In the post smallpox era, vaccinia virus (VACV) has emerged as an important candidate vaccine vector. As yet, the binding receptors and entry mechanisms utilised by the two infectious forms, IMV and EEV, in dendritic cells (DCs) are unknown. We have investigated the interactions between VACV and C-type lectin receptors (CLRs) that are known to be utilised by many other viruses for binding and entry in DCs. Using a variety of CLR ligands and inhibitors we were unable to inhibit IMV or EEV binding to MDDCs and we conclude that they do not bind to CLRs. We have also investigated VACV entry in MDDCs and show that both IMV and EEV enter MDDCs via an endocytic pathway. Using a variety of drugs that inhibit cellular processes we found IMV and EEV entry to be actin- and calcium-dependent. EEV entry was also cholesterol- and energy-dependent, whereas IMV entry was only partially dependent on these factors. Both IMV and EEV colocalised with endolysosomal markers. This data suggests that EEV may enter DCs via caveolin-mediated endocytosis whereas IMV entry can occur via multiple complementary mechanisms, including endocytosis and fusion. Macropinocytosis may also constitute a minor route of entry for IMV as entry was partially inhibited by dimethyl amiloride and the virus colocalised with dextran. Finally we have provided a comprehensive flow cytometric analysis of Toll-like receptor (TLR) expression at the protein level in MDDCs and monocyte-derived Langerhans cells (MDLCs) as models for different myeloid DC subsets. We found TLR expression to be cell type-specific and MDDCs expressed the full repertoire of TLRs 1-9, including small amounts of TLR8 and TLR9 on the cell surface. The expression of these TLRs that recognise nucleic acids on the surface of cells may constitute an early warning system for signalling the presence of viral invaders that would normally subvert the function of DCs. We also found TLR expression in mature cells to be dependent on the nature of the maturation stimulus (lipopolysaccharide versus cytokine/prostaglandin cocktail) and VACV infection induced profound down-regulation of all TLRs. These findings will have important implications for the rational design of VACV-vectored vaccines.

The Mater Medical Research Institute proposes to undertake a Phase I clinical trial using CMRF-56 immunoselected blood dendritic cells (BDC) loaded with control and myeloma-associated tumour peptide antigens for the treatment of multiple myeloma (MM) patients with minimal residual disease. This thesis describes some of the fundamental pre-clinical in vitro experiments undertaken in preparation for this trial so as to maximise the potential of this vaccine to induce myeloma-specific immune responses. These experiments involved determining the parameters for optimal activation of the CMRF-56 immunoselected cell preparation and exploring the potential of novel myeloma peptide antigens to induce anti-myeloma cytotoxic T lymphocyte (CTL) responses. CMRF-56 immunoselected cell preparations, containing predominately myeloid BDC, monocytes and B cells, were prepared from both healthy donors and myeloma patients. Activation of this preparation with granulocyte macrophage colony stimulating factor (GM-CSF) was found to increase co-stimulatory molecule expression by and survival of BDC, improve peptide- and lysate-specific CTL induction, and, in combination with prostaglandin E2 (PGE2), improve chemokine-specific migration of BDC. Following optimisation of in vitro CTL generation protocols, GM-CSF activated CMRF-56 immunoselected cells were examined for their ability to induce myeloma-specific immunity. Using lysate from myeloma cell line U266 as an antigen source, a polyclonal T cell pool was generated within which peptide specific CTL recognising myeloma antigens Muc1, HM1.24/BST2, DKK-1 and CT-7/MAGE-C1 could be identified. Furthermore, GM-CSF activated CMRF-56 immunoselected cells pulsed with HLA-A*201 restricted peptides derived from Muc1, HM1.24/BST2 and CT-7/MAGE-C1 could induce CTL capable of lysing both peptide- and myeloma cell line targets in both healthy donors and myeloma patients. These results provide the first evidence of immunogenic HLA-A*201 restricted epitopes of novel myeloma antigen CT-7/MAGE-C1. The data collected in this study supports the application of GM-CSF activated CMRF-56 immunoselected cells loaded with defined myeloma peptide antigens for the therapeutic vaccination of MM patients with minimal residual disease.

Acute graft versus host disease (aGVHD) affects more than 40% of patients undergoing haematopoietic stem cell transplantation. aGVHD occurs after transplantation of donor haematopoietic cells into hosts incapable of rejecting the donor cells, when donor T cells attack host tissue. Despite extensive efforts, aGVHD remains problematic to prevent and difficult to control. Current therapies to prevent aGVHD induce profound immunosuppression, leaving patients at increased risk of infection and leukaemic relapse. Dendritic cells (DC) are professional antigen presenting cells of haematopoietic origin and are the primary stimulators of the immune system, uniquely being able to activate naïve T cells. A growing body of evidence suggests that DC are responsible for the stimulation of the donor T cells which cause aGVHD. I have used a model of aGVHD which utilizes conditioned severe combined immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMC). In this model human CD4+ T cells appear to be responsible for an aGVHD-like syndrome which results in death 15-30 days post transplant. I have shown, using in vitro depletion of individual populations, that other subpopulations of human PBMC did not affect the survival of the mice. I have also demonstrated that human DC are required for the induction of aGVHD in the majority of mice. This novel finding validated the use of this model to test the primary hypothesis; that antibody mediated depletion of DC would prevent aGVHD. The murine IgM monoclonal antibody (Mab), CMRF-44 Mab, is specific for an unknown molecule expressed on the surface of activated human DC. Previous work had shown that when mixed lymphocyte reaction stimulator cells were depleted of CMRF-44+ cells, there was a significant reduction in the proliferation of responder cells. Here I tested the efficacy of CMRF-44 as a therapy for the prevention of aGVHD in the model. CMRF-44 Mab did not improve survival of mice treated with human PBMC, despite recent data showing that CMRF-44 expression on DC was predictive of aGVHD in patients. In vitro depletion of CMRF-44+ cells from human PBMC prior to transplantation also did not reduce incidence of aGVHD. An alternate target for the depletion of human DC was CD83 which is also expressed on the surface of activated human DC. I generated a rabbit polyclonal antibody using a human CD83 fusion protein, which was then affinity purified in a multi-step process which yielded only antibody specific for human CD83. Treatment with this antibody greatly improved survival of transplanted mice. Further experiments showed that anti-CD83 treatment did not abrogate human leucocytes including CD8+ memory T cells suggesting that a therapy using an anti-CD83 antibody has the potential to prevent aGVHD without the immunosuppression associated with current anti-aGVHD therapies. The work described here has validated the use of a human mouse chimeric model as an in vivo assay of human DC function and shown that targeting CD83 has the potential to reduce the incidence of clinical aGVHD whilst preserving donor memory T cells.

Interest in cellular immunotherapy has increased with the recognition of the pivotal role that dendritic cells (DC) play in the adaptive immune system. The preparation of DC to present tumour antigens and subsequent induction of tumour specific T cells have been widely documented. This thesis studied the ability of cord blood (CB) stem cells to differentiate into functional CD34+DC, followed by the optimisation of electroporation of RNA into these cells. Total RNA derived from a leukaemic cell line and a primary human leukaemic sample was electroporated into CD34+DC DC and we were able to generate anti-leukaemic cytotoxic T lymphocytes (CTL). The CTL specifically targeted leukaemia but not normal cells. While the in vitro data showed promising results of the CTL specificity, a NOD-SCID model of human ALL was established to allow the CTL to be tested in vivo. We established a reproducible model of human ALL in NOD-SCID mouse using four primary human ALL samples. The adoptively transferred anti-leukaemic CTL into the ALL bearing NOD-SCID mice showed that ALL engraftment was significantly delayed. However, the addition of total RNA loaded CD34+DC DC did not enhance the in vivo CTL effect. Lastly, by dissecting the CTL response, we found that the polyclonal CTL were targeting survivin, HM1.24 and CT-7 antigens. The CTL clones generated from these polyclonal CTL showed high specificity for leukaemia but not normal cells. In conclusion, these preliminary data support the use of total RNA electroporated CD34+DC as a means of inducing anti-leukaemic CTL, and have demonstrated the efficacy of the CTL in a NOD-SCID model of ALL. This study has also provided insight into the polyclonal CTL response and future studies will likely continue along this path.

[Truncated abstract] A paradox exists in tumour immunology whereby progressive tumour growth exists in parallel with an anti-tumour T cell response. This defective T cell response is thought to result from the induction of T cell tolerance and/or tumour induced immunosuppression, which act to inhibit the activation, differentiation and function of tumour-specific CD8+ T cells. Dendritic cells (DCs) are professional antigen presenting cells (APCs) that are critical to the generation of effective CTL; however their function and phenotype is often defective or altered in tumour-bearing hosts, which may limit their capacity to mount an effective tumour specific T cell response. In this thesis, the role of DCs in the cross-presentation of tumour antigen was assessed in terms of their APC function, migration and location. In doing so the intention was to gain insight into the early processes that potentially contribute to the development of an ineffective anti-tumour immune response. This study examined cross-presentation of the nominal tumour antigen, influenza A hemagglutinin (HA) expressed by the murine malignant mesothelioma cell line, AB1-HA. Cross-presentation was predominantly restricted to the local draining lymph nodes throughout tumour growth and was mediated by CD8a+ and CD8a- DCs. This results in an ineffective CTL response due to the lack of DC activation and the presence of potentially immunosuppressive B7 molecules. However, the capacity of the CD8a- DC subset to cross-present antigen suggested a role for migratory tumour-resident DCs in this process. Analysis of tumour infiltrating DCs showed that they were paralysed in their capacity to cross-present tumour antigen and were immobilised at the tumour site. Conversely, cross-presentation of tumour antigen in the local draining lymph node was dependent on the continuous traffic of antigen from the tumour microenvironment. In this vein, small numbers of metastatic tumour cells were detected in the draining lymph nodes, however their isolation was dependent on the removal of DCs and T cells, suggesting immune control of metastatic spread. Thus, tumour cells may be the source of antigen for cross-presentation by DCs in the tumour draining lymph nodes. .... In conclusion, the results presented in this thesis support a role for DCs in the generation of tumour-specific T cell responses that fail to control tumour growth. In addition the results provide a basis for further investigation into the effects of chemotherapy on the source and form of tumour antigen for cross-presentation by specific DC subsets in the tumour bearing host. These findings may have important implications for the development of future anti-cancer immune therapies targeting DCs.

The Mater Medical Research Institute proposes to undertake a Phase I clinical trial using CMRF-56 immunoselected blood dendritic cells (BDC) loaded with control and myeloma-associated tumour peptide antigens for the treatment of multiple myeloma (MM) patients with minimal residual disease. This thesis describes some of the fundamental pre-clinical in vitro experiments undertaken in preparation for this trial so as to maximise the potential of this vaccine to induce myeloma-specific immune responses. These experiments involved determining the parameters for optimal activation of the CMRF-56 immunoselected cell preparation and exploring the potential of novel myeloma peptide antigens to induce anti-myeloma cytotoxic T lymphocyte (CTL) responses. CMRF-56 immunoselected cell preparations, containing predominately myeloid BDC, monocytes and B cells, were prepared from both healthy donors and myeloma patients. Activation of this preparation with granulocyte macrophage colony stimulating factor (GM-CSF) was found to increase co-stimulatory molecule expression by and survival of BDC, improve peptide- and lysate-specific CTL induction, and, in combination with prostaglandin E2 (PGE2), improve chemokine-specific migration of BDC. Following optimisation of in vitro CTL generation protocols, GM-CSF activated CMRF-56 immunoselected cells were examined for their ability to induce myeloma-specific immunity. Using lysate from myeloma cell line U266 as an antigen source, a polyclonal T cell pool was generated within which peptide specific CTL recognising myeloma antigens Muc1, HM1.24/BST2, DKK-1 and CT-7/MAGE-C1 could be identified. Furthermore, GM-CSF activated CMRF-56 immunoselected cells pulsed with HLA-A*201 restricted peptides derived from Muc1, HM1.24/BST2 and CT-7/MAGE-C1 could induce CTL capable of lysing both peptide- and myeloma cell line targets in both healthy donors and myeloma patients. These results provide the first evidence of immunogenic HLA-A*201 restricted epitopes of novel myeloma antigen CT-7/MAGE-C1. The data collected in this study supports the application of GM-CSF activated CMRF-56 immunoselected cells loaded with defined myeloma peptide antigens for the therapeutic vaccination of MM patients with minimal residual disease.

Thesis (MScMed)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 5 uppsatser.