The ability of dendritic cells (DCs) to boost an antigen-specific immune response is utilized in several cancer immunotherapy strategies including therapeutic vaccination using long peptides. Naked peptides are rapidly degraded and oil-based delivery strategies trap immune cells to unwanted sites or have inappropriate adjuvant properties. To enhance the uptake of cancer or viral T cell epitopes and subsequent activation of DCs we make use of circulating antibodies to mount cellular responses against tumor antigens of interest by conjugating a B cell epitope to a T cell epitope. The conjugation of the two greatly improves antigen uptake and concomitant activation of the same DC in contrast to naked long peptides which are not conjugated. Our identified B cell epitope of choice is derived from tetanus toxin and can be targeted by tetanus-specific antibodies boosted by a standard tetanus toxoid vaccine. We have applied a modified chandler loop model preserving intact cascade systems to characterize how the tetanus-peptide conjugated vaccine is targeted to human immune cells. The B cell-T cell conjugate is taken up by human monocytes and blood DCs in an antibody-dependent manner. Rather than FcγRs, the internalization of the antigen appears to be partly mediated through the classical pathway of the complement system. Tetanus-CMV conjugates, containing a T cell epitope from the pp65 protein of cytomegalovirus (CMV), strongly reactivates memory T cells when analyzed in blood from donors with CMV-specific T cells. The CMV-specific T cells rapidly produce IFNγ and TNF in response to the conjugate illustrating that the uptake of the conjugate leads to activation of antigen-specific T cells. Uptake as well as T cell activation occurs at low concentrations of the SLP conjugate, superior to a conjugate lacking the tetanus-sequence as well as to SLPs with or without additional adjuvant (LPS). Of importance, when the B and T cell epitopes are separate entities but mixed, CMV-specific T cells are not activated, illustrating the requirement of conjugating the two. Our data show that we have a unique delivery system for peptide based vaccines that can aid induction of human T cell responses, and may potentiate immune responses in cancer patients.We are now actively working on a prostate cancer vaccine candidate using this novel loading/adjuvant technology. Citation Format: Erika A. Fletcher, Wendy van Maren, Robert Cordfunke, Jasper Dinkelaar, Ricardo Castelli, Jeroen Codee, Gijs van de Marel, Cornelis J. Melief, Ferry Ossendorp, Jan Wouter Drijfhout, Sara Mangsbo. T cell responses to peptide-epitopes can be boosted by immune complexes of circulating anti-tetanus antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1693. doi:10.1158/1538-7445.AM2017-1693
A new type of cytocentrifuge has been developed in which the sedimentation process of the cells onto the slides is separated from the draining of the sedimentation fluid. This is realised by electrically controlled valves which can be closed and opened while the centrifuge is running. Sedimentation is carried out with closed valves, draining of adhering medium with open valves. The preparations, freed of adhering medium by the centrifugal force can be taken out and the cells can be fixed. Alternatively the valves can be closed again and fixative can be introduced through a central well, the cells still being under the influence of the centrifugal force. With subsequent draining of the fixative and introduction of washing and staining solutions through the central well, the whole process from sedimentation to staining can be carried out in the running centrifuge. The process seems well suited for complete automation. Using dilution series from a suspension of human buffy coat cells counted in a Buerker chamber, the cell counts in the centrifuge preparations showed virtually total recovery of cells, with no apparent selection or specific distribution of cell types. Draining of the sedimentation and fixative fluids at a slow rate was found to be vital for optimal recovery of cells. The morphology of different cell types sedimented on the slide was excellent. The flattening of nuclei thorugh gravity was studied by cytophotometry of Feulgen-stained leucocytes. The nuclear area of these cells was found to be approximately double that from cells in identically stained classical smears. With this type of valve-centrifuge a quantitative and unbiased recovery of uniformly spread and flattened cells on coverslips or slides may be obtained, thus making the procedure well suited to automated analysis based on cytophotometric principles and morphometric pattern recognition.
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