Ordinarily, MHC class II molecules present epitopes derived from endocytosed protein cargo, while MHC class I molecules present epitopes derived from cytosolic protein degradation. These observations, however, were based on classic studies done in 1980s and 1990s, when for the purpose of simplification, research scientists were using "nominal", non-replicative proteins. But what about protein presentation during live, replicating viral infection? Will it follow the same rule [I-in/II-out]?
New study in Nature Medicine tried to answer this question. Here, the authors showed that live viral infection elicited broader CD4 T cell epitope response and that to some extent, this broader CD4 T cell response to live viral infection was driven via non-classical class II presentation.
This is surprisingly quite simple study [done in a manner characteristic to research from early 2000s], and I am not entirely sure whether it really has answers for its claims. Lets examine it.
First, the authors showed that unlike priming with inactivated influenza virus (BLP-PR8), i.n. infection with live influenza virus (live PR8) elicited broader and stronger CD4 T cell ex vivo IFN-γ response.
Direct, in vitro exposure of BmDCs to live or inactivated influenza virus confirmed that live infection produced broader response (notable for NA-25 and NP-47 epitopes).
More importantly, i.m. or s.c. priming with live PR8 or inactivated PR8 produced similar results: live PR8 infection produced stronger and broader immune [CD4 or Ab] response. Note, i.m. priming does not support live influenza virus replication, so it could be assumed that antigen load should be very similar here between live and inactivated PR8.
To rule out the role of inflammatory milieu [produced during live infection] affected the strength of immune response to inactivated PR8 infection, mice were challenged with inactivated PR8 virus in combination with non-cross-reactive live B/Lee virus. However even the presence of live B/Lee virus did not modify immune response to inactivated PR8 virus.
Next, the authors conducted several in vitro experiments to further evaluate antigen presentation pathways. In first set of experiments, MHC-II negative fibroblast were infected and then exposed to BmDCs and T cell hybridomas [in presence of anti-influenza Ab that supposedly prevents direct infection of BmDCs]. Here, out of six T cell hybridomas tested, only one hybridoma, specific for NP-47, showed response in this classical MHC II presentation assay.
In second set of experiments, MHC II-positive cells were harvested ex vivo from live PR8 infected donors, sorted into surface viral antigen HA+ [indicative of live infection] or HA- populations and cultured with primed CD4 T cells or CD4 T cell hybridomas. Here too, classical exogenous MHC II presentation pathway [HA- population] produced minimal response.
Afterwards, significance of rest of the data are not clear: (a) the authors showed that cells from both H2-DMa-/- and CD74-/- hosts that lack the classical MHC II presentation pathway molecules could still present CD4 T cell epitopes after live viral infection; (b) however, TAP-/- hosts that lack the classical MHC I presentation pathway molecule were capable of producing CD4 T cell epitopes as well; (c) proteosome inhibitor epoxomicin could reduce presentation of some of the epitopes from live PR8 viral infection.
However, none of these above mentioned experiments, strictly speaking, provide a definite answer that epitope presentation during live viral infection goes through endogenous cytosolic pathway. First, experiments are missing with the positive control epitope(s) from classical, exogenous, H2-DMa and CD74-dependent pathways. Second, live viral infection in vivo could infect or taken up by different cell or DC population compared to inactivated virus [even BmDC culture used here does not represent a homogeneous cell system at all]. This could introduce variability in antigen presentation pathway not directly addressed by this study.
David Usharauli
Direct, in vitro exposure of BmDCs to live or inactivated influenza virus confirmed that live infection produced broader response (notable for NA-25 and NP-47 epitopes).
More importantly, i.m. or s.c. priming with live PR8 or inactivated PR8 produced similar results: live PR8 infection produced stronger and broader immune [CD4 or Ab] response. Note, i.m. priming does not support live influenza virus replication, so it could be assumed that antigen load should be very similar here between live and inactivated PR8.
To rule out the role of inflammatory milieu [produced during live infection] affected the strength of immune response to inactivated PR8 infection, mice were challenged with inactivated PR8 virus in combination with non-cross-reactive live B/Lee virus. However even the presence of live B/Lee virus did not modify immune response to inactivated PR8 virus.
Next, the authors conducted several in vitro experiments to further evaluate antigen presentation pathways. In first set of experiments, MHC-II negative fibroblast were infected and then exposed to BmDCs and T cell hybridomas [in presence of anti-influenza Ab that supposedly prevents direct infection of BmDCs]. Here, out of six T cell hybridomas tested, only one hybridoma, specific for NP-47, showed response in this classical MHC II presentation assay.
In second set of experiments, MHC II-positive cells were harvested ex vivo from live PR8 infected donors, sorted into surface viral antigen HA+ [indicative of live infection] or HA- populations and cultured with primed CD4 T cells or CD4 T cell hybridomas. Here too, classical exogenous MHC II presentation pathway [HA- population] produced minimal response.
Afterwards, significance of rest of the data are not clear: (a) the authors showed that cells from both H2-DMa-/- and CD74-/- hosts that lack the classical MHC II presentation pathway molecules could still present CD4 T cell epitopes after live viral infection; (b) however, TAP-/- hosts that lack the classical MHC I presentation pathway molecule were capable of producing CD4 T cell epitopes as well; (c) proteosome inhibitor epoxomicin could reduce presentation of some of the epitopes from live PR8 viral infection.
However, none of these above mentioned experiments, strictly speaking, provide a definite answer that epitope presentation during live viral infection goes through endogenous cytosolic pathway. First, experiments are missing with the positive control epitope(s) from classical, exogenous, H2-DMa and CD74-dependent pathways. Second, live viral infection in vivo could infect or taken up by different cell or DC population compared to inactivated virus [even BmDC culture used here does not represent a homogeneous cell system at all]. This could introduce variability in antigen presentation pathway not directly addressed by this study.
David Usharauli
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