T Cell Repertoire after Alpha/Beta-T Cell Depleted Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Patients

Allogeneic transplantation of hematopoietic cells (HSCT) is an established method to treat different hematologic malignancies and disorders of hematopoietic and lymphoid system. Graft-versus-host-disease is one of the main risk factor for success of the procedure. Simultaneous depletion of alpha-beta T-cells and CD19+ cells in graft is the promising way to reduce the risk. The approach was recently introduced in clinical practice and many aspects of immune system reconstitution are still unknown. We applied improved technology for T cell receptor (TCR) repertoire sequencing to study origin and dynamics of T cell clones during 1 year follow-up period after allogeneic TCRαβ/CD19-depleted HSCT in children.

We performed TCR repertoire sequencing for peripheral blood samples of patients before HSCT, at 2, 6 and 12 months after HSCT (n=21, 21, 17, 16 respectively), and for respective donor blood apheresis samples before abT/CD19 depletion. Twelve of the patients were diagnosed with acute leukemia and the others with non-malignant inherited and acquired blood disorders. For each patient data on recipient's T cell chimerism and counts of CD3+, naïve CD3+, alpha-beta T-cells and recent thymic emigrants (RTE) have been collected during 1 year follow-up period.

Barcoding of each original TCR mRNA molecule passed to massive parallel sequencing allowed us to: (1) reduce sample preparation biases and quantitatively reconstruct of TCR repertoires; (2) equalize repertoire data analysis depth which is absolutely necessary for correct comparison of samples; (3) prevent risk of cross-contamination between samples and increase confidence of T clone origin determination.

Two months after TCRαβ/CD19-depleted HSCT T cell repertoire mostly consists of several hundreds highly abundant clones. For patients with low recipient T cell chimerism from 13 to 504 largest T-cell clones (median 255, IQR 219, n=9, T cell chimerism <=20%) represented 80% of all T cells in peripheral blood. For comparison in healthy age-matched donors we found from 32,000 to 47,000 largest T-cell clones in identical analysis (median 43191, IQR 6493, n=14, data from Britanova O.V. et.al., JI 2014). The overall diversity at d60 after HSCT was also much less compared with the healthy subjects. We also found that most expanded T cell clones at d60 do not represent just a replica of the most expanded clones in graft samples, originating from low-abundant graft T cell clones.

The diversity of T repertoire early after HSCT positively correlated with recipient T cell chimerism (the diversity was higher for those patients with higher percentage of recipient's T cells). Also patients with low chimerism had higher number of T clones originating from the graft than from d0 pre-transplant recipient repertoire in contrast to the patients with high T cell chimerism who had inverse ratio (median number of patient's clonotypes shared between graft and d0 was 56 or 3 for patients with low or absent chimerism (IQR = 24 or 19.25, n = 5) and 21.5 or 321.5 for patients with T cell chimerism >2% (IQR = 46.5 or 724.75, n = 10)). In addition CD4+ RTE count was higher for patients with high T cell chimerism. This observation was additionally confirmed by analysis of flow cytometry data for the expanded cohort of 105 patients at d60 after αβT-cell depleted HSCT (Wilcoxon rank sum test p-value = 0.002).

Our results demonstrate that early after αβT-cell depleted HSCT repertoire of T cells are extremely skewed and unlikely able protect recipient efficiently. Observed recovery of T cell count mostly results from expansion of a few clones that have to divide intensely for the whole 60 days period in order to achieve the observed counts. Early reconstitution of TCR diversity and RTE counts in patients with substantial recipient T cell chimerism is mostly explained by surviving recipient T cells and intrathymic T cell progenitors, respectively.

This work was supported by the Russian Science Foundation project №14-35-00105. Zvyagin I. is supported by grant MK-4583.2015.4.