Dendritic cells (DC) are specialised leukocytes that comprise less than 1% of peripheral blood mononuclear cells (PBMC) and have the capacity to prime and direct a cytotoxic T lymphocyte (CTL) response against tumour-associated antigens (TAA). Their use as cellular adjuvants is being investigated for cancer immunotherapy. Multiple myeloma (MM) is a bone marrow-derived cancer, which may be amenable to DC immunotherapy. Many DC preparations have been considered for clinical immunotherapy protocols, including monocyte-derived DC, CD34+ stem cell-derived DC and blood DC (BDC). Relatively few studies have used BDC as the immunotherapeutic adjuvant. This is principally due to difficulties encountered in BDC isolation. The monoclonal antibody, CMRF-56, when used in a novel immunomagnetic positive selection procedure, has allowed the isolation of a BDC preparation, incorporating predominantly CD11c+ BDC and some activated B lymphocytes and monocytes. This thesis provides the first preliminary data to develop the CMRF-56+ BDC preparation as a clinically applicable immunotherapeutic modality for MM.
The cornerstone of a successful immune response against MM is the induction of a CTL response against TAA on the malignant plasma cells. In vitro induction of TAA-specific CTL responses is biologically and experimentally demanding and has not been widely reported after priming with immunomagnetically selected BDC. One obstacle is the reported poor in vitro viability of BDC. Initial studies in this thesis investigate the factors affecting BDC survival, purity, activation and cytokine secretion in the CMRF-56+ BDC preparation.
To optimise CTL priming by the CMRF-56+ BDC preparation, in vitro models of CTL generation were developed using HLA-A*0201 -restricted peptides derived from recall (influenza matrix protein (FMP)) and TAA (human telomerase reverse transcriptase (hTERT), MelanA/Mart1 ). Models incorporating CD4+ T lymphocyte-, poly I:C- and cognate antigen-activated and nonactivated CMRF-56+ BDC preparations were investigated for their ability to rapidly induce a TAA-specific CTL response after a single stimulation. No single model was found to be consistently superior to the others. A CTL priming model involving a single stimulation of autologous PBMC with MelanA/Mart 1 peptide-pulsed poly I:C activated or non-activated CMRF-56+ BDC preparations was found to be the most practical. The use of the TAA, hTERT peptide, in this model was found to be impractical as the antigen-specific responses were inconsistent and below the level of readout sensitivity.
The preferred model was used to compare the capacity of poly I:C-activated and non-activated CD11c+ BDC and MoDC to induce CTL responses to peptide recall (FMP) and TAA (MelanA/Mart1). Both DC preparations were equally effective as evaluated by tetramer, IFN-ϒ ELISPOT and 51Cr release assays. Differences were however noted in the effects of poly 1:C activation and NK and NKT cell populations in the responding PBMC. Sorted CMRF-56+ APC and MoDC were similarly investigated for their capacity to induce MelanA/Mart 1 peptide-specific responses. Again, both preparations were found to be equally effective, validating the development of CMRF-56+ BDC preparations as therapeutic vaccines for MM.
The use of RNA as an antigen source was investigated in healthy donors. Loading of the CMRF- 56+ BDC preparation with enhanced green fluorescent protein (EGFP)-encoding mRNA was established and optimised. It was shown that CMRF-56+ BDC have a specialised capacity to translate transfected EGFP mRNA into EGFP protein antigen in comparison to the contaminating B lymphocytes and monocytes in the CMRF-56+ BDC preparation. Different methods of activating and loading the preparation with FMP mRNA were evaluated for their effect on the presentation of processed FMP peptide antigen to FMP-specific CTL clones. FMP mRNA-loaded CMRF-56+ BDC preparations were used to expand FMP peptide-specific CTL responses in whole PBMC populations. Priming of autologous CTL with U266 MM. cell line total tumour RNA-loaded CMRF-56+ BDC preparations was unsuccessful.
Finally, the function of BDC in CMRF-56+ BDC preparations from healthy donors was compared with those from MM patients. TruCOUNTTM technology was used to evaluate the BDC subset composition of the two donor groups. It was found that CD11c+/CD16- and CD11c-/CD123+ BDC subsets were reduced in MM. patients. In addition, CM.RF-56 antigen upregulation was impaired on BDC in MM patients compared to healthy donors. Despite these findings, CMRF-56+ BDC preparations could be generated from MM patients and were able to induce recall, naive and allogeneic proliferative MHC class II-restricted and recall and TAA-specific MHC class 1- restricted immune responses in MM patients. This research forms the rationale for an ongoing Phase I clinical trial ofCMRF-56+ BDC immunotherapy in MM patients.