A CTCF-binding site in the Mdm1-Il22-Ifng locus shapes cytokine expression profiles and plays a critical role in early Th1 cell fate specification.

Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.

TGFß prevents IgE-mediated allergic disease by restraining T follicular helper 2 differentiation.

Allergic diseases are common, affecting more than 20% of the population. Genetic variants in the TGFß pathway are strongly associated with atopy. To interrogate the mechanisms underlying this association, we examined patients and mice with Loeys-Dietz syndrome (LDS) who harbor missense mutations in the kinase domain of TGFΒR1/2. We demonstrate that LDS mutations lead to reduced TGFß signaling and elevated total and allergen-specific IgE, despite the presence of wild-type T regulatory cells in a chimera model. Germinal center activity was enhanced in LDS and characterized by a selective increase in type 2 follicular helper T cells (TFH2). Expression of Pik3cg was increased in LDS TFH cells and associated with reduced levels of the transcriptional repressor SnoN. PI3Kγ/mTOR signaling in LDS naïve CD4+ T cells was elevated after T cell receptor cross-linking, and pharmacologic inhibition of PI3Kγ or mTOR prevented exaggerated TFH2 and antigen-specific IgE responses after oral antigen exposure in an adoptive transfer model. Naïve CD4+ T cells from nonsyndromic allergic individuals also displayed decreased TGFß signaling, suggesting that our mechanistic discoveries may be broadly relevant to allergic patients in general. Thus, TGFß plays a conserved, T cell-intrinsic, and nonredundant role in restraining TFH2 development via the PI3Kγ/mTOR pathway and thereby protects against allergic disease.

Selectivity, efficacy and safety of JAKinibs: new evidence for a still evolving story.

Fundamental insight gained over the last decades led to the discovery of cytokines as pivotal drivers of inflammatory diseases such as rheumatoid arthritis, psoriasis/psoriasis arthritis, inflammatory bowel diseases, atopic dermatitis and spondylarthritis. A deeper understanding of the pro-inflammatory and anti-inflammatory effects of various cytokines has prompted new cytokine-targeting therapies, which revolutionised the treatment options in the last years for patients with inflammatory disorders. Disease-associated immune responses typically involve a complex interplay of multiple cytokines. Therefore, blockade of one single cytokine does not necessarily lead to a persistent remission in all patients with inflammatory disorders and fostered new therapeutic strategies targeting intracellular pathways shared by multiple cytokines. By inhibiting JAK-STAT signalling pathways common to families of cytokines, JAK-inhibitors (JAKinibs) have created a new paradigm for the treatment of inflammatory diseases. Multiple agents have been approved for various disorders and more are being investigated for several new indications. Second-generation selective JAKinibs have been devised with the aim to achieve an increased selectivity and a possible reduced risk of side effects. In the current review, we will summarise the current body of evidence of pan versus selective JAKinibs and the most recent insights on new side effects and indications, including COVID-19.

Microbial ligand-independent regulation of lymphopoiesis by NOD1.

Aberrant differentiation of progenitor cells in the hematopoietic system is known to severely impact host immune responsiveness. Here we demonstrate that NOD1, a cytosolic innate sensor of bacterial peptidoglycan, also functions in murine hematopoietic cells as a major regulator of both the generation and differentiation of lymphoid progenitors as well as peripheral T lymphocyte homeostasis. We further show that NOD1 mediates these functions by facilitating STAT5 signaling downstream of hematopoietic cytokines. In steady-state, loss of NOD1 resulted in a modest but significant decrease in numbers of mature T, B and natural killer cells. During systemic protozoan infection this defect was markedly enhanced, leading to host mortality. Lack of functional NOD1 also impaired T cell-dependent anti-tumor immunity while preventing colitis. These findings reveal that, in addition to its classical role as a bacterial ligand receptor, NOD1 plays an important function in regulating adaptive immunity through interaction with a major host cytokine signaling pathway.

Complete heart block is a significant predictor of mortality in immune checkpoint inhibitor myocarditis.

BACKGROUND: Immune checkpoint inhibitor (ICI) myocarditis is associated with significant mortality risk. Electrocardiogram (ECG) changes in ICI myocarditis have strong prognostic value. However the impact of complete heart block (CHB) is not well defined. This study sought to evaluate the impact of CHB on mortality in ICI myocarditis, and to identify clinical predictors of mortality and CHB incidence. METHODS: We conducted a retrospective cohort study of patients with ICI myocarditis at three Mayo Clinic sites from 1st January 2010 to 31st September 2022 to evaluate mortality rates at 180 days. Clinical, laboratory, ECG, echocardiographic, and cardiac magnetic resonance imaging (CMR) characteristics were assessed. Cox and logistic regression were performed for associations with mortality and CHB respectively. RESULTS: Of 34 identified cases of ICI myocarditis, 7 (20.6%) had CHB. CHB was associated with higher mortality (HR 7.41, p = 0.03, attributable fraction 86.5%). Among those with CHB, troponin T (TnT) < 1000 ng/dL, low white blood cell count and high ventricular rate at admission were protective. There was trend towards increased survival among patients who underwent permanent pacemaker insertion (p = 0.051), although most experienced device lead complications. Factors associated with development of CHB included prolonged PR and QRS intervals and low Sokolow Lyon Index. Where these were normal and TnT was < 1000 ng/dL, no deaths occurred. Impaired myocardial longitudinal strain was sensitive for ICI myocarditis but was not prognostically significant. CONCLUSION: There is a strong temporal association between CHB and early mortality in people with ICI myocarditis. Focusing on arrhythmogenic complications can be helpful in predicting outcomes for this group of critically ill individuals.

T cell intrinsic STAT1 signaling prevents aberrant Th1 responses during acute toxoplasmosis.

Infection-induced T cell responses must be properly tempered and terminated to prevent immuno-pathology. Using transgenic mice, we demonstrate that T cell intrinsic STAT1 signaling is required to curb inflammation during acute infection with Toxoplasma gondii. Specifically, we report that mice lacking STAT1 selectively in T cells expel parasites but ultimately succumb to lethal immuno-pathology characterized by aberrant Th1-type responses with reduced IL-10 and increased IL-13 production. We also find that, unlike STAT1, STAT3 is not required for induction of IL-10 or suppression of IL-13 during acute toxoplasmosis. Each of these findings was confirmed in vitro and ChIP-seq data mining showed that STAT1 and STAT3 co-localize at the Il10 locus, as well as loci encoding other transcription factors that regulate IL-10 production, most notably Maf and Irf4. These data advance basic understanding of how infection-induced T cell responses are managed to prevent immuno-pathology and provide specific insights on the anti-inflammatory properties of STAT1, highlighting its role in shaping the character of Th1-type responses.

Infectious Aortitis Due to Salmonella Species Masquerading as Lung Malignancy.

Infectious aortitis is a rare but devastating vascular infection with mortality exceeding 40%. Early diagnosis is crucial but often hampered by radiographic mimickers. We report a patient who was thought to have lung cancer but ultimately found to have an infected aortic aneurysm and bacteremia owing to Salmonella species. Owing to surgical contraindications, he was treated palliatively with an initial regimen of intravenous ampicillin/sulbactam followed by lifelong oral antibiotic suppression. He ultimately rejected his diagnosis, discontinued medications, and was lost to follow-up. (Level of Difficulty: Intermediate.).

Transcription factor TCF-1 regulates the functions, but not the development, of lymphoid tissue inducer subsets in different tissues.

Lymphoid tissue inducer (LTi) cells, a subset of innate lymphoid cells (ILCs), play an essential role in the formation of secondary lymphoid tissues. However, the regulation of the development and functions of this ILC subset is still elusive. In this study, we report that the transcription factor T cell factor 1 (TCF-1), just as GATA3, is indispensable for the development of non-LTi ILC subsets. While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer's patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indirectly regulate Lta, including through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during T cell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells.

TGF-ß uncouples glycolysis and inflammation in macrophages and controls survival during sepsis.

Changes in metabolism of macrophages are required to sustain macrophage activation in response to different stimuli. We showed that the cytokine TGF-ß (transforming growth factor-ß) regulates glycolysis in macrophages independently of inflammatory cytokine production and affects survival in mouse models of sepsis. During macrophage activation, TGF-ß increased the expression and activity of the glycolytic enzyme PFKL (phosphofructokinase-1 liver type) and promoted glycolysis but suppressed the production of proinflammatory cytokines. The increase in glycolysis was mediated by an mTOR-c-MYC-dependent pathway, whereas the inhibition of cytokine production was due to activation of the transcriptional coactivator SMAD3 and suppression of the activity of the proinflammatory transcription factors AP-1, NF-κB, and STAT1. In mice with LPS-induced endotoxemia and experimentally induced sepsis, the TGF-ß-induced enhancement in macrophage glycolysis led to decreased survival, which was associated with increased blood coagulation. Analysis of septic patient cohorts revealed that the expression of PFKL, TGFBRI (which encodes a TGF-ß receptor), and F13A1 (which encodes a coagulation factor) in myeloid cells positively correlated with COVID-19 disease. Thus, these results suggest that TGF-ß is a critical regulator of macrophage metabolism and could be a therapeutic target in patients with sepsis.

Transcription factor EGR2 controls homing and pathogenicity of TH17 cells in the central nervous system.

CD4+ T helper 17 (TH17) cells protect barrier tissues but also trigger autoimmunity. The mechanisms behind these opposing processes remain unclear. Here, we found that the transcription factor EGR2 controlled the transcriptional program of pathogenic TH17 cells in the central nervous system (CNS) but not that of protective TH17 cells at barrier sites. EGR2 was significantly elevated in myelin-reactive CD4+ T cells from patients with multiple sclerosis and mice with autoimmune neuroinflammation. The EGR2 transcriptional program was intricately woven within the TH17 cell transcriptional regulatory network and showed high interconnectivity with core TH17 cell-specific transcription factors. Mechanistically, EGR2 enhanced TH17 cell differentiation and myeloid cell recruitment to the CNS by upregulating pathogenesis-associated genes and myelomonocytic chemokines. T cell-specific deletion of Egr2 attenuated neuroinflammation without compromising the host's ability to control infections. Our study shows that EGR2 regulates tissue-specific and disease-specific functions in pathogenic TH17 cells in the CNS.

Protein kinases: drug targets for immunological disorders.

Protein kinases play a major role in cellular activation processes, including signal transduction by diverse immunoreceptors. Given their roles in cell growth and death and in the production of inflammatory mediators, targeting kinases has proven to be an effective treatment strategy, initially as anticancer therapies, but shortly thereafter in immune-mediated diseases. Herein, we provide an overview of the status of small molecule inhibitors specifically generated to target protein kinases relevant to immune cell function, with an emphasis on those approved for the treatment of immune-mediated diseases. The development of inhibitors of Janus kinases that target cytokine receptor signalling has been a particularly active area, with Janus kinase inhibitors being approved for the treatment of multiple autoimmune and allergic diseases as well as COVID-19. In addition, TEC family kinase inhibitors (including Bruton's tyrosine kinase inhibitors) targeting antigen receptor signalling have been approved for haematological malignancies and graft versus host disease. This experience provides multiple important lessons regarding the importance (or not) of selectivity and the limits to which genetic information informs efficacy and safety. Many new agents are being generated, along with new approaches for targeting kinases.

Variant STAT4 and Response to Ruxolitinib in an Autoinflammatory Syndrome.

BACKGROUND: Disabling pansclerotic morphea (DPM) is a rare systemic inflammatory disorder, characterized by poor wound healing, fibrosis, cytopenias, hypogammaglobulinemia, and squamous-cell carcinoma. The cause is unknown, and mortality is high. METHODS: We evaluated four patients from three unrelated families with an autosomal dominant pattern of inheritance of DPM. Genomic sequencing independently identified three heterozygous variants in a specific region of the gene that encodes signal transducer and activator of transcription 4 (STAT4). Primary skin fibroblast and cell-line assays were used to define the functional nature of the genetic defect. We also assayed gene expression using single-cell RNA sequencing of peripheral-blood mononuclear cells to identify inflammatory pathways that may be affected in DPM and that may respond to therapy. RESULTS: Genome sequencing revealed three novel heterozygous missense gain-of-function variants in STAT4. In vitro, primary skin fibroblasts showed enhanced interleukin-6 secretion, with impaired wound healing, contraction of the collagen matrix, and matrix secretion. Inhibition of Janus kinase (JAK)-STAT signaling with ruxolitinib led to improvement in the hyperinflammatory fibroblast phenotype in vitro and resolution of inflammatory markers and clinical symptoms in treated patients, without adverse effects. Single-cell RNA sequencing revealed expression patterns consistent with an immunodysregulatory phenotype that were appropriately modified through JAK inhibition. CONCLUSIONS: Gain-of-function variants in STAT4 caused DPM in the families that we studied. The JAK inhibitor ruxolitinib attenuated the dermatologic and inflammatory phenotype in vitro and in the affected family members. (Funded by the American Academy of Allergy, Asthma, and Immunology Foundation and others.).

Medical Information Delivering Improved Customer Experience: A Guide.

Customer experience (CX) is essential in any business. In the pharmaceutical industry, the Medical Information Contact Center is a customer-facing unit that provides evidence-based, scientifically balanced information to healthcare professionals and patients in response to unsolicited inquiries. The purpose of this paper is to provide analysis and guidance for designing and measuring interactions in the Medical Information Contact Center to facilitate the delivery of a superior and continuously improving CX. Surveys were conducted to establish current trends in CX among a diverse group of CX professionals and members of phactMI, a non-profit collaboration of Medical Information leaders from the pharmaceutical industry. The top three observations from the CX professionals survey centered on establishing a clear CX strategy, use of technology, and frequency of sharing results. Three potential areas for improvement focus on CX strategy, measurements of CX, and sharing of results. An analysis of quality monitoring results of customer interactions in the pharmaceutical industry from Centerfirst, a contact center quality monitoring service provider, was also reviewed. This analysis found a positive correlation between CX and three agent skills: taking the lead, empathy, and strong compliance skills. Based on these results, a CX guide was developed and specifically tailored for the pharmaceutical industry. This tool may be used to help identify, assess, and possibly improve CX.

FURIN regulates cytotoxic T-lymphocyte effector function and memory cell transition in mice.

The proprotein convertase subtilisin/kexins (PCSKs) regulate biological actions by cleaving immature substrate proteins. The archetype PCSK, FURIN, promotes the pathogenicity of viruses by proteolytically processing viral proteins. FURIN has also important regulatory functions in both innate and adaptive immune responses but its role in the CD8+ CTLs remains enigmatic. We used a T-cell-specific FURIN deletion in vivo to demonstrate that FURIN promotes host response against the CTL-dependent lymphocytic choriomeningitis virus by virtue of restricting viral burden and augmenting interferon gamma (IFNG) production. We also characterized Furin KO CD8+ T cells ex vivo, including after their activation with FURIN regulating cytokines IL12 or TGFB1. Furin KO CD8+ T cells show an inherently activated phenotype characterized by the upregulation of effector genes and increased frequencies of CD44+ , TNF+ , and IFNG+ cells. In the activated CTLs, FURIN regulates the productions of IL2, TNF, and GZMB and the genes associated with the TGFBR-signaling pathway. FURIN also controls the expression of Eomes, Foxo1, and Bcl6 and the levels of ITGAE and CD62L, which implies a role in the development of CTL memory. Collectively, our data suggest that the T-cell expressed FURIN is important for host responses in viral infections, CTL homeostasis/activation, and memory development.

Biologic Versus Small Molecule Therapy for Treating Moderate to Severe Atopic Dermatitis: Clinical Considerations.

The U.S. Food and Drug Administration approval of dupilumab for moderate-to-severe atopic dermatitis shifted the paradigm from use of broad, systemic immunosuppressants to a safer, targeted treatment and led to the emergence of newer interleukin (IL)-4/IL-13 directed biologics and small molecule therapies, namely Janus kinase (JAK) inhibitors (JAKi). Tralokinumab and emerging (not yet approved) lebrikizumab, which both target IL-13, are alternative biologics to dupilumab. The emerging anti-IL-31 receptor nemolizumab is likely to be used second-line to other biologics, primarily for pruritus. Three JAKi are currently in use for treating atopic dermatitis, 2 of which, abrocitinib and upadacitinib, are U.S. Food and Drug Administration-approved. This review provides an in-depth, practical discussion on use of these biologics and JAKi that are approved or have completed phase 3 clinical trials in pediatric patients and adults, comparing the groups of medications based on available efficacy and safety data.

From thymus to tissues and tumors: A review of T-cell biology.

T cells are critical orchestrators of the adaptive immune response that optimally eliminate a specific pathogen. Aberrant T-cell development and function are implicated in a broad range of human disease including immunodeficiencies, autoimmune diseases, and allergic diseases. Accordingly, therapies targeting T cells and their effector cytokines have markedly improved the care of patients with immune dysregulatory diseases. Newer discoveries concerning T-cell-mediated antitumor immunity and T-cell exhaustion have further prompted development of highly effective and novel treatment modalities for malignancies, including checkpoint inhibitors and antigen-reactive T cells. Recent discoveries are also uncovering the depth and variability of T-cell phenotypes: while T cells have long been described using a subset-based classification system, next-generation sequencing technologies suggest an astounding degree of complexity and heterogeneity at the single-cell level.

A central role for STAT5 in the transcriptional programing of T helper cell metabolism.

Activated lymphocytes adapt their metabolism to meet the energetic and biosynthetic demands imposed by rapid growth and proliferation. Common gamma chain (cγ) family cytokines are central to these processes, but the role of downstream signal transducer and activator of transcription 5 (STAT5) signaling, which is engaged by all cγ members, is poorly understood. Using genome-, transcriptome-, and metabolome-wide analyses, we demonstrate that STAT5 is a master regulator of energy and amino acid metabolism in CD4+ T helper cells. Mechanistically, STAT5 localizes to an array of enhancers and promoters for genes encoding essential enzymes and transporters, where it facilitates p300 recruitment and epigenetic remodeling. We also find that STAT5 licenses the activity of two other key metabolic regulators, the mTOR signaling pathway and the MYC transcription factor. Building on the latter, we present evidence for transcriptome-wide cooperation between STAT5 and MYC in both normal and transformed T cells. Together, our data provide a molecular framework for transcriptional programing of T cell metabolism downstream of cγ cytokines and highlight the JAK-STAT pathway in mediating cellular growth and proliferation.

The JAK-STAT pathway at 30: Much learned, much more to do.

The discovery of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway arose from investigations of how cells respond to interferons (IFNs), revealing a paradigm in cell signaling conserved from slime molds to mammals. These discoveries revealed mechanisms underlying rapid gene expression mediated by a wide variety of extracellular polypeptides including cytokines, interleukins, and related factors. This knowledge has provided numerous insights into human disease, from immune deficiencies to cancer, and was rapidly translated to new drugs for autoimmune, allergic, and infectious diseases, including COVID-19. Despite these advances, major challenges and opportunities remain.