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Schizophrenia is a psychiatric disorder that is still not readily treatable. Pharmaceutical advances in the treatment of schizophrenia have mainly focused on the protein coding part of the human genome. However, the vast majority of the human transcriptome consists of non-coding RNAs. MicroRNAs are small non-coding RNAs that control the transcriptome at the systems level. In the present study we analyzed the microRNAome in blood and postmortem brains of controls and schizophrenia patients and found that miR-99b-5p was downregulated in both the prefrontal cortex and blood of patients. At the mechanistic level we show that inhibition of miR-99b-5p leads to schizophrenia-like phenotypes in mice and induced inflammatory processes in microglia linked to synaptic pruning. The miR-99b-5p-mediated inflammatory response in microglia depended onZ-DNA binding protein 1(Zbp1) which we identified as a novel miR-99b-5p target. Antisense oligos (ASOs) againstZbp1ameliorated the pathological phenotypes caused by miR-99b-5p inhibition. In conclusion, we report a novel miR-99b-5p-Zbp1pathway in microglia that contributes to the pathogenesis of schizophrenia. Our data suggest that strategies to increase the levels of miR-99b-5p or inhibitZbp1could become a novel therapeutic strategy.

It is undisputed that CD8 T cells play a critical role in controlling tumor growth and killing tumor cells, and that tumor specific T cells are essential for clinical responses to immune checkpoint inhibitors. Yet, the induction and maintenance of functional tumor antigen specific T cell responses represent still one of the greatest challenges in cancer immunotherapy. Therapeutic cancer vaccines based on engineered arenavirus vectors have been shown preclinically to overcome self-tolerance to tumor associated antigens (TAA) and mount unprecedented T cell responses, both, in preclinical tumor models as well as HPV16+ cancer patients in an ongoing Phase 1/2 trial. The novel PD-1 targeted IL-2 variant (PD1-IL2v) was created to maximize the potency of IL-2R activation of effector T cells while overcoming the toxicities of wildtype IL-2. Cis binding of PD1-IL2v to PD-1 and IL-2Rβγ on the same cell was recently shown to promote differentiation of stem-like CD8 T cells into better effectors and was therefore identified as an ideal compound for combination therapies with arenavirus vectors. Combination of vectors encoding tumor associated foreign or self-antigens with PD1-IL2v lead to a massive, up to 60-fold, increase of peripheral tumor specific CD8 T cell numbers compared to vector treatment alone. Notably, frequency of peripheral regulatory T cells was not enhanced by this combination. The massive increase of TAA specific CD8 T cells was associated with synergistic anti-tumor efficacy in two independent tumor models, resulting in longer survival times and up to 60% complete responders. Complete tumor clearance resulted in a stable memory T cell population and protection against tumor re-challenge. Ongoing studies will address modifications of the tumor microenvironment and further characterization of tumor infiltrating T cells upon combination therapy. These preclinical data establish the combination of arenavirus vectors and PD1-IL2v as a promising next generation cancer immunotherapy. Citation Format: Judith Strauss, Diana Reckendorfer, Kimberly Pojar, Laura Codarri Deak, Valeria Nicolini, Roger Sutmuller, Christian Klein, Pablo Umaña, Klaus K. Orlinger, Henning Lauterbach. A novel combination therapy of arenavirus vectors and PD1-IL2v strongly potentiates tumor specific T cell responses resulting in synergistic anti-tumor efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5747.

The proinflammatory cytokine interleukin 1 (IL-1) is crucially involved in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Herein, we studied the role of IL-1 signaling in blood–brain barrier (BBB) endothelial cells (ECs), astrocytes and microglia for EAE development, using mice with the conditional deletion of its signaling receptor IL-1R1. We found that IL-1 signaling in microglia and astrocytes is redundant for the development of EAE, whereas the IL-1R1 deletion in BBB-ECs markedly ameliorated disease severity. IL-1 signaling in BBB-ECs upregulated the expression of the adhesion molecules Vcam-1, Icam-1 and the chemokine receptor Darc, all of which have been previously shown to promote CNS-specific inflammation. In contrast, IL-1R1 signaling suppressed the expression of the stress-responsive heme catabolizing enzyme heme oxygenase-1 (HO-1) in BBB-ECs, promoting disease progression via a mechanism associated with deregulated expression of the IL-1-responsive genes Vcam1, Icam1 and Ackr1 (Darc). Mechanistically, our data emphasize a functional crosstalk of BBB-EC IL-1 signaling and HO-1, controlling the transcription of downstream proinflammatory genes promoting the pathogenesis of autoimmune neuroinflammation. Electronic supplementary material The online version of this article (10.1007/s00401-020-02187-x) contains supplementary material, which is available to authorized users.

Angiopoietin-2 (Ang2), a ligand of the endothelial Tie2 tyrosine kinase, is involved in vascular inflammation and leakage in critically ill patients. However, the role of Ang2 in demyelinating central nervous system (CNS) autoimmune diseases is unknown. Here, we report that Ang2 is critically involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis. Ang2 expression was induced in CNS autoimmunity, and transgenic mice overexpressing Ang2 specifically in endothelial cells (ECs) developed a significantly more severe EAE. In contrast, treatment with Ang2-blocking Abs ameliorated neuroinflammation and decreased spinal cord demyelination and leukocyte infiltration into the CNS. Similarly, Ang2-binding and Tie2-activating Ab attenuated the development of CNS autoimmune disease. Ang2 blockade inhibited expression of EC adhesion molecules, improved blood-brain barrier integrity, and decreased expression of genes involved in antigen presentation and proinflammatory responses of microglia and macrophages, which was accompanied by inhibition of α5β1 integrin activation in microglia. Taken together, our data suggest that Ang2 provides a target for increasing Tie2 activation in ECs and inhibiting proinflammatory polarization of CNS myeloid cells via α5β1 integrin in neuroinflammation. Thus, Ang2 targeting may serve as a therapeutic option for the treatment of CNS autoimmune disease.

T cells play a central role in immune responses against cancer. Within the tumor, however, T cells are exposed to a plethora of inactivating factors causing various degrees of dysfunction, changes in metabolism and a generally reduced cellular fitness, eventually leading to tumor progression. To prevent or delay the onset of exhaustion and instead augment effector functions and persistence of functional T cells, costimulatory factors and cytokines are needed. Targeting 4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily, has been shown to represent a promising strategy for inducing an activating signal in CD8+ T cells, resulting in increased pro-inflammatory cytokine secretion, cytotoxic function, and survival. Engineered arenavirus vectors based on lymphocytic choriomeningitis virus (LCMV) or Pichinde virus (PICV) have been shown previously to induce massive infiltration of tumor antigen specific CD8+ T cells into the tumor in several preclinical cancer models. To investigate whether enhanced co-stimulation via 4-1BB further improves T cell responses and/or tumor control, combination therapies with replicating LCMV based vectors (artLCMV) and 4-1BB agonists were explored. A single intravenous treatment of artLCMV encoding the tumor associated antigens (TAA) gp70 or Trp2 in B16.F10 tumor bearing mice induced TAA specific CD8+ T cells in both the periphery and the tumor, resulting in tumor growth delay and some complete responses. Combining artLCMV with agonistic anti-4-1BB significantly improved tumor control and increased the number of complete responders. Analysis of tumor infiltrating lymphocytes revealed higher absolute numbers of TAA specific CD8+ T cells in the combination group compared to the artLCMV alone group. Analyses of the TAA specific cells revealed that more cells expressed granzyme B, Ki67 and Bcl-XL when co-stimulated with anti-4-1BB compared to the group treated with artLCMV alone. Importantly, encoding 4-1BBL in addition to a TAA in artLCMV revealed similar outcomes as just summarized for the combination with agonistic antibodies. Overall, these experiments confirmed the strong antigenicity and T cell inducing capacity of the engineered arenavirus platform, leading to efficient tumor control in a stringent mouse model. Combination with 4-1BB agonists, either in form of antibodies or encoded within the vector genome, was shown to further augment TAA-specific T cell responses within the tumor, leading to better tumor growth control and a higher rate of complete responders. Citation Format: Judith Strauss, Marilies Scheinost, Theresa Kleissner, Diana Reckendorfer, Kimberly Pojar, Mohamed Habbeddine, Sarah Ahmadi-Erber, Daniela Deutschmann, Sarah Schmidt, Josipa Raguz, Igor Matushansky, Christoph Lampert, Klaus K. Orlinger, Henning Lauterbach. Evaluation of a cancer immunotherapy with engineered arenavirus vectors and 4-1BB agonists in a preclinical tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4198.

The meninges, comprising the leptomeninges (pia and arachnoid layers) and the pachymeninx (dura layer), participate in central nervous system (CNS) autoimmunity, but their relative contributions remain unclear. Here we report on findings in animal models of CNS autoimmunity and in patients with multiple sclerosis, where, in acute and chronic disease, the leptomeninges were highly inflamed and showed structural changes, while the dura mater was only marginally affected. Although dural vessels were leakier than leptomeningeal vessels, effector T cells adhered more weakly to the dural endothelium. Furthermore, local antigen-presenting cells presented myelin and neuronal autoantigens less efficiently, and the activation of autoreactive T cells was lower in dural than leptomeningeal layers, preventing local inflammatory processes. Direct antigen application was required to evoke a local inflammatory response in the dura. Together, our data demonstrate an uneven involvement of the meningeal layers in CNS autoimmunity, in which effector T cell trafficking and activation are functionally confined to the leptomeninges, while the dura remains largely excluded from CNS autoimmune processes.

Glucocorticoids (GC) are widely used to treat acute relapses in multiple sclerosis (MS) patients, but their application is accompanied by side effects due to their broad spectrum of action. Here, we report on the therapeutic option to apply GC via inorganic-organic hybrid nanoparticles (IOH-NP) with the composition [ZrO]2+[(BMP)0.9(FMN)0.1]2- (designated BMP-NP with BMP: betamethasone phosphate; FMN: flavinmononucleotide). We found that these BMP-NP have an increased cell type-specificity compared to free GC while retaining full therapeutic efficacy in a mouse model of MS. BMP-NP were preferentially taken up by phagocytic cells and modulated macrophages in vivo more efficiently than T cells. When GC were applied in the form of BMP-NP, treatment of neuroinflammatory disease in mice exclusively depended on the control of macrophage function whereas effects on T cells and brain endothelial cells were dispensable for therapeutic efficacy. Importantly, BMP-NP were not only active in mice but also showed strong activity towards monocytes isolated from healthy human volunteers. We conclude that application of GC via IOH-NP has the potential to improve MS therapy in the future.

Background Quinoline-3-carboxamides, such as laquinimod, ameliorate CNS autoimmunity in patients and reduce tumor cell metastasis experimentally. Previous studies have focused on the immunomodulatory effect of laquinimod on myeloid cells. The data contained herein suggest that quinoline-3-carboxamides improve the immunomodulatory and anti-tumor effects of NK cells by upregulating the adhesion molecule DNAX accessory molecule-1 (DNAM-1). Methods We explored how NK cell activation by laquinimod inhibits CNS autoimmunity in experimental autoimmune encephalomyelitis (EAE), the most utilized model of MS, and improves immunosurveillance of experimental lung melanoma metastasis. Functional manipulations included in vivo NK and DC depletion experiments and in vitro assays of NK cell function. Clinical, histological, and flow cytometric read-outs were assessed. Results We demonstrate that laquinimod activates natural killer (NK) cells via the aryl hydrocarbon receptor and increases their DNAM-1 cell surface expression. This activation improves the cytotoxicity of NK cells against B16F10 melanoma cells and augments their immunoregulatory functions in EAE by interacting with CD155+ dendritic cells (DC). Noteworthy, the immunosuppressive effect of laquinimod-activated NK cells was due to decreasing MHC class II antigen presentation by DC and not by increasing DC killing. Conclusions This study clarifies how DNAM-1 modifies the bidirectional crosstalk of NK cells with CD155+ DC, which can be exploited to suppress CNS autoimmunity and strengthen tumor surveillance. Open-Access-Publikationsfonds 2019

Multiple sclerosis (MS) is caused by T cells that are reactive for brain antigens. In experimental autoimmune encephalomyelitis, the animal model for MS, myelin-reactive T cells initiate the autoimmune process when entering the nervous tissue and become reactivated upon local encounter of their cognate CNS antigen. Thereby, the strength of the T-cellular reactivation process within the CNS tissue is crucial for the manifestation and the severity of the clinical disease. Recently, B cells were found to participate in the pathogenesis of CNS autoimmunity, with several diverse underlying mechanisms being under discussion. We here report that B cells play an important role in promoting the initiation process of CNS autoimmunity. Myelin-specific antibodies produced by autoreactive B cells after activation in the periphery diffused into the CNS together with the first invading pathogenic T cells. The antibodies accumulated in resident antigen-presenting phagocytes and significantly enhanced the activation of the incoming effector T cells. The ensuing strong blood-brain barrier disruption and immune cell recruitment resulted in rapid manifestation of clinical disease. Therefore, myelin oligodendrocyte glycoprotein (MOG)-specific autoantibodies can initiate disease bouts by cooperating with the autoreactive T cells in helping them to recognize their autoantigen and become efficiently reactivated within the immune-deprived nervous tissue.

The antibody response to proteins may be modulated by the presence of preexisting antigen-specific antibodies and the formation of immune complexes (ICs). Effects such as a general increase or decrease of the response as well as epitope-specific phenomena have been described. In this study, we investigated influences of IC immunization on the fine specificity of antibody responses in a structurally well-defined system, using the envelope (E) protein of tick-borne encephalitis (TBE) virus as an immunogen. TBE virus occurs in Europe and Asia and—together with the yellow fever, dengue, West Nile, and Japanese encephalitis viruses—represents one of the major human-pathogenic flaviviruses. Mice were immunized with a dimeric soluble form of E (sE) alone or in complex with monoclonal antibodies specific for each of the three domains of E, and the antibody response induced by these ICs was compared to that seen after immunization with sE alone. Immunoassays using recombinant domains and domain combinations of TBE virus sE as well as the distantly related West Nile virus sE allowed the dissection and quantification of antibody subsets present in postimmunization sera, thus generating fine-specificity patterns of the polyclonal responses. There were substantially different responses with two of the ICs, and the differences could be mechanistically related to (i) epitope shielding and (ii) antibody-mediated structural changes leading to dissociation of the sE dimer. The phenomena described may also be relevant for polyclonal responses upon secondary infections and/or booster immunizations and may affect antibody responses in an individual-specific way. IMPORTANCE Infections with flaviviruses such as yellow fever, dengue, Japanese encephalitis, West Nile, and tick-borne encephalitis (TBE) viruses pose substantial public health problems in different parts of the world. Antibodies to viral envelope protein E induced by natural infection or vaccination were shown to confer protection from disease. Such antibodies can target different epitopes in E protein, and the fine specificities of polyclonal responses can differ between individuals. We conducted a mouse immunization study with TBE E protein alone or complexed to monoclonal antibodies specific for each of the three protein domains. We demonstrated that phenomena such as epitope shielding and antibody-induced structural changes can profoundly influence the fine specificity of antibody responses to the same immunogen. The study thus provided important new information on the potential immunomodulatory role of preexisting antibodies in a flavivirus system that can be relevant for understanding individual-specific factors influencing antibody responses in sequential flavivirus infections and/or immunizations.

Tick-borne encephalitis (TBE) virus is an important human-pathogenic flavivirus endemic in large parts of Europe and Central and Eastern Asia. Neutralizing antibodies specific for the viral envelope protein E are believed to mediate long-lasting protection after natural infection and vaccination. To study the specificity and individual variation of human antibody responses, we developed immunoassays with recombinant antigens representing viral surface protein domains and domain combinations. These allowed us to dissect and quantify antibody populations of different fine specificities in sera of TBE patients and vaccinees. Postinfection and postvaccination sera both displayed strong individual variation of antibody titers as well as the relative proportions of antibodies to different domains of E, indicating that the immunodominance patterns observed were strongly influenced by individual-specific factors. The contributions of these antibody populations to virus neutralization were quantified by serum depletion analyses and revealed a significantly biased pattern. Antibodies to domain III, in contrast to what was found in mouse immunization studies with TBE and other flaviviruses, did not play any role in the human neutralizing antibody response, which was dominated by antibodies to domains I and II. Importantly, most of the neutralizing activity could be depleted from sera by a dimeric soluble form of the E protein, which is the building block of the icosahedral herringbone-like shell of flaviviruses, suggesting that antibodies to more complex quaternary epitopes involving residues from adjacent dimers play only a minor role in the total response to natural infection and vaccination in humans. IMPORTANCE Tick-borne encephalitis (TBE) virus is a close relative of yellow fever, dengue, Japanese encephalitis, and West Nile viruses and distributed in large parts of Europe and Central and Eastern Asia. Antibodies to the viral envelope protein E prevent viral attachment and entry into cells and thus mediate virus neutralization and protection from disease. However, the fine specificity and individual variation of neutralizing antibody responses are currently not known. We have therefore developed new in vitro assays for dissecting the antibody populations present in blood serum and determining their contribution to virus neutralization. In our analysis of human postinfection and postvaccination sera, we found an extensive variation of the antibody populations present in sera, indicating substantial influences of individual-specific factors that control the specificity of the antibody response. Our study provides new insights into the immune response to an important human pathogen that is of relevance for the design of novel vaccines.