Recent developments in the expansion and manipulation of primary NK cells has allowed this source of effective anti-tumour cells to be exploited for cell-based cancer immunotherapies. While ex vivo expanded primary NK cells are highly effective in the treatment of haematological malignancies, their efficacy against the solid tumour has been limited due to the presence of immune-regulatory factors in the tumour microenvironment. These factors can abrogate NK cell function by down regulating the expression of NK activating receptors, thus preventing these highly cytotoxic effector cells from activating in response to tumour challenge. Our work explores whether the expression of a tumour specific chimeric antigen receptor (CAR) on ex vivo expanded primary NK cells would allow the lost activatory signalling to be recouped, and regain their efficacy against the solid tumour. Unfortunately, the use of primary NK cells as effectors in CAR based cell immunotherapies has been hampered by the technical limitations of producing large numbers of CAR positive primary NK cells. This has led many researchers to utilise the NK-92 cell line instead of primary cells. We demonstrate that ex vivo expanded primary CAR NK cells can be produced efficiently and demonstrate higher anti-tumour functionality than CAR NK-92. Finally, due to the intricacies of NK cell biology, they are able to effectively discriminate between healthy and malignant targets thus preventing their cytotoxic function from being directed towards the incorrect target. This could be a key advantage in the use of primary NK cells over T cells as effectors of CAR as the off-tumour/on-target adverse effects seen with CAR T cells has severely hampered this clinical strategy. We have shown that CAR T cells but not CAR NK cells are reactive towards phenotypically non-malignant, clinically relevant, healthy cells expressing the CAR target. / Thesis / Master of Science (MSc)

Indiana University-Purdue University Indianapolis (IUPUI) / Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We previously reported that chemotherapy-treated lymphoma patients acquire a deficiency of Signal Transducer and Activator of Transcription 4 (STAT4), which results in defective IFNy production during clinical immunotherapy. With the goal of further improvement in cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that exhibits immunostimulatory effects on natural killer cells (NKCs). Peripheral blood mononucleated cells (PBMCs) from healthy donors and chemotherapy-treated lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-cultuted NK cells demonstrated synergistic effects in the induction of IFNG and genes involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNy production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines have higher tumoricidal activity than those stimulated with cytokines alone using in vitro tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation at target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.

Indiana University-Purdue University Indianapolis (IUPUI) / Vaccinia virus (VV) is the prototypical member of the orthopoxvirus genus of the Poxviridae family, and is currently being evaluated as a vector for vaccine development and cancer cell-targeting therapy. Despite the importance of studying poxvirus effects on the human immune system, reports of the direct interactions between poxviruses and primary human leukocytes (PHLs) are limited. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias towards binding and infecting monocytes among PHLs. VV binding strongly co-localized with lipid rafts on the surface of these cell types, even when lipid rafts were relocated to the cell uropods upon cell polarization. In humans, monocytic and professional antigen-presenting cells (APCs) have so far only been reported to exhibit abortive infections with VV. We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, were permissive to VV replication. The majority of virions produced in MDMs were extracellular enveloped virions (EEV). Visualization of infected MDMs revealed the formation of VV factories, actin tails, virion-associated branching structures and cell linkages, indicating that infected MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. Classical activation of MDMs by LPS plus IFN-γ stimulation caused no effect on VV replication, whereas alternative activation of MDMs by IL-10 or LPS plus IL-1β treatment significantly decreased VV production. The IL-10-mediated suppression of VV replication was largely due to STAT3 activation, as a STAT3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data indicate that PHL subsets express and share VV protein receptors enriched in lipid rafts. We also demonstrate that primary human macrophages are permissive to VV replication. After infection, MDMs produced EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread.