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Pro-inflammatory autoantigen-specific CD4 + T helper (auto-Th) cells are central orchestrators of autoimmune diseases (AIDs). We aimed to characterize these cells in human AIDs with defined autoantigens by combining human leukocyte antigen (HLA)-tetramer-based and activation-based multidimensional ex vivo analyses. In aquaporin4-antibody-positive neuromyelitis optica spectrum disorder (AQP4-NMOSD) patients, auto-Th cells expressed CD154, but proliferative capacity and pro-inflammatory cytokines were strongly reduced. Instead, exhaustion-associated co-inhibitory receptors were expressed together with FOXP3, the canonical regulatory T cell (Treg) transcription factor. Auto-Th cells responded in vitro to checkpoint inhibition and provided potent B cell help. Cells with the same exhaustion-like (ThEx) phenotype were identified in soluble liver antigen (SLA)-antibody-autoimmune hepatitis and BP180-antibody-positive bullous pemphigoid, AIDs of the liver and skin, respectively. While originally described in cancer and chronic infection, our data point to T cell exhaustion as a common mechanism of adaptation to chronic (self-)stimulation across AID types and link exhausted CD4 + T cells to humoral autoimmune responses, with implications for therapeutic targeting., Competing Interests: Declaration of interests R.N. is a current employee of Cellerys AG. F.L. discloses speaker honoraria from Grifols, Teva, Biogen, Bayer, Roche, Novartis, and Fresenius; travel funding from Merck, Grifols, and Bayer; and is serving on advisory boards for Roche, Biogen, and Alexion. R.M. has received unrestricted grants from Biogen, Novartis, Roche, and Third Rock and honoraria for advisory roles and lectures from Roche, Novartis, Biogen, Genzyme, Neuway, CellProtect, Third Rock, and Teva. He is a patent holder and co-holder on the following patents: daclizumab in MS (held by NIH), JCV VP1 for vaccination against PML; JCV-specific neutralizing antibodies to treat PML; and antigen-specific tolerization with peptide-coupled cells, novel autoantigens in MS, and designer neoantigens for tumor vaccination (all held by University of Zurich). He is a co-founder of Abata Therapeutics, Watertown, MA, USA and co-founder and employee of Cellerys AG, Schlieren, Switzerland., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Aberrant CD4 + T cell reactivity against intestinal microorganisms is considered to drive mucosal inflammation in inflammatory bowel diseases. The disease-relevant microbial species and the corresponding microorganism-specific, pathogenic T cell phenotypes remain largely unknown. In the present study, we identified common gut commensal and food-derived yeasts, as direct activators of altered CD4 + T cell reactions in patients with Crohn's disease (CD). Yeast-responsive CD4 + T cells in CD display a cytotoxic T helper cell (T H 1 cell) phenotype and show selective expansion of T cell clones that are highly cross-reactive to several commensal, as well as food-derived, fungal species. This indicates cross-reactive T cell selection by repeated encounter with conserved fungal antigens in the context of chronic intestinal disease. Our results highlighted a role of yeasts as drivers of aberrant CD4 + T cell reactivity in patients with CD and suggest that both gut-resident fungal commensals and daily dietary intake of yeasts might contribute to chronic activation of inflammatory CD4 + T cell responses in patients with CD., (© 2023. The Author(s).)

Alcohol-associated liver disease is accompanied by intestinal mycobiome dysbiosis, yet the impacts on liver disease are unclear. We demonstrate that Candida albicans-specific T helper 17 (Th17) cells are increased in circulation and present in the liver of patients with alcohol-associated liver disease. Chronic ethanol administration in mice causes migration of Candida albicans (C. albicans)-reactive Th17 cells from the intestine to the liver. The antifungal agent nystatin decreased C. albicans-specific Th17 cells in the liver and reduced ethanol-induced liver disease in mice. Transgenic mice expressing T cell receptors (TCRs) reactive to Candida antigens developed more severe ethanol-induced liver disease than transgene-negative littermates. Adoptively transferring Candida-specific TCR transgenic T cells or polyclonal C. albicans-primed T cells exacerbated ethanol-induced liver disease in wild-type mice. Interleukin-17 (IL-17) receptor A signaling in Kupffer cells was required for the effects of polyclonal C. albicans-primed T cells. Our findings indicate that ethanol increases C. albicans-specific Th17 cells, which contribute to alcohol-associated liver disease., Competing Interests: Declaration of interests B.S. has been consulting for Ambys Medicines, Ferring Research Institute, Gelesis, HOST Therabiomics, Intercept Pharmaceuticals, Mabwell Therapeutics, Patara Pharmaceuticals, and Takeda. B.S.’s institution UC San Diego has received research support from Artizan Biosciences, Axial Biotherapeutics, BiomX, CymaBay Therapeutics, NGM Biopharmaceuticals, Prodigy Biotech, and Synlogic Operating Company. B.S. is the founder of Nterica Bio. UC San Diego has filed several patents with B.S. and Y.D. as inventors related to this work. P.S. received grant support from Gilead Sciences Belgium., (Published by Elsevier Inc.)

The humoral immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination in patients with chronic inflammatory disease (CID) declines more rapidly with tumor necrosis factor-α (TNF-α) inhibition. Furthermore, the efficacy of current vaccines against Omicron variants of concern (VOC) including BA.2 is limited. Alterations within immune cell populations, changes in IgG affinity, and the ability to neutralize a pre-VOC strain and the BA.2 virus were investigated in these at-risk patients. Serum levels of anti-SARS-CoV-2 IgG, IgG avidity, and neutralizing antibodies (NA) were determined in anti-TNF-α patients (n = 10) and controls (n = 24 healthy individuals; n = 12 patients under other disease-modifying antirheumatic drugs, oDMARD) before and after the second and third vaccination by ELISA, immunoblot and live virus neutralization assay. SARS-CoV-2-specific B- and T cell subsets were analysed by multicolor flow cytometry. Six months after the second vaccination, anti-SARS-CoV-2 IgG levels, IgG avidity and anti-pre-VOC NA titres were significantly reduced in anti-TNF-α recipients compared to controls (healthy individuals: avidity: p ≤ 0.0001; NA: p = 0.0347; oDMARDs: avidity: p = 0.0012; NA: p = 0.0293). The number of plasma cells was increased in anti-TNF-α patients (Healthy individuals: p = 0.0344; oDMARDs: p = 0.0254), while the absolute number of SARS-CoV-2-specific plasma cells 7 days after 2nd vaccination were comparable. Even after a third vaccination, these patients had lower anti-BA.2 NA titres compared to both other groups. We show a reduced SARS-CoV-2 neutralizing capacity in patients under TNF-α blockade. In this cohort, the plasma cell response appears to be less specific and shows stronger bystander activation. While these effects were observable after the first two vaccinations and with older VOC, the differences in responses to BA.2 were enhanced., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

SARS-CoV-2 infection and vaccination generates enormous host-response heterogeneity and an age-dependent loss of immune-response quality. How the pre-exposure T cell repertoire contributes to this heterogeneity is poorly understood. We combined analysis of SARS-CoV-2-specific CD4 + T cells pre- and post-vaccination with longitudinal T cell receptor tracking. We identified strong pre-exposure T cell variability that correlated with subsequent immune-response quality and age. High-quality responses, defined by strong expansion of high-avidity spike-specific T cells, high interleukin-21 production, and specific immunoglobulin G, depended on an intact naive repertoire and exclusion of pre-existing memory T cells. In the elderly, T cell expansion from both compartments was severely compromised. Our results reveal that an intrinsic defect of the CD4 + T cell repertoire causes the age-dependent decline of immune-response quality against SARS-CoV-2 and highlight the need for alternative strategies to induce high-quality T cell responses against newly arising pathogens in the elderly., Competing Interests: Declaration of interests E.R. is employee of Evotec SE. O.K. is employee of Peptides & elephants GmbH. O.C. reports grants or contracts from MSD, Pfizer; consulting fees from Biocon, Molecular Partners, Noxxon, PSI; honoraria for lectures from Abbott, Pfizer; participation on a data safety monitoring board or advisory board from Jannsen, PSI. P.K. reports grants or contracts from BMBF, B-FAST, and NAPKON of the Network University Medicine and the State of North Rhine-Westphalia; consulting fees from Ambu GmbH, Gilead Sciences, Noxxon N.V., Pfizer Pharma; honoraria for lectures from Ambu GmbH, Gilead Sciences, MSD Sharp & Dohme GmbH, Pfizer Pharma GmbH, Scilink Comunicación Científica SC; participation on an advisory board from Ambu GmbH, Gilead Sciences, Pfizer Pharma., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The Ars moriendi, which translates to "The Art of Dying," encompasses two Latin texts that gave advice on how to die well and without fear according to the Christian precepts of the late Middle Ages. Given that ten to hundred billion cells die in our bodies every day, it is obvious that the concept of a well and orderly ("regulated") death is also paramount at the cellular level. In apoptosis, as the most well-studied form of regulated cell death, proteases of the caspase family are the central mediators. However, caspases are not the only proteases that act as sculptors of cellular suicide, and therefore, we here provide an overview of the impact of proteases in apoptosis and other forms of regulated cell death., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers., (© 2021 Wiley-VCH GmbH.)

The detection and functional characterization of antigen-reactive T helper (Th) cells has been challenging due to their low frequency and functional heterogeneity. Antigen-reactive T cell enrichment (ARTE) allows the in-depth characterization of antigen-specific Th lymphocytes as a prerequisite for better understanding the role of adaptive immune responses in health and disease. ARTE is based on detection of the activation markers CD154 (CD40L) (expressed on all conventional Th cell subsets, Tcons) and CD137 (4-1BB) (expressed on regulatory T cells, Tregs), which are upregulated on the surface of CD4 + T cells upon short-term (7 h) in vitro stimulation with antigens in the presence of antigen-presenting cells (APCs). To substantially increase the sensitivity for the detection of antigen-specific Th cells, ARTE combines magnetic pre-enrichment of rare antigen-reactive T cells with multiparameter flow cytometry. Using CD154 and CD137 in combination allows the parallel detection of reactive Tcons and Tregs, after stimulation with the antigen. Thus, the ARTE technology now enables to characterize antigen-specific T cells with increased sensitivity of detection allowing even the investigation of antigen-specific Th cells in the naive T cell repertoire and regardless of prior knowledge of MHC alleles or antigenic epitopes.

CD4 + T cells reactive against SARS-CoV-2 can be found in unexposed individuals, and these are suggested to arise in response to common cold coronavirus (CCCoV) infection. Here, we utilized SARS-CoV-2-reactive CD4 + T cell enrichment to examine the antigen avidity and clonality of these cells, as well as the relative contribution of CCCoV cross-reactivity. SARS-CoV-2-reactive CD4 + memory T cells were present in virtually all unexposed individuals examined, displaying low functional avidity and multiple, highly variable cross-reactivities that were not restricted to CCCoVs. SARS-CoV-2-reactive CD4 + T cells from COVID-19 patients lacked cross-reactivity to CCCoVs, irrespective of strong memory T cell responses against CCCoV in all donors analyzed. In severe but not mild COVID-19, SARS-CoV-2-specific T cells displayed low functional avidity and clonality, despite increased frequencies. Our findings identify low-avidity CD4 + T cell responses as a hallmark of severe COVID-19 and argue against a protective role for CCCoV-reactive T cells in SARS-CoV-2 infection., Competing Interests: Declaration of Interests P.B. and A.S. are consultants of Miltenyi Biotec, who own IP rights concerning parts of the ARTE technology. M.J.G.T.V. has received research grants from 3M, Astellas Pharma, Biontech, DaVolterra, Evonik, Gilead Sciences, Glycom, Immunic, MaaT Pharma, Merck/MSD, Organobalance, Seres Therapeutics, and Takeda Pharmaceutical; speakers fees from Astellas Pharma, Basilea, Gilead Sciences, Merck/MSD, Organobalance, and Pfizer; and consultation fees from Alb Fils Kliniken GmbH, Arderypharm, Astellas Pharma, DaVolterra, Farmak International Holding GmbH, Ferring, Immunic AG, MaaT Pharma, Merck/MSD, and SocraTec R&D GmbH. F.L. discloses speaker honoraria from Grifols, Teva, Biogen, Bayer, Roche, Novartis, and Fresenius; travel funding from Merck, Grifols, and Bayer; and serves on advisory boards for Roche, Biogen, and Alexion. P.K. has received non-financial scientific grants from Miltenyi Biotec GmbH and the CECAD, University of Cologne, outside the submitted work. O.A.C. has received research grants from, is an advisor to, or received lecture honoraria from Actelion, Allecra, Amplyx, Astellas, Basilea, Biosys, Cidara, Da Volterra, Entasis, F2G, Gilead, Grupo Biotoscana, IQVIA, Janssen, Matinas, Medicines Company, MedPace, Melinta, Menarini, Merck/MSD, Mylan, Nabriva, Noxxon, Octapharma, Paratek, Pfizer, PSI, Roche Diagnostics, Scynexis, and Shionogi., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Binding of tumor necrosis factor (TNF) to TNF-receptor 1 (TNF-R1) can induce either cell survival or cell death. The selection between these diametrically opposed effects depends on the subcellular location of TNF-R1: plasma membrane retention leads to survival, while endocytosis leads to cell death. How the respective TNF-R1 associated signaling complexes are recruited to the distinct subcellular location is not known. Here, we identify palmitoylation of TNF-R1 as a molecular mechanism to achieve signal diversification., Methods: Human monocytic U937 cells were analyzed. Palmitoylated proteins were enriched by acyl resin assisted capture (AcylRAC) and analyzed by western blot and mass spectrometry. Palmitoylation of TNF-R1 was validated by metabolic labeling. TNF induced depalmitoylation and involvement of APT2 was analyzed by enzyme activity assays, pharmacological inhibition and shRNA mediated knock-down. TNF-R1 palmitoylation site analysis was done by mutated TNF-R1 expression in TNF-R1 knock-out cells. Apoptosis (nuclear DNA fragmentation, caspase 3 assays), NF-κB activation and TNF-R1 internalization were used as biological readouts., Results: We identify dynamic S-palmitoylation as a new mechanism that controls selective TNF signaling. TNF-R1 itself is constitutively palmitoylated and depalmitoylated upon ligand binding. We identified the palmitoyl thioesterase APT2 to be involved in TNF-R1 depalmitoylation and TNF induced NF-κB activation. Mutation of the putative palmitoylation site C248 interferes with TNF-R1 localization to the plasma membrane and thus, proper signal transduction., Conclusions: Our results introduce palmitoylation as a new layer of dynamic regulation of TNF-R1 induced signal transduction at a very early step of the TNF induced signaling cascade. Understanding the underlying mechanism may allow novel therapeutic options for disease treatment in future.

The disintegrin metalloprotease ADAM17 has a critical role in intestinal inflammation and regeneration in mice, as illustrated by the dramatically increased susceptibility of ADAM17 hypomorphic (ADAM17 ex/ex ) mice to dextran sulfate sodium (DSS)-induced colitis. Similarly, necroptosis has been implicated in inflammatory responses in the intestine. In this study, we have investigated the contribution of necroptosis to ADAM17-regulated intestinal inflammation in vivo by crossing ADAM17 ex/ex mice with mice that lack the necroptotic core protein RIPK3. Despite the loss of RIPK3, ADAM17 ex/ex /RIPK3 -/- mice showed the same increased susceptibility as ADAM17 ex/ex mice in both acute and chronic models of DSS-induced colitis. Mice of both genotypes revealed comparable results with regard to weight loss, disease activity index and colitis-associated changes of inner organs. Histopathological analyses confirmed similar tissue destruction, loss of barrier integrity, immune cell infiltration, and cell death; serum analyses revealed similar levels of the pro-inflammatory cytokine KC. Resolving these unexpected findings, ADAM17 ex/ex mice did not show phosphorylation of RIPK3 and its necroptotic interaction partner MLKL during DSS-induced colitis, although both proteins were clearly expressed. Consistent with these findings, murine embryonic fibroblasts derived from ADAM17 ex/ex mice were protected from tumor necrosis factor (TNF)-induced necroptosis and failed to show phosphorylation of MLKL and RIPK3 after induction of necroptosis by TNF, revealing a novel, undescribed role of the protease ADAM17 in necroptosis., Competing Interests: CONFLICTS OF INTEREST The authors of this manuscript state that they do not have any financial conflict of interests and nothing to disclose. All authors have approved the manuscript.

Proteases control most of the physiological processes that occur in a cell. This particularly applies to apoptosis, the most well-studied form of cell death, where proteolysis by cysteine-aspartic proteases (caspases) is the primary mechanism for both initiation and execution of cell suicide. In contrast, the impact of proteolysis on other, non-apoptotic cell death pathways (summarized under the term "regulated necrosis", RN) has long been enigmatic, but has clearly been confirmed by a number of recent groundbreaking discoveries. Here, we review these discoveries and provide an overview on the role of proteolysis in known forms of RN, with a particular focus on necroptosis and pyroptosis, and their regulation by deubiquitinases, apoptotic and inflammatory caspases. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recently, a type of regulated necrosis (RN) called necroptosis was identified to be involved in many pathophysiological processes and emerged as an alternative method to eliminate cancer cells. However, only a few studies have elucidated components of TRAIL-mediated necroptosis useful for anticancer therapy. Therefore, we have compared this type of cell death to tumor necrosis factor (TNF)-mediated necroptosis and found similar signaling through acid and neutral sphingomyelinases, the mitochondrial serine protease HtrA2/Omi, Atg5, and vacuolar H(+)-ATPase. Notably, executive mechanisms of both TRAIL- and TNF-mediated necroptosis are independent of poly(ADP-ribose) polymerase 1 (PARP-1), and depletion of p38α increases the levels of both types of cell death. Moreover, we found differences in signaling between TNF- and TRAIL-mediated necroptosis, e.g., a lack of involvement of ubiquitin carboxyl hydrolase L1 (UCH-L1) and Atg16L1 in executive mechanisms of TRAIL-mediated necroptosis. Furthermore, we discovered indications of an altered involvement of mitochondrial components, since overexpression of the mitochondrial protein Bcl-2 protected Jurkat cells from TRAIL- and TNF-mediated necroptosis, and overexpression of Bcl-XL diminished only TRAIL-induced necroptosis in Colo357 cells. Furthermore, TRAIL does not require receptor internalization and endosome-lysosome acidification to mediate necroptosis. Taken together, pathways described for TRAIL-mediated necroptosis and differences from those for TNF-mediated necroptosis might be unique targets to increase or modify necroptotic signaling and eliminate tumor cells more specifically in future anticancer approaches., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Inflammation and metabolism have been shown to be evolutionary linked and increasing evidence exists that pro-inflammatory factors are involved in the pathogenesis of obesity and type 2 diabetes. Until now, most data suggest that within adipose tissue these factors are secreted by cells of the innate immune system, e. g. macrophages. In the present study we demonstrate that B lymphocyte stimulator (BLyS) is increased in human obesity. In contrast to several pro-inflammatory factors, we found the source of BLyS in human adipose tissue to be the adipocytes rather than immune cells. In grade 3 obese human subjects, expression of BLyS in vivo in adipose tissue is significantly increased (p<0.001). Furthermore, BLyS serum levels are elevated in grade 3 human obesity (862.5+222.0 pg/ml vs. 543.7+60.7 pg/ml in lean controls, p<0.001) and are positively correlated to the BMI (r = 0.43, p<0.0002). In the present study, bariatric surgery significantly altered serum BLyS concentrations. In contrast, weight loss due to a very-low-calorie-formula-diet (800 kcal/d) had no such effect. To examine metabolic activity of BLyS, in a translational research approach, insulin sensitivity was measured in human subjects in vivo before and after treatment with the human recombinant anti-BLyS antibody belimumab. Since BLyS is known to promote B-cell proliferation and immunoglobulin secretion, the present data suggest that adipocytes of grade 3 obese human subjects are able to activate the adaptive immune system, suggesting that in metabolic inflammation in humans both, innate and adaptive immunity, are of pathophysiological relevance.

Background: Female signals of fertility have evolved in diverse taxa. Among the most interesting study systems are those of multimale multifemale group-living primates, where females signal fertility to males through multiple signals, and in which there is substantial inter-specific variation in the composition and reliability of such signals. Among the macaques, some species display reliable behavioural and/or anogenital signals while others do not. One cause of this variation may be differences in male competitive regimes: some species show marked sexual dimorphism and reproductive skew, with males fighting for dominance, while others show low dimorphism and skew, with males queuing for dominance. As such, there is variation in the extent to which rank is a reliable proxy for male competitiveness, which may affect the extent to which it is in females' interest to signal ovulation reliably. However, data on ovulatory signals are absent from species at one end of the macaque continuum, where selection has led to high sexual dimorphism and male reproductive skew. Here we present data from 31 cycles of 19 wild female crested macaques, a highly sexually dimorphic species with strong mating skew. We collected measures of ovarian hormone data from faeces, sexual swelling size from digital images, and male and female behaviour., Results: We show that both sexual swelling size and female proceptivity are graded-signals, but relatively reliable indicators of ovulation, with swelling size largest and female proceptive behaviours most frequent around ovulation. Sexual swelling size was also larger in conceptive cycles. Male mating behaviour was well timed to female ovulation, suggesting that males had accurate information about this., Conclusion: Though probabilistic, crested macaque ovulatory signals are relatively reliable. We argue that in species where males fight over dominance, male dominance rank is surrogate for competitiveness. Under these circumstances it is in the interest of females to increase paternity concentration and assurance in dominants beyond levels seen in species where such competition is less marked. As such, we suggest that it may in part be variation in male competitive regimes that leads to the evolution of fertility signalling systems of different reliability.