Cancer testis antigen Cyclin A1 harbors several HLA-A*02:01-restricted T cell epitopes, which are presented and recognized in vivo.

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.

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

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 ß5i/LMP7 (LMP, low molecular weight protein) or ß1i/LMP2 and ß5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit ß2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation. ß2i/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.

[Nurses in telehealth]. / Expérience infirmière en télésanté.

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.

The efficiency of human cytomegalovirus pp65(495-503) CD8+ T cell epitope generation is determined by the balanced activities of cytosolic and endoplasmic reticulum-resident peptidases.

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 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 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 aminopeptidases 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.

Maturation of human dendritic cells is accompanied by functional remodelling of the ubiquitin-proteasome system.

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.

Proteasome activator and antigen-processing aminopeptidases are regulated by virus-induced type I interferon in the hepatitis C virus-infected liver.

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.