Expression of KIR2DS1 does not significantly contribute to NK cell cytotoxicity in HLA-C1/C2 heterozygous haplotype B donors.

NK cells are functionally controlled by the killer immunoglobulin-like receptor (KIR) family that comprises inhibitory (iKIR) and activating (aKIR) members. Genetic association studies suggest that donors expressing aKIRs next to iKIRs will be superior donors in the setting of hematopoietic stem cell transplantation of patients with leukemia. However, contrary evidence states that aKIR expression may be irrelevant or even detrimental. Using a complex methodology incorporating KIR-Q-PCR, double fluorescence and viSNE analysis, we characterized subset distribution patterns and functionality in haplotype A donors which lack aKIRs and haplotype B donors that express a variety of B-specific genes. Here, we show that the alloreactive KIR2DS1+ NK cell subset in HLA-C1/C2 donors is highly responsive towards C2-expressing targets but quantitatively small and as such does not significantly contribute to cytotoxicity. Thus, we fail to find a direct link between haplotype allocation status and NK cell cytotoxicity at least in HLA-C1/C2 heterozygous donors.

Both mature KIR+ and immature KIR- NK cells control pediatric acute B-cell precursor leukemia in NOD.Cg-Prkdcscid IL2rgtmWjl/Sz mice.

Therapeutic natural killer (NK)-cell-mediated alloreactivity toward acute myeloid leukemia has largely been attributed to mismatches between killer immunoglobulin-like receptors (KIRs) on NK cells and their ligands, HLA class I molecules, on target cells. While adult acute B-cell precursor leukemia (BCP-ALL) appears to be resistant to NK-cell-mediated lysis, recent data indicate that pediatric BCP-ALL might yet be a target of NK cells. In this study, we demonstrate in a donor-patient-specific NOD.Cg-Prkdc(scid) IL2rg(tmWjl)/Sz (NSG) xenotransplantation model that NK cells mediate considerable alloreactivity toward pediatric BCP-ALL in vivo. Notably, both adoptively transferred mature KIR(+) NK cells and immature KIR(-) NK cells arising early posttransplantation in humanized NSG mice exerted substantial antileukemic activity. Low-dose and long-term treatment of humanized NSG mice with the DNA-demethylating agent 5-aza-cytidine distinctly enhanced the antitumor response, interestingly without inducing common inhibitory KIR expression but rather by promoting the differentiation of various NK-cell precursor subsets. Collectively, these data indicate that the future design of innovative therapy protocols should consider further exploitation of NK-cell-mediated immune responses for poor prognosis pediatric BCP-ALL patients.

An Fc-optimized NKG2D-immunoglobulin G fusion protein for induction of natural killer cell reactivity against leukemia.

Recruitment of Fc-receptor-bearing effector cells, such as natural killer (NK) cells, is a feature critical for the therapeutic success of antitumor antibodies and can be improved by the modifications of an antibody's Fc part. The various ligands of the activating immunoreceptor NKG2D, NKG2DL) are selectively expressed on malignant cells including leukemia. We here took advantage of the tumor-associated expression of NKG2DL for targeting leukemic cells by NKG2D-immunoglobulin G (IgG)1 fusion proteins containing modified Fc parts. Compared to NKG2D-Fc containing a wild-type Fc part (NKG2D-Fc-WT), our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell Fc receptor, respectively. Functional analyses with allogenic as well as autologous NK cells and primary malignant cells of leukemia patients revealed that NKG2D-Fc-KO significantly reduced NK reactivity by blocking immunostimulatory NKG2D-NKG2DL interaction. NKG2D-Fc-WT already enhanced antileukemia reactivity by inducing antibody-dependent cellular cytotoxicity (ADCC) with NKG2D-Fc-ADCC mediating significantly stronger effects. Parallel application of NKG2D-Fc-ADCC with Rituximab caused additive effects in lymphoid leukemia. In line with the tumor-associated expression of NKG2DL, no NK cell ADCC against resting healthy blood cells was induced. Thus, NKG2D-Fc-ADCC potently enhances NK antileukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL and may constitute an attractive means for immunotherapy of leukemia.

High glutathione and glutathione peroxidase-2 levels mediate cell-type-specific DNA damage protection in human induced pluripotent stem cells.

Pluripotent stem cells must strictly maintain genomic integrity to prevent transmission of mutations. In human induced pluripotent stem cells (iPSCs), we found that genome surveillance is achieved via two ways, namely, a hypersensitivity to apoptosis and a very low accumulation of DNA lesions. The low apoptosis threshold was mediated by constitutive p53 expression and a marked upregulation of proapoptotic p53 target genes of the BCL-2 family, ensuring the efficient iPSC removal upon genotoxic insults. Intriguingly, despite the elevated apoptosis sensitivity, both mitochondrial and nuclear DNA lesions induced by genotoxins were less frequent in iPSCs compared to fibroblasts. Gene profiling identified that mRNA expression of several antioxidant proteins was considerably upregulated in iPSCs. Knockdown of glutathione peroxidase-2 and depletion of glutathione impaired protection against DNA lesions. Thus, iPSCs ensure genomic integrity through enhanced apoptosis induction and increased antioxidant defense, contributing to protection against DNA damage.