The proteasome immunosubunits, PA28 and ER‐aminopeptidase 1 protect melanoma cells from efficient MART‐1_26‐35‐specific T‐cell recognition

Abstract

Eur. J. Immunol. 2015. 45: 3257-3268 IntroductionThe differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (thereafter named MART-1) has been a major target in antigen-specific immunotherapy of malignant melanoma and in particular its immunodominant HLA-A*0201-restricted CD8 + T-cell epitope MART-1 26(27)-35 [1,2]. But clinical efficacy of different MART-1-specific therapies has only been limited. In vitro studies employing high-affinity MART-1-specific CD8 + T-cell clones demonstrated that some melanoma cell lines, despite expression of MART-1 and the corresponding HLA allele, were only barely recognized by specific T cells [3]. While different variants of the MART-1 epitope (including the MART-1 [26][27][28][29][30][31][32][33][34][35] 10-mer and the MART-1 27-35 9-mer) have been identified, only the 9-mer epitope can be eluted in significant amounts from the cell surface of melanoma cells [4]. This suggests that mechanisms related to antigen processing interfere with efficient generation of the 10-mer MART-1 26-35 epitope in tumor cells. It has been demonstrated that MART-1 26-35 epitope processing is controlled by the proteasome, the major proteolytic machinery of the cytosol [5]. The standard proteasome (SP) contains the catalytic subunits β1, β2, and β5, whereas exposure of cells to IFN-γ enhances expression and incorporation of the immunosubunits β1i/LMP2 (LMP, low molecular weight protein), β2i/MECL-1 (multicatalytic endopeptidase complex-like 1), and β5i/LMP7 into nascent proteasome complexes. Immunosubunit-containing proteasomes represent catalytic properties that are different from that of SPs [6]. Interestingly, IFN-γ has been described to interfere with efficient MART-1 26-35 epitope generation due to the activity of the proteasome immunosubunits β1i/LMP2 and β5i/LMP7 [5,7,8].To degrade cellular proteins in an ubiquitin-dependent manner, 20S catalytic core particles are connected to 19S regulatory complexes. In the presence of IFN-γ, the expression of PA28α/β (PA, proteasome activator), an activator complex that in combination with the 19S regulatory complex and the 20S proteasome forms so-called PA28-20S-19S hybrid proteasome complexes, is induced [9,10]. The PA28 has been associated with positive effects on epitope processing due to facilitating the access of substrates to the active sites of the proteasome. Generation of the TRP2 360-368 epitope derived from the melanoma antigen tyrosinase-related protein 2 (TRP2) was shown to be completely dependent on PA28 activity by influencing the proteasomal structure and cleavage specificity [11]. Moreover, IFN-γ induces the expression of the ER-resident aminopeptidases ERAP1/2 that trim epitope precursor peptides released into the ER to bind MHC class I molecules [12][13][14][15]. In this context, increased epitope generation has been preferentially attributed to ERAP1, whereas the role of ERAP2 in epitope processing is still under debate [16]. Varying expression levels of ERAP1/2 have been described in different tumor enti...

ER‐aminopeptidase 1 determines the processing and presentation of an immunotherapy‐relevant melanoma epitope

“…Upon exposure to IFN‐γ, ERAP1 expression was enhanced in both Ma‐Mel‐86a cells and Ma‐Mel‐63a cells (Fig. D) and also in UKRV‐Mel‐15a cells as shown before . Since ERAP activity could be influenced by the various polymorphisms that these enzymes have, we analyzed the ERAP1 alleles present in the investigated cell lines.…”

Immunoproteasomes edit tumors, which then escapes immune recognition

“…Taken together, the report by Keller et al. indicates that a combination of proteolytic activities induced by IFN‐γ can result in immunoediting of tumors and promote immune evasion. This form of immunoediting might explain the curious case of a patient, identified as VMM5, who had a melanoma recur twice over a 12‐year period .…”

“…In additional experiments, Keller et al. found that β1i‐ and/or β2i‐containing HeLa cells poorly activated MART‐1 26—35 ‐specific CD8 + T cells. Accordingly, 20S proteasomes isolated from these cells yielded low‐to‐no MART‐1 26—35 epitope, owing to poor liberation of the carboxyl‐terminus by β2i, as well as the amino‐terminus by both β1i and β2i (Fig.…”

“…Incidentally, chemical inhibition of ERAP‐1 in UKRV‐Mel‐15a cells or downregulation of ERAP1 by RNA interference was shown to enhance MART‐1 26–35 recognition by specific CD8 + T cells suggesting that under these conditions, ERAP‐1 destroyed the MART‐1 26—35 epitope. Accordingly, recombinant ERAP1 cleaved the MART‐1 15—40 substrate to generate the MART‐1 26–35 epitope in vitro, but eventually destroyed it . But whether eventual degradation of the MART‐1 26—35 epitope was due to the absence of peptide receptive A*02;01 in the in vitro cleavage assay remains unclear.…”

PA28 modulates antigen processing and viral replication during coxsackievirus B3 infection

“…24 h post seeding cells were transfected with (i) PA28α and PA28β siRNA (each 30nM, ON-TARGET plus—SMART pool, Dharmacon) or with (ii) control siRNA (60 nM, ON-TARGET plus—SMART pool, Dharmacon) using X-tremeGENE siRNA transfection reagent (Roche) and cultured for 72 hours. For overexpression studies, HeLa cells were seeded into 12-well plates at a density of 1.5 x 10 5 cells per well and transfected the following day with (i) hPA28α-pcDNA3.1 and hPA28β-pcDNA3.1 (0.25 μg each/well) [35] or (ii) mPA28α-pSG5 and mPA28β-pSG5 (0.25 μg each/well) for 24 hours using polyethylenimine (Polysciences). Empty pcDNA3.1 and pSG5 served as a control, respectively.…”