Recovery of Specific Subsets of Natural Killer and T Cells Highly Associated with Graft-Versus-Host Disease after Haploidentical Stem Cell Transplantation in Acute Leukemia

Background In the allogeneic hematopoietic stem cell transplantation, recent studies showed that T cell and natural killer (NK) cells recovery are implicated in the graft-versus-host disease (GVHD) and graft versus leukemia (GVL) effects. However, the significance of specific subsets of NK and T cell recovery in relation to transplantation outcomes remains to be elucidated in the haploidentical stem cell transplantation (haploSCT).

Methods Clinical data of patients with acute myeloid leukemia (n = 21) and acute lymphoblastic leukemia (n = 24) who underwent their first haploSCT between September 2009 and December 2017 were analyzed. Peripheral blood mononuclear cells obtained from 27 patients were examined by multiparametric flow cytometric analysis. PD-1 and Tim-3 expression were examined in CD4⁺ and CD8⁺ T-cells and NK cell receptor (NKG2D, NKG2A, NKG2C, DNAM1 and NKp46) expression were analyzed in NK cells, respectively, at the 3 determined times (immediate prior to conditioning therapy, 28 and 90 days after haploSCT).

Results Median age at haploSCT was 38 years (range, 21-62) and median follow-up duration was 31.6 months. Myeloablative conditioning was used for 32% and reduced intensity regimen for 68% of patients. GVHD prophylaxis was based on post-transplant cyclophosphamide for 8 (18%) or on anti-thymocyte-globulin for 36 (82%) plus standard prophylaxis. Incidence of grade II-IV acute GVHD was 50%, gastrointestinal tract (GIT) GVHD was 55.6%, non-GIT acute GVHD 35.7%, and chronic GVHD was 52.4%. Longitudinal analysis of immune reconstitution after haploSCT showed that the incidence of acute GVHD was associated with a delayed expansion of the NK cell population and incidence of chronic GVHD was associated with the extent of CD4⁺ T cell reconstitution. The incidence of acute GVHD was significantly higher in patients with lower counts of CD56^bright CD16^neg cell (100% for patients with less than 30 cells/uL at day 28 vs 50% for patients with higher counts, P = 0.026), particularly in NKG2A (P = 0.002) and DNAM1 (P = 0.027)-positive NK cell subsets. In univariate analysis, early CMV replication (P < 0.001), chronic GVHD (P = 0.001), donor age ≥ 28years (P = 0.018), CD4/CD8 ratio of product ≥ 2.4 (P = 0.033), and dose of infused T cells ≥ 3.91 x 10⁸ /kg (P = 0.022) were significantly associated with lower 3-year cumulative incidence of relapse after haploSCT. Donor age ≥ 28years was significantly associated with high incidence of chronic GVHD (P = 0.002). Dose of infused T cells ≥ 3.91 x 10⁸ /kg (HR, 0.088; CI, 0.009 to 0.823; P = 0.033) were independent factors for reducing leukemia relapse after adjustment in multivariate analysis. Chronic GVHD was an independent prognostic factor for higher leukemia-free survival rate (72.7% versus 20.1%, P = 0.008). Longitudinal analysis of T cell reconstitution after haploSCT showed that the high dose of infused T cells was associated with the increased expansion of CD4⁺PD-1^- T cells (P = 0.031 at day 28 and P = 0.017 at day 90). Of note, The incidence of chronic GVHD was significantly higher in patients with higher counts of CD4⁺ T cell at day 28 (100% for patients with over than 150 cells/uL at day 28 vs 38.8% for patients with lower counts, P = 0.008), particularly in CD4⁺PD-1^- subsets (P = 0.008). Among CD4⁺ T cell, PD-1^-/PD-1⁺ ratio over than 4.5 was significantly associated with increased chronic GVHD (P = 0.005). In 22 patients with chronic GVHD, GIT GVHD was adverse prognostic factor for overall survival (59.5 % in GIT GHVD vs 100% in patients without GIT GVHD, P = 0.063). The incidence of GIT GVHD was significantly higher in patients with lower CD4/CD8 ratio at day 28 (77.8% for patients with less than 1.5 vs 0% for patients with higher ratio, P = 0.045).

Conclusions Our findings suggest that high CD56^brightCD16^neg NK cell count at day 28 after hasploSCT was significantly associated with decreased incidence of acute GVHD. High dose of infused T cells was associated with increased reconstitution of CD4⁺ PD-1^- T cells and high CD4⁺ T cell counts, particularly in PD-1^- subset, are associated with increased development of chronic GVHD. These findings should be further validated for elucidating the roles of these immune effectors cells in the development of GVHD and GVL effect in haploSCT for acute leukemia.