Biology subjects

Pipkin, M. E.

Publications and source records attributed to Pipkin, M. E..

6 recordsLinked to original sources

bioRxiv2026

Selective Glucocorticoid Receptor Modulators of Immune Checkpoint Function

Glucocorticoids (GCs) coordinate immunity, inflammation, and metabolism through allosteric regulation of the glucocorticoid receptor (GR) transcription factor. GCs are indispensable anti-inflammatory drugs yet linking specific ligand-receptor structural states to specific biological outcomes has remained a major barrier to designing safer, more selective therapies. Using structure-based design, we developed selective glucocorticoid receptor modulators (SGRMs) of immune function by extending a steroidal scaffold from the ligand-binding pocket into an adjacent solvent channel. These SGRMs suppressed T cell pro-inflammatory cytokines and promoted differentiation of memory precursor T cells while showing minimal induction of M2 macrophage polarization or T cell checkpoint proteins PD-1 and CTLA-4, all key targets of immunotherapy. Molecular dynamics simulations revealed that solvent-channel substituents function as a lever arm to drive dynamic oscillations in the steroid core, thereby allosterically tuning GR activity states. Systematic perturbation of immune cells with a graded series of ligands enabled a ligand perturbation with machine learning (LPML) framework to map coregulated responses across cell types and identified effector T cell gene networks tightly coupled with immune checkpoint induction. This approach outlines a general strategy for decoding the logic of allosteric drug action, enabling the rational design of SGRMs with tailored immunomodulatory profiles.

systems biology↗

bioRxiv2025

FlAsH-ID: A short peptide tag for live cell photoproximity labeling

Methods to determine how biomolecules interact in a cellular context has led to significant advances in our understanding of biology. Of these, proximity labeling has emerged as a valuable tool for the interrogation protein-protein and protein-RNA interactions. Almost all proximity labeling methods are deployed via large fusion proteins, which while powerful in many contexts, can disrupt native biology or obscure important epitopes on the protein under study. This challenge is especially prominent for small transcriptionally active proteins where the protein cistrome is vital for correct localization and function. Here, we have developed a photoproximity labeling method that is localized to a short 6 amino acid peptide sequence that employs the combination of the Tsien tetracysteine tag with FlAsH dye for localized singlet oxygen generation that we term FlAsH-ID. We demonstrate this method is non toxic and can be employed to label several cellular compartments, and generate high quality interactomics data around H3.1. Furthermore, we show that FlAsH-ID can be used to probe dynamic chnages in protein movement in response to immune stimuli, identifying several RNA-binding protiens that are trafficked to the nucleus following poly(I:C) treatment. Finally we demonstrate FlAsH-ID can be used for proximity proteomics in primary CD8+ T cells by focusing on the transcription factor RUNX3. RUNX3-tetracysteine, but not RUNX3-TurboID, recaptiulated the function of wildtype RUNX3 and rescued the phenotype of Runx3-deficeint CD8+ T cells in response to viral infection in vivo. In primary CD8+ T cells, FlAsH-ID identified RUNX3 in proximity to the nucleosome remodeler ARID1A and we show that both factors act concertedly during initial naive CD8+ T cell activation to drive chromatin accessibility in cis-regulatory regions that induce differentiation of effector and long-lived memory CD8+ T cells. Our results suggest this technique will expand the field of proximity labeling to small, functionally sensitive proteins in primary cells without perturbing their function.

molecular biology↗

bioRxiv2023

Mixed Lineage Leukemia (Mll1) establishes global histone H3K4 trimethylation and stem like memory Cd8 T cell formation

CD8+ T cells with stem cell-like properties (TSCM) sustain adaptive immunity to intracellular pathogens and tumors. However, the developmental origins and chromatin regulatory factors (CRFs) that establish their differentiation are unclear. Using an RNA interference screen of all CRFs we discovered the histone methylase Mll1 was required during T cell receptor (TCR) stimulation for development of a TSCM precursor state and mature memory (TMEM) cells, but not short-lived or transitory effector cell-like states, in response to viral infections and tumors. Mll1 was essential for widespread de novo deposition of histone H3 lysine 4 trimethylation (H3K4me3) upon TCR stimulation, which accounted for 70% of all activation-induced sites in mature TMEM cells. Mll1 promoted both H3K4me3 deposition and reduced TCR-induced Pol II pausing at genes whose single-cell transcriptional dynamics explained trajectories into nascent TSCM precursor states during viral infection. Our results suggest Mll1-dependent control of Pol II elongation and H3K4me3 establishes and maintains differentiation of CD8+ TSCM cell states.

immunology↗

bioRxiv2022

Polycomb Repressive Complex 1 subunit Cbx4 positively regulates effector responses in CD8 T cells

CD8 T cell differentiation is controlled by the crosstalk of various transcription factors and epigenetic modulators. Uncovering the different players in regulating this process is fundamental to improving immunotherapy and designing novel therapeutic approaches. Here, we show that Polycomb Repressive Complex (PRC)1 subunit Chromobox (Cbx)4 favors differentiation to effector CD8 T cells. Cbx4 deficiency in CD8 T cells induced transcriptional signature and phenotype of memory cells, increasing the formation of memory population during acute viral infection. It has been previously shown that besides chromodomain-mediated binding to H3K27me3, Cbx4 function as a SUMO E3 ligase in a SUMO interacting motifs (SIM)-dependent way. The overexpression of Cbx4 mutants in distinct domains showed that this protein regulates CTL differentiation primarily in a SIM-dependent way and partially through its chromodomain. Our data revealed a novel role of a Polycomb group protein Cbx4 controlling CD8 T lymphocyte differentiation and indicates the SUMOylation process as a key molecular mechanism connected to chromatin modification in this process. SummaryUnderstanding the epigenetic control of CTL differentiation is critical for the manipulation of these cells in immunotherapy protocols. This article demonstrates a novel role for Cbx4, a Polycomb-group protein, in supporting CD8 T cell commitment to an effector cell phenotype.

immunology↗

bioRxiv2021

Single-cell lineage trajectories and chromatin regulators that initialize antiviral CD8 T cell ontogeny

Individual naive CD8 T cells activated in lymphoid organs differentiate into functionally diverse and anatomically distributed T cell phylogenies in response to intracellular microbes. During infections that resolve rapidly, including live viral vaccines1, distinct effector (TEFF) and memory (TMEM) cell populations develop that ensure long term immunity2. During chronic infections, responding cells progressively become dysfunctional and "exhaust"3. A diverse taxonomy of TEFF, TMEM and exhausted (TEX) CD8 T cell populations is known, but the initial developmental basis of this phenotypic variation remains unclear4-10. Here, we defined single-cell trajectories and identified chromatin regulators that establish antiviral CD8 T cell heterogeneity using unsupervised analyses of single-cell RNA dynamics11-13 and an in vivo RNAi screen14. Activated naive cells differentiate linearly into uncommitted effector-memory progenitor (EMP) cells, which initially branch into an analogous manifold during either acute or chronic infection. Disparate RNA velocities in single EMP cells initiate divergence into stem, circulating, and tissue-resident memory lineages that generate diverse TMEM and TEX precursor states in specific developmental orders. Interleukin-2 receptor (IL-2R) signals are essential for formation and transcriptional heterogeneity of EMP cells, and promote trajectories toward TEFF rather than TEX states. Nucleosome remodelers Smarca4 and Chd7 differentially promote transcription that delineates divergent TMEM lineages before cooperatively driving terminal TEFF cell differentiation. Thus, the lineage architecture is established by specific chromatin regulators that stabilize diverging transcription in uncommitted progenitors.

immunology↗

bioRxiv2020

Constitutive androstane receptor directs T cell adaptation to bile acids in the small intestine

Bile acids (BAs) are lipid emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, BAs can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain BA homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high BA concentrations in the small intestine lamina propria (siLP). We previously reported that CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1/ABCB1 in the siLP to prevent BA toxicity and suppress Crohns disease-like small bowel inflammation4. Here, we identify the nuclear xenobiotic receptor, constitutive androstane receptor (CAR/NR1I3), as a regulator of MDR1 expression in T cells, and safeguard against BA toxicity and inflammation in the small intestine. CAR was activated and induced large-scale transcriptional reprograming in Teff cells infiltrating the siLP, but not the colon. CAR induced expression of detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine, Il10. Accordingly, CAR-deficiency in T cells exacerbated, whereas pharmacologic CAR activation suppressed, BA-driven ileitis in T cell-reconstituted Rag-/- mice. These data suggest that CAR acts locally in small intestinal T cells to detoxify BAs and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohns disease, and provides evidence of lymphocyte sub-specialization within the small intestine.

immunology↗