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Natural killer cells (NK) are essential for the elimination of resistant acute myeloid and acute lymphoblastic leukemia (AML and ALL) cells. NK cell-based immunotherapies have already successfully entered for clinical trials, but limitations due to immune escape mechanisms were identified. Therefore, we extended our established NK cell protocol by integration of the previously investigated powerful trispecific immunoligand ULBP2-aCD19-aCD33 [the so-called triplebodies (TBs)] to improve the anti-leukemic specificity of activated NK cells. IL-2-driven expansion led to strongly elevated natural killer group 2 member D (NKG2D) expressions on donor NK cells which promote the binding to ULBP2+ TBs. Similarly, CD33 expression on these NK cells could be detected. Dual-specific targeting and elimination were investigated against the B-cell precursor leukemia cell line BV-173 and patient blasts, which were positive for myeloid marker CD33 and B lymphoid marker CD19 exclusively presented on biphenotypic B/myeloid leukemia’s. Cytotoxicity assays demonstrated improved killing properties of NK cells pre-coated with TBs compared to untreated controls. Specific NKG2D blocking on those NK cells in response to TBs diminished this killing activity. On the contrary, the observed upregulation of surface CD33 on about 28.0% of the NK cells decreased their viability in response to TBs during cytotoxic interaction of effector and target cells. Similar side effects were also detected against CD33+ T- and CD19+ B-cells. Very preliminary proof of principle results showed promising effects using NK cells and TBs against primary leukemic cells. In summary, we demonstrated a promising strategy for redirecting primary human NK cells in response to TBs against leukemia, which may lead to a future progress in NK cell-based immunotherapies.

BK polyomavirus (BKV)-associated nephropathy is a threat to kidney allograft survival affecting up to 15% of renal transplant patients. Previous studies revealed that tubular epithelial cells (TEC) show a limited response towards BKV infection. Here we investigated the interplay between BKV and TEC in more detail. In particular, we questioned whether BKV suppresses and/or evades antiviral responses. Human primary TEC and peripheral blood mononuclear cells were infected with BKV Dunlop strain or other viruses. Moreover, TEC were stimulated with genomic double-stranded (ds)DNA or IFN. Viral replication and cellular responses were measured using quantitative real time PCR and multiplex assay. BKV infection of primary human TEC did not induce an antiviral response, whereas infection with influenza A virus, herpes simplex virus 1, or cytomegalovirus induced a strong antiviral response measured by upregulation of interferon-stimulated genes, such as CXCL10 and DAI. In addition, intracellular delivery of dsDNA or stimulation with IFN did elicit a rapid and pronounced response. However, BKV infection did not affect dsDNA-induced gene expression, indicating BKV did not modulate the antiviral response. Prestimulation of primary TEC with IFNα or dsDNA did not hamper replication of BKV, whereas influenza and herpes simplex virus 1 replication were clearly reduced. In contrast, BKV infection of leukocytes did elicit an antiviral response. BKV specifically evades innate immunity in TEC and is not susceptible to an intrinsic interferon response, which may facilitate latent presence of the virus in this cell type