Clinical Determinants of T-Cell Receptor Diversity after Allogeneic Hematopoietic Stem Cell Transplantation for Acute Myeloid Leukemia

T-cell reconstitution after allogeneic haematopoietic stem cell transplantation (alloSCT) is critical for protection against infection and to mediate the graft versus leukemia (GVL) effect against hematological malignancies including acute myeloid leukemia (AML). T-cell reconstitution post-alloSCT is significantly impacted by exogenous factors including T-cell depleting strategies, immunosuppressive medications and the prohibitive effects of graft versus host disease (GVHD) and infection. The early T-cell repertoire post-alloSCT is oligoclonal and clinical events such as infection and GVHD may adversely impact recovery of a diverse TCR repertoire. The objectives of this study were to investigate clinical determinants of TCR diversity at day 100 after alloSCT and the impact of TCR diversity on risk of early AML relapse after alloSCT.

Methods

Twenty-nine patients who underwent HLA-matched sibling or unrelated donor alloSCT were included in this cohort comprising 16 patients with AML relapse at day 100 to 180 post-alloSCT and 13 control patients who did not relapse post-alloSCT. All patients received unmanipulated peripheral blood or bone marrow stem cells. Anti-thymocyte globulin was administered to all patients receiving unrelated donor stem cells as per institutional practice. Surveillance for cytomegalovirus (CMV) viremia in peripheral blood (plasma) was monitored twice weekly using polymerase chain reaction (PCR) and pre-emptive therapy with intravenous ganciclovir or oral valganciclovir was commenced in patients with plasma viral load of 400 copies/mL or greater. Peripheral blood samples were obtained at day 100 (early time-point). Eleven patients had follow-up samples 1-2 years post-transplant (late time-point). T-cells were isolated using immunomagnetic separation. Following DNA extraction, TCRβ loci deep amplicon sequencing was performed using LymphoTrack TRB. Sequence assembly, annotation and error correction was performed by MiXCR. TCR diversity was quantified using inverse Simpson's diversity index (1/D).

Results

TCRβ sequencing of the entire cohort of 29 patients was performed with a mean of 454516 sequence reads per patient. Median time from transplant for the early post-alloSCT timepoint was 99 days. Median TCR repertoire diversity (1/D) early post-transplant was 104.3 (IQR 46.5-398.4). TCR diversity was significantly greater in patients who received T-cell replete transplants from matched sibling donors compared with T-cell depleted transplants from unrelated donors (siblings 130.1 [IQR 54-1017] vs unrelated donors 64 [IQR 28.9-96.9]; P=0.04). Early TCR diversity was significantly reduced in recipients who were CMV seropositive prior to transplant compared with seronegative patients (77.5 [IQR 42.4-127.5] vs 718.8 [IQR 75.5-1884]; P=0.01). Twenty patients (69%) developed CMV viremia, defined as any detectable CMV virus in peripheral blood, prior to day 100 post-alloSCT. Early TCR diversity was significantly reduced in patients with CMV viremia within the first 100 days post-alloSCT (83.5 [IQR 42.4-131.5] vs 964.4 [IQR 71.7-2399]; P=0.02). There was no significant difference in TCR diversity at day 100 in patients who had prior acute GVHD compared to those who did not. There was no significant difference in early TCR diversity at the time of AML relapse compared with patients who remained in remission (78.4 [IQR 38-799.2] vs 132.8 [IQR 59.5-753.6]; P=0.22), suggesting that a restricted TCR repertoire early post-transplant is not a mechanism of AML relapse. Eleven patients had serial samples analysed at early (day 100) and late (between 1-2 years post-transplant) timepoints. All patients remained free of leukemia relapse between these two timepoints. Patients with early CMV viremia (prior to day 100) continued to have a significantly reduced TCR diversity late post-transplant compared with patients who did not have early CMV reactivation (33.2 [IQR 27.3-61.4] vs 3868 [IQR 1421-4565]; P=0.006), indicating that early CMV viremia had a persistent effect on post-transplant T-cell recovery extending to 1-2 years post-transplant.

Conclusion

T-cell depletion and CMV viremia are key determinants of early TCR repertoire diversity post-alloSCT. CMV viremia has persistent and deleterious effects on TCR repertoire late post-transplant. TCR diversity does not impact early AML relapse post-alloSCT.