Biology subjects

Berg, L. J.

Publications and source records attributed to Berg, L. J..

6 recordsLinked to original sources

bioRxiv2026

An ITK - SLP-76 Y173 Axis Regulates TCR Signal Persistence and Thymic Development

The TCR-induced tyrosine kinase cascade results in ITK-mediated phosphorylation of at least two substrates: PLC-{gamma}1 Y783, an essential mediator of TCR signaling, and SLP-76 Y173. We previously provided evidence that SLP-76 Y173 supports proximal TCR and Fc{varepsilon}RI signaling; but its role in thymocyte development was never explored. Here we demonstrate that SLP-76 Y173 was required for TCR/CD28-induced degradation of I{kappa}B; moreover, both ITK and SLP-76 Y173 were required for persistent TCR-induced phosphorylation of PLC-{gamma}1, ERK and p38. To explore whether SLP-76 Y173 may mediate ITK-dependent events in vivo, we replaced SLP 76 Y173 with phenylalanine in a mouse model. Like ITK-deficient thymocytes, SLP-76 Y173F thymocytes exhibited reduced thymic cellularity, reduced CD4/CD8 ratios, and reduced surface expression of CD5. In ex vivo-stimulated thymocytes, mutation of SLP-76 Y173 or inhibition of ITK reduced the persistence of TCR-induced calcium flux; moreover, TCR-induced phosphorylation of ERK and p38 depended on SLP-76 Y173. Like ITK-deficient thymi, SLP-76 Y173F thymocytes exhibited decreased production of CD4- iNKT1 thymocytes, coupled with increased production of innate-like CD4+ and CD8+ SP thymocytes. Our results provide strong evidence that the ITK substrate site, SLP-76 Y173, promotes TCR signal persistence that is required to mediate the thymic developmental effects of ITK.

immunology↗

bioRxiv2025

Protracted fate acquisition and epigenetic de-aging during induced neural stem cell conversion of human blood cells

Transcription factor-based direct conversion of somatic cells represents an interesting avenue for generating induced neural stem cells (iNSCs) from peripheral blood without transit through a pluripotent stage. While this paradigm has been shown to be associated with epigenetic de-aging, the dynamics of this process have remained unclear. Here, we used overexpression of the two reprogramming factors SOX2 and cMYC to generate iNSC from erythroid progenitors of donors ranging from neonatal to 101 years of age. Using an epigenetic clock algorithm, we corroborated our previous finding that iNSCs generated from aged donors show pronounced epigenetic de-aging, preserving around 13 % and 5 % of the original donor age at low and high passages, respectively. Studying the dynamic of epigenetic de-aging during iNSC conversion across time, we found that this process is largely protracted, continuing for several weeks and even beyond forced neuronal differentiation of iNSCs. Transcriptomic differences between young and old donor-derived iNSCs dissipate with extended time in conversion, too. Concordant with this observation, established iNSC lines lack age-associated cellular hallmarks, similar to induced pluripotent stem cells and their derivatives. Interestingly, time course analysis of DNA methylation and RNA sequencing data revealed that acquisition of a bona fide NSC signature extends greatly beyond the time point when proliferative PAX6-positive iNSCs emerge. The unexpected slow dynamics of these processes makes iNSC conversion an attractive model for dissecting the mechanisms underlying somatic transdifferentiation and de-aging. Statement of Ethical ApprovalsThe collection of human somatic material (i.e., blood) from healthy donors for iPSC reprogramming and iNSC conversion was conducted in accordance with German law and approved by the Ethics Committee of the University of Bonn Medical Center (approval number: 275/08). All subjects gave written informed consent.

neuroscience↗

bioRxiv2025

Regulation of TCR-induced Transcriptional Kinetics by Interleukin 2-inducible T Cell Kinase (ITK) in CD8+ T cells

The initial activation of naive CD8+ T cells induces three major transcription factor pathways, NFAT, NF{kappa}B, and AP-1, predominantly regulated by T-cell receptor (TCR) signaling. Downstream of the TCR, the Tec family tyrosine kinase ITK modulates the transcriptional response in T cells by differentially affecting the kinetics and magnitude of activation of these three transcription factors. How signaling through ITK regulates the duration and/or termination of TCR signaling has not been investigated. To address this, we utilized the "Nr4a3-Tocky" reporter mouse which provides information on the kinetics of TCR signaling over time courses from hours to days. OT-I CD8+ Nr4a3-Tocky T cells were stimulated with peptide ligands of varying affinities, and cells were assayed for a panel of surface and intracellular markers of activation and differentiation at multiple time points post-stimulation. As previously reported, at early time points ITK signaling enhanced the kinetics and magnitude of expression of these markers. At later time points the absence of ITK led to persistent expression of several proteins, indicating an important role for ITK in the termination of TCR-dependent transcriptional responses. Using a dual ITK/ RLK inhibitor (PRN694) at 24h post OVA peptide stimulation it was found that this function of ITK was required in the first 24 hours. These findings reveal a possible key negative regulatory mechanism that is programmed in the first day of activation but impacts CD8+ T cell gene expression patterns days later. One Sentence Summary StatementThe Tec family tyrosine kinase ITK differentially regulates early and late T-cell receptor transcriptional responses.

immunology↗

bioRxiv2025

Myeloid cell IL-15 production in the brain supports Bystander CD8+ T-Cell Neuropathic Immune Responses following Virus infection

The central nervous system (CNS) includes a uniquely regulated immune response that supports homeostasis, response to injury, and response to pathogens. Recent work has shown that virus-associated immune responses in the CNS may contribute to neuronal injury and long-term outcomes such as neurocognitive decline. However, the fundamental mechanisms that regulate acute infiltration of immune cells from vascular compartments into the CNS are not well defined. Using an attenuated Venezuelan equine encephalitis virus TC83 (referred to as TC83) to inoculate using olfactory and intracranial injections, we show that infection in the CNS and olfactory pathways results in rapid infiltration of both CD4+ and CD8+ T-cells as early as 3- and 5-days post-infection. CNS-infiltrating CD8+ T-cells exhibit a bystander, memory phenotype (CD49d+, Tbet+, NKG2D+, Eomes+), are cytotoxic, and are recruited independent of antigen specific responses. We show that infiltration of CD8+ bystander T-cells is supported by microglia and infiltrating macrophage expression of IL-15 and interferon expression in the CNS. These innate antiviral immune signals support activation of bystander CD8+ T-cells in the CNS that contribute to tissue injury independent of virus replication at early time points post-infection. These data support a mechanism by IL-15 stimulates bystander memory CD8+ T-cells to enter the CNS and contribute to injury independent of antigen-specific stimulation. ImportancePrior studies have shown that virus infections in the respiratory and olfactory nerve pathways can result in long term injury in the brain. However, the mechanisms that link virus infection in the olfactory neurons and brain injury are not understood. We show that virus infection of olfactory neurons results in immune stimulation in the brain of resident immune cells to release a cytokine called IL-15 and interferon. This results in infiltration and activation of non-specific T-cells that cause injury of neurons in the brain. This may be an important mechanism by which respiratory viruses and other viruses cause inflammation and injury in the brain.

immunology↗

bioRxiv2020

Optimized detection of acute MHV68 infection with a reporter system identifies large peritoneal macrophages as a dominant target of primary infection

Investigating the dynamics of virus-host interactions in vivo remains an important challenge, often limited by the ability to directly identify virally-infected cells. Here, we combine detection of a beta-lactamase activated fluorescent substrate with full spectrum flow cytometry to identify primary targets of murine gammaherpesvirus 68 (MHV68) infection in the peritoneal cavity. By optimizing substrate and detection conditions, we were able to achieve multiparameter characterization of infected cells and the ensuing host response. MHV68 infection leads to a pronounced increase in immune cells, with CD8+ T cells increasing by 3 days, and total infiltrate peaking around 8 days post-infection. MHV68 infection results in near elimination of large peritoneal macrophages by 8 days post-infection, and a concordant increase in small peritoneal macrophages and monocytes. Infection is associated with prolonged changes to myeloid cells, with a distinct population of MHC IIhigh large peritoneal macrophages emerging by 14 days. Targets of MHV68 infection could be readily detected. Between 1 to 3 days post-infection, MHV68 infects ~5-10% of peritoneal cells, with >75% being large peritoneal macrophages. By 8 days post-infection, the frequency of MHV68 infection is reduced at least 10-fold, with infection primarily in small peritoneal macrophages, with few infected dendritic cells and B cells. MHV68 infection at 3 days post-infection contains both lytic and latent infection, consistent with the identification of cells with active reporter gene expression. Our findings demonstrate the utility of the beta-lactamase MHV68 reporter system for high throughput single-cell analysis and identify dynamic changes during primary gammaherpesvirus infection. ImportanceIdentifying virally-infected cells in vivo is key to tracking viral infection and understanding host-pathogen interactions. The ability to further characterize and phenotype virally-infected cells is technically challenging. We use a mouse gammaherpesvirus, MHV68, expressing a reporter gene to identify infected cells during primary infection via flow cytometry. Optimization using this reporter system allowed us to further characterize infected cells via multiparameter full spectrum flow cytometry. Our study provides a technical model for high throughput single-cell immunophenotyping methods in the context of gammaherpesvirus infection. Furthermore, we show that acute MHV68 infection in the peritoneal cavity dramatically changes the immune landscape of this tissue, results in a high number of infected macrophages at early times, and is characterized by both lytic and latent infection within immune cells.

microbiology↗

bioRxiv2020

The Tec kinase ITK differentially optimizes NFAT, NF-κB, and MAPK signaling during early T cell activation to regulate graded gene induction

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-associated proteins like IRF4. To regulate T cell activation programming, the TCR and the TCR proximal kinase ITK simultaneously trigger many biochemically separate TCR signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through unequal activation of disparate signaling pathways, we examined Erk1/2 activation and NFAT, NF-{kappa}B translocation in naive OT-I CD8+ cell nuclei. We observed consistent digital activation of NFAT1 and Erk-MAPK, but NF-{kappa}B displayed dynamic, graded activation in response to variation in TCR signal strength and was tunable by treatment with an ITK inhibitor. Inhibitor-treated cells showed dampened induction of AP-1 factors Fos and Fosb, NF-{kappa}B response gene transcripts, and survival factor Il2 transcripts. ATAC-seq analysis also revealed genomic regions most sensitive to ITK inhibition were enriched for NF-{kappa}B and AP-1 motifs. Specific inhibition of NF-{kappa}B during peptide stimulation tuned expression of early gene products like c-Fos. Together, these data indicate a key role for ITK in orchestrating optimal activation of separate TCR downstream pathways, specifically aiding NF-{kappa}B activation. More broadly, we revealed a mechanism by which variation in TCR signal strength can produce patterns of graded gene expression in activated T cells.

immunology↗