Stimulation of cells with pro-inflammatory cytokines or IFNγ upregulates the expression of the proteasome immunosubunits and of PA28. While the immunosubunits are known to play an important role in epitope processing and to shape the immunodominance hierarchy of pathogen-specific CD8 T cell responses, the impact of PA28 on immune defense still remains enigmatic. We here demonstrate that both the proteasome immunosubunits and PA28 fulfill a central function in the production of MHC class I ligands in vivo
. Importantly, we find that spleen-derived lymphoid cells of mice that are gene-deficient for PA28 display lower MHC class I expression levels than wt
mice, and observe a similar reduction in MHC class I levels on cells of PA28+β5i/LMP7+β2i/MECL-1-deficient mice, in comparison to mice gene deficient for the immunosubunits only (, ). These differences in MHC class I cell surface levels become even more apparent after infection of the different mouse strains with rLM, a pathogen that targets the spleen () and induces the secretion of Th1 cytokines, or after stimulation of B cells from uninfected wt
or proteasome component-deficient mice with LPS, in cell culture (). Thus, PA28 plays an important role in the production of MHC class I binding peptides in lymphoid cells. Thereby, in quantitative terms, PA28 is the second most important component of the proteasome system that contributes to the production of MHC class I ligands, surpassed only by β5i/LMP7, whose absence is responsible for the reduction in class I levels observed in mice that lack β5i/LMP7+β2i/MECL-1 [29
]. Although β2i/MECL-1 (and β1i/LMP2) play an important role in the processing of a variety of epitopes [35
], their effects are not substantial enough to influence overall MHC class I expression [35
]. Thus, both the proteasome immunosubunits and PA28 should be considered as integral components of the MHC class I antigen processing machinery. They play an important role in the production of high-affinity peptides required to maintain the basal MHC class I cell surface levels on lymphoid cells, as well as in providing additional ligands to stabilize newly synthesized MHC class I molecules following cytokine exposure.
The finding that PA28-deficiency leads to reduced MHC class I cell surface levels both on immunoproteasome-positive and -negative cells, on which MHC expression is diminished already due to the absence of immunosubunits (), indicates that the effects of the immunosubunits and PA28 are additive. Thus, these proteasome components enhance the production of different MHC class I ligands. This is consistent with the different working mechanisms of PA28 and the catalytic immunosubunits. PA28 binds to the 20S complex, leading to coordinated double cleavages and apparently thereby an enhanced generation of MHC class I ligands [23
], whereas the immunosubunits change the 20S cleavage site preferences. In particular the replacement of the constitutive subunit β5 by β5i/LMP7 leads to enhanced cleavage at the C-terminus of hydrophobic residues, which are critical anchors for binding to MHC class I molecules [33
]. In addition, enhanced proteolytic activity in the presence of immunoproteasomes may lead to enhanced antigenic peptide generation [21
], although our previous studies failed to show any differences in kinetics of presentation of two immunoproteasome-independent epitopes between rLM-infected β5i/LMP7+β2i/MECL-1-deficient and -sufficient DC in vitro
Remarkably, deficiency of PA28+β5i/LMP7+β2i/MECl-1 reduces MHC class I expression only to approximately 53 % on unstimulated cells compared to wt
and these levels can still be upregulated by exposure to LPS or cytokines. Thus, IFNγ-induced expression of other components of the antigen processing pathway such as of β1i/LMP2 (which however poorly assembles into 20S complexes in the absence of β5i/LMP7 [45
]) or of aminopeptidases may lead to the production of additional peptides that stabilize the newly synthesized MHC class I molecules on stimulated PA28+β5i/LMP7+β2i/MECL-1-deficient cells. Alternatively, the increase in numbers of cell surface MHC class I /peptide complexes may result from enhanced synthesis of MHC class I heavy chains and thus a renewed balance between gain and loss of MHC class I molecules, complexed with low affinity peptides, from the cell surface. In support of this, addition of synthetic ova257-264
to LPS blast cultures restores the H2-Kb
levels on PA28+β5i/LMP7+β2i/MECL-1-deficient cells to nearly wt
levels (not shown), indicating that MHC class I molecules traffick to the cell surface also in cells that lack these proteasome components.
Importantly, in contrast to the profound effects of immunosubunits on the generation of specific epitopes and the fine-specificity of pathogen-specific CD8 T cell responses, the contributions of PA28 to peptide presentation had little impact on the sizes of the responses to Influenza- and rLM-derived epitopes. Taken together, all of four dominant CD8 T cell epitopes tested triggered robust responses in PA28-deficient mice. Previous studies have shown that PA28 enhances 20S-mediated processing of the ovalbumin-derived ova257-264
]. However, our data do not show any significant differences in responses to this epitope in PA28-deficient compared to PA28-positive mice (). Thus, reduced ova257-264
presentation in the absence of PA28 has no or only minor effects on CD8 T cell activation. This contrasts to the dramatic reduction in size of CD8 T cell responses in immunoproteasome-deficient mice to immunoproteasome-dependent epitopes, of which two (E1B192-200
) were included in this study.
It has been suggested that the hsp90 chaperone provides an alternative pathway for epitope supply to MHC class I molecules in cells that lack PA28 expression, and also in PA28-positive lymphoid cell lines unless stimulated with IFNγ [27
]. More recent studies however demonstrated that hsp90 binds proteasome-generated protein fragments to protect these from further degradation, and makes such fragments available for MHC class I loading [47
]. Thus, hsp90 acts downstream of the proteasome, which was confirmed also for PA28-deficient cells [28
]. Also our finding that MHC class I expression levels are reduced on PA28-deficient compared to wt
spleen cells implies that any compensatory effects of hsp90 on the peptide pool available for MHC class I binding in the absence of PA28 are partial at most, and is consistent with the notion that the hsp90 pathway does not produce new MHC class I ligands but enhances the presentation of peptides that are produced by the proteasome.
Taken together, we here show that both PA28 and the immunosubunits play an important and additive role in proteasome-mediated processing of intracellular proteins into peptides that bind to MHC class I molecules. The role of PA28 in the production of class I ligands is much greater than expected, based on previous studies [25
]. We showed previously that the early kinetics of MHC class I antigen processing following infection are strongly influenced by the immunosubunits and determine the immunogenicity of individual antigenic peptides [1
]. Like the immunosubunits, also PA28 enhances MHC class I presentation of specific epitopes (like ova257-264
) at early time points after infection. Nevertheless, consistent with previous findings [25
], our analyses of rLM and Influenza-specific CD8 T cell responses indicate that the effects of PA28 on antigen processing do not have any dramatic effects on the size and fine specificity of CD8 T cell responses to these pathogens. Remarkably, a recent study showed that, like immunoproteasomes, also PA28 is involved in degradation of oxidized proteins. Therefore, these components of the proteasome system may play a role also beyond antigen processing and immune recognition, in cellular adaptation to oxidative stress [21