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Patients with poor risk acute myeloid leukemia (AML) have a dismal outcome. We hypothesized that combining decitabine with a standard non-myeloablative (NMA) conditioning regimen prior to allogeneic hematopoietic cell transplantation (allo HCT), might decrease the relapse incidence. We conducted a multicenter prospective phase II study (NCT02252107) with 10-day decitabine (20 mg/m2/day) integrated in a standard non-myeloablative conditioning regimen (3 days fludarabine 30 mg/m2with 2 Gray total body irradiation (TBI)). Patients with AML ≥ 18 years in 1st (in)complete remission (CR/CRi) with a poor or very poor risk profile, as defined by the HOVON-132 protocol, were eligible. Results: Forty-six patients (median age 60; range 23–74) were included. Median follow up time was 44 months (range 31–65 months). The cumulative 1-year incidence of relapse and NRM were respectively 23% and 11%. Incidence of grade III-IV acute graft-vs-host-disease (GVHD) and severe chronic GVHD were 13% and 20%, respectively. One-year OS was 70%. Application of ELN 2017 risk classification to the study cohort revealed a cumulative one-year relapse rate of respectively 31% and 13% for the adverse and intermediate risk patients. To conclude, the 10-day DEC/FLU/TBI conditioning regimen prior to allo HCT in poor risk AML patients is effective and feasible.

Allogeneic hematopoietic cell transplantation (alloHCT) is a complex, potentially fatal therapy featuring a myriad of complications. Triggering event(s) of such complications vary significantly, but often a so-called “multi-organ failure” (MOF) is reported as the leading cause of death. The identification of the exact trigger of MOF is critical towards early and disease-specific intervention to improve outcome. We examined data from 202 alloHCT patients reported to have died of MOF from the EBMT registry aiming to determine their exact cause of death focusing on veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) due to its life-threatening, often difficult to capture yet preventable nature. We identified a total of 70 patients (35%) for whom VOD/SOS could be considered as trigger for MOF and leading cause of death, among which 48 (69%) were previously undiagnosed. Multivariate analysis highlighted history of hepatic comorbidity or gentuzumab use and disease status beyond CR1 as the only significant factors predictive of VOD/SOS incidence (OR = 6.6; p= 0.001 and OR = 3.3; p= 0.004 respectively). VOD/SOS-related MOF was widely under-reported, accounting for 27% of deaths attributed to MOF of unknown origin without a previous VOD/SOS diagnosis. Our results suggest most missed cases developed late VOD/SOS beyond 21 days post-alloHCT, highlighting the importance of the newly revised EBMT criteria.

Acute myeloid leukemia (AML) is an immune-susceptible malignancy, as demonstrated by its responsiveness to allogeneic stem cell transplantation (alloSCT). However, by employing inhibitory signaling pathways, including PD-1/PD-L1, leukemia cells suppress T cell-mediated immune attack. Notably, impressive clinical efficacy has been obtained with PD-1/PD-L1 blocking antibodies in cancer patients. Yet, these systemic treatments are often accompanied by severe toxicity, especially after alloSCT. Here, we investigated RNA interference technology as an alternative strategy to locally interfere with PD-1/PD-L1 signaling in AML. We demonstrated efficient siRNA-mediated PD-L1 silencing in HL-60 and patients’ AML cells. Importantly, WT1-antigen T cell receptor+PD-1+2D3 cells showed increased activation toward PD-L1 silenced WT1+AML. Moreover, PD-L1 silenced AML cells significantly enhanced the activation, degranulation, and IFN-? production of minor histocompatibility antigen-specific CD8+T cells. Notably, PD-L1 silencing was equally effective as PD-1 antibody blockade. Together, our study demonstrates that PD-L1 silencing may be an effective strategy to augment AML immune-susceptibility. This provides rationale for further development of targeted approaches to locally interfere with immune escape mechanisms in AML, thereby minimizing severe toxicity. In combination with alloSCT and/or adoptive T cell transfer, this strategy could be very appealing to boost graft-versus-leukemia immunity and improve outcome in AML patients.

Combining natural killer (NK) cell adoptive transfer with hypomethylating agents (HMAs) is an attractive therapeutic approach for patients with acute myeloid leukemia (AML). However, data regarding the impact of HMAs on NK cell functionality are mostly derived from in vitro studies with high nonclinical relevant drug concentrations. In the present study, we report a comparative study of azacitidine (AZA) and decitabine (DAC) in combination with allogeneic NK cells generated from CD34+ hematopoietic stem and progenitor cells (HSPC-NK cells) in in vitro and in vivo AML models. In vitro, low-dose HMAs did not impair viability of HSPC-NK cells. Furthermore, low-dose DAC preserved HSPC-NK killing, proliferation, and interferon gamma production capacity, whereas AZA diminished their proliferation and reactivity. Importantly, we showed HMAs and HSPC-NK cells could potently work together to target AML cell lines and patient AML blasts. In vivo, both agents exerted a significant delay in AML progression in NOD/SCID/IL2Rgnull mice, but the persistence of adoptively transferred HSPC-NK cells was not affected. Infused NK cells showed sustained expression of most activating receptors, upregulated NKp44 expression, and remarkable killer cell immunoglobulin-like receptor acquisition. Most importantly, only DAC potentiated HSPC-NK cell anti-leukemic activity in vivo. Besides upregulation of NKG2D- and DNAM-1–activating ligands on AML cells, DAC enhanced messenger RNA expression of inflammatory cytokines, perforin, and TRAIL by HSPC-NK cells. In addition, treatment resulted in increased numbers of HSPC-NK cells in the bone marrow compartment, suggesting that DAC could positively modulate NK cell activity, trafficking, and tumor targeting. These data provide a rationale to explore combination therapy of adoptive HSPC-NK cells and DAC in patients with AML.

Immunomodulating regulatory T-cell (Treg) therapy is a promising strategy in autoimmunity and transplantation. However, to achieve full clinical efficacy, better understanding of in vivo human Treg biology is warranted. Here, we demonstrate that in contrast to blood and bone marrow Tregs, which showed a resting phenotype, the majority of CD4posCD25posCD127negFoxP3pos Tregs in secondary lymphoid organs were proliferating activated CD69posCD45RAneg cells with a hyperdemethylated FOXP3 gene and a broad T-cell receptor–Vβ repertoire, implying polyclonal activation. Activated CD69pos Tregs were distributed over both T-cell and B-cell areas, distant from Airepos and CD11cpos cells. In contrast to the anergic peripheral blood Tregs, lymphoid organ Tregs had significant ex vivo proliferative capacity and produced cytokines like interleukin-2, while revealing similar suppressive potential. Also, next to Treg-expressing chemokine receptors important for a prolonged stay in lymphoid organs, a significant part of the cells expressed peripheral tissue–associated, functional homing markers. In conclusion, our data suggest that human secondary lymphoid organs aid in the maintenance and regulation of Treg function and homeostasis. This knowledge may be exploited for further optimization of Treg immunotherapy, for example, by ex vivo selection of Tregs with capacity to migrate to lymphoid organs providing an in vivo platform for further Treg expansion.

ABSTRACTAdoptive T cell therapy has shown clinical potential for patients with cancer, though effective treatment is dependent on longevity and potency of the exploited tumor-reactive T cells. Previously, we showed that ex vivoinhibition of AKT using the research compound Akt-inhibitor VIII retained differentiation and improved functionality of minor histocompatibility antigen (MiHA)-specific CD8+T cells. Here, we compared a panel of clinically applicable AKT-inhibitors with an allosteric or adenosine triphosphate-competitive mode of action. We analyzed phenotype, functionality, metabolism and transcriptome of AKT-inhibited CD8+T cells using different T cell activation models. Most inhibitors facilitated T cell expansion while preserving an early memory phenotype, reflected by maintenance of CD62L, CCR7 and CXCR4 expression. Moreover, transcriptome profiling revealed that AKT-inhibited CD8+T cells clustered closely to naturally occurring stem cell-memory CD8+T cells, while control T cells resembled effector-memory T cells. Interestingly, AKT-inhibited CD8+T cells showed enrichment of hypoxia-associated genes, which was consistent with enhanced glycolytic function. Notably, AKT-inhibition during MiHA-specific CD8+T cell priming uncoupled preservation of early memory differentiation from ex vivoexpansion. Furthermore, AKT-inhibited MiHA-specific CD8+T cells showed increased polyfunctionality with co-secretion of IFN-γ and IL-2 upon antigen recall. Together, these data demonstrate that AKT-inhibitors with different modality of action promote the ex vivogeneration of stem cell memory-like CD8+T cells with a unique metabolic profile and retained polyfunctionality. Akt-inhibitor VIII and GDC-0068 outperformed other inhibitors, and are therefore promising candidates for ex vivogeneration of superior tumor-reactive T cells for adoptive immunotherapy in cancer patients.