A Novel Modular Antigen Delivery System for Immuno Targeting of Human 6-sulfo LacNAc-Positive Blood Dendritic Cells (SlanDCs)

Previously, we identified a major myeloid-derived proinflammatory subpopulation of human blood dendritic cells which we termed slanDCs (e.g. Schäkel et al. (2006) Immunity 24, 767-777). The slan epitope is an O-linked sugar modification (6-sulfo LacNAc, slan) of P-selectin glycoprotein ligand-1 (PSGL-1). As slanDCs can induce neoantigen-specific CD4+ T cells and tumor-reactive CD8+ cytotoxic T cells, they appear as promising targets for an in vivo delivery of antigens for vaccination. However, tools for delivery of antigens to slanDCs were not available until now. Moreover, it is unknown whether or not antigens delivered via the slan epitope can be taken up, properly processed and presented by slanDCs to T cells.Single chain fragment variables were prepared from presently available decavalent monoclonal anti-slan IgM antibodies but failed to bind to slanDCs. Therefore, a novel multivalent anti-slanDC scaffold was developed which consists of two components: (i) a single chain bispecific recombinant diabody (scBsDb) that is directed on the one hand to the slan epitope and on the other hand to a novel peptide epitope tag, and (ii) modular (antigen-containing) linker peptides that are flanked at both their termini with at least one peptide epitope tag. Delivery of a Tetanus Toxin-derived antigen to slanDCs via such a scBsDb/antigen scaffold allowed us to recall autologous Tetanus-specific memory T cells.In summary our data show that (i) the slan epitope can be used for delivery of antigens to this class of human-specific DCs, and (ii) antigens bound to the slan epitope can be taken up by slanDCs, processed and presented to T cells. Consequently, our novel modular scaffold system may be useful for the development of human vaccines

Mitochondrial function in neuronal cells depends on p97/VCP/Cdc48-mediated quality control

Maintaining mitochondrial function is essential for neuronal survival and offers protection against neurodegeneration. Ubiquitin-mediated, proteasome-dependent protein degradation in the form of outer mitochondrial membrane associated degradation (OMMAD) was shown to play roles in maintenance of mitochondria on the level of proteostasis, but also mitophagy and cell death. Recently, the AAA-ATPase p97/VCP/Cdc48 was recognized as part of OMMAD acting as retrotranslocase of ubiquitinated mitochondrial proteins for proteasomal degradation. Thus, p97 likely plays a major role in mitochondrial maintenance. Support for this notion comes from mitochondrial dysfunction associated with amyotrophic lateral sclerosis and hereditary inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) caused by p97 mutation. Using SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ treated with mitochondrial toxins rotenone, 6-OHDA, or Aβ-peptide as model for neuronal cells suffering from mitochondrial dysfunction, we found mitochondrial fragmentation under normal and stress conditions was significantly increased upon inactivation of p97. Furthermore, inactivation of p97 resulted in loss of mitochondrial membrane potential and increased production of reactive oxygen species (ROS). Under additional stress conditions, loss of mitochondrial membrane potential and increased ROS production was even more pronounced. Loss of mitochondrial fidelity upon inactivation of p97 was likely due to disturbed maintenance of mitochondrial proteostasis as the employed treatments neither induced mitophagy nor cell death. This was supported by the accumulation of oxidatively-damaged proteins on mitochondria in response to p97 inactivation. Dysfunction of p97 under normal and stress conditions in neuron-like cells severely impacts mitochondrial function, thus supporting for the first time a role for p97 as a major component of mitochondrial proteostasis

A Novel Modular Antigen Delivery System for Immuno Targeting of Human 6-sulfo LacNAc-Positive Blood Dendritic Cells (SlanDCs)

Background Previously, we identified a major myeloid-derived proinflammatory subpopulation of human blood dendritic cells which we termed slanDCs (e.g. Schäkel et al. (2006) Immunity 24, 767–777). The slan epitope is an O-linked sugar modification (6-sulfo LacNAc, slan) of P-selectin glycoprotein ligand-1 (PSGL-1). As slanDCs can induce neoantigen-specific CD4+ T cells and tumor-reactive CD8+ cytotoxic T cells, they appear as promising targets for an in vivo delivery of antigens for vaccination. However, tools for delivery of antigens to slanDCs were not available until now. Moreover, it is unknown whether or not antigens delivered via the slan epitope can be taken up, properly processed and presented by slanDCs to T cells. Methodology/Principal Findings Single chain fragment variables were prepared from presently available decavalent monoclonal anti-slan IgM antibodies but failed to bind to slanDCs. Therefore, a novel multivalent anti-slanDC scaffold was developed which consists of two components: (i) a single chain bispecific recombinant diabody (scBsDb) that is directed on the one hand to the slan epitope and on the other hand to a novel peptide epitope tag, and (ii) modular (antigen-containing) linker peptides that are flanked at both their termini with at least one peptide epitope tag. Delivery of a Tetanus Toxin-derived antigen to slanDCs via such a scBsDb/antigen scaffold allowed us to recall autologous Tetanus-specific memory T cells. Conclusions/Significance In summary our data show that (i) the slan epitope can be used for delivery of antigens to this class of human-specific DCs, and (ii) antigens bound to the slan epitope can be taken up by slanDCs, processed and presented to T cells. Consequently, our novel modular scaffold system may be useful for the development of human vaccines

T Cell Mediated Conversion of a Non-Anti-La Reactive B Cell to an Autoreactive Anti-La B Cell by Somatic Hypermutation

Since the first description of nuclear autoantigens in the late 1960s and early 1970s, researchers, including ourselves, have found it difficult to establish monoclonal antibodies (mabs) against nuclear antigens, including the La/SS-B (Sjögrens’ syndrome associated antigen B) autoantigen. To date, only a few anti-La mabs have been derived by conventional hybridoma technology; however, those anti-La mabs were not bona fide autoantibodies as they recognize either human La specific, cryptic, or post-translationally modified epitopes which are not accessible on native mouse La protein. Herein, we present a series of novel murine anti-La mabs including truly autoreactive ones. These mabs were elicited from a human La transgenic animal through adoptive transfer of T cells from non-transgenic mice immunized with human La antigen. Detailed epitope and paratope analyses experimentally confirm the hypothesis that somatic hypermutations that occur during T cell dependent maturation can lead to autoreactivity to the nuclear La/SS-B autoantigen

Analysis of binding of the SL ScBsDb/linker complex to DD2 positive Jurkat cells.

<p>(A), (a, black graph) IgM isotype negative control. (a, red graph) Binding of the anti-slan IgM mab DD2 to slan(+) positive Jurkat cells. (b, black graph) Anti-penta-HIS, isotype negative control. (b, red graph) Lack of binding of the monovalent SL scBsDb (SL) in the absence of a linker peptide. (c,d black graph) Isotype negative control. (c,d, red graph) Bivalent or trivalent anti-slan scaffolds consisting of the SL scBsDb and the linker peptide l-GFP-l (c, red graph) or l-l-GFP-l (d, red graph). (B), Detection of anti-slan scaffolds on the surface of Jurkat cells by epifluorescence microscopy. GFP (a), phase contrast (b), bar = 10 µm.</p

Immuno targeting of slan(+) Jurkat cells with a multivalent anti-slan scaffold containing a TT-derived T-cell epitope.

<p>(a, black graph) Binding of the anti-slan mab DD2 to slan(+) Jurkat cells. (c,d, red graph) Binding of anti-slan scaffolds consisting of the SL scBsDb and trivalent linker peptides either containing (c) or lacking (d) theTT-derived peptide epitope TT_p. (b,c,d, black graph) Control binding of the single components of the anti-slan scaffolds including the SL scBsDb (b), the trivalent linker l-l-GFP-l (c), and the trivalent linker l- TT_p -l-GFP-l (d).</p

Characterisation of purified linker peptides.

<p>For the formation of bi- or trivalent anti-slan scaffolds peptide linker molecules were constructed containing the l-Tag twice (l-GFP-l) or three times (l-l-GFP-l; l-TT_p-l-GFP-l). The purified peptide linkers were analysed by SDS-PAGE/immunoblotting using the anti-La mab 7B6 directed to the l-Tag. (M) Marker proteins.</p

Construction and characterisation of the SL scBsDb.

<p>(A) A schematic view of the structure and the complete AA sequence of the SL scBsDb. (B) The SL scBsDb was expressed and purified as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016315#s4" target="_blank">Materials and Methods</a>. The flow through fractions, wash fractions (wash1, wash2) and the eluted fractions (elution 1, elution 2) containing the SL scBsDb were analyzed by SDS-PAGE (C) and immunoblotting (B). After SDS-PAGE separated protein(s) were stained with Coomassie brilliant blue (C, CBB), and after transfer the membrane was developed with an anti-penta-HIS antibody (B). M, marker proteins.</p

Binding of the SL scBsDb/linker complex to slan(+) Jurkat cells.

<p>(A) Anti-slan scaffolds were obtained by incubation of the SL scBsDb (SL) with the trivalent linker peptide l-l-GFP-l or the bivalent linker peptide (l-GFP-l, data not shown) at various ratios as indicated and analyzed for binding to slan(+) Jurkat cells. Binding of the maternal IgM anti-slan antibody DD2 was set as 100%. (B) After estimation of the optimal conditions, the binding of the bivalent (SL + l-GFP-l) and trivalent anti-slan scaffolds (SL+l-l-GFP-l) were compared to the binding of the maternal anti-slan IgM antibody DD2 (DD2 IgM). Moreover, the binding of the scaffolds in the presence of the maternal anti-slan IgM antibody DD2 was determined (DD2 IgM+SL+l-l-GFP-l).</p