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lowing allogeneic SCT.8 For example, assessing the recov- ery of the TRb repertoire following allogeneic transplanta- tion revealed a restricted TCR repertoire diversity in patients affected by viral infections.9 We have previously shown that the TRα repertoire becomes oligoclonal and is dominated by a few expanded clonotypes following cytomegalovirus (CMV) infections in transplanted patients.10 Furthermore, GvHD and the relapse of acute myeloid leukemia (AML) correlate with lower TRb diver- sity,11 and the expansion of individual TCR clonotypes was observed in GvHD patients in selected studies.12 Formation of the TCR repertoire starts within months of transplantation, resulting in a more donor-like repertoire after the first year following transplantation.13
T-cell depletion were recruited (SIB-noATG), and samples from the 5 patient donors were analyzed in parallel. Acute GvHD (aGvHD) was defined as GvHD diagnosed within the first 100 days following SCT. In contrast, chronic GvHD (cGvHD) was diagnosed in cases with GvHD after the first 100 days or the typ- ical clinical presentation of cGvHD features.16 CMV reactivation was determined by detection of CMV virus load in the peripheral blood.
Immunophenotyping by flow cytometry
Routine assessment of differential blood counts was used to define the engraftment of neutrophil leukocytes and reconstitu- tion of whole lymphocytes. Samples for immunophenotyping were taken on day 60, day 120 and day 180 following transplan- tation. Twenty healthy stem cell donors were analyzed to define normal ranges (controls).
CD4+ and CD8+ T cells were characterized according to the expression of CCR7 and CD45RA as naïve (CCR7+CD45RA+), central memory (CM, CCR7+CD45RA-), effector memory (EM, CCR7-CD45RA-), and terminally differentiated effector memory (TEMRA, CCR7- CD45RA+) T cells.17 Staining was performed using the following antibodies as previously described:18 CD45- V500, CD3-PerCP-Cy5.5, CD8-APCH7, CCR7-FITC, CD45RA- PE (all BD Biosciences, San Jose, CA, USA) and CD4-eFluor450 (eBioscience, San Diego, CA, USA) (Online Supplementary Figure S1). Immunophenotyping was performed using a BD FACSCanto II (BD Bioscience, San Jose, CA, USA).
Sorting for T-cell receptor-α sequencing
For TRα sequencing, naïve and memory cells were sorted from
CD45+CD3+CD8+ T cells as previously described18 on day 60 and day 180 following transplantation. Sorted memory cells contained all memory subsets (CM, EM, TEMRA). Both T-cell subsets were sorted to a purity of more than 99% as checked by flow cytome- try after sorting. For naïve CD8+ T cells and memory CD8+ T cells, a maximum of 1,000,000 cells were sorted, with a mean of 133,590 and 808,726 sorted cells for naïve and memory CD8+ T cells, respectively.
Library preparation for T-cell receptor-α sequencing Library preparation was performed as previously described.10,18,19 The final PCR product contained the nucleotide sequence for the variable region of the TRα (V and J segments),
including the complementary determining region (CDR3).
Next-generation sequencing
Next-generation sequencing was performed using an Illumina HiSeq 2500 (Illumina, San Diego, CA, USA), generating 150-base- pair reads. Extraction of CDR3 sequences was performed using MiTCR as previously described.19,20 MiTCR used error correction with the highest stringency. Sequencing was performed, calculat- ing 20 reads per cell individually for each sample for normaliza- tion. Samples that failed quality control were excluded from the analyses and indexed as “not available”. TRα chains with identical CDR3 region amino acid (AA) sequences were defined as clono- types. Clonotypes with a TRα read frequency of more than 1% of reads were defined as “dominant” and more than 10% as “highly dominant” clonotypes. Sequence data are available at VDJServer under project UUID 3544765015263285736-242ac11d-0001-012 (https://vdjserver.org).
Statistical analysis
Data analysis was performed using GraphPad Prism (v.5.01, La Jolla, CA, USA), KNIME 2.5.2 and R (v.2.15.2 and Studio v.0.98.945, Boston, MA, USA). Immunophenotyping results were
Little is known about how TCR changes are influenced by the choice of transplant regimen, especially by differ- ent forms of T-cell depletion. One study revealed lower TRb diversity in patients receiving an in vitro T-cell-deplet- ed stem cell graft than in patients who received a non-T- cell-depleted cord blood graft.9 GvHD prophylaxis using post-transplantation cyclophosphamide (PTCy) on day +3 following SCT is an established therapeutic option in patients receiving haploidentical transplantation.14 Furthermore, the application of antithymocyte globulin (ATG) prior to transplantation as part of the conditioning therapy has become a common procedure to prevent GvHD, especially in patients with a mismatched donor.15 However, there have been no studies comparing these dif- ferent regimens of in vivo T-cell depletion (ATG or PTCy) and their impact on the TCR repertoire.
Here we analyzed the TRα repertoire of naïve and memory CD8+ T cells in 25 patients following different forms of allogeneic transplantation. This study addressed the question of whether the recipient TRα repertoire is influenced by anti-T-cell therapies such as ATG or PTCy, and if there are differences in response to haploidentical transplantation between fully matched or mismatched donor transplants. Furthermore, we analyzed to what extent the donor TRα repertoire is transferred to the recip- ient. Finally, the correlation between TRα repertoire diver- sity and the clinical manifestation of GvHD or CMV reac- tivation were addressed.
Methods
Patients
Patients (n=25) and donors were recruited after obtaining writ- ten informed consent and the approval of the local ethical review board (EK-279072013). To qualify, patients needed to have received their first SCT for an underlying hematologic malignancy. Patients who suffered a relapse during the observation period were excluded from the study. All SCTs were performed at Dresden University Hospital. Patients' characteristics are shown in Table 1. Patients were stratified into different groups according to their SCT protocol. In the first group, 5 patients received matched unrelated donor transplants and ATG (UD-ATG) as an addition to conditioning chemotherapy. The second group contained 5 patients who received mismatched unrelated donor transplants (9/10 allele match) and ATG (mmUD-ATG). Group three con- tained 5 patients who received transplants from matched unrelat- ed donors without the application of ATG (UD-noATG), whereas the fourth group (Haplo-PTCy) was made up of patients who underwent haploidentical transplantation and the use of PTCy. Lastly, 5 patients with matched related donors without any use of
haematologica | 2019; 104(3)
T-cell receptors following SCT
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