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Journal of Translational Medicine

, 12:100

Immunobiology and immunotherapy

Abstract

BackgroundAutophagy regulates innate and adaptive immune responses to pathogens and tumors. We have reported that autophagosomes derived from tumor cells after proteasome inhibition, DRibbles Defective ribosomal products in blebs, were excellent sources of antigens for efficient cross priming of tumor-specific CD8 T cells, which mediated regression of established tumors in mice. But the activity of DRibbles in human has not been reported.

MethodsDRibbles or cell lysates derived from HEK293T or UbiLT3 cell lines expressing cytomegalovirus CMV pp65 protein or transfected with a plasmid encoding dominant HLA-A2 restricted CMV, Epstein-Barr virus EBV, and Influenza Flu epitopes CEF were loaded onto human monocytes or PBMCs and the response of human CMV pp65 or CEF antigen-specific CD4 and CD8 memory T cells was detected by intracellular staining. The effect of cytokines GM-CSF, IL-4, IL-12, TNF-α, IFN-α and IFN-γ TLR agonists Lipopolysaccharide, Polyinosinic-polycytidylic acid polyI:C, M52-CpG, R848, TLR2 ligand and CD40 ligand on the cross-presentation of antigens contained in DRibbles or cell lysates was explored.

ResultsIn this study we showed that purified monocytes, or human PBMCs, loaded with DRibbles isolated from cells expressing CMV or CEF epitopes, could activate CMV- or CEF-specific memory T cells. DRibbles were significantly more efficient at stimulating CD8 memory T cells compared to cell lysates expressing the same antigenic epitopes. We optimized the conditions for T-cell activation and IFN-γ production following direct loading of DRibbles onto PBMCs. We found that the addition of PolyI:C, CD40 ligand, and GM-CSF to the PBMCs together with DRibbles significantly increased the level of CD8 T cell responses.

ConclusionsDRibbles containing specific viral antigens are an efficient ex vivo activator of human antigen-specific memory T cells specific for those antigens. This function could be enhanced by combining with PolyI:C, CD40 ligand, and GM-CSF. This study provides proof-of-concept for applying this strategy to activate memory T cells against other antigens, including tumor-specific T cells ex vivo for immunological monitoring and adoptive immunotherapy, and in vivo as vaccines for patients with cancer.

KeywordsAutophagosome Cross-presentation Immunological monitoring Immunotherapy biomarker Viral vaccine Cancer vaccine Memory antigen-specific T cells Proteasome AbbreviationsCTLCytotoxic T lymphocyte

TAATumor-associated antigens

pAPCProfessional antigen presenting cell

DCDendritic cell

DRiPsDefective ribosomal products

CMVCytomegalovirus

NSCLCNon-small cell lung cancer

CEFCytomegalovirus, Epstein-Barr Virus, influenza virus

GM-CSFGranulocyte- macrophage colony stimulating factor

ILInterleukin

IFNInterferon

TLRToll-like receptor

TNFTumor necrosis factor

PBMCPeripheral blood mononuclear cell

BCABicinchoninic acid

HPVHuman papillomavirus.

Electronic supplementary materialThe online version of this article doi:10.1186-1479-5876-12-100 contains supplementary material, which is available to authorized users.

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Fuente: https://link.springer.com/







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