Neutralizing antibody titers over 12 months after SARS-CoV-2 mRNA vaccine booster in patients with relapsing multiple sclerosis continuously treated with ofatumumab.

Booster vaccinations against SARS-CoV-2 are recommended 6-12 months after the last dose or infection in elderly and high-risk groups. The present analysis aims to evaluate whether an interval shorter than 12 months is required in multiple sclerosis patients receiving ofatumumab. Neutralizing antibody status over 1 year in patients receiving booster vaccination in the non-interventional, multicenter KYRIOS study under continued ofatumumab treatment was analyzed. Fifteen patients were included. At the time of the first booster vaccination, ten patients were seropositive for neutralizing antibodies, four patients were seronegative, and for one patient, no baseline levels were available. All patients who were seropositive at baseline showed >2-fold increase in neutralizing antibody titers after the first booster and two patients (20%) showed a >10-fold increase. Among seronegative patients, three (75%) had a >10-fold increase in neutralizing antibody titers. Seropositivity was maintained in almost all patients until month 12. One initially seronegative patient had less than 2-fold increase in neutralizing antibody titers after the booster vaccination and can be considered a non-responder. Most patients with continued ofatumumab treatment are able to maintain permanent seropositivity and therefore presumably constant protection against severe courses of COVID-19 if repeated booster vaccinations are applied.

A20 regulates lymphocyte adhesion in murine neuroinflammation by restricting endothelial ICOSL expression in the CNS.

A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20ΔCNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL to be expressed by A20-deficient CNS-ECs, which we found to function as adhesion molecules. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20ΔCNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in WT mice. In addition, blocking of ICOSL on primary mouse brain microvascular ECs impaired the adhesion of T cells in vitro. Taken together, we propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation.

Externalized histones fuel pulmonary fibrosis via a platelet-macrophage circuit of TGFß1 and IL-27.

Externalized histones erupt from the nucleus as extracellular traps, are associated with several acute and chronic lung disorders, but their implications in the molecular pathogenesis of interstitial lung disease are incompletely defined. To investigate the role and molecular mechanisms of externalized histones within the immunologic networks of pulmonary fibrosis, we studied externalized histones in human and animal bronchoalveolar lavage (BAL) samples of lung fibrosis. Neutralizing anti-histone antibodies were administered in bleomycin-induced fibrosis of C57BL/6 J mice, and subsequent studies used conditional/constitutive knockout mouse strains for TGFß and IL-27 signaling along with isolated platelets and cultured macrophages. We found that externalized histones (citH3) were significantly (P < 0.01) increased in cell-free BAL fluids of patients with idiopathic pulmonary fibrosis (IPF; n = 29) as compared to healthy controls (n = 10). The pulmonary sources of externalized histones were Ly6G+CD11b+ neutrophils and nonhematopoietic cells after bleomycin in mice. Neutralizing monoclonal anti-histone H2A/H4 antibodies reduced the pulmonary collagen accumulation and hydroxyproline concentration. Histones activated platelets to release TGFß1, which signaled through the TGFbRI/TGFbRII receptor complex on LysM+ cells to antagonize macrophage-derived IL-27 production. TGFß1 evoked multiple downstream mechanisms in macrophages, including p38 MAPK, tristetraprolin, IL-10, and binding of SMAD3 to the IL-27 promotor regions. IL-27RA-deficient mice displayed more severe collagen depositions suggesting that intact IL-27 signaling limits fibrosis. In conclusion, externalized histones inactivate a safety switch of antifibrotic, macrophage-derived IL-27 by boosting platelet-derived TGFß1. Externalized histones are accessible to neutralizing antibodies for improving the severity of experimental pulmonary fibrosis.

Immune Response to Initial and Booster SARS-CoV-2 mRNA Vaccination in Patients Treated with Siponimod-Final Analysis of a Nonrandomized Controlled Clinical Trial (AMA-VACC).

BACKGROUND: Evidence on SARS-CoV-2 mRNA vaccination under siponimod treatment is rare. METHODS: AMA-VACC is a prospective, open-label clinical study on SARS-CoV-2 mRNA vaccination during ongoing siponimod treatment (cohort 1), during siponimod interruption (cohort 2), or during treatment with other disease-modifying therapies or without therapy (cohort 3). SARS-CoV-2-specific antibodies and T-cell reactivity were measured six months after the initial vaccination and one month after the booster. RESULTS: 41 patients were recruited into cohort 1 (n = 17), cohort 2 (n = 4), and cohort 3 (n = 20). Seroconversion for SARS-CoV-2 neutralizing antibodies was reached by 50.0%, 100.0%, and 90.0% of patients at month 6 and by 81.3%, 100.0%, and 100.0% one month after booster (cohorts 1, 2, and 3, respectively). Antibody levels in cohort 1 increased after the booster compared to month 6 but remained lower compared to cohorts 2 and 3. T-cell responses were seen in 28.5%, 25.0%, and 73.7% at month 6 and in 28.6%, 50.0%, and 83.3% after the booster (cohorts 1, 2, and 3, respectively). In cohort 1, the extent of T-cell response was lower at month 6 compared to cohorts 2 and 3 but reached almost similar levels after the booster. CONCLUSIONS: The antibody and T-cell responses support SARS-CoV-2 (booster) vaccines in siponimod-treated patients.

Ubiquitination and cell-autonomous immunity.

Cell-autonomous immunity is the first line of defense by which cells recognize and contribute to eliminating invasive pathogens. It is composed of immune signaling networks that sense microbial pathogens, promote pathogen restriction, and stimulate their elimination, including host cell death. Ubiquitination is a pivotal orchestrator of these pathways, by changing the activity of signal transducers and effector proteins in an efficient way. In this review, we will focus on how ubiquitin connects the pathways that sense pathogens to the cellular responses to invaders and shed light on how ubiquitination impacts the microenvironment around the infected cell, stimulating the appropriate immune response. Finally, we discuss therapeutic options directed at favoring cell-autonomous immune responses to infection.

Results on SARS-CoV-2 mRNA Vaccine Booster from an Open-Label Multicenter Study in Ofatumumab-Treated Participants with Relapsing Multiple Sclerosis.

BACKGROUND: Few data exist on how ofatumumab treatment impacts SARS-CoV-2 booster vaccination response. METHODS: KYRIOS is an ongoing prospective open-label multicenter study on the response to initial and booster SARS-CoV-2 mRNA vaccination before or during ofatumumab treatment in relapsing MS patients. The results on the initial vaccination cohort have been published previously. Here, we describe 23 patients who received their initial vaccination outside of the study but booster vaccination during the study. Additionally, we report the booster results of two patients in the initial vaccination cohort. The primary endpoint was SARS-CoV-2-specific T-cell response at month 1. Furthermore, serum total and neutralizing antibodies were measured. RESULTS: The primary endpoint was reached by 87.5% of patients with booster before (booster cohort 1, N = 8) and 46.7% of patients with booster during ofatumumab treatment (booster cohort 2, N = 15). Seroconversion rates for neutralizing antibodies increased from 87.5% at baseline to 100.0% at month 1 in booster cohort 1 and from 71.4% to 93.3% in booster cohort 2. Of note, 3 of 4 initially seronegative patients in booster cohort 2 and one seronegative patient in the initial vaccination cohort seroconverted after the booster during ofatumumab treatment. CONCLUSIONS: Booster vaccinations increase neutralizing antibody titers in ofatumumab-treated patients. A booster is recommended in ofatumumab-treated patients.

Modulation of cellular transcriptome and proteome composition by azidohomoalanine-implications on click chemistry-based secretome analysis.

The analysis of the secretome provides important information on proteins defining intercellular communication and the recruitment and behavior of cells in specific tissues. Especially in the context of tumors, secretome data can support decisions for diagnosis and therapy. The mass spectrometry-based analysis of cell-conditioned media is widely used for the unbiased characterization of cancer secretomes in vitro. Metabolic labeling using azide-containing amino acid analogs in combination with click chemistry facilitates this type of analysis in the presence of serum, preventing serum starvation-induced effects. The modified amino acid analogs, however, are less efficiently incorporated into newly synthesized proteins and may perturb protein folding. Combining transcriptome and proteome analysis, we elucidate in detail the effects of metabolic labeling with the methionine analog azidohomoalanine (AHA) on gene and protein expression. Our data reveal that 15-39% of the proteins detected in the secretome displayed changes in transcript and protein expression induced by AHA labeling. Gene Ontology (GO) analyses indicate that metabolic labeling using AHA leads to induction of cellular stress and apoptosis-related pathways and provide first insights on how this affects the composition of the secretome on a global scale. KEY MESSAGES: Azide-containing amino acid analogs affect gene expression profiles. Azide-containing amino acid analogs influence cellular proteome. Azidohomoalanine labeling induces cellular stress and apoptotic pathways. Secretome consists of proteins with dysregulated expression profiles.

The Role of LFA-1 for the Differentiation and Function of Regulatory T Cells-Lessons Learned from Different Transgenic Mouse Models.

Regulatory T cells (Treg) are essential for the maintenance of peripheral tolerance. Treg dysfunction results in diverse inflammatory and autoimmune diseases with life-threatening consequences. ß2-integrins (CD11a-d/CD18) play important roles in the migration of leukocytes into inflamed tissues and cell signaling. Of all ß2-integrins, T cells, including Treg, only express CD11a/CD18, termed lymphocyte function-associated antigen 1 (LFA-1), on their surface. In humans, loss-of-function mutations in the common subunit CD18 result in leukocyte adhesion deficiency type-1 (LAD-1). Clinical symptoms vary depending on the extent of residual ß2-integrin function, and patients may experience leukocytosis and recurrent infections. Some patients can develop autoimmune diseases, but the immune processes underlying the paradoxical situation of immune deficiency and autoimmunity have been scarcely investigated. To understand this complex phenotype, different transgenic mouse strains with a constitutive knockout of ß2-integrins have been established. However, since a constitutive knockout affects all leukocytes and may limit the validity of studies focusing on their cell type-specific role, we established a Treg-specific CD18-floxed mouse strain. This mini-review aims to delineate the role of LFA-1 for the induction, maintenance, and regulatory function of Treg in vitro and in vivo as deduced from observations using the various ß2-integrin-deficient mouse models.

NFATc1 induction by an intronic enhancer restricts NKT γδ cell formation.

In thymus, the ablation of T cell receptor (TCR)-activated transcription factor NFATc1 or its inducible isoforms during the double-negative (DN) stages of thymocyte development leads to a marked increase in γδ thymocytes whereas the development of αß thymocytes remains mostly unaffected. These γδ thymocytes are characterized by the upregulation of the promyelocytic leukemia zinc-finger factor (PLZF), the "master regulator" of natural killer T (NKT) cell development, and the acquisition of an NKT γδ cell phenotype with higher cell survival rates. The suppressive function of NFATc1 in NKT γδ cell formation critically depends on the remote enhancer E2, which is essential for the inducible expression of NFATc1 directed by its distal promoter P1. Thus, the enhancer deciphers a strong γδ TCR signal into the expression of inducible NFATc1 isoforms resulting in high levels of NFATc1 protein that are essential to control the numbers of NKT γδ cells.

IL18 Receptor Signaling Regulates Tumor-Reactive CD8+ T-cell Exhaustion via Activation of the IL2/STAT5/mTOR Pathway in a Pancreatic Cancer Model.

Intratumoral cytotoxic CD8+ T cells (CTL) enter a dysfunctional state characterized by expression of coinhibitory receptors, loss of effector function, and changes in the transcriptional landscape. Even though several regulators of T-cell exhaustion have been identified, the molecular mechanisms inducing T-cell exhaustion remain unclear. Here, we show that IL18 receptor (IL18R) signaling induces CD8+ T-cell exhaustion in a murine pancreatic cancer model. Adoptive transfer of Il18r-/- OT-1 CD8+ CTLs resulted in enhanced rejection of subcutaneous tumors expressing ovalbumin (OVA) as a model antigen (PancOVA), compared with wild-type OT-1 CTLs. Transferred intratumoral IL18R-deficient CTLs expressed higher levels of effector cytokines TNF and IFNγ and had reduced expression of coinhibitory receptors (PD-1, TIM-3, 2B4, LAG-3) and the transcription factors Eomes and TOX. Lower expression of coinhibitory receptors and TOX on IL18R-deficient versus IL18R-sufficient CD8+ T cells were confirmed in an orthotopic KPC model. IL18R-induced T-cell exhaustion was regulated by IL2/STAT5 and AKT/mTOR pathways, as demonstrated in an in vitro exhaustion assay. Concordantly, mice deficient in NLRP3, the molecular complex activating IL18, had decreased expression of coinhibitory receptors on intratumoral T cells and similar changes in signaling pathways at the transcriptome level. Thus, molecular pathways promoting T-cell exhaustion indicate an involvement of an NLRP3-expressing tumor microenvironment, which mediates IL18 release. The Cancer Genome Atlas analysis of patients with pancreatic carcinoma showed an association between NLRP3-mediated IL18 signaling and shorter survival. These findings indicate NLRP3-mediated IL18R signaling as a regulator of intratumoral T-cell exhaustion and a possible target for immunotherapy. See related Spotlight by Stromnes, p. 400.

IL-17A-producing CD8+ T cells promote PDAC via induction of inflammatory cancer-associated fibroblasts.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant desmoplastic stroma composed of cancer-associated fibroblasts (CAF) and interspersed immune cells. A non-canonical CD8+ T-cell subpopulation producing IL-17A (Tc17) promotes autoimmunity and has been identified in tumours. Here, we evaluated the Tc17 role in PDAC. DESIGN: Infiltration of Tc17 cells in PDAC tissue was correlated with patient overall survival and tumour stage. Wild-type (WT) or Il17ra-/- quiescent pancreatic stellate cells (qPSC) were exposed to conditional media obtained from Tc17 cells (Tc17-CM); moreover, co-culture of Tc17-CM-induced inflammatory (i)CAF (Tc17-iCAF) with tumour cells was performed. IL-17A/F-, IL-17RA-, RAG1-deficient and Foxn1nu/nu mice were used to study the Tc17 role in subcutaneous and orthotopic PDAC mouse models. RESULTS: Increased abundance of Tc17 cells highly correlated with reduced survival and advanced tumour stage in PDAC. Tc17-CM induced iCAF differentiation as assessed by the expression of iCAF-associated genes via synergism of IL-17A and TNF. Accordingly, IL-17RA controlled the responsiveness of qPSC to Tc17-CM. Pancreatic tumour cells co-cultured with Tc17-iCAF displayed enhanced proliferation and increased expression of genes implicated in proliferation, metabolism and protection from apoptosis. Tc17-iCAF accelerated growth of mouse and human tumours in Rag1-/- and Foxn1nu/nu mice, respectively. Finally, Il17ra-expressed by fibroblasts was required for Tc17-driven tumour growth in vivo. CONCLUSIONS: We identified Tc17 as a novel protumourigenic CD8+ T-cell subtype in PDAC, which accelerated tumour growth via IL-17RA-dependent stroma modification. We described a crosstalk between three cell types, Tc17, fibroblasts and tumour cells, promoting PDAC progression, which resulted in poor prognosis for patients.

Guidelines for mouse and human DC functional assays.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Immune Response to SARS-CoV-2 mRNA Vaccines in an Open-Label Multicenter Study in Participants with Relapsing Multiple Sclerosis Treated with Ofatumumab.

BACKGROUND: It is unclear whether multiple sclerosis (MS) patients receiving ofatumumab mount an immune response after SARS-CoV-2 mRNA vaccination. METHODS: KYRIOS is an ongoing, multicenter, open-label, prospective clinical study on immune responses in MS patients after initial or booster SARS-CoV-2 mRNA vaccination prior to (cohort 1) or during (cohort 2) ofatumumab treatment. We report one-week and one-month results of the initial vaccination. A comparison with patients vaccinated while receiving beta-interferon, glatiramer acetate, dimethyl fumarate, teriflunomide or no treatment was included (cohort 3). RESULTS: In total, 11 patients received their initial vaccination during the study. The primary endpoint of SARS-CoV-2-specific T-cells at month 1 was reached by 80.0% of patients in cohort 1 (N = 6) and 100.0% in cohort 2 (N = 5). T-cell reactivity peaked at week 1. All cohort 1 patients reached seroconversion for SARS-CoV-2 neutralizing antibodies at week 1 and month 1. In cohort 2, neutralizing antibodies increased in all patients and exceeded the cut-off for seropositivity in 40.0% of patients at week 1 and 25.0% at month 1. Immune responses in cohort 3 were comparable to cohort 1. CONCLUSION: Presence of T-cell response and increase in levels of neutralizing antibodies, although less pronounced compared to controls, suggest that MS patients receiving ofatumumab are able to mount an immune response to SARS-CoV-2 mRNA vaccination.

Impaired Treg-DC interactions contribute to autoimmunity in leukocyte adhesion deficiency type 1.

Leukocyte adhesion deficiency type 1 (LAD-1) is a rare disease resulting from mutations in the gene encoding for the common ß-chain of the ß2-integrin family (CD18). The most prominent clinical symptoms are profound leukocytosis and high susceptibility to infections. Patients with LAD-1 are prone to develop autoimmune diseases, but the molecular and cellular mechanisms that result in coexisting immunodeficiency and autoimmunity are still unresolved. CD4+FOXP3+ Treg are known for their essential role in preventing autoimmunity. To understand the role of Treg in LAD-1 development and manifestation of autoimmunity, we generated mice specifically lacking CD18 on Treg (CD18Foxp3), resulting in defective LFA-1 expression. Here, we demonstrate a crucial role of LFA-1 on Treg to maintain immune homeostasis by modifying T cell-DC interactions and CD4+ T cell activation. Treg-specific CD18 deletion did not impair Treg migration into extralymphatic organs, but it resulted in shorter interactions of Treg with DC. In vivo, CD18Foxp3 mice developed spontaneous hyperplasia in lymphatic organs and diffuse inflammation of the skin and in multiple internal organs. Thus, LFA-1 on Treg is required for the maintenance of immune homeostasis.

Assessing the immune response to SARS-CoV-2 mRNA vaccines in siponimod-treated patients: a nonrandomized controlled clinical trial (AMA-VACC).

Background: Systematic data are lacking on the immune response toward SARS-CoV-2 mRNA vaccination in SPMS patients on disease-modifying therapies (DMTs). Objective: The AMA-VACC clinical trial was designed to characterize immune responses to SARS-CoV-2 mRNA vaccines in siponimod-treated SPMS patients. Design: AMA-VACC is an ongoing three-cohort, multicenter, open-label, prospective clinical study. Methods: The study included patients at risk for SPMS or patients with SPMS diagnosis. Patients received SARS-CoV-2 mRNA vaccine as part of their clinical routine during ongoing siponimod treatment (cohort 1), during siponimod treatment interruption (cohort 2), or while on dimethyl fumarate, glatiramer acetate, beta-interferons, teriflunomide, or no current therapy (cohort 3). SARS-CoV-2-specific neutralizing antibodies and T-cell responses were measured 1 week and 1 month after the second dose of vaccination. Results: In total, 17 patients, 4 patients, and 20 patients were recruited into cohorts 1, 2, and 3, respectively. The primary endpoint of seroconversion for SARS-CoV-2-neutralizing antibodies at week 1 was reached by 52.9%, 75.0%, and 90.0% of patients in cohorts 1, 2, and 3, respectively. For 64.7% of patients in cohort 1, all patients in cohort 2, and 95% of patients in cohort 3, seroconversion was observed at either week 1 or month 1 or both time points. After 1 week, 71.4% of cohort 1, 75.0% of cohort 2, and 85.0% of cohort 3 were positive for either SARS-CoV-2-neutralizing antibodies or SARS-CoV-2-specific T-cells or both. After 1 month, the rates were 56.3%, 100.0%, and 95.0%, respectively. Conclusion: The study shows that the majority of siponimod patients mount humoral and cellular immune response under continuous siponimod treatment. The data do not sufficiently support interruption of treatment for the purpose of vaccination. Registration: EU Clinical Trials Register: EudraCT 2020-005752-38 (www.clinicaltrialsregister.eu); ClinicalTrials.gov: NCT04792567 (https://clinicaltrials.gov).

ß2 Integrins on Dendritic Cells Modulate Cytokine Signaling and Inflammation-Associated Gene Expression, and Are Required for Induction of Autoimmune Encephalomyelitis.

Heterodimeric ß2 integrin surface receptors (CD11a-d/CD18) are specifically expressed by leukocytes that contribute to pathogen uptake, cell migration, immunological synapse formation and cell signaling. In humans, the loss of CD18 expression results in leukocyte adhesion deficiency syndrome (LAD-)1, largely characterized by recurrent severe infections. All available mouse models display the constitutive and ubiquitous knockout of either α or the common ß2 (CD18) subunit, which hampers the analysis of the cell type-specific role of ß2 integrins in vivo. To overcome this limitation, we generated a CD18 gene floxed mouse strain. Offspring generated from crossing with CD11c-Cre mice displayed the efficient knockdown of ß2 integrins, specifically in dendritic cells (DCs). Stimulated ß2-integrin-deficient splenic DCs showed enhanced cytokine production and the concomitantly elevated activity of signal transducers and activators of transcription (STAT) 1, 3 and 5, as well as the impaired expression of suppressor of cytokine signaling (SOCS) 2-6 as assessed in bone marrow-derived (BM) DCs. Paradoxically, these BMDCs also showed the attenuated expression of genes involved in inflammatory signaling. In line, in experimental autoimmune encephalomyelitis mice with a conditional DC-specific ß2 integrin knockdown presented with a delayed onset and milder course of disease, associated with lower frequencies of T helper cell populations (Th)1/Th17 in the inflamed spinal cord. Altogether, our mouse model may prove to be a valuable tool to study the leukocyte-specific functions of ß2 integrins in vivo.

IRF4 deficiency vulnerates B-cell progeny for leukemogenesis via somatically acquired Jak3 mutations conferring IL-7 hypersensitivity.

The processes leading from disturbed B-cell development to adult B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) remain poorly understood. Here, we describe Irf4-/- mice as prone to developing BCP-ALL with age. Irf4-/- preB-I cells exhibited impaired differentiation but enhanced proliferation in response to IL-7, along with reduced retention in the IL-7 providing bone marrow niche due to decreased CXCL12 responsiveness. Thus selected, preB-I cells acquired Jak3 mutations, probably following irregular AID activity, resulting in malignant transformation. We demonstrate heightened IL-7 sensitivity due to Jak3 mutants, devise a model to explain it, and describe structural and functional similarities to Jak2 mutations often occurring in human Ph-like ALL. Finally, targeting JAK signaling with Ruxolitinib in vivo prolonged survival of mice bearing established Irf4-/- leukemia. Intriguingly, organ infiltration including leukemic meningeosis was selectively reduced without affecting blood blast counts. In this work, we present spontaneous leukemogenesis following IRF4 deficiency with potential implications for high-risk BCP-ALL in adult humans.

Proinflammatory CD20+ T cells contribute to CNS-directed autoimmunity.

The origin and function of CD20+ T cells are poorly understood. Here, we characterized CD20+ T cells in mice and humans and investigated how they are affected by anti-CD20 antibody treatment. We report that murine CD20+ T cells are unable to endogenously express the B cell lineage marker CD20; the development of CD20+ T cells in rodents requires the presence of CD20-expressing B cells. Our results demonstrated that both murine and human T cells acquire CD20 from B cells via trogocytosis while being activated by an antigen-presenting B cell. In patients with multiple sclerosis (MS) and mice with experimental autoimmune encephalomyelitis (EAE), expression of CD20 on T cells is associated with an up-regulation of activation markers, proinflammatory cytokines, and adhesion molecules, suggesting high pathogenic potential. Supporting this hypothesis, CD20+ T cells expand during active EAE in rodents; furthermore, adoptive transfer of CD20+ T cells into EAE-diseased mice worsened histological and clinical severity. Of direct therapeutic relevance, we demonstrate that the exclusive therapeutic elimination of CD20+ T cells effectively ameliorates EAE, independent of B cells. The results support the hypothesis that CD20+ T cells arise upon B cell-T cell interaction and that depletion of CD20+ T cells might contribute to the success of anti-CD20 antibody therapies in MS and other inflammatory disorders.

Neutrophil extracellular traps and their histones promote Th17 cell differentiation directly via TLR2.

Neutrophils perform critical functions in the innate response to infection, including through the production of neutrophil extracellular traps (NETs) - web-like DNA structures which are extruded from neutrophils upon activation. Elevated levels of NETs have been linked to autoimmunity but this association is poorly understood. By contrast, IL-17 producing Th17 cells are a key player in various autoimmune diseases but are also crucial for immunity against fungal and bacterial infections. Here we show that NETs, through their protein component histones, directly activate T cells and specifically enhance Th17 cell differentiation. This modulatory role of neutrophils, NETs and their histones is mediated downstream of TLR2 in T cells, resulting in phosphorylation of STAT3. The innate stimulation of a specific adaptive immune cell subset provides an additional mechanism demonstrating a direct link between neutrophils, NETs and T cell autoimmunity.

K2P18.1 translates T cell receptor signals into thymic regulatory T cell development.

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K2P18.1 is a relevant regulator. Here, we identify K2P18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K2P18.1 upregulation in tTreg progenitors. K2P18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K2P18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K2P18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K2P18.1 variant that is associated with poor clinical outcomes indicate that K2P18.1 also plays a role in human Treg development. Pharmacological modulation of K2P18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K2P18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K2P18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.