novel-protocol-for-generating-physiologic-immunogenic-dendritic-cells

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells

Extracorporeal photochemotherapy (ECP) is a widely used cancer immunotherapy for cutaneous T cell lymphoma (CTCL), operative in over 350 university ...

Yale University School of Medicine

Ex vivo exposure of malignant human T cells to photoactivated 8-methoxypsoralen (8-MOPa), followed by their i.v. return, appears to vaccinate patients against tumor-associated antigens of cutaneous T cell lymphoma in a procedure termed photopheresis. The molecular basis of this Food and Drug Administration-approved therapy, administered in 100 centers worldwide, is unclear. Most of the attention to the mechanism of action of the drug has focused on its capacity to form covalent cross-links with pyrimidine bases of DNA, thereby inhibiting cellular proliferation. Because immunologic factors appear to be important in the clinical response and could potentially serve as a model for immunotherapy of other malignancies, we explored the possibility that 8-MOP-treated cells display increased quantities of antigenic peptides at their cell surface. In this work, human B-lymphoblastoid tissue culture lines were exposed to 8-MOPa and expression of cell surface class I major histocompatibility complex proteins assessed, since CD8 T cells recognize antigenic moieties in the context of class I molecules. A peak 200-300% increase in MHC class I expression in 8-MOPa-treated cells occurred at 20 hr. 8-MOPa was far more effective in inducing this increase in class I MHC than other modalities, including mitomycin C, gamma-irradiation, ultraviolet B or heat or cold shock. This increase in surface class I MHC molecules appears to be driven by the degradation of cytoplasmic proteins into small peptides, followed by the transport of these peptides to MHC class I molecules in the endoplasmic reticulum. The data suggest that 8-MOPa treatment may augment the immunogenicity of tumor and/or antigen-presenting cells by enhancing processing and transport of class I MHC antigenic peptides.

Photoactivated 8-methoxypsoralen treatment causes a peptide-dependent increase in antigen display by transformed lymphocytes.

Here we report a "configuration-dependent" mechanism of action for IL-15:IL-15Rα (heterodimeric IL-15 or hetIL-15) where the manner by which IL-15:IL-15Rα molecules are presented to target cells significantly affects its function as a vaccine adjuvant. Although the cellular mechanism of IL-15 trans-presentation via IL-15Rα and its importance for IL-15 function have been described, the full effect of the IL-15:IL-15Rα configuration on responding cells is not yet known. We found that trans-presenting IL-15:IL-15Rα in a multivalent fashion on the surface of antigen-encapsulating nanoparticles enhanced the ability of nanoparticle-treated dendritic cells (DCs) to stimulate antigen-specific CD8(+) T cell responses. Localization of multivalent IL-15:IL-15Rα and encapsulated antigen to the same DC led to maximal T cell responses. Strikingly, DCs incubated with IL-15:IL-15Rα-coated nanoparticles displayed higher levels of functional IL-15 on the cell surface, implicating a mechanism for nanoparticle-mediated transfer of IL-15 to the DC surface. Using artificial antigen-presenting cells to highlight the effect of IL-15 configuration on DCs, we showed that artificial antigen-presenting cells presenting IL-15:IL-15Rα increased the sensitivity and magnitude of the T cell response, whereas IL-2 enhanced the T cell response only when delivered in a paracrine fashion. Therefore, the mode of cytokine presentation (configuration) is important for optimal immune responses. We tested the effect of configuration dependence in an aggressive model of murine melanoma and demonstrated significantly delayed tumor progression induced by IL-15:IL-15Rα-coated nanoparticles in comparison with monovalent IL-15:IL-15Rα. The novel mechanism of IL-15 transfer to the surface of antigen-processing DCs may explain the enhanced potency of IL-15:IL-15Rα-coated nanoparticles for antigen delivery. 

Configuration-dependent Presentation of Multivalent IL-15:IL-15Rα Enhances the Antigen-specific T Cell Response and Anti-tumor Immunity.

Extracorporeal photochemotherapy (ECP) is a cancer immunotherapy for cutaneous T-cell lymphoma (CTCL) operative in more than 350 centers worldwide. Although its efficacy and favorable safety profile have driven its widespread use, elucidation of its underlying mechanism has been difficult. In this study, we identify the principal contributors to the anticancer immunotherapeutic effects of ECP, with the goal of enhancing potency and broadening applicability to additional malignancies. First, we scaled down the clinical ECP leukocyte-processing device to mouse size. Second, we used that miniaturized device to produce a cellular vaccine that regularly initiated therapeutic antimelanoma immunity. Third, we individually subtracted key factors from either the immunizing inoculum or the treated animal to ascertain their contribution to the  antimelanoma response. Platelet-signaled monocyte-to-dendritic cell (DC) differentiation followed by sorting/processing/presentation of tumor antigens derived from internalized apoptotic tumor cells were absolute requirements. As in clinical ECP, immunogenic cell death of tumor cells was finely titrated by DNA cross-linkage mediated by photoactivated 8-methoxypsoralen (8-MOPA). ECP-induced tumor-loaded DC were effective immunotherapeutic agents only if they were spared exposure to 8-MOPA, indicating that healthy DC are required for ECP. Infusion of responder T cells into naïve tumor-challenged mice established the protective role of stimulated T-cell antitumor immunity. Collectively, these results reveal that selective antitumor effects of ECP are initiated by tumor antigen-loaded, ECP-induced DC, which promote potent collaboration between CD4 and CD8 tumor-specific T cells. These mechanistic insights suggest potential therapeutic applicability of ECP to solid tumors in addition to CTCL. These findings identify principal cellular contributors to the anticancer immunotherapeutic impact of ECP and suggest this treatment may be applicable to a broad spectrum of immunogenic malignancies. .

Article	Year
Photoactivated 8-methoxypsoralen treatment causes a peptide-dependent increase in antigen display by transformed lymphocytes. International journal of cancer\| PubMed ID: 9724096	1998
Configuration-dependent Presentation of Multivalent IL-15:IL-15Rα Enhances the Antigen-specific T Cell Response and Anti-tumor Immunity. The Journal of biological chemistry\| PubMed ID: 26719339	2016
Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity. Cancer research\| PubMed ID: 29764863	2018

Douglas Hanlon

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