PLoS One. 2011;6(11):e27351. doi: 10.1371/journal.pone.0027351. Epub 2011 Nov 9.

IL-2 stimulated but not unstimulated NK cells induce selective disappearance of peripheral blood cells: concomitant results to a phase I/II study.

Brehm C, Huenecke S, Quaiser A, Esser R, Bremm M, Kloess S, Soerensen J, Kreyenberg H, Seidl C, Becker PS, Mühl H, Klingebiel T, Bader P, Passweg JR, Schwabe D, Koehl U.

Source

Pediatric Hematology and Oncology, Laboratory for Stem Cell Transplantation and Immunotherapy, Johann Wolfgang Goethe-University Hospital, Frankfurt, Germany. Claudia.Brehm@kgu.de

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.

PMID:: 22096557; [PubMed - indexed for MEDLINE]
PMCID:: PMC3212563

Free PMC Article

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Figure 1

NK-DLI_{IL-2 stim} but not NK-DLI_unstim led to a considerable disappearance of NK cells from PB.

A) Absolute number of NK cells in the PB of the patients was significantly reduced 10 min post NK-DLI_{IL-2 stim} (grey) in contrast to NK-DLI_unstim (white), which showed minimal influence only. 24 h after NK-DLI_{IL-2 stim} absolute number of NK cells recovered to the level before DLI. Only freshly applied NK-DLIs infused around day +40 post SCT are shown and were used for statistical calculations (n = 7 NK-DLI_{IL-2 stim}, n = 6 NK-DLI_unstim). Box and whiskers plots show minimum, lower quartile, median, upper quartile and maximum of all measured data. For the 4 h level post NK-DLI_unstim two values were available, only. p<0.05 indicated as *. B) Mean and SEM of all freshly applied NK-DLI_{IL-2 stim} (grey) and NK-DLI_unstim (white) applied around day +40 post SCT. Striped bars indicate estimated patient's PB volume and peripheral NK cell count at the time point of NK-DLI infusion. Left graph shows a mean 5.5-fold reduction of absolute NK cell count in the PB as early as 10 min post NK-DLI_{IL-2 stim} compared to a 1.2-fold decrease for NK-DLI_unstim. Middle graph shows relation of NK-DLI volume to total blood volume of patients (NK-DLI_{IL-2 stim} volume: 825 ml±249, compared to PB volume: 3060 ml±789; NK-DLI_unstim volume: 128 ml±19, compared to PB volume 3093 ml±749). DLI volume did not artificially lead to the reduced absolute NK cell count, since NK-DLI_{IL-2 stim} volumes made up maximally around ¼ of patient's PB volume. Right graph shows mean NK-DLI cell dose ×10⁶/kg BW (NK-DLI_{IL-2 stim} 19.2±4.5; NK-DLI_unstim 18.8±5.1) in relation to patient's PB NK cells newly reconstituted post haplo-SCT around d +40 prior to NK-DLI (NK-DLI_{IL-2 stim} 26.0±8.5; NK-DLI_unstim 31.9±4.0). Applied NK dose compromised about 80% of PB NK cells, illustrating the high dose of administered NK-DLI. C) Density plots (CD56 vs. CD16) and box and whiskers plots show a significant change in the distribution of the cytotoxic CD56^dimCD16⁺ and immune regulatory CD56^brightCD16^dim/− NK cell subsets. This was due to an absolute reduction of the CD56^brightCD16^dim/− NK cell subpopulation in the PB 10 min after freshly applied NK-DLI_{IL-2 stim} applications infused around d +40 (left; n = 7). This could not be shown after NK-DLI_unstim (right; n = 6). Plots are gated on CD56⁺CD3⁻ NK cells. For the 4 h level post NK-DLI_unstim 2 values were available, only. p<0.05 indicated as *.

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

PLoS One. 2011;6(11):e27351.