IL-2 Stimulated but Not Unstimulated NK Cells Induce Selective Disappearance of Peripheral Blood Cells: Concomitant Results to a Phase I/II Study

Abstract

In an ongoing clinical phase I/II study, 16 pediatric patients suffering from high risk leukemia/tumors received highly purified donor natural killer (NK) cell immunotherapy (NK-DLI) at day (+3) +40 and +100 post haploidentical stem cell transplantation. However, literature about the influence of NK-DLI on recipient's immune system is scarce. Here we present concomitant results of a noninvasive in vivo monitoring approach of recipient's peripheral blood (PB) cells after transfer of either unstimulated (NK-DLI(unstim)) or IL-2 (1000 U/ml, 9–14 days) activated NK cells (NK-DLI(IL-2 stim)) along with their ex vivo secreted cytokine/chemokines. We performed phenotypical and functional characterizations of the NK-DLIs, detailed flow cytometric analyses of various PB cells and comprehensive cytokine/chemokine arrays before and after NK-DLI. Patients of both groups were comparable with regard to remission status, immune reconstitution, donor chimerism, KIR mismatching, stem cell and NK-DLI dose. Only after NK-DLI(IL-2 stim) was a rapid, almost complete loss of CD56(bright)CD16(dim/−) immune regulatory and CD56(dim)CD16(+) cytotoxic NK cells, monocytes, dendritic cells and eosinophils from PB circulation seen 10 min after infusion, while neutrophils significantly increased. The reduction of NK cells was due to both, a decrease in patients' own CD69(−) NCR(low)CD62L(+) NK cells as well as to a diminishing of the transferred cells from the NK-DLI(IL-2 stim) with the CD56(bright)CD16(+/−)CD69(+)NCR(high)CD62L(−) phenotype. All cell counts recovered within the next 24 h. Transfer of NK-DLI(IL-2 stim) translated into significantly increased levels of various cytokines/chemokines (i.e. IFN-γ, IL-6, MIP-1β) in patients' PB. Those remained stable for at least 1 h, presumably leading to endothelial activation, leukocyte adhesion and/or extravasation. In contrast, NK-DLI(unstim) did not cause any of the observed effects. In conclusion, we assume that the adoptive transfer of NK-DLI(IL-2 stim) under the influence of ex vivo and in vivo secreted cytokines/chemokines may promote NK cell trafficking and therefore might enhance efficacy of immunotherapy.

Pre-emptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective phase II study in two centers

“…Very recently, we could show that IL-2-stimulated NK-DLI but not unstimulated NK-DLI promote NK cell trafficking and changes in the distribution of leukocyte sub-populations in the peripheral blood. 35 Interestingly, stimulated NK-DLI led to a rapid loss of CD56(bright)CD16(dim) immune regulatory and CD56(dim)CD16( þ ) cytotoxic NK cells, monocytes, dendritic cells and eosinophils from patients' peripheral blood circulation 10 min after infusion. We could demonstrate that the reduction of NK cells was due to both, a decrease in patients' own CD69(2) NCR(low)CD62L( þ ) NK cells and to a diminishing of the transferred cells from the stimulated NK-DLI.…”

Low blood lymphocyte count at 30 days post transplant predicts worse acute GVHD and survival but not relapse in a large retrospective cohort

“…Cellular therapy interventions that might ameliorate the increased GVHD associated with low ALC30 include post-transplant infusions of rapamycin-resistant CD4 T-cells (enriched for regulatory T cells) or NK-cell DLI, both of which have the potential to maintain the GVL effect while limiting aGVHD. [47][48][49] Further studies are needed to define the critical cell phenotype of ALC30 and to better define the mechanism by which high ALC30 protects AHSCT recipients from aGVHD and NRM, thereby improving transplant outcomes. Also adjusted for donor gender, donor CMV status, recipient gender, recipient CMV status, relapse risk, intensity and any blank cells (also NS).…”

Clinical Applications of Natural Killer Cells

“…Because only a minor fraction of circulating NK cells is reactive to target cells (tumor cells) in vitro, primary NK cells show insufficient cytotoxicity [14]. Various types of stimulation have thus been reported to enable NK cells to achieve their full effector potential, such as interleukin (IL)-15 produced by dendritic cells (DCs) [15] or macrophages [16], IL-2 [17], IL-12 [18], IL-18 [19] and IL-21 [20]. Currently, the additional cytokines used in the cultivation of NK cells include IL-2, IL-15, and IL-21.…”

“…+ is enriched after the removal of CD3 + T cells [17,90,91]. In cases of an allogeneic type, HLA typing and confirmation of KIR by flow cytometry are carried out, particularly in order to select the optimum donor.…”