Endogenously Expressed Antigens Bind Mammalian RNA via Cationic Domains that Enhance Priming of Effector CD8 T Cells by DNA Vaccination

Abstract

Hepatitis B virus (HBV) core (HBV-C) antigens with homologous or heterologous HIV-tat48-57-like (HBV-C149tat) cationic domains non-specifically bind cellular RNA in vector-transfected cells. Here, we investigated whether RNA-binding to cationic domains influences the immunogenicity of endogenously expressed antigens delivered by DNA vaccination. We initially evaluated induction of HBV-C (K b /C93)-specific CD8 + T cell responses in C57BL/6J (B6) and 1.4HBV-S mut transgenic (tg) mice that harbor a replicating HBV genome in hepatocytes by DNA immunization. RNA-binding HBV-C and HBV-C149tat antigens moderately enhanced K b /C93-specific CD8 + T cells in B6 mice as compared with RNA-free HBV-C149 antigen (lacking cationic domains). However, only the RNA-binding antigens elicited K b /C93-specific CD8 + T cells that inhibited HBV replication in 1.4HBV-S mut tg mice. Moreover, RNA-binding to designer antigens, which express a K b /p15E epitope from an endogenous murine leukemia virus-derived tumor-specific gp70 protein, was crucial to prime tumor-rejecting effector CD8 + T cells in B6 mice. Antigen-bound endogenous RNAs function as a Toll-like receptor 7 (TLR-7) ligand and stimulated priming of K b /p15E-specific CD8 + T cells in B6, but not TLR-7 −/− , mice. Antigen-bound cellular RNAs thus function as an endogenous natural adjuvant in in vivo vector-transfected cells, and thus are an attractive tool to induce and/or enhance effector CD8 + T cell responses directed against chronic viral infections or tumor self-antigens by DNA vaccination.

Thrombospondin 2/Toll-Like Receptor 4 Axis Contributes to HIF-1α-Derived Glycolysis in Colorectal Cancer

“…In CRC, the role of TLRs is particularly important due to a close association between CRC and intestinal microorganism, including bacteria, fungi, and viruses (29)(30)(31)(32)(33). In addition to PAMPs, TLRs also recognize endogenous ligands, such as endogenous RNA or DNA and heat shock proteins (HSPs) (34,35). TLRs also interact with the other molecules in the cell membrane, including CD36 and EGFR (36)(37)(38).…”

IFN-γ treatment protocol for MHC-Ilo/PD-L1+ pancreatic tumor cells selectively restores their TAP-mediated presentation competence and CD8 T-cell priming potential

“…Priming of gp70:K b /p15E and Cr-1:K b /cr 16-24 -specific CD8 T cells thus differed substantially, depending on expression of tumor antigens in the ER versus the cytosol (figures 1 and 2; online supplementary table 1). Furthermore, Cr-1:K b / cr 16-24 -and gp70:K b /p15E-specific CD8 T cells were functional in B6 mice and almost quantitatively and specifically eliminated CFSE-labeled APCs pulsed with Cr-1:K b / cr 16-24 (online supplementary figure S3) or gp70:K b /p15E peptides 30 in in vivo cytotoxicity assays. This suggested that priming of these TAA-specific CD8 T cells in B6 mice was not restricted by central and peripheral tolerance mechanisms.…”

“…Expression of vectorencoded antigens was tested in transiently transfected HEK-293 cells as described previously. 30 Immunization of mice and tumor models Mice were immunized intramuscularly into both tibialis anterior muscles with 100 µg/mouse DNA or transplanted Open access subcutaneously into the left/right flank with 2.5×10 5 tumor cells (in 100 µL PBS). For cell-based immunizations, tumor cells were pretreated with recombinant mouse IFN-γ (20 ng/mL, cat.…”

“…Determination of antigen-specific CD8 T-cell frequencies by flow cytometry (FCM) was carried out as described previously. 30 Gene expression analyses Total RNA was isolated from five individual cell lines. The quality was analyzed with a bioanalyzer (Agilent Technologies, Santa Clara, USA).…”

“…Confirmatory, RNA-binding stgp70 designer antigens (eg, stgp70tat, a fusion antigen composed of stgp70 and a cationic HIVtat domain) induced TLR-7-dependent innate 'help' and primed gp70:K b /p15E-specific CD8 T cells by DNA immunization. 30 Furthermore, considering that a proline (P) at position 3 of the K b /p15E epitope (KSPWFTTL) greatly hindered its TAP-dependent transport and efficient presentation by K b molecules, 39 we think that these adjuvant-like immune-stimulatory immune responses might improve epitope presentation. It is generally assumed that cross-presentation of tumor cells or tumor cell debris by professional APCs is a prerequisite to prime functional effector CD8 T-cell responses.…”