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Summary Proteasome-catalyzed peptide splicing (PCPS) generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8 + T cells in L. monocytogenes -infected mice. While reacting to the spliced epitopes, these CD8 + T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8 + T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design. [ABSTRACT FROM AUTHOR]

The proteasome system is the major source for the generation of viral antigens and tumor antigens presented by major histocompatibility complex class I (MHC class I) molecules. A specific feature of the proteasomal antigen processing machinery is that five of its components are inducible by IFN-γ. Two of these are the α and β subunits of the proteasome activator PA28. Our results show that PA28 selectively up-regulates the presentation of viral MHC class I epitopes and that down regulation PA28 in tumor cells results in impaired presentation of a human TRP2 tumor antigen. [Copyright &y& Elsevier]

Proteasomes are the main producers of the epitopes presented on MHC-I molecules to CD8+ T cells. The epitopes could be produced by peptide-bond hydrolysis or by proteasome-catalysed peptide splicing, which are driven by partially different factors. The frequency and immunological relevance of spliced peptides has been a bellicose matter of debate in the last few years. Some studies show that spliced peptides represent around 20-30% of the MHC-I immunopeptidomes. They extend the antigenic landscape of tumour and non-tumour cells to antigens that otherwise would be neglected by the immune system. These antigens are preferentially longer, more hydrophobic and basic than those represented by non-spliced peptides. The immunological relevance of proteasome-generated spliced epitopes in cancer is known since their discovery in 2004 and it has been further confirmed by recent outcomes. In addition, in the last few years, their recognition by CD8+ T cells in Type 1 Diabetes patients and during Listeria monocytogenes infection has also been demonstrated. Therefore, more and more evidences suggest that spliced epitopes can be targets of immunotherapies against cancer, autoimmunity and infections exploiting the theoretical large spliced peptide sequence variety. [ABSTRACT FROM AUTHOR]

Most vaccines are based on protective humoral responses while for intracellular pathogens CD8 + T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8 + T cell priming, hampering vaccine efficacy. The multistage cDNA vaccine H56, encoding three secreted Mycobacterium tuberculosis antigens, was used to test a complete strategy to enhance vaccine’ immunogenicity. Potential CD8 + T cell epitopes in H56 were predicted using the NetMHC3.4/ANN program. Mice were immunized with H56 cDNA using dermal DNA tattoo immunization and epitope candidates were tested for recognition by responding CD8 + T cells in ex vivo assays. Seven novel CD8 + T cell epitopes were identified. H56 immunogenicity could be substantially enhanced by two strategies: (i) fusion of the H56 sequence to cDNA of proteins that modify intracellular antigen processing or provide CD4 + T cell help, (ii) by substitution of the epitope’s hydrophobic C-terminal flanking residues for polar glutamic acid, which facilitated their proteasome-mediated generation. We conclude that this whole strategy of in silico prediction of potential CD8 + T cell epitopes in novel antigens, followed by fusion to sequences with immunogenicity-enhancing properties or modification of epitope flanking sequences to improve proteasome-mediated processing, may be exploited to design novel vaccines against emerging or ‘hard to treat’ intracellular pathogens. [ABSTRACT FROM AUTHOR]

UVB irradiation (290-320 nm) is used to treat skin diseases like psoriasis and atopic dermatitis, and is known to suppress contact hypersensitivity (CHS) reactions in mouse models. Regulatory T cells (Treg cells) have been shown to be responsible for this UVB-induced suppression of CHS. The epidermal growth factor (EGF)-like growth factor amphiregulin (AREG) engages EGFR on Treg cells and, in different disease models, it was shown that mast cell-derived AREG is essential for optimal Treg cell function in vivo. Here we determined whether AREG has a role in UVB-induced, Treg cell-mediated suppression of CHS reactions in the skin. Our data show that AREG is essential for UVB-induced CHS suppression. In contrast to the general assumption, however, mast cells were dispensable for UVB-induced immune suppression, whereas basophil-derived AREG was essential. These data reveal, to our knowledge, a previously unreported function for basophils in the homeostasis of immune responses in the skin. Basophils thus fulfill a dual function: they contribute to the initiation of effective type 2 immune responses and, by enhancing the suppressive capacity of local Treg cell populations, also to local immune regulation in the skin. [ABSTRACT FROM AUTHOR]

Abstract: Pneumoviruses such as pneumonia virus of mice (PVM), bovine respiratory syncytial virus (bRSV) or human (h)RSV are closely related pneumoviruses that cause severe respiratory disease in their respective hosts. It is well-known that T-cell responses are essential in pneumovirus clearance, but pneumovirus-specific T-cell responses also are important mediators of severe immunopathology. In this study we determined whether memory- or pre-existing, transferred virus-specific CD8+ T-cells provide protection against PVM-induced disease. We show that during infection with a sublethal dose of PVM, both natural killer (NK) cells and CD8+ T-cells expand relatively late. Induction of CD8+ T-cell memory against a single CD8+ T-cell epitope, by dendritic cell (DC)-peptide immunization, leads to partial protection against PVM challenge and prevents Th2 differentiation of PVM-induced CD4 T-cells. In addition, adoptively transferred PVM-specific CD8+ T-cells, covering the entire PVM-specific CD8+ T-cell repertoire, provide partial protection from PVM-induced disease. From these data we infer that antigen-specific memory CD8+ T-cells offer significant protection to PVM-induced disease. Thus, CD8+ T-cells, despite being a major cause of PVM-associated pathology during primary infection, may offer promising targets of a protective pneumovirus vaccine. [Copyright &y& Elsevier]

Tumor necrosis factor receptor 2 (TNFR2) has gained much research interest in recent years because of its potential pivotal role in autoimmune disease and cancer. However, its function in regulating different immune cells is not well understood. There is a need for well-characterized reagents to selectively modulate TNFR2 function, thereby enabling definition of TNFR2-dependent biology in human and mouse surrogate models. Here, we describe the generation, production, purification, and characterization of a panel of novel antibodies targeting mouse TNFR2. The antibodies display functional differences in binding affinity and potency to block TNFα. Furthermore, epitope binding showed that the anti-mTNFR2 antibodies target different domains on the TNFR2 protein, associated with varying capacity to enhance CD8+ T-cell activation and costimulation. Moreover, the anti-TNFR2 antibodies demonstrate binding to isolated splenic mouse Tregs ex vivo and activated CD8+ cells , reinforcing their potential use to establish TNFR2-dependent immune modulation in translational models of autoimmunity and cancer. • We have generated a diverse library of anti-mouse TNFR2 antibodies. • Developed anti-mouse TNFR2 antibodies show binding to regulatory T cells (Tregs) and activated CD8+ cells. • Some anti-mouse TNFR2 antibodies costimulate CD8+ cells. [ABSTRACT FROM AUTHOR]

Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens. The interferon-γ-inducible proteasome activator PA28 plays an important role in the generation of MHC ligands by proteasomes. Generation of the HLA-A*0201 restricted melanoma antigen TRP2360-368 by the proteasome has been shown to be dependent on the function of PA28 in vitro and in vivo (Sun, Y., Sijts, A. J., Song, M., Janek, K., Nussbaum, A. K., Kral, S., Schirle, M., Stevanovic, S., Paschen, A., Schild, H., Kloetzel, P. M., and Schadendorf, D. (2002) Cancer Res. 62, 2875-2882). Here we analyzed the role of the epitope sequence environment in determining this PA28 dependence. Experiments using the melanoma TRP2288-296 epitope and the murine cytomegalovirus-derived pp89 epitope precursor peptide for epitope replacement revealed that the TRP2360-368 flanking sequences can transfer PA28 dependence onto otherwise PA28 independent epitopes. Moreover, the N-terminal flanking sequence is sufficient to establish PA28 dependence of an epitope by allowing PA28-induced coordinated dual cleavages. These results show that N-terminal flanking sequences strongly influence epitope generation efficiency and that PA28 function is particularly relevant for the generation of normally poorly excised peptide products. [ABSTRACT FROM AUTHOR]

Due to the technical innovations in generating bispecific antibodies (BsAbs) in recent years, BsAbs have become important reagents for diagnostic and therapeutic applications. However, the difficulty of producing a heterodimer consisting of two different arms with high yield and purity constituted a major limitation for their application in academic and clinical settings. Here, we describe a novel Fc-containing BsAb format (Fab × sdAb-Fc) composed of a conventional antigen-binding fragment (Fab), and a single domain antibody (sdAb), which avoids heavy-light chain mis-pairing during antibody assembly. In this study, the Fab x sdAb-Fc BsAbs were efficiently produced by three widely used heavy-heavy chain heterodimerization methods: Knobs-into-holes (KIH), Charge-pairs (CP) and controlled Fab-arm exchange (cFAE), respectively. The novel Fab x sdAb-Fc format provided a rapid and efficient strategy to generate BsAb with high purity and a unique possibility to further purify desired BsAbs from undesired antibodies based on molecular weight (MW). Compared to conventional BsAb formats, the advantages of Fab x sdAb-Fc format may thus provide a straightforward opportunity to apply bispecific antibody principles to research and development of novel targets and pathways in diseases such as cancer and autoimmunity. • Bispecific antibodies with format Fab x sdAb-Fc constructed using cFAE, CP and KIH. • Bispecific antibodies with format Fab x sdAb-Fc avoid heavy-light chain mis-pairing. • Bispecific antibodies with format Fab x sdAb-Fc provide additional purification methods. [ABSTRACT FROM AUTHOR]