Bacterial Ribosomes Induce Plasticity in Mouse Adult Fibroblasts.

The incorporation of bacterial ribosome has been reported to induce multipotency in somatic and cancer cells which leads to the conversion of cell lineages. Queried on its universality, we observed that bacterial ribosome incorporation into trypsinized mouse adult fibroblast cells (MAF) led to the formation of ribosome-induced cell clusters (RICs) that showed strong positive alkaline phosphatase staining. Under in vitro differentiation conditions, RICs-MAF were differentiated into adipocytes, osteoblasts, and chondrocytes. In addition, RICs-MAF were able to differentiate into neural cells. Furthermore, RICs-MAF expressed early senescence markers without cell death. Strikingly, no noticeable expression of renowned stemness markers like Oct4, Nanog, Sox2, etc. was observed here. Later RNA-sequencing data revealed the expression of rare pluripotency-associated markers, i.e., Dnmt3l, Sox5, Tbx3 and Cdc73 in RICs-MAF and the enrichment of endogenous ribosomal status. These observations suggested that RICs-MAF might have experienced a non-canonical multipotent state during lineage conversion. In sum, we report a unique approach of an exo-ribosome-mediated plastic state of MAF that is amenable to multi-lineage conversion.

Unveiling epithelial plasticity regulation in lung cancer: Exploring the cross-talk among Tks4 scaffold protein partners.

The epithelial-to-mesenchymal transition (EMT) represents a hallmark event in the evolution of lung cancer. This work aims to study a recently described EMT-regulating protein, Tks4, and to explore its potential as a prognostic biomarker in non-small cell lung cancer. In this study, we used CRISPR/Cas9 method to knockout (KO) Tks4 to study its functional roles in invadopodia formation, migration, and regulation of EMT marker expressions and we identified Tks4-interacting proteins. Tks4-KO A549 cells exhibited an EMT-like phenotype characterized by elongated morphology and increased expression of EMT markers. Furthermore, analyses of a large-scale lung cancer database and a patient-derived tissue array data revealed that the Tks4 mRNA level was decreased in more aggressive lung cancer stages. To understand the regulatory role of Tks4 in lung cancer, we performed a Tks4-interactome analysis via Tks4 immunoprecipitation-mass spectrometry on five different cell lines and identified CAPZA1 as a novel Tks4 partner protein. Thus, we propose that the absence of Tks4 leads to disruption of a connectome of multiple proteins and that the resulting undocking and likely mislocalization of signaling molecules impairs actin cytoskeleton rearrangement and activates EMT-like cell fate switches, both of which likely influence disease severity.

Decoding cellular plasticity and niche regulation of limbal stem cells during corneal wound healing.

BACKGROUND: Dysfunction or deficiency of corneal epithelium results in vision impairment or blindness in severe cases. The rapid and effective regeneration of corneal epithelial cells relies on the limbal stem cells (LSCs). However, the molecular and functional responses of LSCs and their niche cells to injury remain elusive. METHODS: Single-cell RNA sequencing was performed on corneal tissues from normal mice and corneal epithelium defect models. Bioinformatics analysis was performed to confirm the distinct characteristics and cell fates of LSCs. Knockdown of Creb5 and OSM treatment experiment were performed to determine their roles of in corneal epithelial wound healing. RESULTS: Our data defined the molecular signatures of LSCs and reconstructed the pseudotime trajectory of corneal epithelial cells. Gene network analyses characterized transcriptional landmarks that potentially regulate LSC dynamics, and identified a transcription factor Creb5, that was expressed in LSCs and significantly upregulated after injury. Loss-of-function experiments revealed that silencing Creb5 delayed the corneal epithelial healing and LSC mobilization. Through cell-cell communication analysis, we identified 609 candidate regeneration-associated ligand-receptor interaction pairs between LSCs and distinct niche cells, and discovered a unique subset of Arg1+ macrophages infiltrated after injury, which were present as the source of Oncostatin M (OSM), an IL-6 family cytokine, that were demonstrated to effectively accelerate the corneal epithelial wound healing. CONCLUSIONS: This research provides a valuable single-cell resource and reference for the discovery of mechanisms and potential clinical interventions aimed at ocular surface reconstruction.

Changing behind the scenes.

Th17 cell plasticity is associated with pathogenicity in chronic inflammation. In a model of periodontitis, McClure et al. (https://doi.org/10.1084/jem.20232015) describe location-dependent divergence in Th17 plasticity, with surprisingly limited conversion in inflamed gingiva but emergence of protective exTh17-TfH cells in draining LN that enhance protective antibody.

Type II innate lymphoid cell plasticity contributes to impaired reconstitution after allogeneic hematopoietic stem cell transplantation.

Type II innate lymphoid cells (ILC2s) maintain homeostasis and barrier integrity in mucosal tissues. In both mice and humans, ILC2s poorly reconstitute after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Determining the mechanisms involved in their impaired reconstitution could improve transplant outcomes. By integrating single-cell chromatin and transcriptomic analyses of transplanted ILC2s, we identify a previously unreported population of converted ILC1-like cells in the mouse small intestine post-transplant. Exposure of ILC2s to proinflammatory cytokines resulted in a mixed ILC1-ILC2 phenotype but was able to convert only a small population of ILC2s to ILC1s, which were found post-transplant. Whereas ILC2s protected against acute graft-versus-host disease (aGVHD) mediated mortality, infusion of proinflammatory cytokine-exposed ILC2s accelerated aGvHD. Interestingly, murine ILC2 reconstitution post-HSCT is decreased in the presence of alloreactive T cells. Finally, peripheral blood cells from human patients with aGvHD have an altered ILC2-associated chromatin landscape compared to transplanted controls. These data demonstrate that following transplantation ILC2s convert to a pro-pathogenic population with an ILC1-like chromatin state and provide insights into the contribution of ILC plasticity to the impaired reconstitution of ILC2 cells, which is one of several potential mechanisms for the poor reconstitution of these important cells after allo-HSCT.

Early human fetal lung atlas reveals the temporal dynamics of epithelial cell plasticity.

Studying human fetal lungs can inform how developmental defects and disease states alter the function of the lungs. Here, we sequenced >150,000 single cells from 19 healthy human pseudoglandular fetal lung tissues ranging between gestational weeks 10-19. We capture dynamic developmental trajectories from progenitor cells that express abundant levels of the cystic fibrosis conductance transmembrane regulator (CFTR). These cells give rise to multiple specialized epithelial cell types. Combined with spatial transcriptomics, we show temporal regulation of key signalling pathways that may drive the temporal and spatial emergence of specialized epithelial cells including ciliated and pulmonary neuroendocrine cells. Finally, we show that human pluripotent stem cell-derived fetal lung models contain CFTR-expressing progenitor cells that capture similar lineage developmental trajectories as identified in the native tissue. Overall, this study provides a comprehensive single-cell atlas of the developing human lung, outlining the temporal and spatial complexities of cell lineage development and benchmarks fetal lung cultures from human pluripotent stem cell differentiations to similar developmental window.

Highlighting fibroblast plasticity in lung fibrosis: the WI-38 cell line as a model for investigating the myofibroblast and lipofibroblast switch.

Background: Myofibroblasts (MYFs) are generally considered the principal culprits in excessive extracellular matrix deposition and scar formation in the pathogenesis of lung fibrosis. Lipofibroblasts (LIFs), on the other hand, are defined by their lipid-storing capacity and are predominantly found in the alveolar regions of the lung. They have been proposed to play a protective role in lung fibrosis. We previously reported that a LIF to MYF reversible differentiation switch occurred during fibrosis formation and resolution. In this study, we tested whether WI-38 cells, a human embryonic lung fibroblast cell line, could be used to study fibroblast differentiation towards the LIF or MYF phenotype and whether this could be relevant for idiopathic pulmonary fibrosis (IPF). Methods: Using WI-38 cells, Fibroblast (FIB) to MYF differentiation was triggered using TGF-ß1 treatment and FIB to LIF differentiation using Metformin treatment. We also analyzed the MYF to LIF and LIF to MYF differentiation by pre-treating the WI-38 cells with TGF-ß1 or Metformin respectively. We used IF, qPCR and bulk RNA-Seq to analyze the phenotypic and transcriptomic changes in the cells. We correlated our in vitro transcriptome data from WI-38 cells (obtained via bulk RNA sequencing) with the transcriptomic signature of LIFs and MYFs derived from the IPF cell atlas as well as with our own single-cell transcriptomic data from IPF patients-derived lung fibroblasts (LF-IPF) cultured in vitro. We also carried out alveolosphere assays to evaluate the ability of the proposed LIF and MYF cells to support the growth of alveolar epithelial type 2 cells. Results: WI-38 cells and LF-IPF display similar phenotypical and gene expression responses to TGF-ß1 and Metformin treatment. Bulk RNA-Seq analysis of WI-38 cells and LF-IPF treated with TGF-ß1, or Metformin indicate similar transcriptomic changes. We also show the partial conservation of the LIF and MYF signature extracted from the Habermann et al. scRNA-seq dataset in WI-38 cells treated with Metformin or TGF-ß1, respectively. Alveolosphere assays indicate that LIFs enhance organoid growth, while MYFs inhibit organoid growth. Finally, we provide evidence supporting the MYF to LIF and LIF to MYF reversible switch using WI-38 cells. Conclusions: WI-38 cells represent a versatile and reliable model to study the intricate dynamics of fibroblast differentiation towards the MYF or LIF phenotype associated with lung fibrosis formation and resolution, providing valuable insights to drive future research.

Variations in cell plasticity and proliferation underlie distinct modes of regeneration along the antero-posterior axis in the annelid Platynereis.

The capacity to regenerate lost tissues varies significantly among animals. Some phyla, such as the annelids, display substantial regenerating abilities, although little is known about the cellular mechanisms underlying the process. To precisely determine the origin, plasticity and fate of the cells participating in blastema formation and posterior end regeneration after amputation in the annelid Platynereis dumerilii, we developed specific tools to track different cell populations. Using these tools, we find that regeneration is partly promoted by a population of proliferative gut cells whose regenerative potential varies as a function of their position along the antero-posterior axis of the worm. Gut progenitors from anterior differentiated tissues are lineage restricted, whereas gut progenitors from the less differentiated and more proliferative posterior tissues are much more plastic. However, they are unable to regenerate the stem cells responsible for the growth of the worms. Those stem cells are of local origin, deriving from the cells present in the segment abutting the amputation plane, as are most of the blastema cells. Our results favour a hybrid and flexible cellular model for posterior regeneration in Platynereis relying on different degrees of cell plasticity.

Cellular adaptation to cancer therapy along a resistance continuum.

Advancements in precision oncology over the past decades have led to new therapeutic interventions, but the efficacy of such treatments is generally limited by an adaptive process that fosters drug resistance1. In addition to genetic mutations2, recent research has identified a role for non-genetic plasticity in transient drug tolerance3 and the acquisition of stable resistance4,5. However, the dynamics of cell-state transitions that occur in the adaptation to cancer therapies remain unknown and require a systems-level longitudinal framework. Here we demonstrate that resistance develops through trajectories of cell-state transitions accompanied by a progressive increase in cell fitness, which we denote as the 'resistance continuum'. This cellular adaptation involves a stepwise assembly of gene expression programmes and epigenetically reinforced cell states underpinned by phenotypic plasticity, adaptation to stress and metabolic reprogramming. Our results support the notion that epithelial-to-mesenchymal transition or stemness programmes-often considered a proxy for phenotypic plasticity-enable adaptation, rather than a full resistance mechanism. Through systematic genetic perturbations, we identify the acquisition of metabolic dependencies, exposing vulnerabilities that can potentially be exploited therapeutically. The concept of the resistance continuum highlights the dynamic nature of cellular adaptation and calls for complementary therapies directed at the mechanisms underlying adaptive cell-state transitions.

Pink adipose tissue: A paradigm of adipose tissue plasticity.

Adipose tissue is highly plastic, as illustrated mainly by the transdifferentiation of white adipocytes into beige adipocytes, depending on environmental conditions. However, during gestation and lactation in rodent, there is an amazing phenomenon of transformation of subcutaneous adipose tissue into mammary glandular tissue, known as pink adipose tissue, capable of synthesizing and secreting milk. Recent work using transgenic lineage-tracing experiments, mainly carried out in Saverio Cinti's team, has demonstrated very convincingly that this process does indeed correspond to a transdifferentiation of white adipocytes into mammary alveolar cells (pink adipocytes) during gestation and lactation. This phenomenon is reversible, since during the post-lactation phase, pink adipocytes revert to the white adipocyte phenotype. The molecular mechanisms underlying this reversible transdifferentiation remain poorly understood.

FOXA2 rewires AP-1 for transcriptional reprogramming and lineage plasticity in prostate cancer.

FOXA family proteins act as pioneer factors by remodeling compact chromatin structures. FOXA1 is crucial for the chromatin binding of the androgen receptor (AR) in both normal prostate epithelial cells and the luminal subtype of prostate cancer (PCa). Recent studies have highlighted the emergence of FOXA2 as an adaptive response to AR signaling inhibition treatments. However, the role of the FOXA1 to FOXA2 transition in regulating cancer lineage plasticity remains unclear. Our study demonstrates that FOXA2 binds to distinct classes of developmental enhancers in multiple AR-independent PCa subtypes, with its binding depending on LSD1. Moreover, we reveal that FOXA2 collaborates with JUN at chromatin and promotes transcriptional reprogramming of AP-1 in lineage-plastic cancer cells, thereby facilitating cell state transitions to multiple lineages. Overall, our findings underscore the pivotal role of FOXA2 as a pan-plasticity driver that rewires AP-1 to induce the differential transcriptional reprogramming necessary for cancer cell lineage plasticity.

From stem cells to regulatory T cells: A tale of plasticity.

In this issue, Yano et al.1 present a method to obtain suppressive regulatory T (Treg) cells from human induced pluripotent stem cells (hiPSCs). This approach has the potential to address the low Treg cell yields of current ex vivo Treg cell expansion and induction protocols, an unmet challenge for autologous Treg cell treatments.

Metabolic Plasticity of Regulatory T Cells in Health and Autoimmunity.

Immunometabolism has been demonstrated to control immune tolerance and the pathogenic events leading to autoimmunity. Compelling experimental evidence also suggests that intracellular metabolic programs influence differentiation, phenotype, proliferation, and effector functions of anti-inflammatory CD4+CD25+Foxp3+ regulatory T (Treg) cells. Indeed, alterations in intracellular metabolism associate with quantitative and qualitative impairments of Treg cells in several pathological conditions. In this review, we summarize the most recent advances linking how metabolic pathways control Treg cell homeostasis and their alterations occurring in autoimmunity. Also, we analyze how metabolic manipulations could be employed to restore Treg cell frequency and function with the aim to create novel therapeutic opportunities to halt immune-mediated disorders.

Many Faces of Regulatory T Cells: Heterogeneity or Plasticity?

Regulatory T cells (Tregs) are essential for maintaining the immune balance in normal and pathological conditions. In autoimmune diseases and transplantation, they restrain the loss of self-tolerance and promote engraftment, whereas in cancer, an increase in Treg numbers is mostly associated with tumor growth and poor prognosis. Numerous markers and their combinations have been used to identify Treg subsets, demonstrating the phenotypic diversity of Tregs. The complexity of Treg identification can be hampered by the unstable expression of some markers, the decrease in the expression of a specific marker over time or the emergence of a new marker. It remains unclear whether such phenotypic shifts are due to new conditions or whether the observed changes are due to initially different populations. In the first case, cellular plasticity is observed, whereas in the second, cellular heterogeneity is observed. The difference between these terms in relation to Tregs is rather blurred. Considering the promising perspectives of Tregs in regenerative cell-based therapy, the existing confusing data on Treg phenotypes require further investigation and analysis. In our review, we introduce criteria that allow us to distinguish between the heterogeneity and plasticity of Tregs normally and pathologically, taking a closer look at their diversity and drawing the line between two terms.

Targeting T cell plasticity in kidney and gut inflammation by pooled single-cell CRISPR screening.

Pro-inflammatory CD4+ T cells are major drivers of autoimmune diseases, yet therapies modulating T cell phenotypes to promote an anti-inflammatory state are lacking. Here, we identify T helper 17 (TH17) cell plasticity in the kidneys of patients with antineutrophil cytoplasmic antibody-associated glomerulonephritis on the basis of single-cell (sc) T cell receptor analysis and scRNA velocity. To uncover molecules driving T cell polarization and plasticity, we established an in vivo pooled scCRISPR droplet sequencing (iCROP-seq) screen and applied it to mouse models of glomerulonephritis and colitis. CRISPR-based gene targeting in TH17 cells could be ranked according to the resulting transcriptional perturbations, and polarization biases into T helper 1 (TH1) and regulatory T cells could be quantified. Furthermore, we show that iCROP-seq can facilitate the identification of therapeutic targets by efficient functional stratification of genes and pathways in a disease- and tissue-specific manner. These findings uncover TH17 to TH1 cell plasticity in the human kidney in the context of renal autoimmunity.

Plasticity and lineage commitment of individual TH1 cells are determined by stable T-bet expression quantities.

T helper 1 (TH1) cell identity is defined by the expression of the lineage-specifying transcription factor T-bet. Here, we examine the influence of T-bet expression heterogeneity on subset plasticity by leveraging cell sorting of distinct in vivo-differentiated TH1 cells based on their quantitative expression of T-bet and interferon-γ. Heterogeneous T-bet expression states were regulated by virus-induced type I interferons and were stably maintained even after secondary viral infection. Exposed to alternative differentiation signals, the sorted subpopulations exhibited graded levels of plasticity, particularly toward the TH2 lineage: T-bet quantities were inversely correlated with the ability to express the TH2 lineage-specifying transcription factor GATA-3 and TH2 cytokines. Reprogramed TH1 cells acquired graded mixed TH1 + TH2 phenotypes with a hybrid epigenetic landscape. Continuous presence of T-bet in differentiated TH1 cells was essential to ensure TH1 cell stability. Thus, innate cytokine signals regulate TH1 cell plasticity via an individual cell-intrinsic rheostat to enable T cell subset adaptation to subsequent challenges.

Cancer cell plasticity defines response to immunotherapy in cutaneous squamous cell carcinoma.

Immune checkpoint blockade (ICB) approaches have changed the therapeutic landscape for many tumor types. However, half of cutaneous squamous cell carcinoma (cSCC) patients remain unresponsive or develop resistance. Here, we show that, during cSCC progression in male mice, cancer cells acquire epithelial/mesenchymal plasticity and change their immune checkpoint (IC) ligand profile according to their features, dictating the IC pathways involved in immune evasion. Epithelial cancer cells, through the PD-1/PD-L1 pathway, and mesenchymal cancer cells, through the CTLA-4/CD80 and TIGIT/CD155 pathways, differentially block antitumor immune responses and determine the response to ICB therapies. Accordingly, the anti-PD-L1/TIGIT combination is the most effective strategy for blocking the growth of cSCCs that contain both epithelial and mesenchymal cancer cells. The expression of E-cadherin/Vimentin/CD80/CD155 proteins in cSCC, HNSCC and melanoma patient samples predicts response to anti-PD-1/PD-L1 therapy. Collectively, our findings indicate that the selection of ICB therapies should take into account the epithelial/mesenchymal features of cancer cells.

Not so aDAMant after all: Plasticity of phagocytic microglia.

Phagocytic microglia such as proliferative region-associated microglia and disease-associated microglia appear in the brain transiently during development and across various brain pathologies, but their function and degree of plasticity remain unclear. In this issue of Immunity, Barclay et al. established a novel Clec7a-CreERT2 mouse line to uncover the plasticity of this cell state and its role in a model of myelin injury.

PROX1 drives neuroendocrine plasticity and liver metastases in prostate cancer.

With the widespread use of anti-androgen therapy, such as abiraterone and enzalutamide, the incidence of neuroendocrine prostate cancer (NEPC) is increasing. NEPC is a lethal form of prostate cancer (PCa), with a median overall survival of less than one year after diagnosis. In addition to the common bone metastases seen in PCa, NEPC exhibits characteristics of visceral metastases, notably liver metastasis, which serves as an indicator of a poor prognosis clinically. Key factors driving the neuroendocrine plasticity of PCa have been identified, yet the underlying mechanism behind liver metastasis remains unclear. In this study, we identified PROX1 as a driver of neuroendocrine plasticity in PCa, responsible for promoting liver metastases. Mechanistically, anti-androgen therapy alleviates transcriptional inhibition of PROX1. Subsequently, elevated PROX1 levels drive both neuroendocrine plasticity and liver-specific transcriptional reprogramming, promoting liver metastases. Moreover, liver metastases in PCa induced by PROX1 depend on reprogrammed lipid metabolism, a disruption that effectively reduces the formation of liver metastases.