This study reveals that CD4+
T cell activation in vaccine-induced resistance to A. fumigatus
is contingent upon the nature of the fungal vaccine, the involvement of distinct innate receptor signaling pathways, and the mode of antigen routing in DCs (Figure ). Vaccine-induced resistance required IFN-γ, a cytokine with effector and immunoregulatory activity against fungi (15
), and, partially, IL-17RA signaling, a finding pointing to a degree of similarity among respiratory fungal pathogens (55
). MHC class II–restricted CD4+
Th1 cells provided long-lasting antifungal protection and disease control in CGD mice, in which MHC class I–restricted CD8+
T cells were not activated due to defective autophagy and endosomal alkalinization. Thus, understanding memory at basic levels may lead to a vaccination approach in CGD patients. As in CGD mice, CD4-dependent but not CD8-dependent memory responses were observed in BALB/c mice (data not shown), a finding indicating that the activation of either one of antifungal memory response is also contingent upon the host’s genetic background.
Distinct pathways of intracellular antigen routing in CD4+ and CD8+ T cell vaccination to Aspergillus fumigatus.
Activation of CD4+
T cells occurred through distinct antigen uptake and presentation pathways. Purified fungal proteins were apparently routed to the endosome/lysosome-dependent MHC class II presentation pathway via MyD88 and the involvement of distinct upstream TLRs known to enhance the uptake of phagocytosed but also soluble antigens and thus potentiate antigen presentation (28
). Further confirming the routing toward the MHC class II presentation pathway, treatment with nocodazole, which interferes with microtubule-dependent motility of early endosomes toward maturing endosomes, also blocked CD4+
T cell activation to fungal antigens (data not shown). Thus, similar to pinocytosed and scavenger receptor endocytosed antigens (14
), fungal peptides are targeted toward a common pool of endosomes, which mature toward late endosomes and fuse with lysosomes, in which peptides for MHC class II–restricted presentation can be generated. Although primarily expressed on the plasma membrane, TLR2, TLR4, and TLR6 could also recycle through early endosomes; target and be activated by (56
) the Rab family; and control the phagosomal pH (28
). Moreover, intracellular TLRs, to signal efficiently, need to be proteolytically activated (29
). Thus, it is likely that TLR trafficking to the diverse organelles may explain the involvement of distinct TLRs in the presentation of the different fungal antigens. However, precisely how this occurs is not known.
En route to lysosomes, purified fungal antigens also targeted to the mildly acidic stable early endosomal compartment, where the uptake by MR leads to presentation on MHC class I molecules and Th1 polarization (32
). Mannosylated fungal antigens (both N
-linked and O
-linked mannosylated) exhibited increased immunogenicity compared with proteins lacking mannosylation, were taken up by mannose-specific C-type lectin receptors, and colocalized in MHC class II+
). We found that deglycosylated Pep1p no longer acted as a fungal vaccine capable of activating CD4-dependent memory to the fungus (data not shown), a finding confirming the importance of MR in the rapid internalization and concentration of a variety of glycosylated fungal antigens (57
In contrast to soluble antigens, live conidia or soluble antigens delivered in a particulate form activated CD8-dependent memory through the TLR3/TRIF-dependent pathway. DC subsets are known to be capable of exporting internalized antigens from the endocytic compartment to the cytosol (58
). In this regard, Aspergillus
-derived antigens may be exported from endocytic compartment to cytosol for conventional MHC class I presentation, as CD8+
T cell activation was dependent on proteasomal degradation. However, the inhibition by brefeldin A, known to inhibit the alternate vacuolar MHC class I processing (33
), suggests that Aspergillus
conidia may exploit multiple class I presentation pathways for CD8+
T cell activation. Cross-priming itself involves a number of distinct mechanisms. How Aspergillus
antigens are delivered to the cytosol or to the ER-resident, vacuolar-associated MHC class I molecules is presently unknown. We found that autophagy, while dispensable for the transit to the Lamp1 compartment, was instead required for diversion from the early endosome to the Rab14+
endosomal storage compartment, in which yeast interaction with MHC class I molecules has been described (47
). Thus, like yeasts, conidia likely interact with MHC class I molecules in the Rab14+
compartment. Whether this actually occurs and the effects of conidia residency in this compartment versus the cytosolic compartment for CD8+
T cell responses are presently unknown but under investigation.
Primarily associated with MHC class II presentation, by delivering intracellular material for lysosomal degradation, macroautophagy has been recently shown to present tumor (59
) and viral antigens (39
) on MHC class I, through the classical and vacuolar antigen presentation pathways (38
). Indeed, by assisting TLRs in encountering their cognate ligands, autophagy contributed to the CD8+
T cell response to pathogens localized in autophagosomes containing components of the MHC class I presentation pathway (61
). Irrespective of the mechanisms through which autophagy mediates Aspergillus
sorting for MHC class I presentation, promoting autophagy in vivo provided CD8-dependent antifungal memory. Indeed, TLR3, known to activate DCs for cross-priming (62
), activated both autophagy and CD8+
T cell memory in CGD mice. Although the net contribution of autophagy to the handling of the fungus and fungal control in infection will likely be more complex, our results are the first to our knowledge to show a role for autophagy in adaptive immunity to A. fumigatus
and suggest that TLR3 adjuvants may optimize vaccination strategies in aspergillosis. Along the same line of reasoning, it will be of interest to assess the therapeutic potential of chloroquine, known to favor CD8+
T cell activation (63
), as well as of inhibitors of cysteine and aspartic proteases to define whether excessive epitope degradation also contributes to defective cross-presentation of fungal antigens in CGD.
Overall, the present study shows that defective cross-presentation in the absence of NOX2 may contribute to the inability of CGD patients to successfully combat A. fumigatus
. Because viral infections are not common in CGD (18
), and CGD mice were not more susceptible to MMCV infection (the present study), it is likely that mechanisms of cross-presentation of viral versus fungal antigens are different, as has been speculated (31
). From an immunological standpoint, our study highlights how understanding molecular and cellular mechanisms of memory at basic levels could be exploited to perfect vaccination strategies against inflammatory fungal diseases through the appropriate selection of fungal antigens and adjuvants and the targeting of DC pathways that enhance vaccine potency.