR None A&W
8 19 / M HL MUD Alemtuzumab none 1×107/m2 Free of CMV/EBV/Adv None A&W
9 17 months/ M WAS MUD ATG, Cytoxan, CSA, MTX EBV (post-infusion) 2×107/ m2 EBV-CR None A&W
10 5 months/ M SCID MMRD (maternal) MMF CMV 5×106/m2 × 2 doses CMV-PR None A&W
11 23 / F HL MMRD (paternal) Cytoxan CMV 5×106/m2 CMV-CR None Relapsed, underwent 2nd transplant followed by third-party VST, A&W
12 4 / F HLH MUD AlemtuzumabFK506 CMV, EBV, Adv (post-infusion) 1×107/m2 × 2 doses CMV-CR,EBV-CR, ADV-CR Grade I skin/gut, mild LFT elevation, cGVHD (skin) A&W
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A&W: alive and well; Adv: adenovirus; AE: adverse events; ALL: acute lymphoblastic leukaemia; AML: acute myeloid leukaemia; ATG: antithymocyte globulin; cGVHD: chronic graft-versus-host disease; CMV: cytomegalovirus; CR: complete response; CSA: ciclosporin; DLI: donor lymphocyte infusion; EBV: Epstein–Barr virus; F: female; HL: Hodgkin lymphoma; HLH: haemophagocytic lymphohistiocytosis; LFT: liver function tests; M: male; MDS: myelodysplastic syndrome; MMF: mycophenylate mofetil; MMRD: mismatched related donor; MRD: matched related donor; MTX: methotrexate; MUD: matched unrelated donor; NR: no response; PR: partial response; SAA: severe aplastic anaemia; VST: virus-specific T cells; WAS: Wiskott-Aldrich syndrome

Of 12 patients evaluated, 7 underwent HSCT for malignant disease, and 5 for non-malignant conditions (three for primary immunodeficiency, one for thalassaemia, and for severe aplastic anaemia). Median patient age was 8 years (range 5 months to 56 years). HSCT donors included matched related donors (n=1), matched unrelated donors (n=6), and mismatched related donors (n=5). Ten of 12 patients received anti-T cell serotherapy (antithymocyte globulin [ATG] or alemtuzimab) as GVHD prophylaxis. Subjects were followed for up to four years after infusion.

VST Production and Characterization

VSTs were generated from stem cell donors using one of two previously described methods.(Gerdemann, et al 2012, Leen, et al 2006) For Patients 1–8, VSTs were generated by stimulation of peripheral blood mononuclear cells (PBMC) with donor-derived lymphoblastoid cell lines that were transduced with an adenoviral vector expressing CMV-pp65 (Ad5f35pp65). T cells were cultured and re-stimulated with irradiated, Ad5f35pp65-transduced EBV-lymphoblastoid cell lines derived from the donor. Following 3–4 stimulations, VSTs were frozen. For Patients 9–12, VST were generated via a rapid expansion protocol in which PBMC were stimulated with overlapping 15-mer peptide pools encompassing viral antigens from CMV (pp65 and IE1), EBV (EBNA1 and LMP2), and adenovirus (Hexon and Penton) (JPT Peptide Technologies, Berlin, Germany). Following 10–12 days of culture, VSTs were frozen. VST characterization, including immunophenotyping, identification via HLA typing, and sterility testing, were performed as previously described.(Leen, et al 2006)

Infusion and Clinical Monitoring

Subjects received VST doses in inpatient or outpatient settings and were monitored for infusion-related reactions. VST doses ranged between 5×106 and 1×108 VST/m2, and were given at a median time of 76 days post-HSCT (range 39 to 289).

Subjects underwent monitoring of blood counts and chemistry weekly, as well as standard clinical assessments for acute and chronic GVHD at regular intervals for up to four years post-infusion. Viral loads were monitored in blood and other relevant body fluids by PCR.(Boeckh and Ljungman 2009) In patients with viral infection or reactivation at the time of infusion, responses were defined as complete if the viraemia fully resolved, partial if there was a sustained >50% decrease in viral load, and non-response if there was a <50% decrease in viral load or a greater but transient decrease.

Pentamer and Functional Cell Sorting

Pentamer staining of class I-restricted viral epitopes for VST products was performed as previously described (Leen, et al 2006) using available pentamers for CMV, EBV and adenoviral class I epitopes corresponding to the patient’s HLA class I alleles (Proimmune, Sarasota, FL, USA). Briefly, 1×106 VSTs were incubated with unlabelled pentamers at 2 μl for 15 min at room temperature, washed, and then stained with an Pro5 APC fluorotag as well as other surface antibodies for CD3, CD19, and CD8 (Miltenyi Biotec, San Diego, CA, USA).

Functional flow cytometry using CD107A expression was performed as follows: VSTs were thawed and rested overnight in medium containing 45% RPMI, 45% Click’s medium, 10% fetal calf serum, and interleukin 2 at 50 u/ml, and were stimulated with 1 μg of CMV viral pepmixes (pp65 and IE1) in the presence of Brefeldin A (GolgiPlug, BD, San Diego, CA, USA). Actin pepmix and Staphylococcal enterotoxin B (Fisher Scientific, Hampton, NC, USA) were utilized as controls. CD107A-APC antibody (BioLegend, San Diego, CA, USA) was added to the culture immediately after stimulation. After four hours, samples were harvested, co-stained with other surface markers (CD3, CD4, CD8) and analysed (Figure S1). Stained samples were run on a MACSQuant flow cytometer (Miltenyi Biotec). Pentamer+/CD8+/CD3+ populations and CD107A+/CD3+/CD8+ populations were sorted using an Influx cell sorter (BD).

TCRB Library Preparation for Sequencing

RNA extraction, cDNA production and TRB mRNA amplification were performed as previously described.(Gros, et al 2014) Briefly, sorted VSTs or PBMC from subjects were pelleted and suspended in RNAzol. RNA isolation was performed via water and isopropanol extraction followed by washing in 70% ethanol. RNA Index Number was determined using an Agilent 2200 Tapestation (Agilent, Santa Clara, CA, USA) (Table SI). cDNA synthesis was performed using Invitrogen Superscript II reverse transcriptase with a synthesized 5’ CDS Oligo dT primer and SMARTER II-A primer. The cDNA was cleaned with a modified version of the Agencourt AMPure XP kit (Beckman Coulter, Brea, CA, USA). The CDR3 region of TRB was amplified by using the KAPA Real Time Library Amplification kit (Kapa Biosystems, Woburn, MA, USA) with TRB constant primers (Figure S2). Each cDNA library underwent a second PCR for addition of the P5 and P7 Illumina adaptors (Illumina, San Diego, CA, USA). The PCR product was cleaned by using the Agencourt AMPure XP kit. The libraries were quantified with the Kapa Library quantification kit (Illumina).

TRB Sequencing

The libraries were clustered according to protocol, and sequencing was performed in an Illumina HiSeq instrument using a modified protocol for 150 paired end reads. PhiX library was utilized as a control with pooled samples. The minimum coverage of the sampled populations was approximately 10 reads per input cell.

Sequencing Pipeline and Statistical Analysis

TRB annotation was performed by combining a custom Java program written in house and the National Center for Biotechnology Information’s BLAST1 program. Briefly, BLAST1 was used to identify the V and J germline genes of a TCRB read. The CDR3 was then determined by finding the conserved cysteine at the 5’ end of the CDR3 and the conserved phenylalanine at the 3’ end of the CDR3. Unique TCRB species, in which species is defined as a unique TRB V–CDR3 (nucleotide)–J combination, were then collapsed to determine the count for each species. For each unique species, the number of nucleotides within the CDR3 contributed by the germline V, J, and D genes was calculated, as was the number of nucleotide additions. The numbers of nucleotide additions were determined by taking the length of the CDR3 nucleotides and subtracting the number of nucleotides encoded by the V, J, and D genes. The germline index was calculated by dividing the number of nucleotides in the CDR3 encoded by V, J, and D genes by the length of the CDR3, producing a value of between 0.0 and 1.0.

In an effort to identify potential sequencing errors, TCRB species represented by a single sequencing read (i.e., having a count of 1) were discarded. Additionally, the coverage was calculated for each sequenced sample in which coverage was defined as follows: (Total count of annotated reads/Cell count used for library generation).

TCRB species represented by fewer reads than half of the coverage were then discarded so that if a TCRB species is represented by 4 reads and the coverage is 103, that TCRB species will be discarded. PCR amplification error was addressed by identifying species in which the same V and J genes are used and the CDR3 is of the same length but the CDR3s vary by only 1 nucleotide. The count of the species pair was then probed, and if the count of species A was found to be less than 5% of the count of species B, then species A would be discarded as the likely product of PCR amplification error. Replicates from three separate CD81 T-cell memory populations from 2 different healthy donors were compared to determine the variability of sequences from sample run to sample run. Clonotype frequencies correlated with a P value of less than 0.0001 and an r2 median value of 0.85 (SD, 0.19). Shannon entropy, species richness, and species evenness were calculated for each TCRB repertoire by using the R package Vegan.(Rempala and Seweryn 2013) Shannon entropy and species richness were normalized by calculating the maximum Shannon entropy and maximum species richness for each repertoire based on the cell count used for library generation. The calculated Shannon entropy and species richness were then divided by their respective maximums to return a number between 0.0 and 1.0. To compare overlap between samples, the Morisita-Horn index was utilized.(Magurran 2005)

For each repertoire, the average and SD were calculated for germline index, CDR3 length, and number of nucleotide additions. This was done twice. In one scenario, all TCRB species were given the same weight (unique sequence analysis), and in the other scenario the relative frequency of each clone within a repertoire was considered (total sequence analysis).

Custom Perl scripts were used to calculate the distribution of CDR3 length, V-J pairing percentage, and amino acid compositions of each CDR3 position, as well as whole CDR3s from all of the annotated TRB sequence reads in each subject and group. Each TCRB percentage of each subject was used for clonally expanded analysis. For sorted T-cell populations, clonotype frequencies that were less prevalent in the sorted samples than in the bulk T-cell populations were discarded as non-specific in absence of purification. The clonotypes with higher frequency in the sorted T-cell populations than in the bulk population were designated as virus-specific for the given sorting condition. Tracking of virus-associated clonotypes in patient cells was performed by comparing the frequencies of the identified clonotypes in the peripheral blood of the patients over time.

All of the figures and various statistical analyses were performed with GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA), SAS 9.4 (SAS Institute, Cary, NC, USA) or computing environment R software; additional packages (reshape, ggplot2, and pheatmap) were taken from the Comprehensive R Archive Network (http://cran.r-project.org). All of the TRB sequence reads from each subject in the same group were taken into account when comparing nucleotide addition in V(D)J junctional regions, CDR3 length, and germline index among groups. Analysis of Shannon entropy indices versus CMV viral loads was performed as follows: subjects were dichotomized into two groups: low risk (CMV=0), and high risk (CMV>0). A logistical regression model was used to predict whether CMV log >0 or CMV log=0 using Shannon entropy index and time as predictors.

RESULTS

Safety of VST Infusions

None of the 12 patients had any immediate reactions to VST infusion. Two patients had new or recurring acute GVHD in the 45 days following VST infusion, all of which was grade I skin and/or gut disease. One patient developed grade I chronic GVHD of the skin, controlled with calcineurin inhibitors and topical steroids. Three patients developed transient transaminase elevation (grade I in Patients 2 and 9, grade III in Patient 12).

Antiviral Efficacy

Ten of 12 patients had one or more active viral infections at the time of VST infusion, and two patients were treated prophylactically and remained virus-free. Patients 1 and 6 were treated for CMV and had subsequent reactivations of EBV, which were transient and did not require treatment. Of the 10 patients with active viral infection at the time of infusion, six had early decrease in viral load, while another three had >50% decrease in viral load with eventual clearance. Patient 12 had a late adenovirus infection at day +179 following the first infusion, which resolved 23 days after a second VST infusion. Median time to 100-fold reduction of viral load was 56 days (Figure S3). Responses to individual viruses were 85% to CMV (n=5/6), 88% to EBV (n=7/8), and three of three patients with adenovirus. Patient 5 died of progressive CMV, while Patient 11 had a relapse of her underlying malignancy requiring a second bone marrow transplant. Overall survival at 1 year was 92%.

Virus-specific clonotypes demonstrate expansion and persistence

VST products were polyclonal based on analysis of TRBV and TRBJ gene segment usage (median Shannon entropy index 0.659), while multimer-sorted VST samples showed a narrower number of clonotypes (median 97 clonotypes) and TCRB diversity (median Shannon entropy index 0.413, p=0.0009), as did CD107A-sorted VST samples (median 124 clonotypes, median Shannon entropy index 0.419, p=0.0105, Figure 1A–C).

Figure 1: TRB gene segment usage demonstrates polyclonality of VST products.

Figure 1:

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A. T-cell receptor diversity of bulk virus-specific T cell (VST) products following 10-day ex vivo expansion demonstrates greater polyclonality (median Shannon entropy index 0.659), compared with Multimer-sorted VST samples corresponding to immunodominant cytomegalovirus (CMV) epitopes (median Shannon entropy index 0.413, * p=0.0009) or CD107A-sorted VST following CMV pepmix restimulation (median Shannon entropy index 0.419, ** p=0.0105). B. Bulk VST products generally contained more unique T-cell receptor beta chain (TCRB) clonotypes (median 1477) in comparison to multimer sorted VST samples (median 97 clonotypes, not significant [ns]) or CD107A-sorted VST samples (median 124 clonotypes, ns). Lines/ranges: median/95% confidence interval. C. TRBV and TRBJ gene segment usage of the bulk product from Patient 8 (P8) versus multimer-sorted cells based on binding of a CMV-pp65/YSE pentamer. Colour coding denotes frequency of gene segment usage within the overall population.

Following VST infusion, the peripheral TCRB repertoire in 8 patients showed an expansion of CMV/EBV-specific clonotypes that were present in the administered VSTs within three months of infusion (Figure 2). The total proportion of the repertoire corresponding to CMV/EBV-specific clonotypes differed between individuals at early time points (median 0.47% at 1–3 months, range 0.057–55.1%, Figure 2). CMV/EBV epitopes corresponded to a range of clonotypes, with several individual clonotypes that were detectable at high frequencies at timepoints following infusion (median 0.630%, range 0–4.73%, Table SII) Seven of the eight patients with detectable expansion of CMV/EBV-specific clonotypes achieved complete responses against the targeted virus. In responding patients, CMV/EBV-specific clonotypes represented a median of 0.03% of the peripheral TCRB repertoire at six months (range 0.003 – 3.7%), 0.07% at 12 months (range 0 – 7.25%, Figure S4), and remained detectable at four years post-infusion in Patient 1. Patient 8 was treated prophylactically, and received VSTs that had CD8+ T-cells corresponding to the adenovirus TYF epitope that is HLA-A24:02 restricted. There was no detectable expansion of the Adv-associated clonotypes in the 13 months after infusion (data not shown), but the subject also remained free of adenoviral infection. Patient 10, who received a maternal non-conditioned transplant for severe combined immunodeficiency disease (SCID), had a partial antiviral response to CMV, and showed a high clonotype frequency corresponding to a CMV-pp65 TPR epitope that is restricted through HLA-B07:02. This was present at a high level prior to T-cell infusion, and gradually decreased over time.

Figure 2: Virus-associated TCRB clonotypes expand longitudinally in the majority of responding patients.

Figure 2:

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Frequency of virus-associated T-cell receptor beta chain (TCRB) clonotypes in patient peripheral blood over time demonstrated expansion of clonotypes associated with cytomegalovirus/Epstein–Barr virus (CMV/EBV) epitopes in 8 of 11 evaluable patients within three months of virus-specific T cells (VST) infusion. Virus-associated clonotypes were detectable for up to four years post-infusion in Patient 1 (P1). Patient 5 (P5) had no antiviral response in spite of detectable expansion of clonotypes corresponding to the CMV-pp65/NLV epitope. Patient 10 (P10) had a high TCRB clonotype frequency corresponding to CMV-pp65/TPR prior to VST infusion, probably representing T cells from the maternal graft.

Patient 5 demonstrated a subtle expansion of CMV-specific clonotypes at 4 weeks post-infusion (0.28%), but had progressive, fatal CMV pneumonitis. Pentamer staining from peripheral blood at 3 weeks post-infusion showed a large population of T cells recognizing a CMV-pp65 NLV epitope, restricted through HLA-A02:01 (Figure 3). Sequencing of CMV isolate from patient plasma showed no mutations in the NLV epitope of the pp65 protein (Figure S5).

Figure 3: CMV-pp65/NLV-specific T cells are detectable in a non-responding patient.

Figure 3:

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Multimer sorting of the virus-specific T cell (VST) product and peripheral blood in Patient 5 (P5) demonstrated the presence of cytomegalovirus (CMV)-specific CD8+ T cells recognizing the HLA-A02:01 restricted CMV-pp65/NLV epitope, which represented a third of CD8+ T cells in the VST product and 2.96% of CD8+ T cells in the peripheral blood of the patient at three weeks post-infusion.

Absolute CD8+ clonotype numbers were available only for a subset of patient timepoints, and levels varied significantly at early time points (Figure S6). No overlap in virus-specific clonotypes was seen between different individuals, despite overlap in HLA alleles between many patients (data not shown).

TCRB repertoires resemble CMV/EBV-specific subsets of VST products after infusion.

TCRB repertoire from the pre-infusion samples shared few to no clonotypes with the infused VST products in all patients (median Morisita-Horn index: 0.9996, range: 0.9866 to 1). In 8 of 11 evaluable patients (Patients 1, 2, 3, 5, 6, 7, 9, and 11), there was increasing similarity between the TCRB repertoire of the peripheral blood and the CMV/EBV-associated clonotypes from the utilized VST products within 3 months following infusion (median Morisita-Horn Index 0.9922 at 1–3 month; range 0.6603 to 1). In 5 of 9 patients who had complete responses (Patients 1, 2, 6, 9 and 11), there was a trend toward greater similarity with the virus-associated clonotypes (as determined by pentamer or CD107A sorting) than with the bulk VST population at early time points (mean Morisita-Horn Index 0.912 and 0.992 (respectively) at 1 month post-infusion; Figure 4), though differences were non-significant (p≥0.14). Patient 10 showed a high degree of similarity between the pre-infusion repertoire and the virus-specific clonotypes, which reflects a high prevalence of CMV-specific clonotypes in the circulation before therapy. Patient 5 demonstrated over a log-fold greater similarity to the bulk VST clonotypes than CMV-pp65 NLV-specific clonotypes at 4 weeks, suggesting that other unidentified T-cells had persisted, in spite of the presence of NLV-specific T-cells by pentamer staining.

Figure 4: TCRB repertoires show similarity to CMV/EBV-associated clonotypes.

Figure 4:

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Similarity indices of the T-cell receptor beta chain (TCRB) repertoire comparing patient peripheral blood mononuclear cells with sorted or unsorted virus-specific T cell (VST) populations over time showed early repertoire changes in 8 of 11 evaluated patients. Five of nine responding patients (Patients 1, 2, 6, 9 and 11) displayed increasing similarity to the CMV/EBV pentamer-associated clonotypes after infusion, and Patient 8 had similarity to adenovirus pentamer-associated clonotypes at later timepoints. MH: Morisita-Horn Index.

TCRB diversity inversely correlates with CMV viral load.

Results of TRB sequencing from pre-infusion time points demonstrated oligoclonal TCRB repertoires in seven of 12 patients (Patients 1, 4, 5, 7, 8, 10 and 12) at the time of infusion (Figure S7). Following VST infusion, the overall repertoire diversity increased in nine of 12 patients as determined by the normalized Shannon entropy index, with a mean increase of 48.1% between the pre-infusion and final values (range −28.9 to 190.4%, Figure 5A). Patient 10 had partial response to CMV, and showed prolonged oligoclonality of the TCRB repertoire and a decrease over time in Shannon entropy index. Patient 12 had a decrease in TRB diversity corresponding to treatment with corticosteroids for GVHD. For the 10 patients treated for active viral infections, normalized Shannon entropy index was predictive of having a CMV load > log 3 versus those with associated with lower or undetectable CMV viral loads (Figure 5B, Table SIII, p=0.0071), whereas time did not correlate with CMV status. No statistically significant trend was noted for EBV.

Figure 5: TCRB repertoire diversity increases in most responding patients and correlates with CMV control.

Figure 5:

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. A. Normalized Shannon entropy index of the T-cell receptor beta chain (TCRB) repertoire increased over time in 9 of 12 evaluable patients, including 8 of 10 patients with complete responses. Patient 10 had a partial antiviral response to cytomegalovirus (CMV) with decreasing Shannon entropy index over time, and Patient 12 had a decrease in Shannon entropy index corresponding to corticosteroid treatment for GVHD. B. Shannon entropy index of high risk (CMV log>0, red) time points differed from low risk (CMV log 0, blue) time points in patients following virus-specific T cell (VST) infusion based on a logistical regression model (p=0.0071).

DISCUSSION

Adoptive immunotherapy with VSTs has been used with increasing success in many trials over the past two decades for prevention or treatment of viral reactivation post-stem cell transplantation.(O’Reilly, et al 2016, Saglio, et al 2014) In gene marking studies, long-term persistence of donor-derived VSTs has been previously demonstrated, indicating that they contribute to the T-cell receptor (TCR) repertoire long after transplantation. However, the precise viral-specific T-cell clones that mediate clinical responses are not well defined. Here, we demonstrate that ex vivo expanded VSTs are polyclonal based on their TRB diversity, but adoptively transferred clonotypes corresponding to immunodominant CMV/EBV epitopes tend to predominate in patients receiving VST infusion for treatment of CMV and/or EBV after HSCT. This probably reflects T-cell stimulation from viral epitopes persisting during latency or reactivation.

Our results demonstrate a spectrum of TRB sequences associated with immunodominant CMV and EBV epitopes, and further showed that these clonotypes can be tracked longitudinally in vivo for long periods. Prior studies have similarly tied specific CDR3 sequences to epitopes on various pathogens.(Glanville, et al 2017, Iglesias, et al 2011, Klinger, et al 2013) Curiously, none of the epitope-specific clonotypes were shared between individuals in this study, despite the presence of shared MHC alleles. In comparing the subjects’ clonotypes to a public database (VDJdb, http://vdjdb.cdr3.net),(Shugay, et al 2017) only a single clonotype (CASSDNYGYTF) was identified as a previously-described, public clonotype. Given the low number of patients sequenced, our findings do not rule out the existence of public clonotypes, and differing methodologies of TRB sequencing may have limited our ability to detect these sequences, as we were unable to detect similar public clonotypes within our own laboratory that were sequenced using different techniques.(Hanley, et al 2015) Prior studies have demonstrated the existence of public CDR3 motifs targeting viral antigens in CMV, EBV and human immunodeficiency virus (HIV) (Benati, et al 2016, Lim, et al 2000, Trautmann, et al 2005).

Suessmuth et al (2015) demonstrated that CMV reactivation following HSCT has an adverse impact on the reconstitution of the peripheral TCRB repertoire by skewing dominant clonotypes and eliminating minor clonal populations. In our study, we found that T-cell diversity correlated with control of CMV post-VST infusion. Most patients experienced improvement in their TCRB repertoire diversity after infusion, suggesting that successful VST therapy may improve T-cell reconstitution. In patients with poorly controlled CMV (Patients 5 and 10), overall T-cell diversity was lower than in patients with complete responses. It has been previously demonstrated that CMV infects thymic epithelial cells.(Numazaki, et al 1989) If virus-mediated damage to the thymus is the underlying mechanism of repertoire skewing, then it is possible that earlier CMV control may avert deleterious effects on repertoire recovery.

Two of the 12 patients evaluated by TRB sequencing failed to rapidly respond to VST infusion. In Patient 5, progressive CMV pneumonitis occurred in spite of detectable VST expansion. No escape mutations in CMV-pp65 were detected in the peripheral blood. CMV displays a phenomenon known as compartmentalization, in which organ-specific viral isolates differ in mutation patterns,(Renzette, et al 2013) and it is possible that the patient may have had CMV mutations restricted to the lungs which were not detected in the peripheral blood. Unfortunately, other tissue samples were unavailable for testing. Patient 10 had prolonged viraemia after VST therapy, which eventually cleared, but had poor TCR repertoire diversity 1-year post-infusion. TCRB sequencing showed the presence of CMV-specific T cells in the peripheral blood prior to VST infusion, which probably represent T cells transferred with the unconditioned maternal graft. In this setting, T-cell exhaustion or viral epitope escape may have accounted for delayed CMV viral control following HSCT and subsequent maternal VST infusion. Further study of antigen-specificity and function of transplacentally-transferred lymphocytes, as well as longitudinal viral sequencing, would be worthwhile to identify factors contributing to viral persistence in SCID and other severe forms of primary immunodeficiency.

Limitations of this study include the low number of patients evaluated and the heterogenous nature of their clinical courses in terms of viral infections/reactivation. They were also heterogenous in terms of HLA types, which prevents any conclusions regarding the hierarchy of antiviral restrictions in clinical responders. However, eight of the evaluated patients had expansion of CMV/EBV-specific clonotypes within three months of infusion. TRB sequencing also cannot distinguish whether detected clonotypes originate from infused VSTs or develop from the new donor marrow graft in the recipient. Given that 10 of 12 subjects who were sequenced received either alemtuzumab or ATG, and VSTs were given at a median of 76 days post-HSCT, it is very likely that the detected virus-specific clonotypes expanded from the infused VSTs rather than the new marrow graft.

In summary, we confirmed, by TCRB clonotype tracking, long term persistence of adoptively transferred VST cells, and showed preliminary evidence that T-cell diversity appears to correlate with CMV viral load post-HSCT. The identification of CDR3 sequences corresponding to highly conserved viral epitopes could enable future studies utilizing recombinant paired α/β TCR libraries to imbue epitope specificity on a T-cell population. Larger studies using TRB sequencing of multiple T-cell subsets will be needed to determine whether specific clonotypes, or patterns thereof, are critical for viral control in immunocompromised patients.

Supplementary Material

Supp TableS1-3
Supp figS1-7

Acknowledgements:

We thank our patients and their families for participation in this study, as well as the staff of the Center for Cell and Gene Therapy, the Vaccine Research Center and the Center for Cancer and Immunology Research. This work was supported by funding from the National Institutes of Health (U10HL108945–05 to HH/CB, an NIH Bench-to-Bedside award to HH/DD/AB/CB, K23-HL136783–01 to MDK), the Children’s Cancer Foundation, and the Jeffrey Modell Foundation. was supported by the NHLBI PACT grant.

Footnotes

Trial Registration:

Conflicts of Interests Disclosure: MDK, SD, HL, AR, CAL, YW, BJD, JH, RA, AJB, CR, DD have no relevant financial conflicts of interest to disclose. HEH is a founder of Viracyte and Marker Therapeutics and has received research support from Cell Medica and Tessa Therapeutics. CMB is a founder of Mana Therapeutics and received research support from Cellectis and NexImmune. PJH is a founder of Mana Therapeutics.

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Supplementary Materials

Supp TableS1-3
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Supp figS1-7
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