Biology subjects

Wells, K. L.

Publications and source records attributed to Wells, K. L..

9 recordsLinked to original sources

bioRxiv2026

Beta cell reactivity defines disease-relevant pancreatic CD8 T cells in type 1 diabetes

Type 1 diabetes (T1D) is characterized by immune-mediated destruction of pancreatic beta cells, yet the properties that distinguish disease-associated CD8 T cells from other pancreatic resident T cells remain incompletely defined. In this study, we analyzed CD8 T cell receptor (TCR) clonotypes isolated from the pancreas of organ donors with and without T1D and assessed their reactivity to beta cells using stem cell-derived beta-like cells. We found that highly beta cell-reactive CD8 T cells were selectively present in the pancreas of T1D donors but were largely absent from donors without T1D. In contrast, virus-specific CD8 T cells were detected in pancreata of donors with and without T1D and showed no evidence of cross-reactivity to beta-like cells, indicating that pancreatic residency alone does not confer beta cell specificity. Among beta cell-reactive CD8 T cells in T1D, reactivity to native peptides from major islet proteins other than preproinsulin was rare. Thus, despite beta cell specificity as a hallmark of T1D, T cells reactive to native islet proteins other than preproinsulin do not infiltrate the islets. These results identify beta cell reactivity as a key functional feature separating T1D-associated CD8 T cells from other pancreatic T cells. This functional definition of pathogenic T cells offers a framework for understanding selective beta cell loss and for developing approaches to monitor and therapeutically target disease-relevant CD8 T cells.

immunology↗

bioRxiv2025

Human NKX2.2 influences islet endocrine cell fate choices through regulation of WNT pathway genes

Transcriptional regulation is a key central mechanism of cell fate determination in developing tissues. The homeobox transcription factor NKX2.2 is an essential regulator of mouse and human pancreatic endocrine development, however its precise molecular role in a human system has not been previously investigated. In this study we generated NKX2.2 null (NKX2.2KO) human embryonic stem cell (hESC) lines using CRISPR/Cas9 technologies and differentiated them towards a pancreatic {beta} cell fate using a stem cell-derived {beta} cell differentiation protocol. Functional and transcriptomic analyses of the hESC-derived pancreatic endocrine cells lacking NKX2.2 revealed similarities and differences compared to the molecular functions of NKX2.2 in mice. In the absence of NKX2.2, the {beta} cell differentiations result in reduced numbers of insulin-producing cells, and the differentiations become skewed towards polyhormonal fates, including cells co-expressing insulin, ghrelin and somatostatin. Deletion of NKX2.2 also eliminates the off-target formation of enterochromaffin cells. Single cell transcriptome analysis of the early endocrine cell population revealed a marked disruption of metabolic pathways that was confirmed by comparative metabolite tracing, providing novel insights into the regulation of early endocrine lineage decisions. Furthermore, NKX2.2 directly regulates several genes in the WNT signaling pathway, suggesting this is a key molecular mechanism through which NKX2.2 regulates these islet cell fate decisions in the human system.

developmental biology↗

bioRxiv2025

The Chromatin Remodeler Protein CHD4 Cooperates With NKX2.2 to Regulate Pancreatic Beta Cell Integrity

NKX2.2 is a transcription factor that regulates pancreatic islet beta ({beta}) cell identity and function; however, cofactor proteins that modulate the functional activity of NKX2.2 in {beta} cells are relatively unexplored. An unbiased proteomics screen identified chromodomain helicase DNA-binding protein 4 (CHD4) as an NKX2.2 interacting partner. CHD4 is a nucleosome remodeler that directs the appropriate differentiation, maturation and function of many cell types. To characterize the roles of CHD4 in {beta} cells, we generated Chd4 {beta}KO mice. Deletion of Chd4 substantially impaired the function of {beta} cells. The Chd4 {beta}KO mice became diabetic due to the disruption of islet integrity, calcium signaling and downregulation of essential {beta} cell regulatory genes. We also discovered CHD4 is required to bind at and repress non-beta cell genes, including Kcnj5, the gene that encodes the GIRK4 potassium channel in {beta} cells. Aberrant upregulation of GIRK4 causes impaired glucose-stimulated insulin secretion. These studies demonstrate that CHD4 is an essential transcriptional cofactor of NKX2.2 that is required for the proper maturation and function of pancreatic {beta} cells. Article HighlightsO_LINKX2.2 interacts with the Nucleosome Remodeling and Deacetylase (NuRD) complex through its interaction with CHD4. C_LIO_LIDeletion of CHD4 from developing pancreatic {beta} cells in mice causes diabetes due to a loss of islet integrity, disrupted calcium signaling and impaired insulin secretion. C_LIO_LIBeta cells lacking CHD4 inappropriately upregulate the GIRK4 potassium channel; inhibition of GIRK4 rescues the insulin secretion defect. C_LI

developmental biology↗

bioRxiv2025

Optimizing Single-Cell Long-Read Sequencing for Enhanced Isoform Detection in Pancreatic Islets

Alternative splicing is an essential mechanism for generating protein diversity by producing distinct isoforms from a single gene. Dysregulation of splicing that affects pancreatic function and immune tolerance has been linked to both type 1 and type 2 diabetes. Next-generation sequencing technologies, with their short read lengths, are limited in their ability to accurately detect splice variants. Long-read sequencing technologies offer the potential to overcome these limitations by providing full-length transcript information; however, their application in single-cell RNA sequencing has been hindered by technical challenges, including insufficient read lengths and higher error rates. Furthermore, cell types that produce high levels of a single transcript, such as islet endocrine cells, can obscure identification of lower abundance transcripts. In this study, we optimized a protocol for single-cell long-read sequencing in pancreatic islets to improve read length and transcript detection. Our findings demonstrate that 5 library preparation protocols outperform 3 protocols, resulting in better transcript identification. Furthermore, we show that targeted depletion of insulin transcripts enhances the detection of informative reads, highlighting the utility of transcript depletion strategies. This optimized protocol enables isoform-specific gene expression analysis and reveals differential transcript usage across the various cell types in pancreatic islets. By leveraging this approach, we gain deeper insights into the transcriptomic complexity and cellular heterogeneity within pancreatic islets. Article HighlightsO_LIThis study addresses the limitations of current single-cell long-read RNA-sequencing (sclrRNA-seq) technologies in detecting full-length transcripts and isoform diversity, particularly in pancreatic islets. C_LIO_LIWe demonstrate that optimizing single-cell library preparation protocols reproducibly enhances read length and transcript identification in pancreatic islets. C_LIO_LI5 capture methods, combined with targeted insulin depletion and extended reverse transcription, significantly improved read length and isoform detection compared to standard protocols, while maximizing the number of informative reads. C_LIO_LIThese improvements yield longer reads in single-cell experiments, substantially enhancing transcript identification and enabling more accurate analysis of isoform diversity. C_LI

genomics↗

bioRxiv2024

NKX2.2 and KLF4 cooperate to regulate alpha cell identity

Transcription factors (TFs) are indispensable for maintaining cell identity through regulating cell-specific gene expression. Distinct cell identities derived from a common progenitor are frequently perpetuated by shared TFs; yet the mechanisms that enable these TFs to regulate cell-specific targets are poorly characterized. We report that the TF NKX2.2 is critical for the identity of pancreatic islet cells by directly activating cell genes and repressing alternate islet cell fate genes. When compared to the known role of NKX2.2 in islet {beta} cells, we demonstrate that NKX2.2 regulates cell genes, facilitated in part by cell specific DNA binding at gene promoters. Furthermore, we have identified the reprogramming factor KLF4 as having enriched expression in cells, where it co-occupies NKX2.2-bound cell promoters, is necessary for NKX2.2 promoter occupancy in cells and co-regulates many NKX2.2 cell transcriptional targets. Misexpression of Klf4 in {beta} cells is sufficient to manipulate chromatin accessibility, increase binding of NKX2.2 at cell specific promoter sites, and alter expression of NKX2.2-regulated cell-specific targets. This study identifies KLF4 is a novel cell factor that cooperates with NKX2.2 to regulate cell identity.

developmental biology↗

bioRxiv2024

Islet-antigen reactive B cells display a unique phenotype and BCR repertoire in autoantibody positive and recent-onset type 1 diabetes patients

Autoreactive B cells play an important but ill-defined role in autoimmune type 1 diabetes (T1D). To better understand their contribution, we performed single cell gene and BCR-seq analysis on pancreatic islet antigen-reactive (IAR) B cells from the peripheral blood of nondiabetic (ND), autoantibody positive prediabetic (AAB), and recent-onset T1D individuals. We found that the frequency of IAR B cells was increased in AAB and T1D. IAR B cells from these donors had altered expression of B cell signaling, pro-inflammatory, infection, and antigen processing and presentation genes. Both AAB and T1D donors demonstrated a significant increase in certain heavy and light chain V genes, and these V genes were enriched in islet-reactivity. Public clones of IAR B cells were restricted almost entirely to AAB and T1D donors. IAR B cells were clonally expanded in the autoimmune donors, particularly the AAB group. Notably, a substantial fraction of IAR B cells in AAB and T1D donors appeared to be polyreactive, which was corroborated by analysis of recombinant monoclonal antibodies. These results expand our understanding of autoreactive B cell activation during T1D and identify unique BCR repertoire changes that may serve as biomarkers for increased disease risk. One Sentence SummaryPancreatic islet antigen-reactive B cells from individuals with prediabetes and recently diagnosed with type 1 diabetes display a unique phenotype and BCR repertoire compared to non-diabetic donors.

immunology↗

bioRxiv2022

Uterine injury during diestrus leads to embryo spacing defects and perturbations in the COX pathway in subsequent pregnancies

Uterine injury from procedures such as Cesarean sections (C-sections) often have severe consequences on subsequent pregnancy outcomes, leading to disorders such as placenta previa, placenta accreta, and infertility. With rates of C-section at approximately 30% of deliveries in the US and that are projected to continue to climb, a deeper understanding of the mechanisms by which these pregnancy disorders arise and opportunities for intervention are needed. However, there are no animal models to date that comprehensively assess the consequences of uterine injury. Here we describe a rodent model of uterine injury on subsequent in utero outcomes. We observed three distinct phenotypes: increased rates of resorption and death, embryo spacing defects, and placenta accreta-like features of reduced decidua and expansion of invasive trophoblasts. We show that the appearance of embryo spacing defects depends entirely on the phase of estrous cycle at the time of injury. Using RNA-seq, we identified perturbations in the expression of components of the COX/prostaglandin pathway after recovery from injury, a pathway that has previously been demonstrated to play an important role in embryo spacing. Therefore, we demonstrate that uterine damage in this mouse model causes morphological and molecular changes, most notably perturbed expression of COX/prostaglandin pathway-related genes, that ultimately lead to placental and embryonic developmental defects.

developmental biology↗

bioRxiv2021

Integrated single-cell transcriptomics and epigenomics reveals strong germinal center-associated etiology of autoimmune risk loci

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting auto-reactive B cells. Dysfunction in these processes can lead to defects in immune response to pathogens or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type-specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC cell populations. Together, these analyses provide a powerful new cell type-resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease. One sentence summarySingle-cell chromatin accessibility landscapes of immune cell subsets reveal regulatory potential of autoimmune-associated genetic variants during the germinal center response.

genomics↗

bioRxiv2020

Combined transient ablation and single cell RNA sequencing reveals the development of medullary thymic epithelial cells

Medullary thymic epithelial cells (mTECs) play a critical role in central immune tolerance by mediating negative selection of autoreactive T cells through the collective expression of the peripheral self-antigen compartment, including tissue-specific antigens (TSAs). Recent work has shown that gene expression patterns within the mTEC compartment are remarkably heterogenous and include multiple differentiated cell states. To further define mTEC development and medullary epithelial lineage relationships, we combined lineage tracing and recovery from transient in vivo mTEC ablation with single cell RNA-sequencing. The combination of bioinformatic and experimental approaches revealed a non-stem transit-amplifying population of cycling mTECs that preceded Aire expression. Based on our findings, we propose a branching model of mTEC development wherein a heterogeneous pool of transit-amplifying cells gives rise to Aire- and Ccl21a-expressing mTEC subsets. We further use experimental techniques to show that within the Aire-expressing developmental branch, TSA expression peaked as Aire expression decreased, implying Aire expression must be established before TSA expression can occur. Collectively, these data provide a higher order roadmap of mTEC development and demonstrate the power of combinatorial approaches leveraging both in vivo models and high-dimensional datasets.

immunology↗