Human placental hematopoietic stem cell-derived natural killer cells (CYNK) recognize and eliminate influenza A virus-infected cells.

Influenza A virus (IAV) infections are a significant recurrent threat to public health and a significant burden on global economy, highlighting the need for developing more effective therapies. Natural killer (NK) cells play a pivotal role in the control of pulmonary IAV infection, however, little is known about the therapeutic potential of adoptively transferred NK cells for viral infections. Here, we investigated the antiviral activity of CYNK, human placental hematopoietic stem cell-derived NK cells, against IAV infection in vitro. Virus infection induced the expression of NK cell activating ligands on respiratory epithelial cells, resulting in enhanced recognition by CYNK cells. Upon co-culture with IAV-infected epithelial cells, CYNK exhibited elevated degranulation and increased production of IFN-γ, TNF-α and GM-CSF in a virus dose-dependent manner. Furthermore, CYNK showed virus dose-dependent cytotoxicity against IAV-infected cells. The antiviral activity of CYNK was mediated by NKp46 and NKG2D. Together, these data demonstrate that CYNK possesses potent antiviral function against IAV and warrant clinical investigations for adoptive NK cell therapies against viral infections.

CBLB ablation with CRISPR/Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy.

BACKGROUND: Tumors often develop resistance to surveillance by endogenous immune cells, which include natural killer (NK) cells. Ex vivo activated and/or expanded NK cells demonstrate cytotoxicity against various tumor cells and are promising therapeutics for adoptive cancer immunotherapy. Genetic modification can further enhance NK effector cell activity or activation sensitization. Here, we evaluated the effect of the genetic deletion of ubiquitin ligase Casitas B-lineage lymphoma pro-oncogene-b (CBLB), a negative regulator of lymphocyte activity, on placental CD34+ cell-derived NK (PNK) cell cytotoxicity against tumor cells. METHODS: Using CRISPR/Cas9 technology, CBLB was knocked out in placenta-derived CD34+ hematopoietic stem cells, followed by differentiation into PNK cells. Cell expansion, phenotype and cytotoxicity against tumor cells were characterized in vitro. The antitumor efficacy of CBLB knockout (KO) PNK cells was tested in an acute myeloid leukemia (HL-60) tumor model in NOD-scid IL2R gammanull (NSG) mice. PNK cell persistence, biodistribution, proliferation, phenotype and antitumor activity were evaluated. RESULTS: 94% of CBLB KO efficacy was achieved using CRISPR/Cas9 gene editing technology. CBLB KO placental CD34+ cells differentiated into PNK cells with high cell yield and >90% purity determined by CD56+ CD3- cell identity. Ablation of CBLB did not impact cell proliferation, NK cell differentiation or phenotypical characteristics of PNK cells. When compared with the unmodified PNK control, CBLB KO PNK cells exhibited higher cytotoxicity against a range of liquid and solid tumor cell lines in vitro. On infusion into busulfan-conditioned NSG mice, CBLB KO PNK cells showed in vivo proliferation and maturation as evidenced by increased expression of CD16, killer Ig-like receptors and NKG2A over 3 weeks. Additionally, CBLB KO PNK cells showed greater antitumor activity in a disseminated HL60-luciferase mouse model compared with unmodified PNK cells. CONCLUSION: CBLB ablation increased PNK cell effector function and proliferative capacity compared with non-modified PNK cells. These data suggest that targeting CBLB may offer therapeutic advantages via enhancing antitumor activities of NK cell therapies.

Human Placenta-Derived Mesenchymal Stromal-Like Cells Enhance Angiogenesis via T Cell-Dependent Reprogramming of Macrophage Differentiation.

Peripheral arterial disease (PAD) is a leading cause of limb loss and mortality worldwide with limited treatment options. Mesenchymal stromal cell (MSC) therapy has demonstrated positive effects on angiogenesis in preclinical models and promising therapeutic efficacy signals in early stage clinical studies; however, the mechanisms underlying MSC-mediated angiogenesis remain largely undefined. Here, we investigated the mechanism of action of human placenta-derived MSC-like cells (PDA-002) in inducing angiogenesis using mice hind limb ischemia model. We showed that PDA-002 improved blood flow and promoted collateral vessel formation in the injured limb. Histological analysis demonstrated that PDA-002 increased M2-like macrophages in ischemic tissue. Analysis of the changes in functional T cell phenotype in the draining lymph nodes revealed that PDA-002 treatment was associated with the induction of cytokine and gene expression signatures of Th2 response. Angiogenic effect of PDA-002 was markedly reduced in Balb/c nude mice compared with wild type. This reduction in efficacy was reversed by T cell reconstitution, suggesting T cells are essential for PDA-002-mediated angiogenesis. Furthermore, effect of PDA-002 on macrophage differentiation was also T cell-dependent as a PDA-002-mediated M2-like macrophage skewing was only observed in wild type and T cell reconstituted nude mice, but not in nude mice. Finally, we showed that PDA-002-treated animals had enhanced angiogenic recovery in response to the second injury when PDA-002 no longer persisted in vivo. These results suggest that PDA-002 enhances angiogenesis through an immunomodulatory mechanism involving T cell-dependent reprogramming of macrophage differentiation toward M2-like phenotype. Stem Cells 2017;35:1603-1613.