Cytotoxic T lymphocytes (CTL) provide protection against pathogens and tumors. In addition, experiments in mouse models have shown that CTL can also kill antigen-presenting dendritic cells (DC), reducing their ability to activate primary and secondary CD8+ T cell responses. In contrast, the effects of CTL-mediated killing on CD4+ T cell responses have not been fully investigated. Here we use adoptive transfer of TCR transgenic T cells and DC immunization to show that specific CTL significantly inhibited CD4+ T cell proliferation induced by DC loaded with peptide or low concentrations of protein antigen. In contrast, CTL had little effect on CD4+ T cell proliferation induced by DC loaded with high protein concentrations or expressing antigen endogenously, even if these DC were efficiently killed and failed to accumulate in the lymph node (LN). Residual CD4+ T cell proliferation was due to the transfer of antigen from carrier DC to host APC, and predominantly involved skin DC populations. Importantly, the proliferating CD4+ T cells also developed into IFN-γ producing memory cells, a property normally requiring direct presentation by activated DC. Thus, CTL-mediated DC killing can inhibit CD4+ T cell proliferation, with the extent of inhibition being determined by the form and amount of antigen used to load DC. In the presence of high antigen concentrations, antigen transfer to host DC enables the generation of CD4+ T cell responses regardless of DC killing, and suggests mechanisms whereby CD4+ T cell responses can be amplified.

The cytokines granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 are frequently used for generating dendritic cells (DCs) for therapeutic vaccination against cancer. These in vitro DCs share several characteristics with inflammatory monocyte-derived DCs in vivo. In contrast, culture of bone marrow cells in Flt3-ligand (Flt3L) generates a heterogeneous population of DCs, which comprise conventional DCs (cDCs) and plasmacytoid DCs similar to the steady-state populations found in vivo. Although previous studies have identified combinations of toll-like receptor ligands (TLR-Ls) that induce optimal activation of GM-CSF/IL-4 DCs in vitro, the conditions for optimal activation of Flt3L-DCs have not been established. In this study, we show that various combinations of the TLR-Ls Pam3Cys, Poly I:C, lipopolysaccharide, and CpG all increased Flt3L-DC maturation, but only the combination of Pam3Cys/Poly I:C showed a trend to enhanced production of IL-12p70 and tumor necrosis factor-α by cDCs. Pam3Cys/Poly I:C-treated cDCs also displayed enhanced capacity to present antigen to CD4(+) T cells, and cross-present to CD8(+) T cells, increasing T-cell proliferation in vitro. Within a prophylactic vaccination setting, cDCs activated with Pam3Cys/Poly I:C conferred tumor protection in mice. However, the numbers of cDCs required for protection were higher than the numbers of optimally activated GM-CSF/IL-4 DCs required for a similar effect. Our results show that combined TLR stimulation can enhance both the phenotypic and functional properties of Flt3L-DCs, but even under conditions of optimal activation these cells are not superior in activity to GM-CSF/IL-4 DCs in vivo.

<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>