Recipient BCL2 inhibition and NK cell ablation form part of a reduced intensity conditioning regime that improves allo-bone marrow transplantation outcomes

Abstract

Allogeneic hematopoietic stem cell transplantation (alloSCT) is used to treat over 15,000 patients with acute myeloid leukemia (AML) per year. Donor graft-versus-leukemia (GVL) effect can prevent AML relapse; however, alloSCT is limited by significant toxicity related to conditioning intensity, immunosuppression, opportunistic infections, and graft-versus-host disease (GVHD). Reducing the intensity of conditioning regimens prior to alloSCT has improved their tolerability, but does not alter the pattern of GVHD and has been associated with increased rates of graft rejection and relapse. Here, using a murine pre-clinical model, we describe a novel recipient conditioning approach combining reduced intensity conditioning with either genetic or pharmacological inhibition of NK cell numbers that permits efficient donor engraftment and promotes GVL without inducing GVHD. We show that NK cell-specific deletion of Bcl2 or Mcl1 in mice, or pharmacological inhibition of BCL2 impairs radio-resistant NK cell-mediated rejection of allogeneic engraftment and allows reduction of conditioning intensity below that associated with GVHD priming. The combination of reduced intensity conditioning and NK cell targeting in mice allowed successful donor T cell engraftment and protective immunity against AML while avoiding GVHD. These findings suggest that reduced conditioning in combination with targeted therapies against recipient NK cells may allow the delivery of effective alloSCT against AML while reducing the toxicities associated with more intensive conditioning including GVHD.

Fig. 1

Dosage-dependent effect of total body irradiation (TBI) on host immune system and transplantation outcome. C57BL/6 mice were irradiated with 2 × 600 or 2 × 400 rad. After 96 h a splenocytes and b bone marrow (BM) cells were analyzed by flow cytometry. Each point represents a single mouse (n = 5, unpaired t-test with Welch’s correction). c Total CD8⁺ T cells, NK, and myeloid cell numbers were enumerated from spleens of untreated, 800 and 1200 rad treated mice, and d fold-reduction for 800 and 1200 rad compared to untreated mice. e Total CD8⁺, NK, and myeloid cell numbers were calculated in BM of untreated, 800 and 1200 rad treated mice, and f fold-reduction for 800 and 1200 rad compared to untreated mice. g C57BL/6 mice were irradiated with 2 × 600 or 2 × 400 rad, and were then injected i.v. with 7.5 × 10⁶ BM cells and 1 × 10⁶ T cells from donor Balb/C (H-2K^d+) mice. Twenty-one days later, CD45⁺ cells in the blood were analyzed for donor hematopoietic cell (H-2K^d+) engraftment. Representative data are shown for one experiment (n = 5/group), from more than five independent experiments. Error bars denote mean±SD. p  < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****)

Fig. 2

Genetic depletion of recipient NK cells permits complete donor engraftment with RIC TBI, in the absence of acute GVHD. C57BL/6 mice, Bcl2^fl/fl, Mcl1^fl/fl, Mcl1^fl/+ mice (all Ncr1-iCre⁺) were irradiated with RIC 2 × 400 rad, and another group of C57BL/6 mice were irradiated with 2 × 600 rad as the myeloablative (positive) control. All recipient mice were transplanted with 7.5 × 10⁶ BM cells and 1 × 10⁶T cells from Balb/C donors. a Body weight and b clinical scores were recorded over 21 days post-alloSCT. c Twenty-one days after transplantation, donor engraftment was analyzed by flow cytometry. d Enumeration of immune cells in spleens. Each data point represents an individual mouse. Error bars denote mean±SD. e At 40 days post-alloSCT, gut tissue was prepared for histology and stained with hematoxylin and eosin (H&E). Representative micrographs from the indicated groups highlight GVHD gut lesions marked with arrows. f Mean pathology scores ±SD are shown for all mice in the indicated groups

Fig. 3

The BCL-2 inhibitor ABT-199 induces NK cell apoptosis both in vitro and in vivo. Human NK cells isolated from peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured with different concentrations of IL-15 and ABT-199 for 48 h. a Fold-change in cell numbers were enumerated, and b mean fluorescence intensity (MFI) of intracellular MCL1 expression by flow cytometry was measured (n = 3). WT C57BL/6 mice were treated with ABT-199 (100 mg/kg) by oral gavage on days −2 and −1. The mice were culled 96 h after the last treatment and c spleens and d BM immune cell subsets were analyzed in addition to e MCL1 expression were analyzed by flow cytometry in NK cells from different organs. Each data point represents an individual mouse. A representative FACS histogram of MCL1 is also shown. Error bars denote mean±SD. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****)

Fig. 4

ABT-199 treatment prior to RIC alloSCT improves long-term donor engraftment, with minimal GVHD. C57BL/6 mice were treated with ABT-199 (100 mg/kg) by oral gavage on two consecutive days (n = 12), or with equal volume of vehicle (n = 12), or were untreated (n = 6). Twenty-four hours after the last treatment, the ABT-199 and vehicle treated mice were irradiated with 2 × 400 rad, and the untreated mice were irradiated with 2 × 600 rad. All the mice were then transplanted with 7.5 × 10⁶ BM cells and 1 × 10⁶T cells from Balb/C donors. a Mice were weighed and b monitored for clinical scores daily during the experiment. c–f Mice were bled at days 14, 40, 80, and 113 post-alloSCT to measure donor cell engraftment by flow cytometry. The “Engrafted” group (green) “Responder” group (yellow), and “Non-engrafted” group (red) were stratified according to their long-term engraftment response. g Longitudinal analysis of engraftment is shown. Each data point represents an individual mouse. Error bars denote mean±SD

Fig. 5

Targeting both MCL1 and BCL2 improves donor cell engraftment. a–c BM cells and d–f Splenocytes were obtained from age and gender matched Mcl1^fl/+ and WT C57BL/6 mice and were analyzed for BIM, MCL1, and BCL2 expression in NK cells by flow cytometry (n = 3). g WT and Mcl1^fl/+ NK cells were enriched from splenocytes and cultured with different concentrations of IL-15 (12.5, 25, or 50 ng/ml) and ABT-199 (32-1024 nM) for 24 h. Viability was measured by PI exclusion and flow cytometry. h Mcl1^fl/+ and C57BL/6 mice were treated twice with ABT-199 at a dose of 100 mg/kg by oral gavage (n = 12), or with an equal volume of vehicle (n = 6), followed by RIC and alloSCT. At days 14 and 28, PBMCs were obtained by retro-orbital bleeding and were analyzed for donor engraftment by flow cytometry. i At day 40, splenic immune cell subsets were analyzed by flow cytometry in different mouse models that had complete donor engraftment. Each data point represents an individual mouse. Error bars denote mean±SD. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****)

Fig. 6

The GVL effect is maintained in NK cell-deficient RIC alloSCT recipients. a Phenotypical analysis of ligands that impact on immune reactivity on MLL-AF9 leukemia and YAC-1 leukemia. Bcl2^fl/fl and WT C57BL/6 mice received RIC 800 rad and were transplanted with 7.5 × 10⁶ BM cells and 1 × 10⁶ T cells from WT Balb/C donors, or 8.5 × 10⁶ BM cells from Rag2^−/−γc^−/− Balb/C donors. After 24 h, all SCT recipients were injected i.v. with 8 × 10⁵ MLL-AF9 AML cells. b Twenty-one days post-SCT (20 days after leukemia inoculation), bioluminescence imaging (BLI) was conducted to identify AML burden, which was measured in radiance units. c AML burden in the blood was measured by flow cytometry at day 21 and d survival curves were determined over 40 days post-SCT; p = 0.025 (*), p  = 0.014 (**) Log-Rank Mantel–Cox test. Bcl2^fl/fl mice received RIC 800 rad and were transplanted with 7.5 × 10⁶ BM cells and 1 × 10⁶ T cells from WT Balb/C donors, or 8.5 × 10⁶ BM cells from Rag2^−/−γc^−/− Balb/C donors. After 24 h, all SCT recipients were injected i.v. with 8 × 10⁵ MLL-AF9 AML cells. At day 21 post-SCT, some cohorts of mice were killed to examine e donor cell engraftment in splenocytes, f AML burden, and g composition of donor immune subsets. Each data point represents an individual mouse. Error bars denote mean±SD. p < 0.05 (*), p < 0.0001 (****)

Fig. 7

The GVL effect was maintained in RIC/ABT-199-conditioned alloSCT recipients. Twenty-four C57BL/6 mice were injected with 0.8 × 10⁶ MLL-AF9 cells on day 0, on days 8 and 9, 12 mice were treated with ABT-199 (100 mg/kg) by oral gavage, 6 mice were treated with equal volume of vehicle, and the remaining 6 mice were untreated. On day 10, the ABT-199 and vehicle-treated mice were irradiated with 2 × 400 rad, and the untreated mice were irradiated with 2 × 600 rad. All the mice were injected i.v. with 7.5 × 10⁶ BM cells and 1 × 10⁶ T cells from WT Balb/C donors. a Mice were monitored daily up to day 60, and killed when AML developed. In an independent experiment at day 20 post-alloSCT; p = 0.06 (*) Log-Rank Mantel–Cox test. b Donor cell engraftment, AML burden in c BM and d spleen were measured by flow cytometry, and stratified between donor cell engrafted and non-engrafted recipients. Each data point represents an individual mouse. Error bars denote mean±SD. p <  0.05 (*), p < 0.01(**), p < 0.0001 (****)