Reconstitution and Licensing of Natural Killer Cells Following T-Cell Replete Haploidentical Stem Cell Transplant with Post-Transplant Cyclophosphamide and Antithymocyte Globulin

Allogeneic hematopoietic stem cell transplantation using a T-cell replete HLA-haploidentical graft (hHSCT) has become possible thanks to the administration of high doses of post-transplant cyclophosphamide (PTCy) which reduces the incidence and severity of acute (a) and chronic graft-versus-host disease (GVHD) by depleting alloreactive graft T-cells in vivo. Addition of antithymocyte globulin (ATG) to PTCy-based hHSCT has been shown to improve the anti-GVHD effect of PTCy alone. Yet, little is known regarding the immune reconstitution after hHSCT using PTCy+ATG as GVHD prophylaxis, especially regarding natural killer cells (NK) reconstitution and functionality.

The main objective of this monocentric study was to evaluate early T and NK reconstitution following hHSCT with a quadruple GVHD prophylaxis combining ciclosporin, mycophenolate mofetil, PTCy and ATG. All patients (pts) gave informed consent and blood samples were collected prospectively at days (d) 30, 60 and 100 post-hHSCT. All patients received a reduced-intensity conditioning (RIC) regimen and peripheral blood stem cells as source of graft. T and NKcells were analyzed by flow cytometry (FC), and the main NK subpopulations identified using the unsupervised clustering (UC) algorithm FlowSOM. NK functionality and cytotoxicity were evaluated by studying NK degranulation (CD107a expression) on d100 in coculture with myeloid (KG1, NB4) or lymphoid (H9, MOLT4) targets, and compared to those of a cohort of 200 healthy donors (HD) of the French Blood Bank. NK reconstitution and cytotoxicity were also evaluated according to donor and recipient HLA genotype and CMV status.

Between May 2017 and November 2023, 42 pts (median age 60, IQR 48-66) could be evaluated, including 17 conditioned by clofarabine-busulfan RIC, 17 by clofarabine-cyclophosphamide-low dose total body irradiation (TBI) RIC and 8 by thiotepa-busulfan-fludarabine RIC. Main diagnoses included AML (55%) and MDS (14%). Median follow-up was 30 months (m), with 24m OS, DFS, NRM, and relapse rate of 75%, 65%, 9.5% and 25%, respectively, d100 grade 2-4 and grade 3-4 aGVHD rates of 9.5% and 2.5%, with no significant differences across conditioning regimens.

NK were the predominant lymphocyte population within the first 3 months post-hHSCT. T and NK reconstitution kinetics were similar between the three conditioning groups, suggesting the absence of influence of clofarabine, thiotepa or TBI by themselves.

UC identified 11 NK clusters in HD and pts. Cluster distribution differed between HD and hHSCT recipients but was identical across conditioning regimens. Predominant clusters in hHSCT recipients were NKG2A⁺/KIR^-/CD57^- (68.8 vs 25.3% in HD, adj p <10^-3), and NKG2A⁺/KIR2DL2-3⁺/CD57^- (7.2 vs 4.23%, adj p <10^-3). This distribution was not influenced by HLA-C genotype in donor or recipient, but correlated with CMV positive (CMV+) serology, which increased the representativity of NKG2C⁺ clusters in both pts and HD.

Median NK degranulation was higher against lymphoid targets (46.8 vs 14.3%, p<10^-3), with a greater degranulation of NKG2A⁺/CD57^-/KIR2DL2-3^+/- cells, resulting in higher total NK degranulation in pts than HD (56.9 vs 45.8%, p<10^-3). Conversely, NK degranulation against myeloid targets was lower in pts (10.3 vs 14.8%, p<10^-3). In both cases, no difference in degranulation was observed across the different conditioning regimens. A donor and/or recipient (D/R) CMV+ status was associated with lower degranulation in several minor NK subsets, without significant impact on overall NK degranulation (22.1 vs 29.4%, p=0.2). In HD, degranulation of KIR2DL2/3⁺ clusters was significantly higher in C1+ individuals, illustrating NK licensing by KIR2DL2/3 - HLA-C1 interactions. Interestingly, in hHSCT recipients, only recipient HLA-C influenced KIR2DL2/3⁺ cluster degranulation, while donor HLA-C had no effect, suggesting that NK licensing depends on non-hematopoietic rather than peripheral blood cells.

NK are the predominant immune cells early after PTCy+ATG hHSCT. The most represented clusters are NKG2A⁺. A D/R CMV+ status influences NK reconstitution. While NK cytotoxicity seems higher against lymphoid targets, their reduced efficacy against myeloid malignancies emphasizes the need for tailored post-transplant immunotherapies in this setting.