Induction of Anti-Tumour Immune Responses by Dendritic Cells Generated with Flt3-Ligand

Abstract

<p>Dendritic cells (DCs) are potent antigen presenting cells that are crucial for the initiation of an immune response. Due to this property, DCs have been used as the basis of cancer vaccines in immunotherapy. In clinical trials, DCs used for vaccination are commonly generated by culturing monocytes from each patients' blood with the growth factors GM-CSF and IL-4 (GMCSF/IL-4 DCs). The DCs generated are reportedly similar to those that arise in vivo during inflammation and trials using these DCs have been met with some success. A recently developed method of generating mouse or human DCs in vitro, involves the culture of bone marrow (BM) precursors with the cytokine Flt3-Ligand (Flt3L-DCs). Flt3L-DCs differ substantially in phenotype from GMCSF/IL-4 DCs and more closely resemble steady-state DCs in vivo. This thesis investigated the suitability of Flt3L-DCs for cancer immunotherapy. Murine BM cells cultured in Flt3L generated three DC subsets. These consisted of plasmacytoid DCs (pDCs) that were CD11c⁺B220⁺, and conventional DCs (cDCs) that were CD11c⁺B220⁻ and could be further subdivided into CD11bhigh and CD24high populations. We observed that cDCs responded to stimulation with a variety of Tolllike receptor (TLR) agonists, as evaluated by the up-regulation of activation markers. However pDCs responded to the agonist CpG at a higher extent compared to all other agonists used. In addition, combining TLR agonists could further enhance the activation of Flt3L-DCs. Among all combinations tested, Pam3Cys/Poly I:C was the most optimal at inducing the secretion of inflammatory cytokines IL-12p70 and TNF-α. Furthermore, Pam3Cys/Poly I:C stimulated Flt3L-cDCs exhibited a greater ability at inducing CD4⁺ T cell proliferation and cross-presentation of soluble antigen to CD8⁺ T cells, compared to Flt3L-cDCs activated with the respective individual agonists. Studies have shown that GM-CSF DCs are highly reliant on glycolytic metabolism during activation in order to up-regulate activation markers. Therefore, we also characterised Flt3L-cDCs for their ability to up-regulate activation markers following stimulation with the agonist LPS and treatment with the glycolysis inhibitor 2-Deoxy-D-glucose (2-DG). In line with previous reports, DCs generated in culture with GMCSF/IL-4 were unable to up-regulate activation markers at all the 2-DG concentrations used. In contrast, Flt3L-cDCs appeared to have a threshold level where only high concentrations of 2-DG inhibited their ability to up-regulate activation markers. This result indicates that steady-state and inflammatory DCs preferentially use different metabolic pathways upon activation. The ability of optimally activated Flt3L-cDCs and GMCSF/IL-4 DCs to confer tumour protection was also examined. While unstimulated Flt3L-cDCs or GMCSF/IL-4 DCs could protect mice from tumour growth, vaccination with activated DCs from either population was required for complete tumour protection. Furthermore, we found that even in optimal conditions of activation, 1x10⁵ Flt3LcDCs were required for maximal tumour protection, whereas 1x10⁴ GMCSF/IL-4 DCs provided sufficient protection. These findings indicate that Flt3L-cDCs can be used as the basis of a therapeutic cancer vaccine, but are not superior to GMCSF/IL- 4 DCs. Further studies are required to establish conditions that can enhance the efficacy of Flt3L-cDCs.</p>