Functional Macroautophagy Induction by Influenza A Virus without a Contribution to Major Histocompatibility Complex Class II-Restricted Presentation

Abstract

Major histocompatibility complex (MHC) class II-presented peptides can be derived from both exogenous (extracellular) and endogenous (biosynthesized) sources of antigen. Although several endogenous antigenprocessing pathways have been reported, little is known about their relative contributions to global CD4؉ T cell responses against complex antigens. Using influenza virus for this purpose, we assessed the role of macroautophagy, a process in which cytosolic proteins are delivered to the lysosome by de novo vesicle formation and membrane fusion. Influenza infection triggered productive macroautophagy, and autophagy-dependent presentation was readily observed with model antigens that naturally traffic to the autophagosome. Furthermore, treatments that enhance or inhibit macroautophagy modulated the level of presentation from these model antigens. However, validated enzyme-linked immunospot (ELISpot) assays of influenza-specific CD4 ؉ T cells from infected mice using a variety of antigen-presenting cells, including primary dendritic cells, revealed no detectable macroautophagy-dependent component. In contrast, the contribution of proteasome-dependent endogenous antigen processing to the global influenza CD4؉ response was readily appreciated. The contribution of macroautophagy to the MHC class II-restricted response may vary depending upon the pathogen. The activation of CD4ϩ T cells depends upon their recognition of peptides (epitopes) associated with major histo compatibility class (MHC) class II molecules. Conventionally, peptide generation involves the degradation of exogenous (extracellular) antigens in the endosomal network by multiple mechanisms, including unfolding, reduction, and proteolytic degradation. It is now clear that epitopes derived from endogenous antigens (synthesized by the cell) also can be presented on MHC class II molecules (12,22,24,38,49,50,60,66). Indeed, endogenous antigen expression appears to be an absolute requirement for the presentation of some epitopes. For example, the UV inactivation of A/PR8/34 influenza virus (PR8) or treatment of infected antigen-presenting cells (APCs) with protein synthesis inhibitors prevents the presentation of the NA79 epitope (12). Similar observations have been made for an epitope derived from influenza matrix protein 1 (24) and an epitope derived from the MHC class I H2-L d molecule (39).Numerous studies now have demonstrated that endogenous antigens can gain access to MHC class II loading compartments via a variety of intracellular pathways (4,38,49,57,66,67). Perhaps the most straightforward route is autophagy, in which cytosolic proteins are delivered to the lysosome via several different mechanisms. Chaperone-mediated autophagy results in the delivery of cytosolic proteins directly to the lysosome based upon the recognition of a KFERQ pentapeptide motif within the target protein, and it was shown to be responsible for the presentation of an epitope derived from glutamate decarboxylase (72). Although 30% of cytosolic proteins contain this motif (70)...

Intersection of autophagy with pathways of antigen presentation

“…The targeting of S1 to autophagosomes, either via S1 conjugation to LC3 or to NeoR, a model antigen, shown previously to be presented by MHCII molecules (Nimmerjahn et al, 2003), does not enhance S1-specific CD4 + T cell responses (Comber et al, 2011). In addition, shRNA knockdown of Atg7 in bone marrow-derived DC does not affect CD4+ T cell interferon-γ (IFNγ) secretion in response to S1 (Comber et al, 2011). Chaperone-mediated autophagy (CMA) is a potential alternative route that may deliver antigen to the MHCII antigen presentation pathway.…”

“…al. have demonstrated that while autophagy is upregulated in influenza A virus infected cells, autophagy does not enhance MHCII antigen presentation of the site 1 (S1) epitope of hemagglutinin (HA) (Comber et al, 2011). The targeting of S1 to autophagosomes, either via S1 conjugation to LC3 or to NeoR, a model antigen, shown previously to be presented by MHCII molecules (Nimmerjahn et al, 2003), does not enhance S1-specific CD4 + T cell responses (Comber et al, 2011). In addition, shRNA knockdown of Atg7 in bone marrow-derived DC does not affect CD4+ T cell interferon-γ (IFNγ) secretion in response to S1 (Comber et al, 2011).…”

HIV-Infected Dendritic Cells Present Endogenous MHC Class II–Restricted Antigens to HIV-Specific CD4+ T Cells

“…Mice with selective impairment of autophagy in conventional DC displayed a reduced capacity to process MHC-II-restricted HSV-1 Ag (30). In contrast, alterations of autophagy in influenza-infected mouse DC or fibroblasts had no impact on the activation of influenza-specific CD4 + T cells (31). Autophagy was initially described as being involved in the processing of intracellular endogenous MHC-II Ag (19).…”

Checking the garbage bin for problems in the house, or how autophagy assists in antigen presentation to the immune system

“…The transposon-derived neomycin phosphotransferase II (NeoR) gets intracellularly processed by lysosomal hydrolysis for MHC class II presentation [18]. This processing is sensitive to pharmacological macroautophagy inhibitors, and influenza hemagglutinin epitope fusion to NeoR augments MHC class II presentation of this epitope [10,18]. Interestingly, forced nuclear localization of NeoR does not compromise macroautophagy mediated processing of this antigen for MHC class II presentation [19].…”

“…Accordingly, LC3 can be used to deliver antigens to autophagosomes, which frequently fuse with MIICs [9]. This targeting enhances antigen presentation on MHC class II molecules up to 20fold [9,10]. A physiological condition, under which this self-antigen processing by macroautophagy seems to play a role is thymic selection.…”