Systemic Toll-Like Receptor Ligation and Selective Killing of Dendritic Cell Subsets Fail To Dissect Priming Pathways for Anti-Vaccinia Virus CD8⁺T Cells

Abstract

CD8؉ T cell responses can be generated by direct or cross-priming mechanisms, and several mouse models have been used to reveal which of these is the most important pathway for various viruses. Among these models is systemic treatment of mice with a CpG-containing oligodeoxynucleotide (CpG) to mature all dendritic cells (DCs), rendering them incapable of cross-presentation. A second is the use of cytochrome c (cytc) as a selective poison of the subsets of DCs able to cross-present antigen. In this study, using two vaccinia virus (VACV) strains, namely, WR and MVA, we found that the CpG and cytc methods gave conflicting data. Moreover, we show for both strains of VACV that treatment of mice with CpG and cytc inhibited CD8 ؉ T cell responses to antigens designed to prime exclusively by direct presentation. Further investigation of the CpG method found that the extent to which priming is inhibited depends on the antigen examined, immunization route, replication ability of the virus, and, crucially, immunization dose. We suggest that greater caution is required when interpreting data using these methods and that priming pathways for antiviral CD8 ؉ T cells are not simply separated according to DC subsets or their maturation state.

Sizing up the key determinants of the CD8+ T cell response

“…In our own studies, it was found that subdominance of the IAV-derived epitopes H2-D b -PB1-F2 62-70 and H2-K b -NS2 [114][115][116][117][118][119][120][121] in the B6 model of IAV infection is associated with the relative paucity of highavidity T cells in the pre-immune and immune repertoires, but not with CTLP numbers or antigen dose 25,38 . In fact, attempts to equalize epitope abundance using a variety of immunization strategies failed to align immune responses with relative CTLP frequency 38 .…”

“…However, this ignores the possibility that these same DC populations might be equally capable of direct presentation 115 . Similarly, paralyzing cross-presentation using systemic maturation of DCs has unanticipated effects on direct presentation, so this tool is not useful for dissecting these pathways 116 . Studies with mice deficient in proteins uniquely required for cross-presentation -for example, interferon-γ-inducible lysosomal thiol reductase (GILT; also known as IFI30) -should offer less complicated interpretations 117 .…”

“…The identification of receptors that promote the cross-presentation of internalized antigen, such as DNGR1 (also known as CLEC9A), offers another possible way to dissect presentation pathways 118,119 , but again the impact of knocking out such receptors on direct presentation needs to be rigorously established. Apart from direct visualization of priming by intravital microscopy 111 , knowledge of the major substrates for direct presentation and cross-presentation may be the most reliable tool for dissecting antigen-presentation pathways 112,116 . Finally, mechanisms such as 'cross-dressing' that allow DCs to acquire pre-formed peptide-MHC complexes need to be considered, although it remains unclear if this is a major mechanism for priming antiviral responses 120 .…”

Activation of CD8 T Lymphocytes during Viral Infections

“…Conversely, it has been reported that CP dominates the CD8 lymphocyte response to the replication-deficient strain of VACV-modified vaccinia Ankara (Gasteiger et al, 2007). However, this finding has been challenged (Wong et al, 2013).…”

The Biology and Underlying Mechanisms of Cross-Presentation of Exogenous Antigens on MHC-I Molecules

“…In vivo, type I IFN may be critical for XPT responses (212). On the other hand, TLR4 and TLR9 agonists can reduce XPT to particulate antigens (163) and systemic administration of TLR-agonists results in a marked inhibition of XPT (213) and possibly direct presentation (214). The stimulatory vs. inhibitory effects of TLR agonists may be influenced by their purity (215).…”