Patients Receiving IL-2 Activated Donor NK Cells Show Lower Incidence of Severe GvHD after Haploidentical SCT.
Authors: Gentilini Chiara , Hilbers Urte , Huppert Volker , Loddenkemper Christoph , Müßig Arne , Kliem Constanze , Lange Thoralf , Basara Nadezda , Uhrberg Marcus , Thiel Eckhard , Niederwieser Dietger W. , Uharek Lutz 
Published: October 2019

Abstract
To improve the antileukemic effectiveness of haploidentical stem cell transplantation (HSCT) and strengthen the cell mediated immune response, we have adoptively transferred high numbers of alloreactive donor NK cells during the early phase after transplantation. In addition, we activated the transferred NK-cell population ex vivo in short term cultures with high doses of IL-2 in order to enhance the antileukemic activity and inhibit the occurrence of severe GvHD.
Method: In a phase-II study, 17 patients (10 AML, 1 MDS, 1 HD, 2 CML, 3 ALL, median age 37 yrs, range 17–48 yrs) were transplanted in late phases of their disease (6 pts. as 2nd transplantation) and received purified NK cells from their haploidentical donors at day +2 after HSCT. Conditioning consisted of 12 Gy fTBI, Thiotepa (10mg/kg), Fludarabine (5 x 30 mg/qm) and OKT3 (day −4 to +2). NK cells were isolated from the CD34- fraction using an automated two-step procedure of CD3+ depletion and subsequent CD56+ selection. Seven patients received activated NK cells and 10 patients received unstimulated NK cells. Cells were activated by 16h incubation with IL-2 (500U/1x107 cells).
Results: After selection and subsequent overnight activation of the NK cells with IL-2 (7 out of 17 patients), a mean number of 8.3 x 106/kg CD56+CD3− NK cells was transferred at day 2 after transplantation. The purity was 76%, due to contaminating CD56+ monocytes. The mean number of contaminating CD3+ cells in the transfused NK product was 2.1 x 104/kg. Activation of donor NK cells with IL-2 resulted in an increase of cytotoxic activity, when cells were tested against target cell lines. No differences in yield or number of contaminating T cells were observed between IL-2 activated and not activated NK cells. No severe acute toxicity attributable to NK cell infusion was observed in both groups of patients. Comparing the rate of high-grade GvHD revealed an interesting result. Whereas only one patient developed GvHD ≥ grade II after treatment with IL-2 activated NK cells, seven out of ten patients showed GvHD ≥ grade II after transfer of non-activated NK cells (p<0.05). When the correlation between GvHD and the presence of a KIR mismatch was analyzed no significant difference was observed. Moreover immunocytometric analysis of lymphocyte subpopulations at different time points after transplant revealed a long-lasting cellular immunodeficiency in all patients, with slow recovery of CD4+ lymphocytes. As for the incidence of GvHD, there was also a striking difference in immune recovery between the patients receiving IL2-activated and those treated with non-activated NK cells. Patients receiving activated NK cells showed significantly lower numbers of NK- and T cells during the first months post transplant, whereas no differences in the number of granulocytes were present between the two groups. Based on these findings we can assume that the use of IL-2 for NK cell activation could play a role in reducing the incidence of severe GvHD after haploidentical HSCT.