SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

Serotonin (5-HT) regulates peristaltic and secretory reflexes in the gut. The serotonin reuptake transporter (SERT; SLC6A4), which inactivates 5-HT, is expressed in the intestinal mucosa and the enteric nervous system. Stool water content is increased and colonic motility is irregular in mice with a targeted deletion of SERT. We tested the hypotheses that 5-HT plays a role in regulating intestinal inflammation and that the potentiation of serotonergic signaling that results from SERT deletion is proinflammatory. Rectal installation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to induce an immune-mediated colitis, which was compared in SERT knockout mice and littermate controls. Intestinal myeloperoxidase and histamine levels were significantly increased, whereas the survival rate and state of health were significantly decreased in TNBS-treated mice that lacked SERT. Deletion of SERT thus increases the severity of TNBS colitis. These data suggest that 5-HT and its SERT-mediated termination play roles in intestinal immune/inflammatory responses in mice.

CD4⁺FOXP3⁺ regulatory T (Treg) cells are essential for maintaining immunological self-tolerance. Treg cell development and function depend on the transcription factor FOXP3, which is present in several distinct isoforms due to alternative splicing. Despite the importance of FOXP3 in the proper maintenance of Treg cells, the regulation and functional consequences of FOXP3 isoform expression remains poorly understood. Here, we show that in human Treg cells IL-1β promotes excision of FOXP3 exon 7. FOXP3 is not only expressed by Treg cells but is also transiently expressed when naïve T cells differentiate into Th17 cells. Forced splicing of FOXP3 into FOXP3Δ2Δ7 strongly favored Th17 differentiation in vitro. We also found that patients with Crohn’s disease express increased levels of FOXP3 transcripts lacking exon 7, which correlate with disease severity and IL-17 production. Our results demonstrate that alternative splicing of FOXP3 modulates T cell differentiation. These results highlight the importance of characterizing FOXP3 expression on an isoform basis and suggest that immune responses may be manipulated by modulating the expression of FOXP3 isoforms, which has broad implications for the treatment of autoimmune diseases.

FOXP3 is the lineage-defining transcription factor of CD4+ CD25+ regulatory T cells. While many aspects of its regulation, interaction, and function are conserved among species, alternatively spliced FOXP3 isoforms are expressed only in human cells. This review summarizes current knowledge about alternative splicing of FOXP3 and the specific functions of FOXP3 isoforms in health and disease. Future perspectives in research and the therapeutic potential of manipulating alternative splicing of FOXP3 are discussed.

Hypercholesterolemia promotes the inflammation against lipoproteins in atherosclerosis. Development of atherosclerosis is affected by the balance between pro-inflammatory effector T cells and anti-inflammatory regulatory T (Treg) cells. However, phenotype and function of T cell subpopulations in hypercholesterolemia remain to be investigated. Here, we found that cholesterol-containing diet increased the expression of the Treg cell lineage-defining transcription factor FoxP3 among thymocytes and splenocytes. Hypercholesterolemia elevated the FoxP3 expression level and population size of peripheral Treg cells, but did not prevent enhanced proliferation of stimulated T cells. Moreover, cholesterol supplementation in diet as well as in cell culture medium promoted T cell antigen receptor (TCR) signaling in CD4+ T cells. Our results demonstrate that hypercholesterolemia enhances TCR stimulation, Treg cell development as well as T cell proliferation. Thus, our findings may help to understand why hypercholesterolemia correlates with altered CD4+ T cell responses.

Rationale:: The liver is the central organ that responds to dietary cholesterol intake and facilitates the release and clearance of lipoprotein particles. Persistent hypercholesterolemia leads to immune responses against lipoprotein particles that drive atherosclerosis. However, the effect of hypercholesterolemia on hepatic T-cell differentiation remains unknown. Objective:: To investigate hepatic T-cell subsets upon hypercholesterolemia. Methods and Results:: We observed that hypercholesterolemia elevated the intrahepatic regulatory T (Treg) cell population and increased the expression of transforming growth factor-β1 in the liver. Adoptive transfer experiments revealed that intrahepatically differentiated Treg cells relocated to the inflamed aorta in atherosclerosis-prone low-density lipoprotein receptor deficient ( Ldlr ^−/− ) mice. Moreover, hypercholesterolemia induced the differentiation of intrahepatic, but not intrasplenic, Th17 cells in wild-type mice, whereas the disrupted liver homeostasis in hypercholesterolemic Ldlr ^−/− mice led to intrahepatic Th1 cell differentiation and CD11b ⁺ CD11c ⁺ leukocyte accumulation. Conclusions:: Our results elucidate a new mechanism that controls intrahepatic T-cell differentiation during atherosclerosis development and indicates that intrahepatically differentiated T cells contribute to the CD4 ⁺ T-cell pool in the atherosclerotic aorta.