The Efficiency of Human Cytomegalovirus pp65495–503 CD8+ T Cell Epitope Generation Is Determined by the Balanced Activities of Cytosolic and Endoplasmic Reticulum-Resident Peptidases

Abstract

Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8+ CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65495–503 is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65495–503 generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65495–503 epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65495–503 epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65495–503-specific T cell activation, indicating the importance of ER aminopeptidases in pp65495–503 generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65495–503 epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency.

The proteasome immunosubunits, PA28 and ER‐aminopeptidase 1 protect melanoma cells from efficient MART‐126‐35‐specific T‐cell recognition

“…In CTL assays, the following target cells were used: UKRV-Mel-15a cells either exposed to 200 U/mL IFN-γ, or exposed to 30 μM leucinethiol (Sigma, Germany; solved in 0.5 mM DTT) for 17 h, HeLa A2+ cell lines, HeLa A2+ cells transfected with pcDNA3.1 plasmid and/or loaded with 5 μg/mL MART-1 [26][27][28][29][30][31][32][33][34][35] peptide for 1 h and T2 cells, as well as Ma-Mel-63a cells, loaded with 0.1 or 1 μg/mL MART-1 [26][27][28][29][30][31][32][33][34][35] peptide for 2 h. HeLa A2+ and HeLa A2+/IP cells were transiently transfected with the expression plasmids encoding PA28α, PA28β, or MART-1 for 24 h according to the manufacturer's instructions (Lipofectamine 2000, Invitrogen, Germany). In order to silence ERAP1/2, UKRV-Mel-15a cells were transfected with specific siRNA (Eurogentec, Germany) at a concentration of 100 nM (ERAP1) or 200 nM (pool of four duplexes ERAP2) for 72 h, as described [24]. As control, nontargeting siRNA from Dharmacon (Germany) was employed.…”

“…As control, nontargeting siRNA from Dharmacon (Germany) was employed. For RT-PCR analysis, primers for ERAP1/2 and GAPDH were used as described as well as MART-1 forward 5´-CATCCTGACAGTGATCCTG-3´and reverse 5´-GGTGGTGACTGTTCTGCAG-3´ [24]. For PA28 knockdown, MART-1 expressing Ma-Mel-63a cells were transfected with 20 nM siRNA oligonucleotides for 72 h targeting either PA28α or PA28β (PA28α/β siRNA, SMARTpool catalogue no.…”

“…These data show that ERAP1/2 negatively contributes to MART-1 [26][27][28][29][30][31][32][33][34][35] epitope generation and that in particular, ERAP1 is responsible for the impairment of epitope presentation by IFN-γ. To study the underlying molecular mechanism, we next performed in vitro trimming experiments using recombinant ERAPs in combination with the N-terminally extended 14-mer epitope precursor peptide MART-1 [22][23][24][25][26][27][28][29][30][31][32][33][34][35] . As shown by reversed-phase HPLC analyses, ERAP1 efficiently trimmed the MART-1 [22][23][24][25][26][27][28][29][30][31][32][33][34][35] precursor peptide.…”

“…2B) cells expressing HLA-A*0201) clones that constitutively overexpressed each immunosubunit alone or in combination. HeLa A2+ cells containing predominantly SPs and HeLa A2+/IP cells expressing immunoproteasomes (IPs) were used as controls [24]. Initially, we analyzed the proteasome composition of the HeLa A2+ transfectants using isolated cellular 20S proteasome complexes.…”

“…Melanoma cells and T2 cells were grown in RPMI1640 (Biochrom AG, Germany). HeLa A2+ cell lines were generated by transfection with the proteasomal immunosubunits (β1i/LMP2, β2i/MECL-1, β5i/LMP7, and β2i-T1A/MECL-1-T1A) as described and were grown in medium supplemented with 2 μg/mL puromycin and 300 μg/mL hygromycin (PAA Laboratories, Germany) [24]. …”