Targeting of the WT191-138 fragment to human dendritic cells improves leukemia-specific T-cell responses providing an alternative approach to WT1-based vaccination.

Due to its immunogenicity and overexpression concomitant with leukemia progression, Wilms tumor protein 1 (WT1) is of particular interest for immunotherapy of AML relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). So far, WT1-specific T-cell responses have mainly been induced by vaccination with peptides presented by certain HLA alleles. However, this approach is still not widely applicable in clinical practice due to common limitations of HLA restriction. Dendritic cell (DC) vaccines electroporated with mRNA encoding full-length protein have also been tested for generating WT1-derived peptides for presentation to T-cells. Alternatively, an efficient and broad WT1 peptide presentation could be elicited by triggering receptor-mediated protein endocytosis of DCs. Therefore, we developed antibody fusion proteins consisting of an antibody specific for the DEC205 endocytic receptor on human DCs and various fragments of WT1 as DC-targeting recombinant WT1 vaccines (anti-hDEC205-WT1). Of all anti-hDEC205-WT1 fusion proteins designed for overcoming insufficient expression, anti-hDEC205-WT110-35, anti-hDEC205-WT191-138, anti-hDEC205-WT1223-273, and anti-hDEC205-WT1324-371 were identified in good yields. The anti-hDEC205-WT191-138 was capable of directly inducing ex vivo T-cell responses by co-incubation of the fusion protein-loaded monocyte-derived mature DCs and autologous T-cells of either healthy or HSCT individuals. Furthermore, the DC-targeted WT191-138-induced specific T-cells showed a strong cytotoxic activity by lysing WT1-overexpressing THP-1 leukemia cells in vitro while sparing WT1-negative hematopoietic cells. In conclusion, our approach identifies four WT1 peptide-antibody fusion proteins with sufficient production and introduces an alternative vaccine that could be easily translated into clinical practice to improve WT1-directed antileukemia immune responses after allo-HSCT.

Cell death triggered by Yersinia enterocolitica identifies processing of the proinflammatory signal adapter MyD88 as a general event in the execution of apoptosis.

Many pathogenic microorganisms have evolved tactics to modulate host cell death or survival pathways for establishing infection. The enteropathogenic bacterium Yersinia enterocolitica deactivates TLR-induced signaling pathways, which triggers apoptosis in macrophages. In this article, we show that Yersinia-induced apoptosis of human macrophages involves caspase-dependent cleavage of the TLR adapter protein MyD88. MyD88 was also cleaved when apoptosis was mediated by overexpression of the Toll-IL-1R domain-containing adapter inducing IFN-ß in epithelial cells. The caspase-processing site was mapped to aspartate-135 in the central region of MyD88. MyD88 is consequently split by caspases in two fragments, one harboring the death domain and the other the Toll-IL-1R domain. Caspase-3 was identified as the protease that conferred the cleavage of MyD88 in in vitro caspase assays. In line with a broad role of caspase-3 in the execution of apoptosis, the processing of MyD88 was not restricted to Yersinia infection and to proapoptotic Toll-IL-1R domain-containing adapter inducing IFN-ß signaling, but was also triggered by staurosporine treatment. The cleavage of MyD88 therefore seems to be a common event in the advanced stages of apoptosis, when caspase-3 is active. We propose that the processing of MyD88 disrupts its scaffolding function and uncouples the activation of TLR and IL-1Rs from the initiation of proinflammatory signaling events. The disruption of MyD88 may consequently render dying cells less sensitive to proinflammatory stimuli in the execution phase of apoptosis. The cleavage of MyD88 could therefore be a means of conferring immunogenic tolerance to apoptotic cells to ensure silent, noninflammatory cell demise.

Beta1 integrin-dependent engulfment of Yersinia enterocolitica by macrophages is coupled to the activation of autophagy and suppressed by type III protein secretion.

Autophagy is a central lysosomal degradation process that is essential for the maintenance of cellular homeostasis. Autophagy has furthermore emerged as integral part of the host immune response. Autophagic processes promote the separation and degradation of intracellular microorganisms which contributes to the development of innate and adaptive immunity. Some pathogenic microbes have therefore evolved mechanisms to evade or impede autophagy. We analyzed the effects of the enteropathogenic bacterium Yersinia enterocolitica on autophagy in macrophages. Yersiniae use a number of defined adhesins and secreted proteins to manipulate host immune responses. Our results showed that Y. enterocolitica defective in type III protein secretion efficiently activated autophagy in macrophages. Autophagy was mediated by the Yersinia adhesins invasin and YadA and particularly depended on the engagement of beta(1) integrin receptors. Several autophagy-related events followed beta(1) integrin-mediated engulfment of the bacteria including the formation of autophagosomes, processing of the marker protein LC3, redistribution of GFP-LC3 to bacteria-containing vacuoles, and the segregation of intracellular bacteria by autophagosomal compartments. These results provide direct evidence for the linkage of beta(1) integrin-mediated phagocytosis and autophagy induction. Multiple microbes signal through integrin receptors, and our results suggest a general principle by which the sensing of an extracellular microbe triggers autophagy. Owing to the importance of autophagy as host defense response, wild-type Y. enterocolitica suppressed autophagy by mobilizing type III protein secretion. The subversion of autophagy may be part of the Y. enterocolitica virulence strategy that supports bacterial survival when beta(1) integrin-dependent internalization and autophagy activation by macrophages are deleterious for the pathogen.

Crosstalk of signalling processes of innate immunity with Yersinia Yop effector functions.

The interaction of microbial pathogens with host cells critically determines the genesis of infectious diseases. Gram-negative, pathogenic bacteria from the genus Yersinia deliver a set of virulence proteins, the so-called Yersinia outer proteins (Yops), inside the eukaryotic cell where the Yops perturb key cellular functions of innate immunity. In our past work, we used Yersinia enterocolitica as a tool to explore the crosstalk between the bacterial pathogen and its host cell. Yersiniae counteract phagocytosis, suppress proinflammatory signalling and trigger apoptosis in macrophages. Macrophage cell death results from the deregulation of Toll-like receptors-dependent conserved signalling pathways by Yersinia infection. We summarize our current understanding about the signals and reactions elicited on both the bacterial and host cell sides that determine the fate of the infected cell along with the innate immune response.

p38MAPK/MK2-dependent phosphorylation controls cytotoxic RIPK1 signalling in inflammation and infection.

Receptor-interacting protein kinase-1 (RIPK1), a master regulator of cell fate decisions, was identified as a direct substrate of MAPKAP kinase-2 (MK2) by phosphoproteomic screens using LPS-treated macrophages and stress-stimulated embryonic fibroblasts. p38MAPK/MK2 interact with RIPK1 in a cytoplasmic complex and MK2 phosphorylates mouse RIPK1 at Ser321/336 in response to pro-inflammatory stimuli, such as TNF and LPS, and infection with the pathogen Yersinia enterocolitica. MK2 phosphorylation inhibits RIPK1 autophosphorylation, curtails RIPK1 integration into cytoplasmic cytotoxic complexes, and suppresses RIPK1-dependent apoptosis and necroptosis. In Yersinia-infected macrophages, RIPK1 phosphorylation by MK2 protects against infection-induced apoptosis, a process targeted by Yersinia outer protein P (YopP). YopP suppresses p38MAPK/MK2 activation to increase Yersinia-driven apoptosis. Hence, MK2 phosphorylation of RIPK1 is a crucial checkpoint for cell fate in inflammation and infection that determines the outcome of bacteria-host cell interaction.