Landscape of Immune Reconstitution after Allogeneic Hematopoietic Cell Transplantation Is Superdominated By Distinct T Cell Populations Bearing T-Cell Receptor Clonotypes Shared Among Different Individuals

Background: Enormous diversity of T-cell receptor (TCR) repertoire is generated by somatic rearrangement of gene segments that encode TCRα and TCRβ subunits. Next-generation TCR sequencing is emerging as a powerful tool to elucidate TCR repertoire at very high resolution. We recently found that a small fraction of TCRs shared among different individuals confers immunologic predominance on T-cell reactivities against some viral antigens (Miyama, Sci Rep 2017). To investigate the role of T cells bearing such "shared" TCRs during immune reconstitution after allogeneic hematopoietic cell transplantation (allo-HCT), we herein performed semi-quantitative NGS analysis of TCR repertoire in peripheral blood (PB) of post-transplant patients.

Methods: This study was conducted according to a protocol approved by the ethics committee of Hiroshima University. We collected PB mononuclear cells from 15 patients who received allo-HCT at our center for hematologic malignancies (acute leukemia=9; myelodysplastic syndrome=3; lymphomas=3) prior to and at different time points (1, 2, 3, 4, 6, 8 weeks and 3, 6, and 12 months) after transplantation. 10 patients received HLA-8/8 or 7/8-matched unrelated-donor bone marrow and 5 received HLA-mismatched single-cord blood unit. Using these samples, unbiased NGS analysis of TRB V-D-J gene segments was performed. We assessed the TCR repertoire diversity of each sample by calculating the Simpson's Reciprocal Index (SRI, 1/D) using NGS data. We defined TCRβ clonotypes found in two or more patients using the same TRBV/TRBJ gene segments and CDR3 amino acid sequences as a "shared" clonotype. To determine whether the read numbers of a shared TCR clonotype in a given sample occupy the higher or lower rank compared with those of unshared clonotypes, we performed the nonparametric Mann-Whitney test. P values less than 0.05 were considered statistically significant.

Results: All patients achieved neutrophil engraftment at a median of 19 days with complete donor-type chimerism. A total of 12 patients developed grade II-IV acute GVHD at a median of 23.5 (range 6-60) days and 10 patients experienced cytomegalovirus reactivation at a median onset of 36 (range 15-56) days after transplantation. Among 36,281,897 TRB sequence reads obtained from the collected samples, 2,782,129 were identified as a unique read that defines a single TCRβ clonotype. Among these unique clonotypes, 42,436 (1.5%) were shared among different patients. Although SRIs in the samples obtained from 2 to 4 weeks post-transplant were nearly equivalent to those in pre-transplant samples, the diversity indices from 6 weeks to 12 months were significantly decreased before transplant (median 1/D; pre-transplant=137, 6w=29.9, 8w=40.5, 3m=35.8, 6m=33.6, 12m=28.3, P<0.05), indicating the persistent reduction of TCR repertoire diversity. Intriguingly, although the proportions of "shared" clonotypes among total TCRβ clonotypes later than 1 week post-transplant were relatively small (median 5.2%, range 0.55-16.9%), the ratio of unique reads encoding shared clonotypes among total TRB reads ranged from 3.6% to 93.3% (median 50.7%), which was significantly higher than that of unshared unique reads (P<0.05). Such predominance of shared TCRβ clonotypes was observed in both CD4+ and CD8+ T cell populations.

Conclusions: T-cell repertoire reconstitution after allo-HCT is characterized by extreme predominance of T cells bearing TCRβ clonotypes shared among different individuals. Our results suggest that a distinct population of T cells expressing "shared" TCRs strongly affects the homeostasis of T cell repertoire after allo-HCT. Functional assays of these superdominant TCRs are strongly warranted in the context of their reactivities against microbial or allogenic antigens.