Abstract Introduction Impaired immune tolerance triggers aberrant or sometimes overshooting immune responses, contributing to disease manifestations and/or exacerbations. Notably, the prevalence of serum cytokine-specific autoantibodies (c-AAbs) has been documented in ‘healthy’ individuals as well as in association with acute and chronic states of immune disorders. Precise detection of c-AAbs, which can either neutralize, stabilize, or modulate the function of cytokines, requires sensitive assays that present the antigen in its native conformation. Methods A novel multiplex flow cytometry-based cellular assay (mFCCA) was devised based on a collection of stable HEK-293T single-cell clones expressing functional interferons and interleukins, for the assessment of c-AAbs in patients’ serum (n = 400), comprising controls, HIV-infected, and those with suspected hyperinflammatory, autoimmune, or malignant disorders characterized by laboratory parameters. The IgG immunoreactivity against 15 cytokine-expressing cell lines was evaluated in a multiplex format, alongside the parental cell line, with specific binding quantified as delta channel geometric mean fluorescence intensity. Subsequently, a cross-validated Random Forest algorithm was applied to discriminate between the five disease phenotypes and the controls. Model performance was gauged by the receiver operating characteristic curves. Results Distinct disease-specific c-AAb signatures were identified across patient groups. For instance, HIV patients exhibited significantly higher frequencies of anti-IFN-α2a, IL-1α, IL-2, and IL-5 autoantibodies compared with controls. The classification model demonstrated high accuracy, with area under the curve values ranging from 0.91 to 0.99, and correct assignment of 331 of 400 samples (83%) to their respective groups. Conclusion The high-throughput approach for screening c-AAb reactivities against surface-expressed cytokines via multiplexed flow cytometry enables rapid immunopathology classification and may assist targeted therapy. Funding Source The work was supported by the Federal State of Lower Austria under the Danube Allergy Research Cluster 2.0 (Danube-ARC 2.0) [grant no. FA624A0404 —Project#13] and the Medical University of Vienna. Topic Categories Basic Autoimmunity (BA)

Eine Immunsuppression unterschiedlicher Genese geht mit einem erhöhten Risiko diverser Infektionen einher. Die Impfprävention besitzt daher bei immunsupprimierten Patientinnen und Patienten einen hohen Stellenwert. Auf Grundlage der verfügbaren Evidenz sowie immunologischer und theoretischer Überlegungen wurden 2016 erstmals Empfehlungen zur Impfversorgung immunsupprimierter Personen formuliert. Diese Empfehlungen wurden nun umfassend aktualisiert und erweitert. Ziel ist die Bereitstellung einer praxisorientierten, indikations- und therapiebezogenen Handlungsanleitung für die klinische Versorgung. Eine Anleitung für die vorliegenden Impfempfehlungen, die gleichzeitig als Entscheidungsbaum für das praktische Vorgehen dienen kann, erleichtert die Orientierung. Die vorliegenden Empfehlungen umfassen einen einführenden Abschnitt zu grundlegenden Aspekten der Immunsuppression sowie zu praxisrelevanten immunologischen und organisatorischen Fragestellungen im Zusammenhang mit Impfplanung, Ermittlung der Impffähigkeit und Überprüfung des Impferfolgs. Darüber hinaus werden aktuelle Entwicklungen zu relevanten Impfstoffen berücksichtigt. Im weiteren Verlauf erfolgt eine systematische Darstellung der in verschiedenen medizinischen Fachgebieten häufig eingesetzten immunsuppressiven Wirkstoffe. Diese werden hinsichtlich ihres Immunsuppressionsgrades und der damit verbundenen Infektionsrisiken eingeordnet. Ergänzend werden standardisierte Impfschemata bereitgestellt, die den jeweiligen Wirkstoffklassen zugeordnet sind und eine strukturierte Orientierung im klinischen Alltag ermöglichen. Die Evaluierung der Impffähigkeit kann immer nur im Zusammenspiel zwischen Grunderkrankung und geplanter oder bestehender Therapie getroffen werden. Besonderen klinischen Situationen – einschließlich jenen, in denen Impfungen nicht durchgeführt werden können sowie Reiseimpfungen im Kontext der Immunsuppression – sind eigene Kapitel gewidmet. Zur Sicherstellung der längerfristigen Aktualität wird an geeigneten Stellen auf aktualisierte externe Quellen verwiesen, die gesammelt am Ende des Dokuments aufgelistet sind. Dieses Dokument ist eine allgemeine Empfehlung, ohne Anspruch auf Vollständigkeit. Das spezifische Vorgehen in der klinischen Praxis kann an die Gegebenheiten in den einzelnen Fachgebieten angepasst werden und es wird auf die jeweiligen Fachgesellschaften verwiesen.

Immunosuppression of various origins is associated with an increased risk of infections and infection-triggered morbidity and mortality. Vaccination, therefore, plays a central role in the care of immunosuppressed individuals by protecting against vaccine-preventable diseases.Based on the available evidence as well as immunological and theoretical considerations, an expert statement on vaccination in immunocompromised individuals was first formulated in 2016. These recommendations have herewith been comprehensively updated and expanded. The aim is to provide a practice-oriented, indication- and therapy-specific guidance document for clinical care. A practical chart accompanying the present vaccination recommendation, which also functions as a decision tree for implementation, facilitates orientation within the document.The document includes an introductory section outlining fundamental aspects of immunosuppression and addressing immunological and organizational issues relevant to vaccination planning, application, and control of vaccination responsiveness. In addition, current developments regarding relevant vaccines are reviewed.Subsequently, a systematic overview of immunosuppressive agents commonly used across medical specialties is provided. These agents are categorized according to their degree of immunosuppression and the associated infection risks. Standardized vaccination schedules are provided and assigned to the respective drug classes to facilitate a structured approach in clinical practice. The decision on vaccination eligibility of these patients can only be made based on a comprehensive assessment of the underlying disease and the planned or ongoing immunosuppressive therapy.Special clinical situations-including circumstances in which vaccination cannot be performed, as well as travel-related vaccinations in the context of immunosuppression-are addressed in separate chapters. To ensure long-term relevance, the document refers to continuously updated external sources throughout, which are compiled at the end.This document acts as general recommendation without a claim of completeness. The specific procedure in clinical practice can be adapted to the clinical context, and the reader is referred to the respective professional societies.

Hypersensitivity reactions are dysregulated and potentially devastating immune responses, characterized by a tendency to become chronic. They target either self-proteins or harmless foreign proteins and are driven by both T and B cells. Although numerous symptomatic treatment options for hypersensitivity reactions have been established over recent decades, only a few antigen-specific, causal approaches capable of specifically targeting the pathogenic autoreactive T and/or B cells have been developed. Among these are cell-based treatment modalities involving chimeric antigen receptor (CAR)- or chimeric autoantibody-receptor (CAAR)-expressing cells. These therapies utilize B- or T-cell antigens, presented as B-cell epitopes or peptide-major histocompatibility complexes (pMHCs) to serve as bait. The latter are coupled to potent activation domains derived from the TCR/CD3 complex itself, such as the zeta or CD3 chains, as well as domains from bona fide co-stimulatory molecules (e.g., CD28, 4-1BB). Recent in vitro and in vivo studies have demonstrated the therapeutic potential of these ATMP-based strategies in eliminating autoreactive lymphocytes and alleviating hypersensitivity reactions. This systematic review provides a comprehensive overview of the current status of antigen-specific CAR and CAAR T-cell therapies, highlighting novel directions as well as the ongoing challenges within this promising research field.

IntroductionDuring the acute-phase of COVID-19, elevated levels of several acute-phase proteins, such as C-reactive protein (CRP), mannose-binding lectin (MBL), pentraxin 3 (PTX-3), serum amyloid A (SAA) and surfactant protein D (SP-D), are associated with severe to fatal clinical outcomes. Typically, these markers return to baseline within days after resolution of the acute infection.MethodsIn this study, we assessed the plasma levels of these proteins in a well-defined cohort of 141 COVID-19 convalescent patients 10 weeks after infection and compared them to 98 non-infected controls. In addition, we performed genetic analyses in a subgroup of patients and related the findings with structural equation modelling to disease severity. ResultsIn contrast to other acute-phase proteins, PTX-3 levels were significantly higher in severe COVID-19 convalescent patients than in the control group. Furthermore, a higher proportion of patients with severe COVID-19 exhibited PTX-3 levels above 5000 pg/ml even 10 months post-infection, compared to those with mild disease. To explore potential genetic influences, a genetic analysis was performed on all severely affected patients (n=36) and on an age- and sex-matched subset of mild COVID-19 patients (n=38). Results revealed a significantly higher frequency (p<0.0001) of the homozygous wildtype genotype of the PTX-3 SNP rs971145291 in severe (15 out of 36) versus mild (1 out of 38) COVID-19 patients. Using structural equation modelling, the association of this PTX-3 genotype and disease severity was shown to be mediated by elevated PTX-3 levels, with no contribution from other analyzed (clinical) confounders. DiscussionIn summary, severe COVID-19 patients show high PTX-3 serum levels which may be influenced by genetic predisposition, specifically the absence of the rs971145291 SNP variant. PTX-3 may thus serve both as a biomarker for tissue damage and/or long-term immune activation and eventually post-COVID-19 complications.

During antiviral immune responses, activated immune cells remodel metabolic pathways towards uptake and utilization of biosynthetic and bioenergetic metabolites. Concurrently, viral infections alter metabolic environments, impacting metabolite availability for the establishment of an effective immune response. Here, we integrated in vivo metabolomics data from murine and human viral infections with in vitro metabolite screens, identifying purine nucleobases as novel immunometabolites that enhance CD8+ T cell effector function. We found that CD8+ T cells can switch from resource-intensive purine de novo synthesis to purine salvage pathway, to produce nucleotides from purine nucleobases. This strategy of metabolic adaptation allows diversion of biosynthetic and bioenergetic resources towards enhancing effector molecule production. Our findings unveil an adaptation strategy by CD8+ T cells to manage metabolic challenges in dynamic organismal environments and suggest pharmacological targets in purine metabolism as potential targets for immunotherapy. ![Figure][1] Graphical Abstract Instead of producing nucleotides via purine de novo synthesis, CD8+ T cells can import and utilize purine nucleobases via the purine salvage pathway to divert bioenergetic and biosynthetic resources towards effector function. By shifting from purine de novo synthesis to the purine salvage pathway, cells save significant resources: 5 moles of the key bioenergetic metabolite ATP, and biosynthetic metabolites including 2 moles of glutamine, 1 mole each of serine or glycine, and 1 mole of aspartate. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, 677006 ANR PRC MetaNiche, ANR-22-CE15-0015-02 [1]: pending:yes

Viral infections, including respiratory diseases such as Coronavirus disease 2019 (COVID-19), are hypothesized to contribute to the onset of autoimmune disorders. Although elevated levels of autoantibodies have been observed following COVID-19, the role of specific autoantibodies linked to autoimmune diseases and their correlation with disease severity remains poorly defined. In this study, we used a comprehensive autoantibody panel to assess the autoantibody production across different cohorts of COVID-19 patients, categorized by disease severity. We also compared patients with severe COVID-19 to a control group with other severe, non-COVID-related diseases. Our findings indicate that the severity of COVID-19 corresponds to the overall production of specific autoantibodies, which are particularly associated with COVID-19. This association might predispose to an increased risk for the development of autoimmune conditions after a severe course of COVID-19.

Virus-like nanoparticles (VNPs) based on Moloney murine leukemia virus represent a well-established platform for the expression of heterologous molecules such as cytokines, cytokine receptors, peptide MHC (pMHC) and major allergens, but their application for inducing protective anti-viral immunity has remained understudied as of yet. Here, we variably fused the wildtype SARS-CoV-2 spike, its receptor-binding domain (RBD) and nucleocapsid (NC) to the minimal CD16b-GPI anchor acceptor sequence for expression on the surface of VNP. Moreover, a CD16b-GPI-anchored single-chain version of IL-12 was tested for its adjuvanticity. VNPs expressing RBD::CD16b-GPI alone or in combination with IL-12::CD16b-GPI were used to immunize BALB/c mice intramuscularly and subsequently to investigate virus-specific humoral and cellular immune responses. CD16b-GPI-anchored viral molecules and IL-12-GPI were well-expressed on HEK-293T-producer cells and purified VNPs. After the immunization of mice with VNPs, RBD-specific antibodies were only induced with RBD-expressing VNPs, but not with empty control VNPs or VNPs solely expressing IL-12. Mice immunized with RBD VNPs produced RBD-specific IgM, IgG2a and IgG1 after the first immunization, whereas RBD-specific IgA only appeared after a booster immunization. Protein/peptide microarray and ELISA analyses confirmed exclusive IgG reactivity with folded but not unfolded RBD and showed no specific IgG reactivity with linear RBD peptides. Notably, booster injections gradually increased long-term IgG antibody avidity as measured by ELISA. Interestingly, the final immunization with RBD-Omicron VNPs mainly enhanced preexisting RBD Wuhan Hu-1-specific antibodies. Furthermore, the induced antibodies significantly neutralized SARS-CoV-2 and specifically enhanced cellular cytotoxicity (ADCC) against RBD protein-expressing target cells. In summary, VNPs expressing viral proteins, even in the absence of adjuvants, efficiently induce functional SARS-CoV-2-specific antibodies of all three major classes, making this technology very interesting for future vaccine development and boosting strategies with low reactogenicity.

Intracellular signaling factors are important targets for immunosuppressive drugs. We report a selective induction of the serine/threonine kinase Provirus Integration site for Moloney murine leukemia virus 3 (PIM3), but not its homologs PIM1 and PIM2, in activated human T cells. Specific pharmacological inhibition and CRISPR/Cas9-mediated knockout of PIM3 in primary human T cells within 2D and 3D cell culture models uncovered essential roles of this kinase in regulating T cell proliferation, viability, migration, metabolic activity, and cytotoxicity. Furthermore, PIM3 inhibition resulted in immunosuppressive effects in human immune organoids. Mass spectrometry-based target protein identification coupled with genome engineering revealed that PIM3 targets the Nucleolar protein Interacting with the FHA domain of MKI67 (NIFK) to control T cell proliferation, establishing a novel regulatory circuit that could be targeted therapeutically in conditions involving T cell hyperproliferation. Elevated PIM3 expression was detected in pathologic T cell infiltrates in human patients and pre-clinical models. Together, these findings identify PIM3 as a promising new target for immunosuppressive therapies. Supported by the Austrian Science Funds (FWF) project P34728-B Immune Response Regulation: Molecular Mechanisms (IRM)

Virus-like nanoparticles (VNP) are regarded as efficient vaccination platforms and have proven to be useful for the non-anaphylactogenic delivery of allergen-specific immunotherapy in preclinical models previously. Herein, we sought to determine the mode of VNP uptake by antigen presenting cells (APC). Accordingly, we screened a collection of substances known to inhibit different uptake pathways by APC. The human leukemia monocytic cell line THP-1 and the murine dendritic cell line DC 2.4 were examined for the uptake of fluorescently labelled VNP in the presence or absence of inhibitors. The inhibitory effect of candidate substances that blocked VNP uptake in APC lines was subsequently evaluated in studies with primary APC present in splenocyte and lung cell homogenates in vitro and upon intratracheal application of VNP in vivo. The uptake of allergen-specific VNP in vitro and in vivo was mainly observed by macrophages and CD103+ dendritic cells and was sensitive to inhibitors that block macropinocytosis, such as hyperosmolarity induced by sucrose or the polyphenol compound Rottlerin at low micromolar concentrations but not by other inhibitors. Also, T-cell proliferation induced by allergen-specific VNP was significantly reduced by both substances. In contrast, substances that stimulate macropinocytosis, such as Heparin and phorbol myristate acetate (PMA), increased VNP-uptake and may, thus, help modulate allergen-specific T-cell responses. We have identified macropinocytosis as the principal uptake mechanism of APC for allergen-specific VNP in vitro and in vivo, paving the way for further improvement of VNP-based therapies, especially those that can be used for tolerance induction in allergy, in the future.

Background: COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a recurrent endemic disease affecting the whole world. Since November 2021, Omicron and its subvariants have dominated in the spread of the disease. In order to prevent severe courses of disease, vaccines are needed to boost and maintain antibody levels capable of neutralizing Omicron. Recently, we produced and characterized a SARS-CoV-2 vaccine based on a recombinant fusion protein consisting of hepatitis B virus (HBV)-derived PreS and two SARS-CoV-2 wild-type RBDs. Objectives: To develop a PreS-RBD vaccine which induces high levels of Omicron-specific neutralizing antibodies. Methods: We designed, produced, characterized and compared strain-specific (wild-type: W-PreS-W; Omicron: O-PreS-O), bivalent (mix of W-PreS-W and O-PreS-O) and chimeric (i.e., W-PreS-O) SARS-CoV-2 protein subunit vaccines. Immunogens were characterized in vitro using protein chemical methods, mass spectrometry, and circular dichroism in combination with thermal denaturation and immunological methods. In addition, BALB/c mice were immunized with aluminum–hydroxide-adsorbed proteins and aluminum hydroxide alone (i.e., placebo) to study the specific antibody and cytokine responses, safety and Omicron neutralization. Results: Defined and pure immunogens could be produced in significant quantities as secreted and folded proteins in mammalian cells. The antibodies induced after vaccination with different doses of strain-specific, bivalent and chimeric PreS-RBD fusion proteins reacted with wild-type and Omicron RBD in a dose-dependent manner and resulted in a mixed Th1/Th2 immune response. Interestingly, the RBD-specific IgG levels induced with the different vaccines were comparable, but the W-PreS-O-induced virus neutralization titers against Omicron (median VNT50: 5000) were seven- and twofold higher than the W-PreS-W- and O-PreS-O-specific ones, respectively, and they were six-fold higher than those of the bivalent vaccine. Conclusion: Among the tested immunogens, the chimeric PreS-RBD subunit vaccine, W-PreS-O, induced the highest neutralizing antibody titers against Omicron. Thus, W-PreS-O seems to be a highly promising COVID-19 vaccine candidate for further preclinical and clinical evaluation.

Background: COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has now become endemic and is currently one of the important respiratory virus infections regularly affecting mankind. The assessment of immunity against SARS-CoV-2 and its variants is important for guiding active and passive immunization and SARS-CoV-2-specific treatment strategies. Methods: We here devised a novel flow cytometry-based diagnostic platform for the assessment of immunity against cell-bound virus antigens. This platform is based on a collection of HEK-293T cell lines which, as exemplified in our study, stably express the receptor-binding domains (RBDs) of the SARS-CoV-2 S-proteins of eight major SARS-CoV-2 variants, ranging from Wuhan-Hu-1 to Omicron. Results: RBD-expressing cell lines stably display comparable levels of RBD on the surface of HEK-293T cells, as shown with anti-FLAG-tag antibodies directed against a N-terminally introduced 3x-FLAG sequence while the functionality of RBD was proven by ACE2 binding. We exemplify the usefulness and specificity of the cell-based test by direct binding of IgG and IgA antibodies of SARS-CoV-2-exposed and/or vaccinated individuals in which the assay shows a wide linear performance range both at very low and very high serum antibody concentrations. In another application, i.e., antibody adsorption studies, the test proved to be a powerful tool for measuring the ratios of individual variant-specific antibodies. Conclusion: We have established a toolbox for measuring SARS-CoV-2-specific immunity against cell-bound virus antigens, which may be considered as an important addition to the armamentarium of SARS-CoV-2-specific diagnostic tests, allowing flexible and quick adaptation to new variants of concern.

IntroductionPancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related deaths worldwide with limited treatment options due to extensive radiation and chemotherapy resistance. Monotherapy with immune checkpoint blockade showed no survival benefit. A combination of immunomodulation and radiotherapy may offer new treatment strategies, as demonstrated for non-small cell lung cancer. Radiation-induced anti-tumour immunity is mediated through cytosolic nucleic acid sensing pathways that drive the expression of interferon beta-1 (IFNB1) and proinflammatory cytokines.MethodsHuman PDAC cell lines (PANC-1, MIA PaCa-2, BxPC-3) were treated with X-rays and protons. Immunogenic cell death was measured based on HMGB1 release. Cytosolic dsDNA and dsRNA were analysed by immunofluorescence microscopy. Cell cycle progression, MHC-I and PD-L1 expression were determined by flow cytometry. Galectin-1 and IFNB1 were measured by ELISA. The expression levels and the phosphorylation status of the cGAS/STING and RIG-I/MAVS signalling pathways were analysed by western blotting, the expression of IFNB1 and proinflammatory cytokines was determined by RT-qPCR and genome-wide by RNA-seq. CRISPR-Cas9 knock-outs and inhibitors were used to elucidate the relevance of STING, MAVS and NF-κB for radiation-induced IFNB1 activation.ResultsWe demonstrate that a clinically relevant X-ray hypofractionation regimen (3x8 Gy) induces immunogenic cell death and activates IFNB1 and proinflammatory cytokines. Fractionated radiation induces G2/M arrest and accumulation of cytosolic DNA in PDAC cells, which partly originates from mitochondria. RNA-seq analysis shows a global upregulation of type I interferon response and NF-κB signalling in PDAC cells following 3x8 Gy. Radiation-induced immunogenic response is regulated by STING, MAVS and NF-κB. In addition to immunostimulation, radiation also induces immunosuppressive galectin-1. No significant changes in MHC-I or PD-L1 expression were observed. Moreover, PDAC cell lines show similar radiation-induced immune effects when exposed to single-dose protons or photons.ConclusionOur findings provide a rationale for combinatorial radiation-immunomodulatory treatment approaches in PDAC using conventional photon-based or proton beam radiotherapy.

BACKGROUND:SARS-CoV-2 has triggered a pandemic and contributes to long-lasting morbidity. Several studies have investigated immediate cellular and humoral immune responses during acute infection. However, little is known about long-term effects of COVID-19 on the immune system. METHODS:We performed a longitudinal investigation of cellular and humoral immune parameters in 106 non-vaccinated subjects ten weeks (10 w) and ten months (10 m) after their first SARS-CoV-2 infection. Peripheral blood immune cells were analyzed by multiparametric flow cytometry, serum cytokines were examined by multiplex technology. Antibodies specific for the Spike protein (S), the receptor-binding domain (RBD) and the nucleocapsid protein (NC) were determined. All parameters measured 10 w and 10 m after infection were compared with those of a matched, noninfected control group (n = 98). RESULTS:Whole blood flow cytometric analyses revealed that 10 m after COVID-19, convalescent patients compared to controls had reduced absolute granulocyte, monocyte, and lymphocyte counts, involving T, B, and NK cells, in particular CD3+CD45RA+CD62L+CD31+ recent thymic emigrant T cells and non-class-switched CD19+IgD+CD27+ memory B cells. Cellular changes were associated with a reversal from Th1- to Th2-dominated serum cytokine patterns. Strong declines of NC- and S-specific antibody levels were associated with younger age (by 10.3 years, p < .01) and fewer CD3-CD56+ NK and CD19+CD27+ B memory cells. Changes of T-cell subsets at 10 m such as normalization of effector and Treg numbers, decline of RTE, and increase of central memory T cell numbers were independent of antibody decline pattern. CONCLUSIONS:COVID-19 causes long-term reduction of innate and adaptive immune cells which is associated with a Th2 serum cytokine profile. This may provide an immunological mechanism for long-term sequelae after COVID-19.