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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.

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

The immunodominant MART-1(26(27)-35) epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART-1/Melan-A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits β5i/LMP7 (LMP, low molecular weight protein) or β1i/LMP2 and β5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit β2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation. β2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-1(26-35) epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-1(26-35) epitope generation even in the absence of IFN-γ. In summary, our results provide first evidence that activities of different antigen-processing components contribute to an inefficient MART-1(26-35) epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope-specific immunotherapies.

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.

Dendritic cell maturation is the process by which immature dendritic cells differentiate into fully competent antigen-presenting cells that initiate T cell responses. Although some mechanistic aspects of DC maturation have begun to be characterised, very little is known about the genetic events regulating the ubiquitin-proteasome system which plays a key role at various levels of the immune response. Therefore, we here investigated the expression of more than 1000 genes related to the ubiquitin-proteasome system in maturing dendritic cells following various stimuli and identified a specific set of transcripts induced by lipopolysaccharide and/or Poly(I:C) which is largely distinct from that induced by CD40 ligand or pro-inflammatory cytokines. This group of genes was dependent on a type I interferon autocrine loop and included E1 and E2 enzymes, E3-ligases, de-ubiquitylating enzymes, proteasome components as well as the ubiquitin-like modifiers ISG15 and FAT10. We further demonstrate that the increased expression of the E2 enzyme UBE2L6 (UbcH8) is required for efficient antigen cross-presentation by dendritic cells. In summary, our data underline the importance of remodelling the ubiquitin-proteasome system for dendritic cell function.

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.

e23080 Background: Cyclin A1 is a promising antigen for T-cell therapy being selectively expressed in ovarian cancer and AML stem cells. For adoptive T-cell therapy, a single epitope has to be selected, which should bind with high affinity to MHC class I and should be adequately processed and presented by malignant cells to trigger full activation of specific T-cells. Methods: Three algorithms were used to predict in silicopeptides of Cyclin A1 with high affinity to HLA A*02:01. Affinities were tested by HLA stabilization assay using TAP-deficient T2 cells. Immunogenicity of peptides was assessed by stimulation of T-cells with peptide pulsed dendritic cells and monocytes. Specificity of T-cells was assessed by intracellular cytokine staining. 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 line THP-1. Data from immunopeptide analysis based on direct isolation of HLA peptides by affinity chromatography and mass spectometry of 20 primary AML samples were analyzed retrospectively to identify naturally presented epitopes of Cyclin A1. Results: Thirteen peptides nine to eleven amino acids of length were predicted. Ten showed stabilization of HLA A*02:01 on T2 cells. By repetitive stimulation of T-cells of two healthy donors with single peptides, 40 T-cell lines were generated, of which eight were specific for the respective peptides. Five peptides were immunogenic. Against two peptides (Cyclin A1 isoform c, p156-164, p410-420) T-cell lines could be generated from both donors. For T-cell clones against two peptides (p111-120, p289-297), specific lysis of THP-1 was shown indicating adequate processing. Peptide p410-420 was found by mass spectroscopy-based ligandome analysis to be presented by AML blasts in vivo. Conclusions: We identified ten peptides with adequate binding to HLA A*02:01. We describe three new HLA A*02:01–restricted epitopes as targets for T-cell therapy. Epitope p410-420 showed high affinity to HLA A*02:01 and immunogenicity in both donors. It proved to be naturally presented on AML blast, and therefore warrants further development for clinical application.

The immunodominant MART-1(26(27)-35) epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART-1/Melan-A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits β5i/LMP7 (LMP, low molecular weight protein) or β1i/LMP2 and β5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit β2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation. β2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-1(26-35) epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-1(26-35) epitope generation even in the absence of IFN-γ. In summary, our results provide first evidence that activities of different antigen-processing components contribute to an inefficient MART-1(26-35) epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope-specific immunotherapies.

The development of telehealth is gathering pace. Advice, telemonitoring, support, as well as teleconsultations or even telediagnosis help to improve access to care across the health territories. The nursing team of a telehealth platform based in Lorraine presents this new area of practice.

International audience; 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 pp65(495-503) is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immuno-dominance 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 pp65(495-503) 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 pp65(495-503) 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 pp65(495-503) epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65(495-503)-specific T cell activation, indicating the importance of ER amino-peptidases in pp65(495-503) generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65(495-503) 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 Journal of Immunology, 2012, 189: 529-538.

The development of telehealth is gathering pace. Advice, telemonitoring, support, as well as teleconsultations or even telediagnosis help to improve access to care across the health territories. The nursing team of a telehealth platform based in Lorraine presents this new area of practice.

Many components of the class I antigen-processing pathway are thought to be regulated solely by interferon-gamma (IFN-gamma). Herein, we report type I IFN-mediated induction of proteasome activator (PA28) subunits alpha and beta, endoplasmic reticulum aminopeptidase 1 (ERAP1), ERAP2, and leucine aminopeptidase (LAP). This mechanism was initiated by either synthetic RNA (poly(I-C)) or by hepatitis C virus (HCV) RNA-mediated induction of type I IFN and abrogated by blocking of type I IFN. In serial liver biopsies of chimpanzees with acute HCV infection, increases in PA28 subunit and aminopeptidase mRNA levels correlated with intrahepatic type I IFN responses and preceded intrahepatic IFN-gamma responses by several weeks. Thus, viral RNA-induced type I IFN regulates the antigen-processing machinery early during viral infection and prior to IFN-gamma response. This mechanism may contribute to the high effectiveness of type I IFN-based therapies if administered early during acute HCV infection.

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.