Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes.

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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...

Summary Persistence of hepatitis B virus (HBV) infection is associated with reducedanti-viral T cell responses. Impaired dendritic cell (DC) function was suggested as the cause of reduced T cell stimulation in chronic HBV carriers. Thus, we compared myeloid (mDC) and plasmacytoid DC (pDC) from chronic HBV carriers and controls. Frequency and phenotype of isolated DC were analysed by fluorescence activated cell sorter staining, DC function by mixed lymphocyte reaction, cytokine bead array, intracellular cytokine staining, enzyme-linked immunosorbent assay and enzyme-linked immunospot. Expression of HBV DNA and mRNA was studied by polymerase chain reaction (PCR). Circulating total DC, mDC or pDC were not reduced in chronic HBV carriers. Isolated mDC and pDC from chronic HBV carriers exhibited similar expression of co-stimulatory molecules and alloreactive T helper cell stimulation as control DC, whether tested directly ex vivo or after in vitro maturation. Secretion of pro-and anti-inflammatory cytokines by CD40 or Toll-like receptor ligand-stimulated patient DC was intact, as was human leucocyte antigen A2-restricted HBV-specific cytotoxic lymphocyte stimulation. Although both DC populations contained viral DNA, viral mRNA was undetectable by reverse transcription-PCR, arguing against viral replication in DC. We found no quantitative, phenotypic or functional impairment of mDC or pDC in chronic hepatitis B, whether studied ex vivo or after in vitro maturation.

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.

Long-term treatment of mouse cancer cells with interferon-alpha (IFN-alpha) converts parental B16 melanoma cells to B16alpha vaccine cells. Inoculation of syngeneic mice with B16alpha vaccine cells triggers immunity to the parental B16 tumor that is mediated by host macrophages, T cells, and natural killer (NK) cells. Lymph node cells from mice inoculated with irradiated B16alpha vaccine cells, but not with irradiated parental cells, proliferate when cultured in vitro, suggesting long-term in vivo activation of lymphoid cells. Long-term IFN-alpha treatment of B16alpha vaccine cells induced both interleukin-15 (IL-15) mRNA and IL-15 protein. The bulk of the induced IL-15 remained cell associated, either cytoplasmic or associated with the cell membrane. Immunofluorescence microscopy studies showed that the cell-associated IL-15 was broadly distributed throughout the cytoplasm. These observations suggest that long-term IFN-alpha treatment may induce primarily the truncated isoform of IL-15. Vaccination with irradiated B16alpha vaccine cells may promote tumor immunity by releasing high levels of cell-associated IL-15 when spontaneously lysed or directly killed by innate immune cells. The release of accumulated cell-associated IL-15 may then trigger a host T cell response to tumor antigens and cause host development of immunity to the B16 tumor cells.

Cyclin A1 is a promising antigen for T cell therapy being selectively expressed in high-grade ovarian cancer (OC) and acute myeloid leukemia (AML) stem cells. For adoptive T cell therapy, a single epitope has to be selected, with high affinity to MHC class I and adequate processing and presentation by malignant cells to trigger full activation of specific T cells. In silico prediction with three algorithms indicated 13 peptides of Cyclin A1 9 to 11 amino acids of length to have high affinity to HLA-A*02:01. Ten of them proved to be affine in an HLA stabilization assay using TAP-deficient T2 cells. Their immunogenicity was assessed by repetitive stimulation of CD8+ T cells from two healthy donors with single-peptide-pulsed dendritic cells or monocytes. Intracellular cytokine staining quantified the enrichment of peptide-specific functional T cells. Seven peptides were immunogenic, three of them against both donors. Specific cell lines were cloned and used in killing assays to demonstrate recognition of endogenous Cyclin A1 in the HLA-A*02:01-positive AML cell line THP-1. Immunopeptidome analysis based on direct isolation of HLA-presented peptides by mass spectrometry of primary AML and OC samples identified four naturally presented epitopes of Cyclin A1. The immunopeptidome of HeLa cells transfected with Cyclin A1 and HLA-A*02:01 revealed six Cyclin A1-derived HLA ligands. Epitope p410–420 showed high affinity to HLA-A*02:01 and immunogenicity in both donors. It proved to be naturally presented on primary AML blast and provoked spontaneous functional response of T cells from treatment naïve OC and, therefore, warrants further development for clinical application.

Many components of the class I-antigen-processing pathway are thought to be regulated solely by IFN-γ. Recently, however, we reported type I IFN-mediated induction of immunoproteasomes in a viral infection of the liver (J Clin Invest 2006, 116:3006). In the present study, we extended this analysis to additional components of the MHC class I antigen-processing machinery. Specifically, we investigated type I IFN-mediated induction of the proteasome activator 28 (PA28) subunits α and β, the ER aminopeptidases-1 (ERAP1), -2 (ERAP2) and leucine aminopeptidase (LAP) in vitro and in vivo in a model of HCV infection. This mechanism was initiated by either synthetic or hepatitis C virus (HCV) RNA and abrogated by blocking of type I IFN. In the liver of chimpanzees with acute HCV infection, increased mRNA levels of PA28 subunits and all aminopeptidases occurred prior to IFN-γ responses and instead, coincided with type I IFN responses. Taken together, viral RNA-induced type I IFN regulates the antigen-processing machinery early during infection. Thus, viral RNA-induced type I IFN does not only exert direct antiviral functions but also enhances antigen processing in virus-infected cells to facilitate recognition by effector CD8+ T cells.