Our results with TKO mice reveal the vital role of immunoproteasomes in creating peptides for MHC class I antigen presentation. The profound defects in antigen presentation that we find in the triple KO APCs, both in vivo and in vitro, are qualitatively and quantitatively much broader than the previously described defects in any of the β1i, β2i or β5i single KO animals and are far greater than the sum of the defects in single knock out animals. The findings suggest that functional overlap between the subunits has masked the true importance of immunoproteasomes.
Of the eleven MHC class I presented antigens we examined, we found clear alterations in nine (i.e. 82%) – eight of which had reduced presentation. In the case of the Smcy antigen, expressed on male cells, there was a significant reduction in presentation by TKO cells even though none of the single KO mice showed a defect. In the case of LCMV GP33 and GP118, there were defects in the response to these epitopes, where none have been previously described with single KO animals11,16
. Similarly there was a partial defect in OVA 257–264 presentation not previously seen in single KOs9,12,15
and also a much more marked defect in the presentation of Influenza NP366–3749,10,14
. We believe, therefore, that data from single KO animals have substantially underestimated the contribution of the immunoproteasomes to generating peptides for MHC class I antigen presentation.
Our finding that many epitopes are presented poorly by cells that completely lack immunoproteasomes are consistent with inefficient generation of optimal peptides by constitutive proteasomes. Presumably this defect in generating peptides accounts for the approximately 50% decrease in MHC class I surface expression in the TKO mice. Although this decrease in MHC class I is similar to that in β5i
single KO animals (our data here and8
), the peptides that the two strains present are almost certainly not identical. Compared to β5i.−/−
animals, TKO mice present many peptides more poorly and more rapidly reject WT cells. The profound difference in Smcy presentation between β5i
KO DCs (which present the antigen normally) and TKO DCs (which present the antigen very poorly) despite the quantitatively similar MHC class I surface expression, suggests that the decreased presentation in the TKO cells cannot be due to the decrease in overall MHC molecules but rather are a result of a poor supply of peptides.
In the absence of immunoproteasomes, there were changes in antigen presentation in vitro
in dendritic cells and, importantly, also in vivo
. During LCMV infection, we observe changes that affect the magnitude of T cell responses, generally substantially reducing responses. GP276, the one epitope for which we found better presentation by TKO cells, has been previously shown to be destroyed by β1i
. The fact that these changes in antigen presentation effect CD8 T cell responses in TKO animals (to the point of altering the immunodominance hierarchy) is impressive in the face of an immunogen as robust as LCMV.
The defects that we observed in vivo selectively affect the MHC class I antigen processing pathway. Loss of immunoproteasomes decreased responses to epitopes from several protein antigens but not from a minigene whose product does not require cleavage for presentation. Moreover, we find no defect in CD4 numbers in response to MHC class II epitopes in vivo, indicating that APC function is not globally decreased. We also conclude that the decreased CD8 T cell responses in TKO mice are not due to pleiotropic effects on T cells, but to antigen presentation defects, since the decreases are found in WT T cells transferred into TKO hosts. Moreover, we also find no defect in CD4 numbers or responses in naïve or LCMV-infected animals.
In addition to the differences we observed in the presentation of 9 of 11 immunogenic epitopes, we found that the peptide repertoire of TKO animals is qualitatively substantially different from WT or any single KO, as evidenced by the robust rejection of WT cells by TKO animals. This is particularly impressive because differences in minor histocompatibility antigens, which are presented peptides that differ between strains due to allelic polymorphisms, stimulate rejection much more slowly. It should be noted that rejection of the WT cells is unlikely to be due to minor histocompatibility differences, because the TKO animals were fully backcrossed. In addition, such histocompatibility differences would be expected to elicit bidirectional responses between the strains, but we found no rejection of TKO cells by WT animals. Instead, this ‘one way’ rejection suggests that cells in WT animals present a substantially different set of peptides than those found in TKO animals, containing epitopes generated by both immunoproteasomes and constitutive ones. Consistent with these results and quite remarkably, comparison of the peptides eluted from matched samples of MHC class I molecules on wild type versus TKO splenocytes revealed that only about one half of the peptides from both Db and Kb were shared between the two strains. This is likely an underestimate because the mass spectrometry analysis detects the presence of abundant peptides but not their precise amount. Therefore, even among the peptides that were in common between the WT and TKO mice there are likely quantitative differences, as we found such differences in the presentation of the majority (82%) of immunogenic epitopes in quantitative assays. When the peptides we identified as unique to immunoproteasome-deficient mice are compared against a larger data set of BL6-presented peptides from the literature, 75–80% are still only present in the TKO pools. The 20–25% of additional ‘shared’ peptides could have been generated by constitutive proteasomes in previous WT preparations and/or by the detection of lower abundance peptides in previous analyses.
Taken together, these results demonstrate the importance of immunoproteasomes in generating peptides for MHC class I antigen presentation, a contribution that has been previously substantially underestimated. A potentially important implication of our findings is that under non-inflammatory conditions the peptides presented by DC, which constitutively express immunoproteasomes, will be substantially different from the ones displayed on parenchymal cells, which contain only constitutive proteasomes. Therefore, T cell responses stimulated by DC may not optimally recognize parenchymal cells until immunoproteasomes are induced in the latter by interferon. This may reduce the effectiveness of CD8 T cell immunity in situations where IFN-γ is not produced. Similarly, this could help pathogenic cells that fail to respond to IFN-γ and/or express immunoproteasomes, such as some tumors or cells infected with viruses that inhibit IFN-γ responses, evade immune responses.