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After allogeneic hematopoietic cell transplantation (Allo-HCT) and conditioning, patients are typically placed in isolated hospital rooms to prevent neutropenic infections. Since 1998, we've offered an alternative: home care for patients living within a one to two-hour drive of the hospital. In Sweden this approach includes daily visits by an experienced nurse and daily phone consultations with a unit physician. When necessary, patients receive transfusions, intravenous antibiotics, and total parenteral nutrition at home. Our initial study report compared 36 home care patients with 54 hospital-treated controls. Multivariate analysis found that home care patients were discharged earlier to outpatient clinics, required fewer days of total parenteral nutrition, had less acute graft-versus-host disease (GVHD) grade II-IV, and lower transplantation-related mortality (TRM) and lower costs. Long-term follow-up showed similar chronic GVHD and relapse rates in both groups, with improved survival rates in the home care group. A subsequent comparison of 146 home care patients with hospital-treated controls indicated that home care and longer home stays were associated with lower grades of acute GVHD. Home care was found to be safe and beneficial for children and adolescents. Over two decades, 252 patients received home care post-Allo-HCT without any fatalities at-home. Ten-year outcomes showed a 14% TRM and a 59% survival rate. In 2020, an independent center confirmed the reduced risk of acute GVHD grades II-IV for patients treated in home care. Here, we report for the first time that home care patients also demonstrate a less inflammatory systemic cytokine profile. We found higher levels of IFN-γ, IL-2, IL-5, IL-13, GM-CSF, and G-CSF, but lower VEGF in hospital-treated patients, which may contribute to acute GVHD grades II-IV. In conclusion, home-based treatment following Allo-HCT yields multiple promising clinical outcomes and improved systemic inflammatory markers, which may contribute to less development of life-threatening GVHD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ringdén, Svahn, Moll and Sadeghi.)

Gamma delta (γδ) T cells represent a minor fraction of human T cell repertoire but play an important role in mediating anti-infectious and anti-tumorous effects in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We performed a prospective study to analyze the effect of different transplant modalities on immune reconstitution of γδ T cells and subsets. CD3, CD4 and CD8 T cells were analyzed in parallel. Secondly, we examined the impact of γδ T cell reconstitution on clinical outcomes including acute Graft-versus-Host-Disease (aGvHD) and viral infections. Our cohort includes 49 pediatric patients who received unmanipulated bone marrow grafts from matched unrelated (MUD) or matched related (MRD) donors. The cohort includes patients with malignant as well as non-malignant diseases. Cell counts were measured using flow cytometry at 15, 30, 60, 100, 180 and 240 days after transplantation. Cells were stained for CD3, CD4, CD8, CD45, TCR αβ , TCRγδ, TCRVδ1, TCRVδ2, HLA-DR and combinations. Patients with a MRD showed significantly higher Vδ2+ T cells than those with MUD at timepoints +30, +60, +100 (p<0.001, respectively) and +180 (p<0.01) in univariate analysis. These results remained significant in multivariate analysis. Patients recovering with a high relative abundance of total γδ T cells and Vδ2+ T cells had a significantly lower cumulative incidence of grade II-IV aGvHD after transplantation (p=0.03 and p=0.04, respectively). A high relative abundance of Vδ2+ T cells was also associated with a lower incidence of EBV infection (p=0.02). Patients with EBV infection on the other hand showed higher absolute Vδ1+ T cell counts at days +100 and +180 after transplantation (p=0.046 and 0.038, respectively) than those without EBV infection. This result remained significant in a multivariate time-averaged analysis (q<0.1). Our results suggest a protective role of γδ T cells and especially Vδ2+ T cell subset against the development of aGvHD and EBV infection after pediatric HSCT. Vδ1+ T cells might be involved in the immune response after EBV infection. Our results encourage further research on γδ T cells as prognostic markers after HSCT and as possible targets of adoptive T cell transfer strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Müller, Alasfar, Preuß, Zimmermann, Streitz, Hundsdörfer, Eggert, Schulte, von Stackelberg and Oevermann.)

Acute cellular rejection remains a significant obstacle affecting successful outcomes of organ transplantation including vascularized composite tissue allografts (VCA). Donor antigen presenting cells (APCs), particularly dendritic cells (DCs), orchestrate early alloimmune responses by activating recipient effector T cells. Employing a targeted approach, we investigated the impact of donor-derived conventional DCs (cDCs) and APCs on the immunogenicity of skin and skin-containing VCA grafts, using mouse models of skin and hind limb transplantation. By post-transplantation day 6, skin grafts demonstrated severe rejections, characterized by predominance of recipient CD4 T cells. In contrast, hind limb grafts showed moderate rejection, primarily infiltrated by CD8 T cells. Notably, the skin component exhibited heightened immunogenicity when compared to the entire VCA, evidenced by increased frequencies of pan (CD11b - CD11c + ), mature (CD11b - CD11c + MHCII + ) and active (CD11b - CD11c + CD40 + ) DCs and cDC2 subset (CD11b + CD11c + MHCII + ) in the lymphoid tissues and the blood of skin transplant recipients. While donor depletion of cDC and APC reduced frequencies, maturation and activation of DCs in all analyzed tissues of skin transplant recipients, reduction in DC activities was only observed in the spleen of hind limb recipients. Donor cDC and APC depletion did not impact all lymphocyte compartments but significantly affected CD8 T cells and activated CD4 T in lymph nodes of skin recipients. Moreover, both donor APC and cDC depletion attenuated the Th17 immune response, evident by significantly reduced Th17 (CD4 + IL-17 + ) cells in the spleen of skin recipients and reduced levels of IL-17E and lymphotoxin-α in the serum samples of both skin and hind limb recipients. In conclusion, our findings underscore the highly immunogenic nature of skin component in VCA. The depletion of donor APCs and cDCs mitigates the immunogenicity of skin grafts while exerting minimal impact on VCA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ashraf, Mengwasser, Reutzel-Selke, Polenz, Führer, Lippert, Tang, Michaelis, Catar, Pratschke, Witzel, Sauer, Tullius and Kern.)

Introduction: Dengue virus infection is a global health problem lacking specific therapy, requiring an improved understanding of DENV immunity and vaccine responses. Considering the recent emerging of new dengue vaccines, here we performed an integrative systems vaccinology characterization of molecular signatures triggered by the natural DENV infection (NDI) and attenuated dengue virus infection models (DVTs)., Methods and Results: We analyzed 955 samples of transcriptomic datasets of patients with NDI and attenuated dengue virus infection trials (DVT1, DVT2, and DVT3) using a systems vaccinology approach. Differential expression analysis identified 237 common differentially expressed genes (DEGs) between DVTs and NDI. Among them, 28 and 60 DEGs were up or downregulated by dengue vaccination during DVT2 and DVT3, respectively, with 20 DEGs intersecting across all three DVTs. Enriched biological processes of these genes included type I/II interferon signaling, cytokine regulation, apoptosis, and T-cell differentiation. Principal component analysis based on 20 common DEGs (overlapping between DVTs and our NDI validation dataset) distinguished dengue patients by disease severity, particularly in the late acute phase. Machine learning analysis ranked the ten most critical predictors of disease severity in NDI, crucial for the anti-viral immune response., Conclusion: This work provides insights into the NDI and vaccine-induced overlapping immune response and suggests molecular markers (e.g., IFIT5, ISG15, and HERC5 ) for anti-dengue-specific therapies and effective vaccination development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This study received funding from IBM. The funder had the following involvement in the study: production of figures, revision, and manuscript editing. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Plaça, Fonseca, Marques, Zaki Pour, Usuda, Baiocchi, Prado, Salgado, Filgueiras, Freire, Rocha, Camara, Catar, Moll, Jurisica, Calich, Giil, Rivino, Ochs, Cabral-Miranda, Schimke and Cabral-Marques.)

Introduction: Epstein-Barr virus (EBV) infection in humans is associated with a wide range of diseases including malignancies of different origins, most prominently B cells. Several EBV latent genes are thought to act together in B cell immortalization, but a minimal set of EBV genes sufficient for transformation remains to be identified., Methods: Here, we addressed this question by transducing human peripheral B cells from EBV-negative donors with retrovirus expressing the latent EBV genes encoding Latent Membrane Protein (LMP) 1 and 2A and Epstein-Barr Nuclear Antigen (EBNA) 2., Results: LMP1 together with EBNA2, but not LMP1 alone or in combination with LMP2A was able to transform human primary B cells. LMP1/EBNA2-immortalized cell lines shared surface markers with EBV-transformed lymphoblastoid cell lines (LCLs). They showed sustained growth for more than 60 days, albeit at a lower growth rate than EBV-transformed LCLs. LMP1/EBNA2-immortalized cell lines generated tumors when transplanted subcutaneously into severely immunodeficient NOG mice., Conclusion: Our results identify a minimal set of EBV proteins sufficient for B cell transformation., Competing Interests: Author MS was employed by the company Experimental Pharmacology and Oncology EPO Berlin-Buch GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Sommermann, Li, Gieselmann, de la Rosa, Stecklum, Klein, Kocks and Rajewsky.)

Non-HLA-directed regulatory autoantibodies (RABs) are known to target G-protein coupled receptors (GPCRs) and thereby contribute to kidney transplant vasculopathy and failure. However, the detailed underlying signaling mechanisms in human microvascular endothelial cells (HMECs) and immune cells need to be clarified in more detail. In this study, we compared the immune stimulatory effects and concomitant intracellular and extracellular signaling mechanisms of immunoglobulin G (IgG)-fractions from kidney transplant patients with allograft vasculopathy (KTx-IgG), to that from patients without vasculopathy, or matched healthy controls (Con-IgG). We found that KTx-IgG from patients with vasculopathy, but not KTx-IgG from patients without vasculopathy or Con-IgG, elicits HMEC activation and subsequent upregulation and secretion of tumor necrosis factor alpha (TNF-α) from HMECs, which was amplified in the presence of the protease-activated thrombin receptor 1 (PAR1) activator thrombin, but could be omitted by selectively blocking the PAR1 receptor. The amount and activity of the TNF-α secreted by HMECs stimulated with KTx-IgG from patients with vasculopathy was sufficient to induce subsequent THP-1 monocytic cell activation. Furthermore, AP-1/c-FOS, was identified as crucial transcription factor complex controlling the KTx-IgG-induced endothelial TNF-α synthesis, and mircoRNA-let-7f-5p as a regulatory element in modulating the underlying signaling cascade. In conclusion, exposure of HMECs to KTx-IgG from patients with allograft vasculopathy, but not KTx-IgG from patients without vasculopathy or healthy Con-IgG, triggers signaling through the PAR1-AP-1/c-FOS-miRNA-let7-axis, to control TNF-α gene transcription and TNF-α-induced monocyte activation. These observations offer a greater mechanistic understanding of endothelial cells and subsequent immune cell activation in the clinical setting of transplant vasculopathy that can eventually lead to transplant failure, irrespective of alloantigen-directed responses., Competing Interests: Author HH was employed by CellTrend GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Moll, Luecht, Gyamfi, da Fonseca, Wang, Zhao, Gong, Chen, Ashraf, Heidecke, Hackel, Dragun, Budde, Penack, Riemekasten, Cabral-Marques, Witowski and Catar.)

Stem cell transplant recipients (SCTR) are imperiled to increased risks after SARS-CoV2 infection, supporting the need for effective vaccination strategies for this vulnerable group. With respect to pediatric patients, data on immunogenicity of SARS-CoV2 mRNA-based vaccination is limited. We therefore comprehensively examined specific humoral, B- and T cell responses in a cohort of 2-19 year old SCTR after the second and third vaccine dose. Only after booster vaccination, transplant recipients reached similar levels of vaccine-specific IgG, IgA and neutralizing antibodies against omicron variant as age-matched controls. Although frequencies of SARS-CoV2 specific B cells increased after the third dose, they were still fourfold reduced in patients compared to controls. Overall, the majority of individuals enrolled mounted SARS-CoV2 Spike protein-specific CD4 + T helper cell responses with patients showing significantly higher portions than controls after the third dose. With respect to functionality, however, SCTR were characterized by reduced frequencies of specific interferon gamma producing CD4 + T cells, along with an increase in IL-2 producers. In summary, our data identify distinct quantitative and qualitative impairments within the SARS-CoV2 vaccination specific B- and CD4 + T cell compartments. More importantly, humoral analyses highlight the need for a booster vaccination of SCTR particularly for development of neutralizing antibodies., Competing Interests: KK received unconditional project funding from Chiesi GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Thole, Tóth, Proß, Siegle, Stahl, Hermsdorf, Knabe, Winkler, Schrezenmeier, Ludwig, Eckert, Eggert, Schrezenmeier, Sattler, Schulte and Kotsch.)

Aims: Expanded hemodialysis (HDx) therapy with improved molecular cut-off dialyzers exerts beneficial effects on lowering uremia-associated chronic systemic microinflammation, a driver of endothelial dysfunction and cardiovascular disease (CVD) in hemodialysis (HD) patients with end-stage renal disease (ESRD). However, studies on the underlying molecular mechanisms are still at an early stage. Here, we identify the (endothelial) transcription factor Krüppel-like factor 2 (KLF2) and its associated molecular signalling pathways as key targets and regulators of uremia-induced endothelial micro-inflammation in the HD/ESRD setting, which is crucial for vascular homeostasis and controlling detrimental vascular inflammation., Methods and Results: First, we found that human microvascular endothelial cells (HMECs) and other typical endothelial and kidney model cell lines (e.g. HUVECs, HREC, and HEK) exposed to uremic serum from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation II (PERCI-II) crossover clinical trial - comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes - exhibited strongly reduced expression of vasculoprotective KLF2 with HF dialyzers, while dialysis with MCO dialyzers led to the maintenance and restoration of physiological KLF2 levels in HMECs. Mechanistic follow-up revealed that the strong downmodulation of KLF2 in HMECs exposed to uremic serum was mediated by a dominant engagement of detrimental ERK instead of beneficial AKT signalling, with subsequent AP1-/c-FOS binding in the KLF2 promoter region, followed by the detrimental triggering of pleiotropic inflammatory mediators, while the introduction of a KLF2 overexpression plasmid could restore physiological KLF2 levels and downmodulate the detrimental vascular inflammation in a mechanistic rescue approach., Conclusion: Uremia downmodulates vasculoprotective KLF2 in endothelium, leading to detrimental vascular inflammation, while MCO dialysis with the novel improved HDx therapy approach can maintain physiological levels of vasculoprotective KLF2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhao, Wu, Gyamfi, Wang, Luecht, Pfefferkorn, Ashraf, Kamhieh-Milz, Witowski, Dragun, Budde, Schindler, Zickler, Moll and Catar.)

Introduction: Mesenchymal Stromal/Stem cells (MSCs) are an essential component of the regenerative and immunoregulatory stem cell compartment of the human body and thus of major importance in human physiology. The MSCs elicit their beneficial properties through a multitude of complementary mechanisms, which makes it challenging to assess their phenotype and function in environmental toxicity screening. We here employed the novel combinatorial assays matrix approach/technology to profile the MSC response to the herbicide Atrazine, which is a common environmental xenobiotic, that is in widespread agricultural use in the US and other countries, but banned in the EU. Our here presented approach is representative for screening the impact of environmental xenobiotics and toxins on MSCs as an essential representative component of human physiology and well-being., Methods: We here employed the combinatorial assay matrix approach, including a panel of well standardized assays, such as flow cytometry, multiplex secretome analysis, and metabolic assays, to define the phenotype and functionality of human-donor-derived primary MSCs exposed to the representative xenobiotic Atrazine. This assay matrix approach is now also endorsed for characterization of cell therapies by leading regulatory agencies, such as FDA and EMA., Results: Our results show that the exposure to Atrazine modulates the metabolic activity, size, and granularity of MSCs in a dose and time dependent manner. Intriguingly, Atrazine exposure leads to a broad modulation of the MSCs secretome (both upregulation and downmodulation of certain factors) with the identification of Interleukin-8 as the topmost upregulated representative secretory molecule. Interestingly, Atrazine attenuates IFNγ-induced upregulation of MHC-class-II, but not MHC-class-I, and early phosphorylation signals on MSCs. Furthermore, Atrazine exposure attenuates IFNγ responsive secretome of MSCs. Mechanistic knockdown analysis identified that the Atrazine-induced effector molecule Interleukin-8 affects only certain but not all the related angiogenic secretome of MSCs., Discussion: The here described Combinatorial Assay Matrix Technology identified that Atrazine affects both the innate/resting and cytokine-induced/stimulated assay matrix functionality of human MSCs, as identified through the modulation of selective, but not all effector molecules, thus vouching for the great usefulness of this approach to study the impact of xenobiotics on this important human cellular subset involved in the regenerative healing responses in humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Uwazie, Pirlot, Faircloth, Patel, Parr, Zastre, Hematti, Moll, Rajan and Chinnadurai.)

During the pandemic of severe respiratory distress syndrome coronavirus 2 (SARS-CoV2), many novel therapeutic modalities to treat Coronavirus 2019 induced disease (COVID-19) were explored. This study summarizes 195 clinical trials of advanced cell therapies targeting COVID-19 that were registered over the two years between January 2020 to December 2021. In addition, this work also analyzed the cell manufacturing and clinical delivery experience of 26 trials that published their outcomes by July 2022. Our demographic analysis found the highest number of cell therapy trials for COVID-19 was in United States, China, and Iran (N=53, 43, and 19, respectively), with the highest number per capita in Israel, Spain, Iran, Australia, and Sweden (N=0.641, 0.232, 0,223, 0.194, and 0.192 trials per million inhabitants). The leading cell types were multipotent mesenchymal stromal/stem cells (MSCs), natural killer (NK) cells, and mononuclear cells (MNCs), accounting for 72%, 9%, and 6% of the studies, respectively. There were 24 published clinical trials that reported on infusions of MSCs. A pooled analysis of these MSC studies found that MSCs provide a relative risk reduction for all-cause COVID-19 mortality of RR=0.63 (95% CI 0.46 to 0.85). This result corroborates previously published smaller meta-analyses, which suggested that MSC therapy demonstrated a clinical benefit for COVID-19 patients. The sources of the MSCs used in these studies and their manufacturing and clinical delivery methods were remarkably heterogeneous, with some predominance of perinatal tissue-derived products. Our results highlight the important role that cell therapy products may play as an adjunct therapy in the management of COVID-19 and its related complications, as well as the importance of controlling key manufacturing parameters to ensure comparability between studies. Thus, we support ongoing calls for a global registry of clinical studies with MSC products that could better link cell product manufacturing and delivery methods to clinical outcomes. Although advanced cell therapies may provide an important adjunct treatment for patients affected by COVID-19 in the near future, preventing pathology through vaccination still remains the best protection to date. We conducted a systematic review and meta-analysis of advanced cell therapy clinical trials as potential novel treatment for COVID-19 (resulting from SARS-CoV-2 coronavirus infection), including analysis of the global clinical trial landscape, published safety/efficacy outcomes (RR/OR), and details on cell product manufacturing and clinical delivery. This study had a 2-year observation interval from start of January 2020 to end of December 2021, including a follow-up period until end of July to identify published outcomes, which covers the most vivid period of clinical trial activity, and is also the longest observation period studied until today. In total, we identified 195 registered advanced cell therapy studies for COVID-19, employing 204 individual cell products. Leading registered trial activity was attributed to the USA, China, and Iran. Through the end of July 2022, 26 clinical trials were published, with 24 out of 26 articles employing intravenous infusions (IV) of mesenchymal stromal/stem cell (MSC) products. Most of the published trials were attributed to China and Iran. The cumulative results from the 24 published studies employing infusions of MSCs indicated an improved survival (RR=0.63 with 95% Confidence Interval 0.46 to 0.85). Our study is the most comprehensive systematic review and meta-analysis on cell therapy trials for COVID-19 conducted to date, clearly identifying the USA, China, and Iran as leading advanced cell therapy trial countries for COVID-19, with further strong contributions from Israel, Spain, Australia and Sweden. Although advanced cell therapies may provide an important adjunct treatment for patients affected by COVID-19 in the future, preventing pathology through vaccination remains the best protection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Couto, Al-Arawe, Filgueiras, Fonseca, Hinterseher, Catar, Chinnadurai, Bersenev, Cabral-Marques, Moll and Verter.)

Introduction: The ubiquitous Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8 + T cell exhaustion and dysregulates CD4 + T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen., Methods: We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain ( TRAC ) locus., Results: Applying upscaled methods with the ExPERT ATx ® MaxCyte system, KI efficacy was ~20% of the total ~2 × 10 8 TCR-knocked-out (KO) generated cells. KO TCR KI CAR-T cells were co-cultured in vitro with the gp350 + CD19 + BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro . CD8 + KI CAR-T cells showed higher persistency than CD4 + KI CAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 × 10 5 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19 KI CAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4 + CD19 KI CAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (T regs ) and TIM-3 + CD4 + T cells. Administration of gp350 KI CAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8 + PD-1 + LAG-3 + gp350 KI CAR-T cells were predominant in the bone marrow., Discussion: The two types of KO TCR KI CAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation., Competing Interests: Author AR-M is employed by MaxCyte Inc., MD, USA. Author RS has filed a patent application for generation of CAR-T cells targeting lytic herpes infections and is a founding shareholder and scientific consultant of BioSyngen/Zelltechs Lpt Ltd. Author DW has filed multiple patent applications on CRISPR-Cas gene editing and adoptive T cell therapy. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Braun, Pruene, Darguzyte, vom Stein, Nguyen, Wagner, Kath, Roig-Merino, Heuser, Riehm, Schneider, Awerkiew, Talbot, Bleich, Figueiredo, Bornhäuser and Stripecke.)

Maternal autoantibodies can be transmitted diaplacentally, with potentially deleterious effects on neurodevelopment. Synapsin 1 (SYN1) is a neuronal protein that is important for synaptic communication and neuronal plasticity. While monoallelic loss of function (LoF) variants in the SYN1 gene result in X-linked intellectual disability (ID), learning disabilities, epilepsy, behavioral problems, and macrocephaly, the effect of SYN1 autoantibodies on neurodevelopment remains unclear. We recruited a clinical cohort of 208 mothers and their children with neurologic abnormalities and analyzed the role of maternal SYN1 autoantibodies. We identified seropositivity in 9.6% of mothers, and seropositivity was associated with an increased risk for ID and behavioral problems. Furthermore, children more frequently had epilepsy, macrocephaly, and developmental delay, in line with the SYN1 LoF phenotype. Whether SYN1 autoantibodies have a direct pathogenic effect on neurodevelopment or serve as biomarkers requires functional experiments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bünger, Makridis, Kreye, Nikolaus, Sedlin, Ullrich, Hoffmann, Tromm, Rasmussen, Milovanovic, Höltje, Prüss and Kaindl.)

Introduction: Despite advances in treating high-risk neuroblastoma, 50-60% of patients still suffer relapse, necessitating new treatment options. Bispecific trifunctional antibodies (trAbs) are a promising new class of immunotherapy. TrAbs are heterodimeric IgG-like molecules that bind CD3 and a tumor-associated antigen simultaneously, whereby inducing a TCR-independent anti-cancer T cell response. Moreover, via their functional Fc region they recruit and activate cells of the innate immune system like antigen-presenting cells potentially enhancing induction of adaptive tumor-specific immune responses., Methods: We used the SUREK trAb, which is bispecific for GD2 and murine Cd3. Tumor-blind trAb and the monoclonal ch14.18 antibody were used as controls. A co-culture model of murine dendritic cells (DCs), T cells and a neuroblastoma cell line was established to evaluate the cytotoxic effect and the T cell effector function in vitro. Expression of immune checkpoint molecules on tumor-infiltrating T cells and the induction of an anti-neuroblastoma immune response using a combination of whole cell vaccination and trAb therapy was investigated in a syngeneic immunocompetent neuroblastoma mouse model (NXS2 in A/J background). Finally, vaccinated mice were assessed for the presence of neuroblastoma-directed antibodies. We show that SUREK trAb-mediated effective killing of NXS2 cells in vitro was strictly dependent on the combined presence of DCs and T cells., Results: Using a syngeneic neuroblastoma mouse model, we showed that vaccination with irradiated tumor cells combined with SUREK trAb treatment significantly prolonged survival of tumor challenged mice and partially prevent tumor outgrowth compared to tumor vaccination alone. Treatment led to upregulation of programmed cell death protein 1 (Pd-1) on tumor infiltrating T cells and combination with anti-Pd-1 checkpoint inhibition enhanced the NXS2-directed humoral immune response., Conclusion: Here, we provide first preclinical evidence that a tumor vaccination combined with SUREK trAb therapy induces an endogenous anti-neuroblastoma immune response reducing tumor recurrence. Furthermore, a combination with anti-Pd-1 immune checkpoint blockade might even further improve this promising immunotherapeutic concept in order to prevent relapse in high-risk neuroblastoma patients., Competing Interests: HLi is CEO of Trion Research and inventor or co-inventor of several trifunctional antibody patents including SUREK. PR is employee at Trion Research. Author PH was employed by company HELIOS Klinikum Berlin. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ivasko, Anders, Grunewald, Launspach, Klaus, Schwiebert, Ruf, Lindhofer, Lode, Andersch, Schulte, Eggert, Hundsdoerfer, Künkele and Zirngibl.)

Introduction: The differentiation of B cells into antibody-secreting plasma cells depends on cell division-coupled, epigenetic and other cellular processes that are incompletely understood., Methods: We have developed a CRISPR/Cas9-based screen that models an early stage of T cell-dependent plasma cell differentiation and measures B cell survival or proliferation versus the formation of CD138+ plasmablasts. Here, we refined and extended this screen to more than 500 candidate genes that are highly expressed in plasma cells., Results: Among known genes whose deletion preferentially or mostly affected plasmablast formation were the transcription factors Prdm1 (BLIMP1), Irf4 and Pou2af1 (OBF-1), and the Ern1 gene encoding IRE1a, while deletion of XBP1, the transcriptional master regulator that specifies the expansion of the secretory program in plasma cells, had no effect. Defective plasmablast formation caused by Ern1 deletion could not be rescued by the active, spliced form of XBP1 whose processing is dependent on and downstream of IRE1a, suggesting that in early plasma cell differentiation IRE1a acts independently of XBP1. Moreover, we newly identified several genes involved in NF-kB signaling (Nfkbia), vesicle trafficking (Arf4, Preb) and epigenetic regulators that form part of the NuRD complex (Hdac1, Mta2, Mbd2) to be required for plasmablast formation. Deletion of ARF4, a small GTPase required for COPI vesicle formation, impaired plasmablast formation and blocked antibody secretion. After Hdac1 deletion plasmablast differentiation was consistently reduced by about 50%, while deletion of the closely related Hdac2 gene had no effect. Hdac1 knock-out led to strongly perturbed protein expression of antagonistic transcription factors that govern plasma cell versus B cell identity (by decreasing IRF4 and BLIMP1 and increasing BACH2 and PAX5)., Discussion: Taken together, our results highlight specific and non-redundant roles for Ern1, Arf4 and Hdac1 in the early steps of plasma cell differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xiong, Popp, Salomon, Mertins, Kocks, Rajewsky and Chu.)

Inflammation and a dysregulated immune system are common denominators of cancer and cardiovascular disease (CVD). Immuno-cardio-oncology addresses the interconnected immunological aspect in both cancer and CVD and the integration of immunotherapies and anti-inflammatory therapies in both distinct disease entities. Building on prominent examples of convergent inflammation (IL-1ß biology) and immune disbalance (CD20 cells) in cancer and CVD/heart failure, the review tackles both the roadblocks and opportunities of repurposed use of IL-1ß drugs and anti-CD20 antibodies in both fields, and discusses the use of advanced therapies e.g. chimeric antigen receptor (CAR) T cells, that can address the raising burden of both cancer and CVD. Finally, it is discussed how inspired by precision medicine in oncology, the use of biomarker-driven patient stratification is needed to better guide anti-inflammatory/immunomodulatory therapeutic interventions in cardiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor SS declared a shared parent affiliation with the authors at the time of review., (Copyright © 2022 Van Linthout and Volk.)

Objectives: To determine the profile of cytokines in patients with severe COVID-19 who were enrolled in a trial of COVID-19 convalescent plasma (CCP)., Methods: Patients were randomized to receive standard treatment and 3 CCP units or standard treatment alone (CAPSID trial, ClinicalTrials.gov NCT04433910). The primary outcome was a dichotomous composite outcome (survival and no longer severe COVID-19 on day 21). Time to clinical improvement was a key secondary endpoint. The concentrations of 27 cytokines were measured (baseline, day 7). We analyzed the change and the correlation between serum cytokine levels over time in different subgroups and the prediction of outcome in receiver operating characteristics (ROC) analyses and in multivariate models., Results: The majority of cytokines showed significant changes from baseline to day 7. Some were strongly correlated amongst each other (at baseline the cluster IL-1ß, IL-2, IL-6, IL-8, G-CSF, MIP-1α, the cluster PDGF-BB, RANTES or the cluster IL-4, IL-17, Eotaxin, bFGF, TNF-α). The correlation matrix substantially changed from baseline to day 7. The heatmaps of the absolute values of the correlation matrix indicated an association of CCP treatment and clinical outcome with the cytokine pattern. Low levels of IP-10, IFN-γ, MCP-1 and IL-1ß on day 0 were predictive of treatment success in a ROC analysis. In multivariate models, low levels of IL-1ß, IFN-γ and MCP-1 on day 0 were significantly associated with both treatment success and shorter time to clinical improvement. Low levels of IP-10, IL-1RA, IL-6, MCP-1 and IFN-γ on day 7 and high levels of IL-9, PDGF and RANTES on day 7 were predictive of treatment success in ROC analyses. Low levels of IP-10, MCP-1 and high levels of RANTES, on day 7 were associated with both treatment success and shorter time to clinical improvement in multivariate models., Conclusion: This analysis demonstrates a considerable dynamic of cytokines over time, which is influenced by both treatment and clinical course of COVID-19. Levels of IL-1ß and MCP-1 at baseline and MCP-1, IP-10 and RANTES on day 7 were associated with a favorable outcome across several endpoints. These cytokines should be included in future trials for further evaluation as predictive factors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Körper, Schrezenmeier, Rincon-Arevalo, Grüner, Zickler, Weiss, Wiesmann, Zacharowski, Kalbhenn, Bentz, Dollinger, Paul, Lepper, Ernst, Wulf, Zinn, Appl, Jahrsdörfer, Rojewski, Lotfi, Dörner, Jungwirth, Seifried, Fürst and Schrezenmeier.)

Selective differentiation of CD4+ T helper (Th) cells into specialized subsets such as Th1 and Th2 cells is a key element of the adaptive immune system driving appropriate immune responses. Besides those canonical Th-cell lineages, hybrid phenotypes such as Th1/2 cells arise in vivo , and their generation could be reproduced in vitro . While master-regulator transcription factors like T-bet for Th1 and GATA-3 for Th2 cells drive and maintain differentiation into the canonical lineages, the transcriptional architecture of hybrid phenotypes is less well understood. In particular, it has remained unclear whether a hybrid phenotype implies a mixture of the effects of several canonical lineages for each gene, or rather a bimodal behavior across genes. Th-cell differentiation is a dynamic process in which the regulatory factors are modulated over time, but longitudinal studies of Th-cell differentiation are sparse. Here, we present a dynamic transcriptome analysis following Th-cell differentiation into Th1, Th2, and Th1/2 hybrid cells at 3-h time intervals in the first hours after stimulation. We identified an early bifurcation point in gene expression programs, and we found that only a minority of ~20% of Th cell-specific genes showed mixed effects from both Th1 and Th2 cells on Th1/2 hybrid cells. While most genes followed either Th1- or Th2-cell gene expression, another fraction of ~20% of genes followed a Th1 and Th2 cell-independent transcriptional program associated with the transcription factors STAT1 and STAT4. Overall, our results emphasize the key role of high-resolution longitudinal data for the characterization of cellular phenotypes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Burt, Peine, Peine, Borek, Serve, Floßdorf, Hegazy, Höfer, Löhning and Thurley.)

Background: Durable vaccine-mediated immunity relies on the generation of long-lived plasma cells and memory B cells (MBCs), differentiating upon germinal center (GC) reactions. SARS-CoV-2 mRNA vaccination induces a strong GC response in healthy volunteers (HC), but limited data is available about response longevity upon rituximab treatment., Methods: We evaluated humoral and cellular responses upon 3rd vaccination in seven patients with rheumatoid arthritis (RA) who initially mounted anti-spike SARS-CoV-2 IgG antibodies after primary 2x vaccination and got re-exposed to rituximab (RTX) 1-2 months after the second vaccination. Ten patients with RA on other therapies and ten HC represented the control groups. As control for known long-lived induced immunity, we analyzed humoral and cellular tetanus toxoid (TT) immune responses in steady-state., Results: After 3 rd vaccination, 5/7 seroconverted RTX patients revealed lower anti-SARS-CoV-2 IgG levels but similar neutralizing capacity compared with HC. Antibody levels after 3rd vaccination correlated with values after 2nd vaccination. Despite significant reduction of circulating total and antigen-specific B cells in RTX re-exposed patients, we observed the induction of IgG+ MBCs upon 3 rd vaccination. Notably, only RTX treated patients revealed a high amount of IgA+ MBCs before and IgA+ plasmablasts after 3 rd vaccination. IgA+ B cells were not part of the steady state TT+ B cell pool. TNF-secretion and generation of effector memory CD4 spike-specific T cells were significantly boosted upon 3 rd vaccination., Summary: On the basis of pre-existing affinity matured MBCs within primary immunisation, RTX re-exposed patients revealed a persistent but atypical GC immune response accompanied by boosted spike-specific memory CD4 T cells upon SARS-CoV-2 recall vaccination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stefanski, Rincon-Arevalo, Schrezenmeier, Karberg, Szelinski, Ritter, Chen, Meisel, Jahrsdörfer, Ludwig, Schrezenmeier, Lino and Dörner.)

Interleukin-6 (IL-6) is an important player in chronic inflammation associated with heart failure and tumor-induced cachexia. Fibroblasts are salient mediators of both inflammation and fibrosis. Whereas the general outcome of IL-6 on the heart's function and muscle wasting has been intensively studied, the influence of IL-6 on fibroblasts of the heart and skeletal muscle (SM) has not been analyzed so far. We illustrate that SM-derived fibroblasts exhibit higher basal mRNA expression of α-SMA, extracellular matrix molecules (collagen1a1/3a1/5a1), and chemokines (CCL2, CCL7, and CX3CL1) as compared to the left ventricle (LV)-derived fibroblasts. IL-6 drives the transdifferentiation of fibroblasts into myofibroblasts as indicated by an increase in α-SMA expression and upregulates NLRP3 inflammasome activity in both LV- and SM-derived fibroblasts. IL-6 increases the release of CCL7 to CX3CL1 in the supernatant of SM-derived fibroblasts associated with the attraction of more pro(Ly6C hi ) versus anti(Ly6C lo ) inflammatory monocytes as compared to unstimulated fibroblasts. IL-6-stimulated LV-derived fibroblasts attract less Ly6C hi to Ly6C lo monocytes compared to IL-6-stimulated SM-derived fibroblasts. In addition, SM-derived fibroblasts have a higher mitochondrial energy turnover and lower glycolytic activity versus LV-derived fibroblasts under basal and IL-6 conditions. In conclusion, IL-6 modulates the inflammatory and metabolic phenotype of LV- and SM-originated fibroblasts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Matz, Pappritz, Springer and Van Linthout.)

Achieving fast immunosuppression blood exposure after kidney transplantation is key to abrogating both preformed and de novo anti-donor humoral and cellular alloresponses. However, while tacrolimus (TAC) is the cornerstone immunosuppressant inhibiting adaptive alloimmunity, its blood exposure is directly impacted by different single-nucleotide polymorphisms (SNPs) in CYP3A TAC-metabolizing enzymes. Here, we investigated how functional TAC-CYP3A genetic variants ( CYP3A4*22 / CYP3A5*3 ) influence the main baseline clinical and immunological risk factors of biopsy-proven acute rejection (BPAR) by means of preformed donor-specific antibodies (DSAs) and donor-specific alloreactive T cells (DSTs) in a large European cohort of 447 kidney transplants receiving TAC-based immunosuppression. A total of 70 (15.7%) patients developed BPAR. Preformed DSAs and DSTs were observed in 12 (2.7%) and 227 (50.8%) patients, respectively. According to the different CYP3A4*22 and CYP3A5*3 functional allele variants, we found 4 differential new clusters impacting fasting TAC exposure after transplantation; 7 (1.6%) were classified as high metabolizers 1 (HM1), 71 (15.9%) as HM2, 324 (72.5%) as intermediate (IM), and 45 (10.1%) as poor metabolizers (PM1). HM1/2 showed significantly lower TAC trough levels and higher dose requirements than IM and PM (p < 0.001) and more frequently showed TAC underexposure (<5 ng/ml). Multivariate Cox regression analyses revealed that CYP3A HM1 and IM pharmacogenetic phenotypes (hazard ratio (HR) 12.566, 95% CI 1.99-79.36, p = 0.007, and HR 4.532, 95% CI 1.10-18.60, p = 0.036, respectively), preformed DSTs (HR 3.482, 95% CI 1.99-6.08, p < 0.001), DSAs (HR 4.421, 95% CI 1.63-11.98, p = 0.003), and delayed graft function (DGF) (HR 2.023, 95% CI 1.22-3.36, p = 0.006) independently predicted BPAR. Notably, a significant interaction between T-cell depletion and TAC underexposure was observed, showing a reduction of the BPAR risk (HR 0.264, 95% CI 0.08-0.92, p = 0.037). Such variables except for DSAs displayed a higher predictive risk for the development of T cell-mediated rejection (TCMR). Refinement of pretransplant monitoring by incorporating TAC CYP3A SNPs with preformed DSAs as well as DSTs may improve current rejection-risk stratification and help induction treatment decision-making., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer L.R has declared a shared affiliation with the author T.J to the handling editor at the time of review., (Copyright © 2022 Crespo, Vidal-Alabró, Jouve, Fontova, Stein, Mocka, Meneghini, Sefrin, Hruba, Gomà, Torija, Donadeu, Favà, Cruzado, Melilli, Moreso, Viklicky, Bemelman, Reinke, Grinyó, Lloberas and Bestard.)

T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first gene-modified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as "off-the-shelf" products. This review addresses important points to consider regarding the status quo , pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells., Competing Interests: RS has filed a patent application for generation of CAR-T cells targeting lytic herpes infections and is a founding shareholder and scientific consultant of BioSyngen/Zelltechs Lpt Ltd. DW has filed multiple patent applications on CRISPR-Cas gene editing and adoptive T cell therapy. CS is consultant for Bristol Myers Squibb, Janssen and Novartis regarding CAR-T cell therapy and is participating in clinical CAR-T studies from Bristol Myers Squibb, Janssen, Novartis and Miltenyi Biotec and is cooperating with Miltenyi Biotec in the production of CAR-T cells. UK states that she is a consultant in immuno-oncology for AstraZeneca, Affimed, Glycostem, GammaDelta and Zelluna, and that she has collaborations with Novartis and Miltenyi Biotec regarding the production of CAR-T cells. MC is co-inventor in granted and filed patents describing CAR-T cells with additional functions to counteract the tumor microenvironment., (Copyright © 2022 Wagner, Koehl, Chmielewski, Scheid and Stripecke.)

Background: Vaccination is considered as most efficient strategy in controlling SARS-CoV-2 pandemic spread. Nevertheless, patients with autoimmune inflammatory rheumatic diseases receiving rituximab (RTX) are at increased risk to fail humoral and cellular responses upon vaccination. The ability to predict vaccination responses is essential to guide adequate safety and optimal protection in these patients., Methods: B- and T- cell data before vaccination were evaluated for characteristics predicting vaccine responses in altogether 15 patients with autoimmune inflammatory rheumatic diseases receiving RTX. Eleven patients with rheumatoid arthritis (RA) on other therapies, 11 kidney transplant recipients (KTR) on regular immunosuppression and 15 healthy controls (HC) served as controls. A multidimensional analysis of B cell subsets via UMAP algorithm and a correlation matrix were performed in order to identify predictive markers of response in patients under RTX therapy., Results: Significant differences regarding absolute B cell counts and specific subset distribution pattern between the groups were identified at baseline. In this context, the majority of B cells from vaccination responders of the RTX group (RTX IgG+) were naïve and transitional B cells, whereas vaccination non-responders (RTX IgG-) carried preferentially plasmablasts and double negative (CD27-IgD-) B cells. Moreover, there was a positive correlation between neutralizing antibodies and B cells expressing HLA-DR and CXCR5 as well as an inverse correlation with CD95 expression and CD21low expression by B cells among vaccination responders., Summary: Substantial repopulation of the naïve B cell compartment after RTX therapy appeared to be essential for an adequate vaccination response, which seem to require the additional capability of antigen presentation and germinal center formation. Moreover, expression of exhaustion markers represent negative predictors of vaccination responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stefanski, Rincon-Arevalo, Schrezenmeier, Karberg, Szelinski, Ritter, Chen, Jahrsdörfer, Ludwig, Schrezenmeier, Lino and Dörner.)

Antibody-secreting cells (ASCs) contribute to immunity through production of antibodies and cytokines. Identification of specific markers of ASC would allow selective targeting of these cells in several disease contexts. Here, we performed an unbiased, large-scale protein screening, and identified twelve new molecules that are specifically expressed by murine ASCs. Expression of these markers, particularly CD39, CD81, CD130, and CD326, is stable and offers an improved resolution for ASC identification. We accessed their expression in germ-free conditions and in T cell deficient mice, showing that at least in part their expression is controlled by microbial- and T cell-derived signals. Further analysis of lupus mice revealed the presence of a subpopulation of LAG-3 - plasma cells, co-expressing high amounts of CD39 and CD326 in the bone marrow. This population was IgM + and correlated with IgM anti-dsDNA autoantibodies in sera. Importantly, we found that CD39, CD81, CD130, and CD326 are also expressed by human peripheral blood and bone marrow ASCs. Our data provide innovative insights into ASC biology and function in mice and human, and identify an intriguing BM specific CD39 ++ CD326 ++ ASC subpopulation in autoimmunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dang, Mohr, Szelinski, Le, Ritter, Hinnenthal, Stefanski, Schrezenmeier, Ocvirk, Hipfl, Hardt, Cheng, Hiepe, Löhning, Dörner and Lino.)

Intestinal parasitic nematodes affect a quarter of the world's population, typically eliciting prominent effector Th2-driven host immune responses. As not all infected hosts develop protection against reinfection, our current understanding of nematode-induced memory Th2 responses remains limited. Here, we investigated the activation of memory Th2 cells and the mechanisms driving early recall responses to the enteric nematode Heligmosomoides polygyrus in mice. We show that nematode-cured mice harbor memory Th2 cells in lymphoid and non-lymphoid organs with distinct transcriptional profiles, expressing recirculation markers like CCR7 and CD62-L in the mesenteric lymph nodes (mLN), and costimulatory markers like Ox40, as well as tissue homing and activation markers like CCR2, CD69 and CD40L in the gut and peritoneal cavity (PEC). While memory Th2 cells persist systemically in both lymphoid and non-lymphoid tissues following cure of infection, peritoneal memory Th2 cells in particular displayed an initial prominent expansion and strong parasite-specific Th2 responses during early recall responses to a challenge nematode infection. This effect was paralleled by a significant influx of dendritic cells (DC) and eosinophils, both also appearing exclusively in the peritoneal cavity of reinfected mice. In addition, we show that within the peritoneal membrane lined by peritoneal mesothelial cells (PeM), the gene expression levels of cell adhesion markers VCAM-1 and ICAM-1 decrease significantly in response to a secondary infection. Overall, our findings indicate that the host peritoneal cavity in particular harbors prominent memory Th2 cells and appears to respond directly to H. polygyrus by an early recall response via differential regulation of cell adhesion markers, marking the peritoneal cavity an important site for host immune responses to an enteric pathogen., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yordanova, Jürchott, Steinfelder, Vogt, Krüger, Kühl, Sawitzki and Hartmann.)

Background: Hemostasis and inflammation are both dysregulated in patients with moderate-to-severe coronavirus disease 2019 (COVID-19). Yet, both processes can also be disturbed in patients with other respiratory diseases, and the interactions between coagulation, inflammation, and disease severity specific to COVID-19 are still vague., Methods: Hospitalized patients with acute respiratory symptoms and with severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)-positive (COV pos ) and SARS-CoV2-negative (COV neg ) status were included. We assessed adenosine diphosphate (ADP)-, thrombin receptor activator peptide 6 (TRAP)-, and arachidonic acid (AA)-induced platelet reactivity by impedance aggregometry, as well as leukocyte subtype spectrum and platelet-leukocyte aggregates by flow cytometry and inflammatory cytokines by cytometric bead array., Results: ADP-, TRAP-, and AA-induced platelet reactivity was significantly higher in COV pos than in COV neg patients. Disease severity, assessed by sequential organ failure assessment (SOFA) score, was higher in COV pos than in COV neg patients and again higher in deceased COV pos patients than in surviving COV pos . The SOFA score correlated significantly with the mean platelet volume and TRAP-induced platelet aggregability. A larger percentage of classical and intermediate monocytes, and of CD4 pos T cells (T H ) aggregated with platelets in COV pos than in COV neg patients. Interleukin (IL)-1 receptor antagonist (RA) and IL-6 levels were higher in COV pos than in COV neg patients and again higher in deceased COV pos patients than in surviving COV pos . IL-1RA and IL-6 levels correlated with the SOFA score in COV pos but not in COV neg patients. In both respiratory disease groups, absolute levels of B-cell-platelet aggregates and NK-cell-platelet aggregates were correlated with ex vivo platelet aggegation upon stimulation with AA and ADP, respectively, indicating a universal, but not a COVID-19-specific mechanism., Conclusion: In moderate-to-severe COVID-19, but not in other respiratory diseases, disease severity was associated with platelet hyperreactivity and a typical inflammatory signature. In addition to a severe inflammatory response, platelet hyperreactivity associated to a worse clinical outcome in patients with COVID-19, pointing to the importance of antithrombotic therapy for reducing disease severity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jakobs, Reinshagen, Puccini, Friebel, Wilde, Alsheik, Rroku, Landmesser, Haghikia, Kränkel and Rauch-Kröhnert.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The nasal epithelium represents the first line of defense against inhaled pathogens, allergens, and irritants and plays a key role in the pathogenesis of a spectrum of acute and chronic airways diseases. Despite age-dependent clinical phenotypes triggered by these noxious stimuli, little is known about how aging affects the structure and function of the airway epithelium that is crucial for lung homeostasis and host defense. The aim of this study was therefore to determine age-related differences in structural and functional properties of primary nasal epithelial cultures from healthy children and non-smoking elderly people. To achieve this goal, highly differentiated nasal epithelial cultures were established from nasal brushes at air-liquid interface and used to study epithelial cell type composition, mucin (MUC5AC and MUC5B) expression, and ion transport properties. Furthermore, we determined age-dependent molecular signatures using global proteomic analysis. We found lower numeric densities of ciliated cells and higher levels of MUC5AC expression in cultures from children vs. elderly people. Bioelectric studies showed no differences in basal ion transport properties, ENaC-mediated sodium absorption, or CFTR-mediated chloride transport, but detected decreased calcium-activated TMEM16A-mediated chloride secretory responses in cultures from children vs. elderly people. Proteome analysis identified distinct age-dependent molecular signatures associated with ciliation and mucin biosynthesis, as well as other pathways implicated in aging. Our data identified intrinsic, age-related differences in structure and function of the nasal epithelium and provide a basis for further studies on the role of these findings in age-dependent airways disease phenotypes observed with a spectrum of respiratory infections and other noxious stimuli., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Balázs, Millar-Büchner, Mülleder, Farztdinov, Szyrwiel, Addante, Kuppe, Rubil, Drescher, Seidel, Stricker, Eils, Lehmann, Sawitzki, Röhmel, Ralser and Mall.)

Peritoneal dialysis (PD) is a valuable 'home treatment' option, even more so during the ongoing Coronavirus pandemic. However, the long-term use of PD is limited by unfavourable tissue remodelling in the peritoneal membrane, which is associated with inflammation-induced angiogenesis. This appears to be driven primarily through vascular endothelial growth factor (VEGF), while the involvement of other angiogenic signaling pathways is still poorly understood. Here, we have identified the crucial contribution of mesothelial cell-derived angiogenic CXC chemokine ligand 1 (CXCL1) to peritoneal angiogenesis in PD. CXCL1 expression and peritoneal microvessel density were analysed in biopsies obtained by the International Peritoneal Biobank (NCT01893710 at www.clinicaltrials.gov), comparing 13 children with end-stage kidney disease before initiating PD to 43 children on chronic PD. The angiogenic potential of mesothelial cell-derived CXCL1 was assessed in vitro by measuring endothelial tube formation of human microvascular endothelial cells (HMECs) treated with conditioned medium from human peritoneal mesothelial cells (HPMCs) stimulated to release CXCL1 by treatment with either recombinant IL-17 or PD effluent. We found that the capillary density in the human peritoneum correlated with local CXCL1 expression. Both CXCL1 expression and microvessel density were higher in PD patients than in the age-matched patients prior to initiation of PD. Exposure of HMECs to recombinant CXCL1 or conditioned medium from IL-17-stimulated HPMCs resulted in increased endothelial tube formation, while selective inhibition of mesothelial CXCL1 production by specific antibodies or through silencing of relevant transcription factors abolished the proangiogenic effect of HPMC-conditioned medium. In conclusion, peritoneal mesothelium-derived CXCL1 promotes endothelial tube formation in vitro and associates with peritoneal microvessel density in uremic patients undergoing PD, thus providing novel targets for therapeutic intervention to prolong PD therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Catar, Bartosova, Kawka, Chen, Marinovic, Zhang, Zhao, Wu, Zickler, Stadnik, Karczewski, Kamhieh-Milz, Jörres, Moll, Schmitt and Witowski.)

Rubella virus (RuV) has recently been found in association with granulomatous inflammation of the skin and several internal organs in patients with inborn errors of immunity (IEI). The cellular tropism and molecular mechanisms of RuV persistence and pathogenesis in select immunocompromised hosts are not clear. We provide clinical, immunological, virological, and histological data on a cohort of 28 patients with a broad spectrum of IEI and RuV-associated granulomas in skin and nine extracutaneous tissues to further delineate this relationship. Combined immunodeficiency was the most frequent diagnosis (67.8%) among patients. Patients with previously undocumented conditions, i.e., humoral immunodeficiencies, a secondary immunodeficiency, and a defect of innate immunity were identified as being susceptible to RuV-associated granulomas. Hematopoietic cell transplantation was the most successful treatment in this case series resulting in granuloma resolution; steroids, and TNF-α and IL-1R inhibitors were moderately effective. In addition to M2 macrophages, neutrophils were identified by immunohistochemical analysis as a novel cell type infected with RuV. Four patterns of RuV-associated granulomatous inflammation were classified based on the structural organization of granulomas and identity and location of cell types harboring RuV antigen. Identification of conditions that increase susceptibility to RuV-associated granulomas combined with structural characterization of the granulomas may lead to a better understanding of the pathogenesis of RuV-associated granulomas and discover new targets for therapeutic interventions., Competing Interests: MA is employed by Sidra Medicine and Hamad Medical Corporation, Qatar. HB is employed by Labor Berlin GmbH, Germany. JH received grants from Immune Deficiency Foundation, the US immunodeficiency network, Chao-physician Scientist award, the Texas Medical Center Digestive Diseases Center and the Jeffrey Modell Foundation. JH received honorarium, consultation fees from Horizon, Pharming, Baxalta, CSL Behring, the National guard, and Al-Faisal University Hospital. TPA received consultation fees from Horizon, Pharming, CSL Behring. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Perelygina, Faisthalab, Abernathy, Chen, Hao, Bercovitch, Bayer, Noroski, Lam, Cicalese, Al-Herz, Nanda, Hajjar, Vanden Driessche, Schroven, Leysen, Rosenbach, Peters, Raedler, Albert, Abraham, Rangarjan, Buchbinder, Kobrynski, Pham-Huy, Dhossche, Cunningham Rundles, Meyer, Theos, Atkinson, Musiek, Adeli, Derichs, Walz, Krüger, von Bernuth, Klein, Icenogle, Hauck and Sullivan.)

Abstract: Systemic chronic microinflammation and altered cytokine signaling, with adjunct cardiovascular disease (CVD), endothelial maladaptation and dysfunction is common in dialysis patients suffering from end-stage renal disease and associated with increased morbidity and mortality. New hemodialysis filters might offer improvements. We here studied the impact of novel improved molecular cut-off hemodialysis filters on systemic microinflammation, uremia and endothelial dysfunction. Human endothelial cells (ECs) were incubated with uremic serum obtained from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation (PERCI-II) crossover clinical trial, comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes, and then assessed for their vascular endothelial growth factor (VEGF) production and angiogenesis. Compared to HF membranes, dialysis with MCO membranes lead to a reduction in proinflammatory mediators and reduced endothelial VEGF production and angiogenesis. Cytokine multiplex screening identified tumor necrosis factor (TNF) superfamily members as promising targets. The influence of TNF-α and its soluble receptors (sTNF-R1 and sTNF-R2) on endothelial VEGF promoter activation, protein release, and the involved signaling pathways was analyzed, revealing that this detrimental signaling was indeed induced by TNF-α and mediated by AP-1/c-FOS signaling. In conclusion, uremic toxins, in particular TNF-signaling, promote endothelial maladaptation, VEGF expression and aberrant angiogenesis, which can be positively modulated by dialysis with novel MCO membranes., Translational Perspective and Graphical Abstract: Systemic microinflammation, altered cytokine signaling, cardiovascular disease, and endothelial maladaptation/dysfunction are common clinical complications in dialysis patients suffering from end-stage renal disease. We studied the impact of novel improved medium-cut-off hemodialysis filters on uremia and endothelial dysfunction. We can show that uremic toxins, especially TNF-signaling, promote endothelial maladaptation, VEGF expression and aberrant angiogenesis, which can be positively modulated by dialysis with novel improved medium-cut-off membranes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Catar, Moll, Kamhieh-Milz, Luecht, Chen, Zhao, Ernst, Willy, Girndt, Fiedler, Witowski, Morawietz, Ringdén, Dragun, Eckardt, Schindler and Zickler.)

Reshaping the immune balance by adoptive transfer of regulatory T-cells (Tregs) has emerged as a promising strategy to combat undesired immune reactions, including in Graft-versus-Host Disease (GvHD), which is the most lethal non-relapse complication of allogeneic hematopoietic stem cell transplantation. Currently however, little is known about the potentially inhibitory in vivo effects of conventional immunosuppressive drugs, which are routinely used to treat GvHD, on adoptively transferred Tregs. Here we demonstrate drug-specific effects of the conventional immunosuppressive drugs Cyclosporine A, Mycophenolate mofetil and methylprednisolone on adoptively transferred Tregs in a humanized NOD/SCID/IL2Rgamma-/- GvHD mouse model. The clinical course of GvHD and postmortem organ histology, including cellular organ infiltration, showed that co-administration of Cyclosporine A and Tregs is highly beneficial as it enhanced Treg accumulation at inflammatory sites like lung and liver. Similarly, co-administration of Mycophenolate mofetil and Tregs improved clinical signs of GvHD. In contrast, co-administration of methylprednisolone and Tregs resulted in reduced Treg recruitment to inflammatory sites and the fast deterioration of some animals. Consequently, when clinical trials investigating safety and efficacy of adjunctive Treg therapy in GvHD are designed, we suggest co-administering Cyclosporine A, whereas high doses of glucocorticosteroids should be avoided., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Landwehr-Kenzel, Zobel, Schmitt-Knosalla, Forke, Hoffmann, Schmueck-Henneresse, Klopfleisch, Volk and Reinke.)

Introduction: High-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (auto-HSCT) represents a standard treatment regime for multiple myeloma (MM) patients. Common and potentially fatal side effects after auto-HSCT are infections due to a severely compromised immune system with hampered humoral and cellular immunity. This study delineates in depth the quantitative and functional B cell defects and investigates underlying extrinsic or intrinsic drivers., Methods: Peripheral blood of MM patients undergoing high-dose chemotherapy and auto-HSCT (before high-dose chemotherapy and in early reconstitution after HSCT) was studied. Absolute numbers and distribution of B cell subsets were analyzed ex vivo using flow cytometry. Additionally, B cell function was assessed with T cell dependent (TD) and T cell independent (TI) stimulation assays, analyzing proliferation and differentiation of B cells by flow cytometry and numbers of immunoglobulin secreting cells in ELISpots., Results: Quantitative B cell defects including a shift in the B cell subset distribution occurred after auto-HSCT. Functionally, these patients showed an impaired TD as well as TI B cell immune response. Individual functional responses correlated with quantitative alterations of CD19+, CD4+, memory B cells and marginal zone-like B cells. The TD B cell function could be partially restored upon stimulation with CD40L/IL-21, successfully inducing B cell proliferation and differentiation into plasmablasts and immunoglobulin secreting cells., Conclusion: Quantitative and functional B cell defects contribute to the compromised immune defense in MM patients undergoing auto-HSCT. Functional recovery upon TD stimulation and correlation with CD4+ T cell numbers, indicate these as extrinsic drivers of the functional B cell defect. Observed correlations of CD4+, CD19+, memory B and MZ-like B cell numbers with the B cell function suggest that these markers should be tested as potential biomarkers in prospective studies., Competing Interests: I-KN declares a conflict of interest through financial support from Shire/Takeda and Octapharma. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Heck, Steiner, Kaebisch, Frentsch, Wittenbecher, Scheibenbogen, Hanitsch, Nogai, le Coutre, Bullinger, Blau and Na.)

Background: Within the bone marrow (BM), mature T cells are maintained under homeostatic conditions to facilitate proper hematopoietic development. This homeostasis depends upon a peculiar elevated frequency of regulatory T cells (Tregs) and immune regulatory activities from BM-mesenchymal stem cells (BM-MSCs). In response to BM transplantation (BMT), the conditioning regimen exposes the BM to a dramatic induction of inflammatory cytokines and causes an unbalanced T-effector (Teff) and Treg ratio. This imbalance negatively impacts hematopoiesis, particularly in regard to B-cell lymphopoiesis that requires an intact cross-talk between BM-MSCs and Tregs. The mechanisms underlying the ability of BM-MSCs to restore Treg homeostasis and proper B-cell development are currently unknown., Methods: We studied the role of host radio-resistant cell-derived CD40 in restoring Teff/Treg homeostasis and proper B-cell development in a murine model of BMT. We characterized the host cellular source of CD40 and performed radiation chimera analyses by transplanting WT or Cd40-KO with WT BM in the presence of T-reg and co-infusing WT or - Cd40-KO BM-MSCs. Residual host and donor T cell expansion and activation (cytokine production) and also the expression of Treg fitness markers and conversion to Th17 were analyzed. The presence of Cd40+ BM-MSCs was analyzed in a human setting in correlation with the frequency of B-cell precursors in patients who underwent HSCT and variably developed acute graft-versus-host (aGVDH) disease., Results: CD40 expression is nearly undetectable in the BM, yet a Cd40-KO recipient of WT donor chimera exhibited impaired B-cell lymphopoiesis and Treg development. Lethal irradiation promotes CD40 and OX40L expression in radio-resistant BM-MSCs through the induction of pro-inflammatory cytokines. OX40L favors Teff expansion and activation at the expense of Tregs; however, the expression of CD40 dampens OX40L expression and restores Treg homeostasis, thus facilitating proper B-cell development. Indeed, in contrast to dendritic cells in secondary lymphoid organs that require CD40 triggers to express OX40L, BM-MSCs require CD40 to inhibit OX40L expression., Conclusions: CD40+ BM-MSCs are immune regulatory elements within BM. Loss of CD40 results in uncontrolled T cell activation due to a reduced number of Tregs, and B-cell development is consequently impaired. GVHD provides an example of how a loss of CD40+ BM-MSCs and a reduction in B-cell precursors may occur in a human setting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bassani, Tripodo, Portararo, Gulino, Botti, Chiodoni, Jachetti, Bolli, Ciciarello, Joehrens, Anagnostopoulos, Na, Curti, Colombo and Sangaletti.)

Acute cellular rejection (ACR) after liver transplantation (LT) goes along with allograft dysfunction, which is diagnosed by liver biopsy and concomitant histological analysis, representing the gold standard in clinical practice. Yet, liver biopsies are invasive, costly, time-intensive and require expert knowledge. Herein we present substantial evidence that blood plasma residing peripheral liver-derived extracellular particles (EP) could be employed to diagnose ACR non-invasively. In vitro experiments showed organ-specific EP release from primary human hepatocytes under immunological stress. Secondly, analysis of consecutive LT patients (n=11) revealed significant heightened EP concentrations days before ACR. By conducting a diagnostic accuracy study (n = 69, DRKS00011631), we explored the viability of using EP as a liquid biopsy for diagnosing ACR following LT. Consequently, novel EP populations in samples were identified using visualization of t-distributed stochastic neighbor embedding (viSNE) and self-organizing maps (FlowSOM) algorithms. As a result, the ASGR1 + CD130 + Annexin V + EP subpopulation exhibited the highest accuracy for predicting ACR (area under the curve: 0.80, 95% confidence interval [CI], 0.70-0.90), with diagnostic sensitivity and specificity of 100% (95% CI, 81.67-100.0%) and 68.5% (95% CI, 55.3-79.3%), respectively. In summary, this new EP subpopulation presented the highest diagnostic accuracy for detecting ACR in LT patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kamali, Schmelzle, Kamali, Brunnbauer, Splith, Leder, Berndt, Hillebrandt, Raschzok, Feldbrügge, Felsenstein, Gaßner, Ritschl, Lurje, Schöning, Benzing, Pratschke and Krenzien.)

Background: The pathogenesis of chronic spontaneous urticaria (CSU) is still insufficiently understood. Recent findings suggest that immunoglobulins, in particular IgE but also IgA, play a role in the development of CSU., Objective: Our aim was to assess differences in clinical and laboratory markers between CSU patients with and without lower levels of serum IgA and IgE., Methods: We analyzed the data of 606 patients with CSU by dividing them into four groups based on their IgA and IgE levels. The groups were compared for their spectrum of symptoms, disease activity, concomitant autoimmunity and routine laboratory markers. Autoreactivity was assessed by basophil activation test (BAT). Moreover, IgE-anti-thyroid peroxidase (TPO) was measured., Results: Of the patients with lower IgE levels, 66.5% also had lower IgA levels (r=0.316, p<0.001). Patients with lower IgA and lower IgE levels showed a higher prevalence of recurrent angioedema (p=0.03, p=0.04) and concomitant autoimmunity (p=0.006, p<0.001). Autoreactivity was also found more frequently in patients with lower IgA and lower IgE levels (p=0.003, p<0.001). Reduced basophil counts were linked to both, lower IgA and lower IgE levels (p<0.001), whereas low eosinophil counts were primarily present in patients with lower IgE levels (p=0.04, p<0.001). Patients with elevated IgE-anti-TPO levels had lower IgA (p=0.007) and IgE levels (p=0.001)., Conclusion: Lower IgA levels in CSU are linked to lower IgE levels and features of autoimmune urticaria. Our findings encourage to screen CSU patients for serum IgA and IgE levels and to further assess their role as disease biomarkers., Competing Interests: PK received personal fees from Novartis and Roche, outside the submitted work. FS received grants and/or personal fees from Allakos, Blueprint, Hyphens, Genentech, Novartis, Pediapharm, and Uriach, outside the submitted work. SA received grants and/or personal fees from Allakos, AstraZeneca, CSL Behring, Moxie and Sanofi, outside the submitted work. MMa received grants and/or personal fees from Allakos, Amgen, Aralez, Argenx, AstraZeneca, Celldex, Centogene, CSL Behring, FAES, Genentech, GIInnovation, Innate Pharma, Kyowa Kirin, Leo Pharma, Lilly, Menarini, Moxie, Novartis, Roche, Sanofi/Regeneron, Third HarmonicBio, UCB, and Uriach, outside the submitted work. MMe received personal fees from Amgen, Aralez, Argenx, Bayer, Moxie, Novartis, Roche, Sanofi and Uriach, outside the submitted work. KK received grants and/or personal fees from Bayer, Berlin Chemie, CSL Behring, Moxie, Novartis, Roche and Shire/Takeda, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sauer, Scheffel, Frischbutter, Kolkhir, Xiang, Siebenhaar, Altrichter, Maurer, Metz and Krause.)

Forkhead box O 3 (FOXO3) is a transcription factor involved in cell metabolism, inflammation and longevity. Here, we investigated if metformin can activate FOXO3 in human immune cells and affects the subsequent level of reactive oxygen/nitrogen species (ROS/RNS) in immune cells. AMP-activated protein kinase (AMPK) and FOXO3 activation were investigated by immunoblot or flow cytometry (FC) analysis, respectively. FOXO3 target gene expression was quantified by real-time PCR. ROS/RNS measurement using dichlorodihydrofluorescein diacetate (DCFH-DA) dye was investigated by FC. The role of the FOXO3 single nucleotide polymorphisms (SNPs) rs12212067 , rs2802292 and rs12206094 on ROS/RNS production was studied using allelic discrimination PCR. Metformin induced activation of AMPK (pT172) and FOXO3 (pS413). ROS/RNS level was reduced in immune cells after metformin stimulation accompanied by induction of the FOXO3 targets mitochondrial superoxide dismutase and cytochrome c. Studies in Foxo3 deficient ( Foxo3 -/- ) mouse splenocytes confirmed that metformin mediates its effects via Foxo3 as it attenuates ROS/RNS in myeloid cells of wildtype (WT) but not of Foxo3 -/- mice. Our results suggest that FOXO3 can be activated by metformin leading to reduced ROS/RNS level in immune cells. This may add to the beneficial clinical effects of metformin observed in large cohort studies on longevity, cardiovascular and cancer risk., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hartwig, Loebel, Steiner, Bauer, Karadeniz, Roeger, Skurk, Scheibenbogen and Sotzny.)

Regulatory Tcells (Treg) are essential components of peripheral immune homeostasis. Adoptive Treg cell therapy has shown efficacy in a variety of immune-mediated diseases in preclinical studies and is now moving from phase I/IIa to larger phase II studies aiming to demonstrate efficacy. However, hurdles such as in vivo stability and efficacy remain to be addressed. Nevertheless, preclinical models have shown that Treg function and specificity can be increased by pharmacological substances or gene modifications, and even that conventional T cells can be converted to Treg potentially providing new sources of Treg and facilitating Treg cell therapy. The exponential growth in genetic engineering techniques and their application to T cells coupled to a large body of knowledge on Treg open numerous opportunities to generate Treg with "superpowers". This review summarizes the genetic engineering techniques available and their applications for the next-generation of Super-Treg with increased function, stability, redirected specificity and survival., Competing Interests: CG, IA, and SB have patents that have been licensed to TxCell S.A., a Sangamo Company. DW, LA, PR, H-DV, and MS-H have filed patent applications on Treg-related diagnostics and therapeutics. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Amini, Greig, Schmueck-Henneresse, Volk, Bézie, Reinke, Guillonneau, Wagner and Anegon.)

The inability of patients with CVID to mount specific antibody responses to pathogens has raised concerns on the risk and severity of SARS-CoV-2 infection, but there might be a role for protective T cells in these patients. SARS-CoV-2 reactive T cells have been reported for SARS-CoV-2 unexposed healthy individuals. Until now, there is no data on T cell immunity to SARS-CoV-2 infection in CVID. This study aimed to evaluate reactive T cells to human endemic corona viruses (HCoV) and to study pre-existing SARS-CoV-2 reactive T cells in unexposed CVID patients. We evaluated SARS-CoV-2- and HCoV-229E and -OC43 reactive T cells in response to seven peptide pools, including spike and nucleocapsid (NCAP) proteins, in 11 unexposed CVID, 12 unexposed and 11 post COVID-19 healthy controls (HC). We further characterized reactive T cells by IFNγ, TNFα and IL-2 profiles. SARS-CoV-2 spike-reactive CD4+ T cells were detected in 7 of 11 unexposed CVID patients, albeit with fewer multifunctional (IFNγ/TNFα/IL-2) cells than unexposed HC. CVID patients had no SARS-CoV-2 NCAP reactive CD4+ T cells and less reactive CD8+ cells compared to unexposed HC. We observed a correlation between T cell reactivity against spike of SARS-CoV-2 and HCoVs in unexposed, but not post COVID-19 HC, suggesting cross-reactivity. T cell responses in post COVID-19 HC could be distinguished from unexposed HC by higher frequencies of triple-positive NCAP reactive CD4+ T cells. Taken together, SARS-CoV-2 reactive T cells are detectable in unexposed CVID patients albeit with lower recognition frequencies and polyfunctional potential. Frequencies of triple-functional reactive CD4+ cells might provide a marker to distinguish HCoV cross-reactive from SARS-CoV-2 specific T cell responses. Our data provides evidence, that anti-viral T cell immunity is not relevantly impaired in most CVID patients., Competing Interests: VC is named together with Euroimmun on a patent application filed recently regarding detection of antibodies against SARS-CoV-2. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Steiner, Sotzny, Bauer, Na, Schmueck-Henneresse, Corman, Schwarz, Drosten, Wendering, Behrends, Volk, Scheibenbogen and Hanitsch.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

After being described in the 1970s as cytotoxic cells that do not require MHC-dependent pre-activation, natural killer (NK) cells remained the sole member of innate lymphocytes for decades until lymphoid tissue-inducer cells in the 1990s and helper-like innate lymphoid lineages from 2008 onward completed the picture of innate lymphoid cell (ILC) diversity. Since some of the ILC members, such as ILC1s and CCR6 - ILC3s, share specific markers previously used to identify NK cells, these findings provoked the question of how to delineate the development of NK cell and helper-like ILCs and how to properly identify and genetically interfere with NK cells. The description of eomesodermin (EOMES) as a lineage-specifying transcription factor of NK cells provided a candidate that may serve as a selective marker for the genetic targeting and identification of NK cells. Unlike helper-like ILCs, NK cell activation is, to a large degree, regulated by the engagement of activating and inhibitory surface receptors. NK cell research has revealed some elegant mechanisms of immunosurveillance, coined "missing-self" and "induced-self" recognition, thus complementing "non-self recognition", which is predominantly utilized by adaptive lymphocytes and myeloid cells. Notably, the balance of activating and inhibitory signals perceived by surface receptors can be therapeutically harnessed for anti-tumor immunity mediated by NK cells. This review aims to summarize the similarities and the differences in development, function, localization, and phenotype of NK cells and helper-like ILCs, with the purpose to highlight the unique feature of NK cell development and regulation., (Copyright © 2020 Stokic-Trtica, Diefenbach and Klose.)

Numerous clinical trials of mesenchymal stromal/stem cells (MSCs) as a new treatment for coronavirus-induced disease (COVID-19) have been registered recently, most of them based on intravenous (IV) infusion. There is no approved effective therapy for COVID-19, but MSC therapies have shown first promise in the treatment of acute respiratory distress syndrome (ARDS) pneumonia, inflammation, and sepsis, which are among the leading causes of mortality in COVID-19 patients. Many of the critically ill COVID-19 patients are in a hypercoagulable procoagulant state and at high risk for disseminated intravascular coagulation, thromboembolism, and thrombotic multi-organ failure, another cause of high fatality. It is not yet clear whether IV infusion is a safe and effective route of MSC delivery in COVID-19, since MSC-based products express variable levels of highly procoagulant tissue factor (TF/CD142), compromising the cells' hemocompatibility and safety profile. Of concern, IV infusions of poorly characterized MSC products with unchecked (high) TF/CD142 expression could trigger blood clotting in COVID-19 and other vulnerable patient populations and further promote the risk for thromboembolism. In contrast, well-characterized products with robust manufacturing procedures and optimized modes of clinical delivery hold great promise for ameliorating COVID-19 by exerting their beneficial immunomodulatory effects, inducing tissue repair and organ protection. While the need for MSC therapy in COVID-19 is apparent, integrating both innate and adaptive immune compatibility testing into the current guidelines for cell, tissue, and organ transplantation is critical for safe and effective therapies. It is paramount to only use well-characterized, safe MSCs even in the most urgent and experimental treatments. We here propose three steps to mitigate the risk for these vulnerable patients: (1) updated clinical guidelines for cell and tissue transplantation, (2) updated minimal criteria for characterization of cellular therapeutics, and (3) updated cell therapy routines reflecting specific patient needs., (Copyright © 2020 Moll, Drzeniek, Kamhieh-Milz, Geissler, Volk and Reinke.)