Identifying the optimal donor for natural killer cell adoptive therapy to treat paediatric B- and T-cell acute lymphoblastic leukaemia.

Objectives: Natural killer (NK) cells are an attractive source of cells for an 'off the shelf' cellular therapy because of their innate capacity to target malignant cells, and ability to be transferred between donors and patients. However, since not all NK cells are equally effective at targeting cancer, selecting the right donor for cellular therapy is critical for the success of the treatment. Recently, cellular therapies utilising NK cells from cytomegalovirus (CMV)-seropositive donors have been explored. However, whether these NK cells are the best source to treat paediatric acute lymphoblastic leukaemia (ALL) remains unclear. Methods: Using a panel of patient-derived paediatric B- and T-ALL, we assessed the ability of NK cells from 49 healthy donors to mount an effective functional response against these two major subtypes of ALL. Results: From this cohort, we have identified a pool of donors with superior activity against multiple ALL cells. While these donors were more likely to be CMV+, we identified multiple CMVneg donors within this group. Furthermore, NK cells from these donors recognised B- and T-ALL through different activating receptors. Dividing functional NK cells into 29 unique subsets, we observed that within each individual the same NK cell subsets dominated across all ALL cells. Intriguingly, this occurred despite the ALL cells in our panel expressing different combinations of NK cell ligands. Finally, we can demonstrate that cellular therapy products derived from these superior donors significantly delayed leukaemia progression in preclinical models of ALL. Conclusions: We have identified a pool of superior donors that are effective against a range of ALL cells, representing a potential pool of donors that can be used as an adoptive NK cell therapy to treat paediatric ALL

Characterisation of small leucine rich proteins gene and protein expression in mesenchymal stem cell differentiation into osteoblasts, adipocytes and chondrocytes

This thesis is directed to understanding the role of Small Leucine Rich Proteins (SLRPs) in the cell biology of mesenchymal tissue in particular bone and cartilage. SLRPs are a family of 17 biologically active macromolecules which form the extracellular matrix in a variety of tissues and may play a role in bone and cartilage biology and diseases, in particular osteoporosis. It was hypothesised that: 1) The gene and protein expression of specific SLRPs will be up-regulated during the development of bone and cartilage. 2) During osteogenesis, the location of these SLRPs shows a pattern of distribution within the extracellular matrix. 3) Osteogenesis related SLRPs are specific to the cell development of that tissue. To investigate the first hypothesis, a bioinformatics study of a human osteosarcoma cell was initially used to determine the gene expression on all 17 SLRP members. The six highest expressed members Lumican, Epiphycan, Tskushi, Biglycan Decorin, and Osteomodulin (OMD) were selected for further analysis. To investigate the second hypothesis, the gene expression of these six selected members were analysed using real time quantitative reverse transcriptase polymerase chain reaction in both long term (up to 28 days) and short term (up to 7 days) osteogenesis of donor matched human adipose and bone marrow mesenchymal stem cells. These results showed the increase in expression of OMD in osteogenic stimulated media. As a result of these studies OMD was selected for further study, as a potential biomarker of osteoblasts. The gene expression of OMD was only increased significantly in osteoblast-like cells compared to other mesenchymal stem cell lineages including cartilage and adipose tissue. Protein expression of OMD was further investigated by western blotting. This was followed by confocal microscopy to further understand the expression of this protein. It was found through both methods that the protein expression of OMD was increased during osteogenesis, reflecting the gene expression previously observed. In conclusion, it was shown that the gene and protein expression of OMD was increased specifically during osteogenesis, and therefore could be used as a marker of osteogenesis of mesenchymal stem cells. Furthermore, its role in osteogenic development should be further studied to understand its role in osteogenesis

Impaired T cell proliferation by ex vivo BET-inhibition impedes adoptive immunotherapy in a murine melanoma model

Activation of naïve CD8+ T cells stimulates proliferation and differentiation into cytotoxic T-lymphocytes (CTLs). Adoptive T Cell Therapy (ACT) involves multiple rounds of ex vivo activation to generate enough CTLs for reinfusion into patients, but this drives differentiation into terminal effector T cells. Less differentiated CTL populations, such as stem cell memory T cells, are more ideal candidates for ACT because of increased self-renewal and persistent properties. Ex vivo targeting of T cell differentiation with epigenetic modifiers is a potential strategy to improve cytotoxic T-lymphocyte (CTL) generation for ACT. We established a pipeline to assess the effects of epigenetic modifiers on CD8+ T cell proliferation, differentiation, and efficacy in a preclinical melanoma model. Single treatment with epigenetic modifiers inhibited T cell proliferation in vitro, producing CD44hiCD62Lhi effector-like T cells rather than a stem cell memory T cell phenotype. Most epigenetic modifying agents had no significant effect on ACT efficacy with the notable exception of the bromodomain and extraterminal (BET)-inhibitor JQ1 which was associated with a decrease in efficacy compared to unmodified T cells. These findings reveal the complexity of epigenetic targeting of T cell differentiation, highlighting the need to precisely define the epigenetic targeting strategies to improve CTL generation for ACT

CD4+ T cell-induced inflammatory cell death controls immune-evasive tumours

Most clinically applied cancer immunotherapies rely on the ability of CD8+ cytolytic T cells to directly recognize and kill tumour cells1,2,3. These strategies are limited by the emergence of major histocompatibility complex (MHC)-deficient tumour cells and the formation of an immunosuppressive tumour microenvironment4,5,6. The ability of CD4+ effector cells to contribute to antitumour immunity independently of CD8+ T cells is increasingly recognized, but strategies to unleash their full potential remain to be identified7,8,9,10. Here, we describe a mechanism whereby a small number of CD4+ T cells is sufficient to eradicate MHC-deficient tumours that escape direct CD8+ T cell targeting. The CD4+ effector T cells preferentially cluster at tumour invasive margins where they interact with MHC-II+CD11c+ antigen-presenting cells. We show that T helper type 1 cell-directed CD4+ T cells and innate immune stimulation reprogramme the tumour-associated myeloid cell network towards interferon-activated antigen-presenting and iNOS-expressing tumouricidal effector phenotypes. Together, CD4+ T cells and tumouricidal myeloid cells orchestrate the induction of remote inflammatory cell death that indirectly eradicates interferon-unresponsive and MHC-deficient tumours. These results warrant the clinical exploitation of this ability of CD4+ T cells and innate immune stimulators in a strategy to complement the direct cytolytic activity of CD8+ T cells and natural killer cells and advance cancer immunotherapies.Title in Web of Science: CD4(+) T cell-induced inflammatory cell death controls immune-evasive tumours</p