Biology subjects

Freedman, T. S.

Publications and source records attributed to Freedman, T. S..

9 recordsLinked to original sources

bioRxiv2026

Dose-dependent activation of Syk and SHIP1 by LynA and LynB at steady state creates a threshold for macrophage signaling in the absence of receptor engagement

LynA-knockout and LynB-knockout mice, each expressing only one of two isoforms of the Src-family kinase (SFK) Lyn, have differential progression to autoimmunity. It is unclear, however, whether isoform-specificity or Lyn dose underlies differential signaling in the single-isoform knockouts. To address this question, we generated a series of Lyn-knockout mice with a varying LynA and LynB expression and tested macrophage signaling in response to pharmacological pan-SFK activation. We found that the magnitude of initiating signaling is a function of the combined basal expression of LynA and LynB, with the two isoforms equally capable of phosphorylating positive-regulatory Syk and negative-regulatory SHIP1. While expression of either isoform restored basal and SFK-initiated downstream signaling, WT-like levels of Erk and Akt signaling were enabled by expression of any amount of Lyn and insensitive to further upregulation of either isoform. Thus, either LynA or LynB expression at steady state leads to balanced activation of positive- and negative-regulatory signaling, setting a maximal response in the absence of a true microbial encounter. Summary SentenceTotal expression of LynA and LynB determines the steady-state phosphorylation of the activating kinase Syk and the inhibitory phosphatase SHIP1, capping signaling in the absence of a microbial encounter.

immunology↗

bioRxiv2025

Discovery Stack Pilot: Feasibility and Outcomes of a Scientist-Designed Peer Review Model Separating Quality and Impact

Peer review serves as the cornerstone of scientific quality control. Yet, the current journal-centric system is hindered by long timelines, high publication costs, inconsistent review quality, systemic biases, and editorial gatekeeping. Notably, the system relies on misaligned measures of impact that are tethered to journal branding and conflate scientific rigor (Quality) with perceived significance (Impact). Here, we report findings from the Discovery Stack Pilot Study, which tested a scientist-designed, journal-independent peer review model. The Discovery Stack model integrates in-line reviewer comments to promote constructive feedback and separately evaluates scientific Quality and Impact using defined criteria. To examine feasibility and effectiveness, manuscripts were reviewed in parallel with traditional journal review. A total of 162 reviews were completed, and survey data from 86 participants were analyzed. The results showed that reviewers effectively evaluated Quality and Impact as separate dimensions, with Quality scores being more consistent across reviewers than Impact scores. Participants strongly supported the core elements of the Discovery Stack model and expressed enthusiasm for its broader adoption to enhance transparency, efficiency, and value in peer review. Future studies will explore integrating this model into a digital platform for reviewing and curating scientific discoveries to improve the production and dissemination of high-quality research.

scientific communication and education↗

bioRxiv2025

Total, not isoform-specific, Lyn expression by macrophages promotes TLR activation and restricts proliferation

Toll-like receptor (TLR) signaling is vital to antimicrobial macrophage function, and its dysregulation is associated with many disease states, including lupus, multiple sclerosis, pulmonary fibrosis, and cancer. The Src-family kinase Lyn plays activating and inhibitory roles downstream of TLRs, yet distinct functions of the Lyn splice variants LynA and LynB in TLR signaling had not been investigated. We used isoform-specific Lyn knockout mice (LynAKO and LynBKO) to interrogate the contribution of each isoform to TLR signaling in bone marrow-derived macrophages. Bulk RNA sequencing and cytokine analyses revealed that complete Lyn deficiency (LynKO) dampens TLR4- and TLR7-induced inflammatory gene expression and TNF production, but enhances the expression of genes responsible for synthesizing the extracellular matrix and promoting proliferation. Despite a reduction in total Lyn levels, the expression of either LynA or LynB alone was sufficient to preserve wild-type transcriptional responses and TNF production in response to the TLR7 agonist R848. However, LyAKO and LynBKO macrophages did have partially impaired TNF production in response to the TLR4 agonist lipopolysaccharide. Additionally, LynAKO and LynBKO macrophages were hyperproliferative, like LynKO cells. These data suggest that Lyn promotes macrophage activation downstream of TLRs and restrains aberrant proliferation and matrix deposition in a dose-dependent rather than isoform-specific manner. Summary SentenceRNA sequencing and functional assays demonstrate that both LynA and LynB restrict macrophage proliferation and drive TLR-induced ECM-remodeling and inflammatory cytokine production.

immunology↗

bioRxiv2025

Steady-state phosphorylation of SHIP1 by Lyn restricts macrophage activation in the absence of a phagocytic synapse

Microscale engagement of the hemi-ITAM-containing receptor Dectin-1 by a fungus-derived particle initiates signaling through the Src-family kinases (SFKs) and Syk that leads to downstream Erk and Akt activation and the macrophage anti-microbial response. To minimize local tissue damage in the absence of a true pathogenic threat, macrophages must remain unresponsive to low-valency receptor ligation by soluble ligands from food or remote infection sites. To investigate how SFKs regulate macrophage sensitivity, we compared signaling in murine bone-marrow-derived macrophages (BMDMs) exposed to depleted zymosan, a particulate and highly multivalent Dectin-1 ligand, with signaling after pharmacologically induced SFK activation without receptor engagement or formation of a phagocytic synapse. We show that high-valency Dectin-1 engagement and formation of a phagocytic synapse restrict phosphorylation of the ITIM-associated phosphatase SHIP1 and promote Erk and Akt signaling. In contrast, SFK activation in the absence of a phagocytic synapse induces phosphorylation of SHIP1 and leads to dampened activation of Erk and Akt. Whereas multiple SFKs are capable of phos-phorylating SHIP1 in principle, the SFK Lyn functions uniquely in maintaining steady-state phosphorylation of SHIP1 and setting tonic and induced levels of Erk and Akt phosphorylation. Consequently, Lyn has a special role in suppressing the Akt pathway and maintaining Erk/Akt pathway balance. Interestingly, formation of a phagocytic synapse circumvents this requirement for Lyn to maintain Erk/Akt balance. These findings highlight the unique function of Lyn in maintaining mac-rophage steady-state signaling and limiting pro-inflammatory responses to disorganized pathway activation and soluble microbial components.

immunology↗

bioRxiv2024

Enhanced IL-15-mediated NK cell activation and proliferation by an ADAM17 function-blocking antibody involves CD16A, CD137, and accessory cells

BackgroundNK cells are being extensively studied as a cell therapy for cancer. Their effector functions are induced by the recognition of ligands on tumor cells and by various cytokines. IL-15 is broadly used to stimulate endogenous and adoptively transferred NK cells in cancer patients. These stimuli activate the membrane protease ADAM17, which then cleaves assorted receptors on the surface of NK cells as a negative feedback loop to limit their activation and function. We have shown that ADAM17 inhibition can enhance IL-15-mediated NK cell proliferation in vitro and in vivo. In this study, we investigated the underlying mechanism of this process. MethodsPBMCs or enriched NK cells from human peripheral blood, either unlabeled or labeled with a cell proliferation dye, were cultured for up to 7 days in the presence of rhIL-15 +/- an ADAM17 function-blocking antibody. Different versions of the antibody were generated; Medi-1 (IgG1), Medi-4 (IgG4), Medi-PGLALA, Medi-F(ab')2, and TAB16 (anti-ADAM17 and anti-CD16 bispecific) to modulate CD16A engagement on NK cells. Flow cytometry was used to assess NK cell proliferation and phenotypic markers, immunoblotting to examine CD16A signaling, and IncuCyte-based live cell imaging to measure NK cell anti-tumor activity. ResultsThe ADAM17 function-blocking mAb Medi-1 markedly increased initial NK cell activation by IL-15. Using different engineered versions of the antibody revealed that the activating Fc{gamma} receptor CD16A, a well-described ADAM17 substrate, was critical for enhancing IL-15 stimulation. Hence, Medi-1 bound to ADAM17 on NK cells can be engaged by CD16A and block its shedding, inducing and prolonging its signaling. This process did not promote evident NK cell fratricide, phagocytosis, or dysfunction. Synergistic activity by Medi-1 and IL-15 enhanced the upregulation of CD137 on CD16A+ NK cells and augmented their proliferation in the presence of PBMC accessory cells. ConclusionsOur data reveal for the first time that CD16A and CD137 underpin Medi-1 enhancement of IL-15-driven NK cell activation and proliferation, respectively. The use of Medi-1 represents a novel strategy to enhance IL-15-driven NK cell proliferation, and it may be of therapeutic importance by increasing the anti-tumor activity of NK cells in cancer patients. What is already known on this topicNK cell therapies are being broadly investigated to treat cancer. NK cell stimulation by IL-15 prolongs their survival in cancer patients. Various stimuli including IL-15 activate ADAM17 in NK cells, a membrane protease that regulates the cell surface density of various receptors as a negative feedback mechanism. What this study addsTreating NK cells with the ADAM17 function-blocking mAb Medi-1 markedly enhanced their activation and proliferation. Our study reveals that the Fc and Fab regions of Medi-1 function synergistically with IL-15 in NK cell activation. Medi-1 treatment augments the upregulation of CD137 by NK cells, which enhances their proliferation in the presence of PBMC accessory cells. How this study might affect research, practice, or policyOur study is of translational importance as Medi-1 treatment in combination with IL-15 could potentially augment the proliferation and function of endogenous or adoptively transferred NK cells in cancer patients. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/593347v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1bf8a19org.highwire.dtl.DTLVardef@39315org.highwire.dtl.DTLVardef@ad5925org.highwire.dtl.DTLVardef@b798cb_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Primary infection with Zika virus provides one-way heterologous protection against Spondweni virus infection in rhesus macaques

Spondweni virus (SPONV) is the closest known relative of Zika virus (ZIKV). SPONV pathogenesis resembles that of ZIKV in pregnant mice, and both viruses are transmitted by Aedes aegypti mosquitoes. We aimed to develop a translational model to further understand SPONV transmission and pathogenesis. We found that cynomolgus macaques (Macaca fascicularis) inoculated with ZIKV or SPONV were susceptible to ZIKV, but resistant to SPONV infection. In contrast, rhesus macaques (Macaca mulatta) supported productive infection with both ZIKV and SPONV and developed robust neutralizing antibody responses. Crossover serial challenge in rhesus macaques revealed that SPONV immunity did not protect against ZIKV infection, whereas ZIKV immunity was fully protective against SPONV infection. These findings establish a viable model for future investigation into SPONV pathogenesis, and suggest the risk of SPONV emergence is low in areas with high ZIKV seroprevalence due to one-way cross-protection between ZIKV and SPONV. TeaserIdentification of asymmetric immune interactions between Zika and Spondweni viruses in macaque monkeys.

microbiology↗

bioRxiv2022

SH3-domain mutations selectively disrupt Csk homodimerization or PTPN22 binding

The kinase Csk is the primary negative regulator of the Src-family kinases (SFKs, e.g., Lck, Fyn, Lyn, Hck, Fgr, Blk, Yes), phosphorylating a tyrosine on the SFK C-terminal tail that mediates autoinhibition. Csk also binds phosphatases, including PTPN12 (PTP-PEST) and immune-cell PTPN22 (LYP/Pep), which dephosphorylate the SFK activation loop to promote autoinhibition. Csk-binding proteins (e.g., CBP/PAG1) oligomerize within membrane microdomains, and high local concentration promotes Csk function. Purified Csk homodimerizes in solution through an interface that overlaps the phosphatase binding footprint. Here we demonstrate that Csk can homodimerize in Jurkat T cells, in competition with PTPN22 binding. We designed SH3-domain mutations in Csk that selectively impair homodimerization (H21I) or PTPN22 binding (K43D) and verified their kinase activity in solution. Disruption of either interaction, however, decreased the negative-regulatory function of Csk in cells. Csk W47A, a substitution previously reported to block PTPN22 binding, also impairs homodimerization. Csk H21I and K43D will be useful tools for dissecting the protein-specific drivers of autoimmunity mediated by the human polymorphism PTPN22 R620W, which impairs interaction with Csk and with the E3 ubiquitin ligase TRAF3. Future investigations of Csk homodimer activity and phosphatase interactions may reveal new facets of SFK regulation in hematopoietic and non-hematopoietic cells.

biochemistry↗

bioRxiv2021

Splice-specific lyn knockout mice reveal a dominant function of LynB in preventing autoimmunity

The unique roles of the Src-family kinases LynA and LynB in immune activating and inhibitory signaling have eluded definition. Here we report that LynB, the shorter splice product of lyn, carries the dominant immunosuppressive function. We used CRISPR/Cas9 gene editing to constrain lyn splicing and expression to a single product: LynAKO or LynBKO mice. While activities of both isoforms regulate homeostatic Lyn expression, only LynB protects against autoimmune disease. LynBKO monocytes and dendritic cells are TLR4-hyper-responsive, and TLR4 expression increases with age in LynBKO myeloid and B cells. These changes are accompanied by the development of an inflammatory disease that resembles human systemic lupus erythematosus (SLE). The interplay between LynB and TLR4 likely underlies the autoimmunity risk associated with LYN hypomorph and TLR4 hypermorph alleles.

immunology↗

bioRxiv2019

An orthogonal c-Cbl recognition mode targets LynA for rapid degradation and builds specificity into the LynA checkpoint

The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is critical factor regulating myeloid-cell activation. In a previous paper (Freedman et al., 2015) we showed in macrophages that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation, functioning as a rheostat regulating ITAM signaling. We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic and biochemical analysis, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via tyrosine 32 in its unique insert region. This orthogonal mode of c-Cbl recognition depresses the steady-state level of macrophage LynA. Mast cells, however, express little c-Cbl and have correspondingly high steady-state levels of LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression therefore builds cell specificity into the LynA checkpoint.

biochemistry↗