title: Cross-presentation of viral antigens in dribbles leads to efficient activation of virus-specific human memory T cells.
abstract: BACKGROUND
  Autophagy 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.
  
  
  METHODS
  DRibbles 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 (poly(I:C), M52-CpG, R848, TLR2 ligand) and CD40 ligand on the cross-presentation of antigens contained in DRibbles or cell lysates was explored.
  
  
  RESULTS
  In 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 Poly(I:C), CD40 ligand, and GM-CSF to the PBMCs together with DRibbles significantly increased the level of CD8⁺ T cell responses.
  
  
  CONCLUSIONS
  DRibbles 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 Poly(I: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.
authors.forename: Wei
authors.initials: W
authors.lastname: Ye
authors.forename: Yun
authors.initials: Y
authors.lastname: Xing
authors.forename: Christopher
authors.initials: C
authors.lastname: Paustian
authors.forename: Rieneke
authors.initials: R
authors.lastname: van de Ven
authors.forename: Tarsem
authors.initials: T
authors.lastname: Moudgil
authors.forename: Traci L
authors.initials: TL
authors.lastname: Hilton
authors.forename: Bernard A
authors.initials: BA
authors.lastname: Fox
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Urba
authors.affiliation: Medical School, Southeast University, 87 Dingjiaqiao Street, 210009 Nanjing, Jiangsu, PR China. zhaoweiseu@126.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.forename: Hong-Ming
authors.initials: HM
authors.lastname: Hu
mesh_terms: D000956:Antigens, Viral / Q000276:immunology*; D015704:CD4 Antigens / Q000276:immunology; D016827:CD8 Antigens / Q000276:immunology; D002460:Cell Line; D003587:Cytomegalovirus / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D007156:Immunologic Memory; D008213:Lymphocyte Activation; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; D015704:CD4 Antigens; D016827:CD8 Antigens
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