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Frey, B. F.

Publications and source records attributed to Frey, B. F..

4 recordsLinked to original sources

IL-23 Licenses Pathogenic Th17 Transdifferentiation via STAT4-dependent Upregulation of IL-12Rβ2

Th17 cells exhibit substantial developmental plasticity, transdifferentiating into pathogenic, interferon-{gamma} (IFN-{gamma})-producing Th1-like effectors during chronic autoimmune inflammation. While both IL-23 and IL-12 are implicated in this process, how these structurally related cytokines coordinate to drive transdifferentiation remains unresolved. Here, we show that IL-23 acts as a developmental licensor rather than a terminal trigger, priming Th17 cells for transdifferentiation upon subsequent IL-12 encounter. IL-23 signals through STAT4 to upregulate the IL-12-specific receptor subunit IL-12R{beta}2, lowering the signalling threshold for IL-12. This licensing can be conferred at both early and late stages of Th17 development and produces a temporal logic in which IL-23-mediated STAT4 signaling functionally arms Th17 cells to terminally transdifferentiate in response to IL-12. Using Stat4 conditional knockouts and a structure-based IL-23 mutein that selectively abolishes IL-23-driven STAT4 signaling while preserving STAT3-dependent lineage maintenance, we genetically establish the IL-23-STAT4-IL-12R{beta}2 axis as a discrete licensing module. Furthermore, using the adoptive Th17 transfer colitis model, we show that host-derived IL-23 and IL-12 are required for Th17-driven colitis, and the inflamed colonic environment reconstitutes transdifferentiation capacity in classically "non-pathogenic" Th17 cells. These findings reframe the pathogenic dichotomy of Th17 cells as one of cumulative cytokine exposure rather than fixed cellular fate, and reveal a stepwise architecture of Th17 plasticity in which IL-23 licensing and IL-12 triggering operate as functionally distinct events. Significance StatementTh17 cells protect mucosal barriers but can convert into pathogenic, IFN-{gamma}-producing effectors that drive inflammatory bowel disease (IBD). Although IL-23 is essential for this conversion, the underlying molecular mechanism remains elusive. We show that IL-23 does not directly trigger transdifferentiation but instead licenses Th17 cells for this conversion, during both early and late stages of their development. By signaling through STAT4, IL-23 upregulates the IL-12-specific receptor subunit IL-12R{beta}2 on Th17 cells, rendering them responsive to subsequent IL-12 exposure. Our findings reframe the classical "pathogenic" versus "non-pathogenic" Th17 distinction and provide a mechanistic basis for the clinical efficacy of IL-23-targeted therapies in IBD.

immunology↗

Barrier Immune Memory is Promoted by Intestinal Epithelial Cell Presentation of Injected Bacterial Antigens

The contributions of antigen compartmentalization to recognition differences between CD4 and CD8 T cells have long been appreciated, but little is known of how subcellular localization of different antigens expressed by a single pathogen impacts T cell immunity. By tracking a clonal CD4 T cell response to its cognate epitope shuttled between different virulence proteins of the enteropathogenic bacterium, Citrobacter rodentium (Cr), we find a remarkable bias in the magnitude and quality of the response contingent on whether antigen remains bacterially associated or is introduced into intestinal epithelial cells colonized by the bacterium. Only proteins injected into the cytosol of colonocytes via the type 3 secretion system (T3SS) of Cr were found to recruit robust antigen-specific T cell responses to the infected mucosa and give rise to CD4 resident memory T (Trm) cells that populate the mucosal epithelium--and this required direct presentation of these antigens by infected epithelial cells. Single-cell transcriptomic analyses revealed that sustained, bidirectional epithelial-T cell communication was required both to elicit epithelial barrier-protective T cell help and to promote transcriptional networks that program a tissue-residency rather than central memory fate. These results establish a central role for antigen presentation by non-professional APCs in controlling memory fate decisions by CD4 T cells, with important implications for development of successful mucosal vaccines.

immunology↗

IL-2-induced Stat3 Signaling is Critical for Effector Treg Cell Programming

Maintenance of immune homeostasis to the intestinal mictrobiota is dependent on a population of effector regulatory T (eTreg) cells that develop from microbiota-reactive induced (i)Treg cells. A cardinal feature of eTreg cells is their production of IL-10, which plays a non-redundant role in immune tolerance of commensal microbes. Here, we identify an unexpected role for IL-2-induced Stat3 signaling to program iTreg cells for eTreg cell differentiation and Il10 transcriptional competency. IL-2 proved to be both necessary and sufficient for eTreg cell development - contingent on Stat3 output of the IL-2 receptor coordinate with IL-2 signaling during early Treg cell commitment. Induction of iTreg cell programming in absence of IL-2-induced Stat3 signaling resulted in impaired eTreg cell differentiation and a failure to produce IL-10. An IL-2 mutein with reduced affinity for the IL-2R{gamma} ({gamma}c) chain was found to have blunted IL-2R Stat3 output, resulting in a deficiency of Il10 transcriptional programming that could not be fully rescued by Stat3 signaling subsequent to an initial window of iTreg cell differentiation. These findings expose a heretofore unappreciated role of IL-2 signaling that acts early to program subsequent production of IL-10 by developing eTreg cells, with broad implications for IL-2-based therapeutic interventions in immune-mediated diseases.

immunology↗

Fixation Before Dissociation Using a Deep Eutectic Solvent Preserves In Vivo States and Phospho-Signaling in Single-Cell Sequencing

Single-cell RNA-sequencing (scRNA-seq) presents an opportunity to deconstruct cellular networks but is limited by the loss of biological information, including in vivo cellular states and phospho-signaling. Herein, we present fixation before dissociation using a deep eutectic solvent (DES), which preserves multiple domains of in vivo biological data, including morphology, RNA, proteins, and post-translational modifications. In scRNA-seq of viable versus DES bone marrow, dissociation induced global stress responses, immune and stromal cell activation, and loss of highly sensitive cell populations, which were prevented with DES. Further, we introduce a validated and flexible method for performing intracellular CITE-seq in DES-fixed cells. Leveraging this approach during Th17 T cell stimulation allowed the simultaneous quantification of transcriptomes and four phosphorylated proteins, leading to the identification of a hyperactivated state in p-ERK/p-FOS double positive cells, which we experimentally validated. We anticipate that DES-based fixatives will allow the accurate reconstruction of in vivo cellular networks and uncover cooperativity amongst intracellular pathways.

immunology↗