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Lung tissue-resident memory T cells (T RM ) are critical for the local control of respiratory tract infections caused by influenza A viruses (IAV). Here we compare T RM populations induced by intranasal adenoviral vector vaccines encoding hemagglutinin and nucleoprotein (NP) with those induced by an H1N1 infection in BALB/c mice. While vaccine-induced T RM express high levels of CD103 and persist longer in the lung parenchyma, short-lived, H1N1-induced T RM have a transcriptome associated with higher cytotoxic potential and distinct transcriptional profile as shown by single-cell RNA sequencing. In both the vaccine and H1N1 groups, NP-specific CD8 + T cells expand during heterologous influenza virus infection and protect the mice from disease. Meanwhile, lung inflammation in response to an infection with unrelated respiratory syncytial virus do not influence the fate of pre-existing T RM . Our preclinical work thus confirms that inflammatory conditions in the tissue shape the phenotypic and functional characteristics of T RM to serve relevant informations for optimizing mucosal vaccines. Lung tissue-resident memory T cells (T RM ) are important for controlling respiratory infections, but how they are regulated is still unclear. Here the author compare mouse lung T RM induced by either intranasal vaccination or direct H1N1 infection to find distinct phenotypes that converge on protecting the mice from H1N1-induced lung pathology.

SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid–based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections

Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in infants and toddlers. Since natural infections do not induce persistent immunity, there is the need of vaccines providing long-term protection. Here, we evaluated a new adenoviral vector (rAd) vaccine based on the rare serotype rAd19a and compared the immunogenicity and efficacy to the highly immunogenic rAd5. Given as an intranasal boost in DNA primed mice, both vectors encoding the F protein provided efficient protection against a subsequent RSV infection. However, intramuscular immunization with rAd19a vectors provoked vaccine-enhanced disease after RSV infection compared to non-vaccinated animals. While mucosal IgA antibodies and tissue-resident memory T-cells in intranasally vaccinated mice rapidly control RSV replication, a strong anamnestic systemic T-cell response in absence of local immunity might be the reason for immune-mediated enhanced disease. Our study highlighted the potential benefits of developing effective mucosal against respiratory pathogens.

Tumor-associated macrophages (TAMs) dynamically influence anti-tumor immunity. Understanding TAM function is therefore critical to design immunotherapies. By combining syngeneic models of colorectal and pancreatic cancer with cell type-specific deletion of the epithelial-to-mesenchymal transition driver Zeb1, which is expressed in subsets of TAMs, we discovered that ZEB1 is an intrinsic regulator of TAM-controlled T cell trafficking and anti-tumor immune responses. ZEB1 supports secretion of a subset of chemokines via the constitutive pathway, including CXCL10, CCL2 and CCL22, by regulating their biosynthesis, vesicular transport and release. This elevates cytotoxic T cell (CTL) recruitment in vitro and fosters immunosurveillance by CTLs in tumors and metastases as well in an organotypic model for therapeutic CD8 + T cell addition. Our study identifies ZEB1 in TAMs as a facilitator of anti-tumor immunity, suggests a window of opportunity for cytokine-guided CTL tropism and reinforces the importance of onco-immunological context, particularly in the design of macrophage- and/or cytokine-depleting strategies. Macrophage-specific genetic ablation of the EMT inducer ZEB1 reveals its pivotal role in intracellular cytokine trafficking, boosting cytotoxic T cell abundance and immune responses, thereby reducing tumor growth and metastatic colonization in mice.

Severe side effects limit the therapeutic potential of checkpoint control and immunomodulatory antibodies in immunotherapy of cancer. A new study demonstrates that bispecific antibodies directing the immunostimulatory activity of CD40-specific antibodies on dendritic cell subsets may allow a greater therapeutic window of opportunity.

While glioblastoma (GBM) is still challenging to treat, novel immunotherapeutic approaches have shown promising effects in preclinical settings. However, their clinical breakthrough is hampered by complex interactions of GBM with the tumor microenvironment (TME). Here, we present an analysis of TME composition in a patient-derived organoid model (PDO) as well as in organotypic slice cultures (OSC). To obtain a more realistic model for immunotherapeutic testing, we introduce an enhanced PDO model. We manufactured PDOs and OSCs from fresh tissue of GBM patients and analyzed the TME. Enhanced PDOs (ePDOs) were obtained via co-culture with PBMCs (peripheral blood mononuclear cells) and compared to normal PDOs (nPDOs) and PT (primary tissue). At first, we showed that TME was not sustained in PDOs after a short time of culture. In contrast, TME was largely maintained in OSCs. Unfortunately, OSCs can only be cultured for up to 9 days. Thus, we enhanced the TME in PDOs by co-culturing PDOs and PBMCs from healthy donors. These cellular TME patterns could be preserved until day 21. The ePDO approach could mirror the interaction of GBM, TME and immunotherapeutic agents and may consequently represent a realistic model for individual immunotherapeutic drug testing in the future.

Extant proteins frequently share sub-domain sized fragments, suggesting that among other mechanisms, proteins evolved new structure and functions via recombination of existing fragments. While the role of protein fragments as evolutionary units is well-established, their biophysical features necessary for generating a well-folded and stable protein are not clearly understood. In order to probe how fragments determine foldability and stability of recombined proteins, we investigated the stability, folding and dynamics of a synthetic chimera created by fusion of fragments of the chemotactic response regulator protein CheY that belongs to the flavodoxin-like fold and imidazole glycerol phosphate synthase from histidine biosynthesis (HisF) which harbors the TIM-barrel fold. The chimera unfolds via an equilibrium intermediate. Mutation of a glycine residue present at the interface of the CheY and HisF fragments to a valine abrogates the equilibrium intermediate while mutation to isoleucine dramatically increases the native state kinetic stability without any significant change in the folding rate. Parts of the fragment interface in the chimera are found to be conformationally dynamic while hydrophobic mutations globally increase its conformational rigidity. We hypothesize that the hydrophobic mutation improves sidechain packing in a large cluster of isoleucine, leucine and valine (ILV) residues that spans the fragment interface. We also extrapolate that inheritance of large ILV clusters from parent proteins could be a key determinant of successful fragment recombination.

[Einleitung ...] Im folgenden Kapitel werden die theoretischen Grundlagen des Experiments dargestellt. Dabei werden zunächst allgemeine Annahmen, die für das Experiment getroffen wurden, erläutert und danach auf die Hintergründe der verschiedenen Strategien, die für das Experiment modelliert wurden, eingegangen. Kapitel 3 zeigt im Anschluss den Aufbau des Experiments. Hierbei wird darauf eingegangen, wie sich das Experiment den Teilnehmern darstellte und was während der Durchführung im Hintergrund passierte. Jedes Treatment wird dazu einzeln erläutert und von den anderen abgegrenzt. Die Arbeit schließt in Kapitel 4 mit dem Fazit und einem Ausblick zur Durchführung des vorgestellten Experiments.

Background. While chimeric antigen receptor (CAR) T cells have achieved encouraging remission rates in hematological malignancies, they demonstrated limited success in treating Glioblastoma (GBM), particularly due to high intra- and intertumoral heterogeneity. In this study, we identified a relevant and preserved target antigen, Podoplanin (PDPN), and evaluated the potential of a PDPN- and GD2-CAR T cell blend to overcome GBM heterogeneity.Methods. Target antigen screening included clinical samples, healthy tissues and cell lines, as well as publicly available RNA sequencing datasets. The anti-tumor function of CAR T cells were examined in co-culture experiments with GBM cell lines and patient-derived organoids (PDOs), and in vivo after locoregional delivery in orthotopic xenograft models. Results. The generated CAR T cells demonstrated strong anti-tumor activity against several cell lines and PDOs from multiple patients. PDPN and GD2 expression was detectable in all PDOs at varying densities and regardless of the antigenic profile, the CAR T cell blend induced significantly higher levels of apoptosis in organoids than single antigen targeting counterparts. In vivo, we observed efficient tumor regression after locoregional administration of monospecific CAR T cells. While heterogeneous orthotopic tumors eventually relapsed in these groups, blended therapy resulted in a significantly increased overall survival and even achieved cure in the majority of mice.Conclusion. This novel PDPN-/GD2-CAR T cell blend demonstrated strong efficacy in advanced preclinical models of glioblastoma. The results suggest that this approach can overcome GBM heterogeneity in clinical application and address previous limitations of single antigen CAR T cell therapies.Competing Interest StatementMH is listed as inventor on patent applications and granted patents related to CAR T technologies that have been filed by the Fred Hutchinson Cancer Research Center, Seattle, WA and that have been, in part, licensed by industry. MH and TN, are listed as inventors on patent applications and granted patents related to CAR T technologies that have been filed by the University of Würzburg, Würzburg, Germany and that have been, in part, licensed by industry. MH is a co-founders and equity owners of T-CURX GmbH, Würzburg, Germany. TN is employed at T-CURX GmbH, Würzburg, Germany. MH received honoraria from BMS, Janssen, and Kite/Gilead. The other authors declare that they have no competing interests.

Tumor-associated macrophages (TAMs) shape the tumor microenvironment (TME) and exert a decisive impact on anti-tumor immunity. Understanding TAM function is therefore critical to understand anti-tumor immune responses and to design immunotherapies. Here, we describe the transcription factor ZEB1, a well-known driver of epithelial-to-mesenchymal transition, as an intrinsic regulator of TAM function in adaptive anti-tumor immunity. By combining cell type-specific deletion of Zeb1 with syngeneic models of colorectal and pancreatic cancer, we discovered an unexpected function of ZEB1 in the TAM-mediated control of T cell trafficking. ZEB1 supports secretion of a subset of chemokines including CCL2 and CCL22 by promoting their transcription and translation as well as by safeguarding protein processing. ZEB1 thereby elevates cytotoxic T cell (CTL) recruitment in vitro and in vivo and fosters immunosurveillance during tumor as well as lung metastatic outgrowth. Our study spotlights ZEB1 as a crucial facilitator of adaptive anti-tumor immunity and uncovers a potential therapeutic window of opportunity for cytokine-guided enhancement of CTL infiltration into tumors and metastases.Competing Interest StatementThe authors have declared no competing interest.