No essential role for tripeptidyl peptidase II for the processing of LCMV‐derived T cell epitopes

Antigenic peptides presented by MHC class I molecules are generated mainly by the proteasome in the cytosol. Several cytosolic aminopeptidases further trim proteasomal products to form mature epitopes or individual amino acids. However, the distinct function of cytosolic aminopeptidases in MHC class I Ag processing remains to be elucidated. In this study, we show that cytosolic aminopeptidases differentially affect the cell surface expression of MHC class I molecules in an allele-dependent manner in human cells. In HeLa cells, knockdown of puromycin-sensitive aminopeptidase (PSA) by RNA interference inhibited optimal peptide loading of MHC class I molecules, and their cell surface expression was correspondingly reduced. In contrast, depletion of bleomycin hydrolase (BH) enhanced optimal peptide loading and cell surface expression of MHC class I molecules. We did not find evidence on the effect of leucine aminopeptidase knockdown on the MHC class I Ag presentation. Moreover, we demonstrated that PSA and BH influence the peptide loading and surface expression of MHC class I in an allele-specific manner. In the absence of either PSA or BH, the surface expression and peptide-dependent stability of HLA-A68 were reduced, whereas those of HLA-B15 were enhanced. The surface expression and peptide-dependent stability of HLA-A3 were enhanced by BH knockdown, although those of HLA-B8 were increased in PSA-depleted conditions.

Section: Discussionmentioning

confidence: 99%

Cytosolic Aminopeptidases Influence MHC Class I-Mediated Antigen Presentation in an Allele-Dependent Manner

Kim

Kwak

Ahn

2009

“…Investigations regarding the proteasome-dependent epitope SIINFEKL derived from OVA also did not prove a dependency on TPPII, because siRNA-mediated inhibition did not reduce epitope presentation (44) and TPP Ϫ/Ϫ cells of a knockout mouse were even better in presenting this epitope (45). Other proteasome-dependent epitopes have also been analyzed regarding TPPII involvement in epitope processing, but no effect could be demonstrated both in in vivo and ex vivo experiments (26,45,46). A different conclusion can be drawn from the so-called unusual epitopes that are presented better with a proteasomal blockade and may be generated by a different protease.…”

Section: Discussionmentioning

confidence: 99%

Processing of Two Latent Membrane Protein 1 MHC Class I Epitopes Requires Tripeptidyl Peptidase II Involvement

Diekmann

Adamopoulou

Beck

et al. 2009

The EBV Ag latent membrane protein 1 (LMP1) has been described as a potential target for T cell immunotherapy in EBV-related malignancies. However, only a few CD8+ T cell epitopes are known, and the benefit of LMP1-specific T cell immunotherapy has not yet been proven. In this work, we studied the processing of the two LMP1 HLA-A02-restricted epitopes, YLLEMLRWL and YLQQNWWTL. We found that target cells endogenously expressing the native LMP1 are not recognized by CTLs specific for these epitopes because the N-terminal part of LMP1 limits the efficiency of epitope generation. We further observed that the proteasome is not required for the generation of both epitopes and that the YLLEMLRWL epitope seems to be destroyed by the proteasome, because blocking of proteasomal activities enhanced specific CTL activation. Activation of LMP1-specific CTLs could be significantly reduced after inhibition of the tripeptidyl peptidase II, suggesting a role for this peptidase in the processing of both epitopes. Taken together, our results demonstrate that the MHC class I-restricted LMP1 epitopes studied in this work are two of very few epitopes known to date to be processed proteasome independently by tripeptidyl peptidase II.

“…Transfections B8 cells overexpressing LMP7 and H-2D b (B8-LMP7/D b ) were transfected with an expression plasmid encoding b1WT-myc/His or b1T1A-myc/His, as previously described, using FuGene 6 (Roche, Basel, Switzerland) (28). Clonal and blasticidin (Invitrogen Life Technologies; 5 mg/ml)-resistant cells were tested for b1WT or b1T1A expression by Western blot.…”

Section: Cloningmentioning

confidence: 99%

Why the Structure but Not the Activity of the Immunoproteasome Subunit Low Molecular Mass Polypeptide 2 Rescues Antigen Presentation

Basler

Lauer

Moebius

et al. 2012

Self Cite

The proteasome is responsible for the generation of most epitopes presented on MHC class I molecules. Treatment of cells with IFN-γ leads to the replacement of the constitutive catalytic subunits β1, β2, and β5 by the inducible subunits low molecular mass polypeptide (LMP) 2 (β1i), multicatalytic endopeptidase complex-like-1 (β2i), and LMP7 (β5i), respectively. The incorporation of these subunits is required for the production of numerous MHC class I-restricted T cell epitopes. The structural features rather than the proteolytic activity of an immunoproteasome subunit are needed for the generation of some epitopes, but the underlying mechanisms have remained elusive. Experiments with LMP2-deficient splenocytes revealed that the generation of the male HY-derived CTL-epitope UTY246–254 was dependent on LMP2. Treatment of male splenocytes with an LMP2-selective inhibitor did not reduce UTY246–254 presentation, whereas silencing of β1 activity increased presentation of UTY246–254. In vitro degradation experiments showed that the caspase-like activity of β1 was responsible for the destruction of this CTL epitope, whereas it was preserved when LMP2 replaced β1. Moreover, inhibition of the β5 subunit rescued the presentation of the influenza matrix 58–66 epitope, thus suggesting that a similar mechanism can apply to the exchange of β5 by LMP7. Taken together, our data provide a rationale why the structural property of an immunoproteasome subunit rather than its activity is required for the generation of a CTL epitope.