Biology subjects

Belkina, A. C.

Publications and source records attributed to Belkina, A. C..

9 recordsLinked to original sources

bioRxiv2025

The altered T cell landscape in Systemic Sclerosis patients is characterized by dysfunctional type 1 immunity.

BackgroundT cells in patients with chronic autoimmunity show features of dysfunction and exhaustion. However, the functionally-defined T cell subsets affected by these changes remain poorly characterized. Here, we sought to reveal aberrations in the composition, phenotype and function of canonical T cell subsets in the blood of Systemic Sclerosis (SSc) patients, compared to healthy subjects and Systemic Lupus Erythematosus (SLE) patients. MethodsWe developed a novel multidimensional flow-cytometry panel to simultaneously detect lineage-defining transcription factors, co-inhibitory receptors and other functional markers to characterize T cell subsets without in vitro restimulation. We compared T cell landscapes in SSc and SLE patients, and healthy subjects, using Optimized t-SNE and PhenoGraph algorithms. Cytokine production in patient samples was determined using intracellular cytokine staining. Transcriptomic analysis was performed to assess the role of IFN-{gamma} in fibroblasts. ResultsThe data show altered distribution and aberrant functional states of transcription factor-defined T cell subsets in patients with autoimmunity, with shared and unique features between diseases and their subtypes. Strikingly, SSc and SLE patients showed a severe deficiency in subsets of CD8+ and CD4-CD8- T cells that expressed T-bet, which is critical for IFN-{gamma}-dependent type 1 immunity, and exhibited features of exhaustion. Moreover, TIGIT+Foxp3+ regulatory T cells, known to suppress Th1 responses, were selectively increased in SSc. Functionally, T cells from SSc patients produced less IFN-{gamma}, a cytokine that suppresses pro-fibrotic gene expression in fibroblasts. ConclusionOur study demonstrates that SSc patients have a defective IFN-{gamma}-producing T cell compartment that may lead to reduced anti-fibrotic T cell activity, enabling chronic fibrosis development. FundingNational Institute of Allergy and Infectious Diseases/NIH, National Institute of Arthritis and Musculoskeletal Diseases/NIH, National Heart, Lung and Blood Institute/NIH, National Scleroderma Foundation, National Jewish Health

immunology↗

bioRxiv2025

Generation of effector CD4+ T cells from Human iPSC

Off the shelf CD4+ T cell therapies, particularly those with immunoregulatory or cell repair functions, could be transformative in CAR therapies for cancers and treatment of chronic inflammatory diseases. However, progress is stunted in this area due to challenges generating human CD4+ T cells from induced pluripotent stem cells. Here we describe a key role for the withdrawal of Notch ligand during the final step of stimulation through the T cell receptor to prompt T cell maturation allowing access to the CD4 lineage in iPSC T cells (iCD4+ T cells). Functional analyses of iCD4+ T cells using a novel high-parameter CyTOF intracellular cytokine staining panel revealed both canonical Th1 cytokine signatures and cells producing varying combinations of other cytokines including IL-4, IL-8, and IL-13. Single cell RNA sequencing of iCD4+ T cells demonstrated a transcriptional signature similar to human blood CD4+ T cells. We believe this robust yet simple platform represents a key step towards the generation of off the shelf iCD4+ T cell therapies with utility for the treatment of a panoply of diseases including cancer and inflammatory autoimmune disorders. HIGHLIGHTST cell receptor stimulation of iPSC CD4+/CD8+ T cell progenitors on coating without Notch ligand allows access to the CD4+ T cell lineage iPSC derived CD4+ T cells express varied cytokines in response to stimulation

immunology↗

bioRxiv2024

Lung CD4+ resident memory T cells use airway secretory cells to stimulate and regulate neutrophilic allergic airways disease.

Neutrophilic asthma is a vexing disease, but mechanistic and therapeutic advancement will require better models of allergy-induced airway neutrophilia. Here, we find that periodic ovalbumin (OVA) inhalation in sensitized mice elicits allergic airway inflammation and pathophysiology mimicking neutrophilic asthma. OVA-experienced murine lungs harbor diverse clusters of CD4+ resident memory TRM cells, including unconventional ROR{gamma}tnegative/low TH17 cells. Acute OVA challenge instigates IL-17A secretion from these TRM cells, driving CXCL5 production from Muc5achigh airway secretory cells, leading to destructive airway neutrophilia. The TRM- and epithelial-cell signals discovered herein are also observed in adult human asthmatic airways. Epithelial antigen presentation regulates this biology by skewing TRM cells towards TH2 and TH1 fates, so that the TH1-related IFN-{gamma} suppresses IL-17A-driven, CXCL5-mediated airway neutrophilia. Concordantly, in vivo IFN-{gamma} supplementation improves disease outcomes. Thus, using our model of neutrophilic asthma we identify lung epithelial-CD4+ TRM cell crosstalk as a key rheostat of allergic airway neutrophilia. HighlightsO_LIRecurrent OVA inhalation experience predisposes mice to allergic airways neutrophilia C_LIO_LINeutrophil-prone lungs harbor CD4+ TRM cells including ROR{gamma}tnegative/low TH17 cells C_LIO_LIMuc5achigh secretory cells instruct CD4+ TRM fates and neutrophilia via MHC-II and CXCL5, respectively C_LIO_LIProphylactic or therapeutic delivery of IFN-{gamma} curbs allergic airway neutrophilia C_LI

immunology↗

bioRxiv2024

A longevity-specific bank of induced pluripotent stem cells from centenarians and their offspring

Centenarians provide a unique lens through which to study longevity, healthy aging, and resiliency. Moreover, models of human aging and resilience to disease that allow for the testing of potential interventions are virtually non-existent. We obtained and characterized over 50 centenarian and offspring peripheral blood samples including those connected to functional independence data highlighting resistance to disability and cognitive impairment. Targeted methylation arrays were used in molecular aging clocks to compare and contrast differences between biological and chronological age in these specialized subjects. Isolated peripheral blood mononuclear cells (PBMCs) were then successfully reprogrammed into high-quality induced pluripotent stem cell (iPSC) lines which were functionally characterized for pluripotency, genomic stability, and the ability to undergo directed differentiation. The result of this work is a one-of-a-kind resource for studies of human longevity and resilience that can fuel the discovery and validation of novel therapeutics for aging-related disease.

molecular biology↗

bioRxiv2022

DE-NOVO HEMATOPOIESIS FROM THE FETAL LUNG

Hemogenic endothelial cells (HECs) are specialized cells that undergo endothelial to hematopoietic transition (EHT) to give rise to hematopoietic progenitors. Though not defined as a hematopoietic organ, the lung houses many resident hematopoietic cells, aids in platelet biogenesis, and is a reservoir for hematopoietic stem and progenitor cells (HSPCs), but lung HECs have never been described. Using explant cultures of murine and human fetal lungs, we demonstrate that the fetal lung is a source of HECs that have the functional capacity to undergo EHT to produce de-novo HSPCs. Flow cytometric and functional assessment of fetal lung explants showed the production of HSPCs that expressed key EHT and pre-HSPC markers. scRNA-Seq and small molecule modulation demonstrated that fetal lung EHT is reliant on canonical EHT signaling pathways. These findings suggest that functional HECs are present in the fetal lung, thus establishing this location as a potential extramedullary site of de-novo hematopoiesis.

developmental biology↗

bioRxiv2020

Multi-Modal Profiling of Human Fetal Liver-Derived Hematopoietic Stem Cells Reveals the Molecular Signature of Engraftment Potential

The human hematopoietic stem cell (HSC) harbors remarkable regenerative potential that can be harnessed therapeutically. During early development, HSCs in the fetal liver (FL) undergo active expansion while simultaneously retaining robust engraftment capacity, yet the underlying molecular program responsible for their efficient engraftment remains unclear. We profiled 26,407 FL cells at both transcriptional and protein levels including over 7,000 highly enriched and functional FL HSCs to establish a detailed molecular signature of engraftment potential. Integration of transcript and linked cell surface marker expression revealed a generalizable signature defining functional FL HSCs and allowed for the stratification of enrichment strategies with high translational potential. This comprehensive, multi-modal profiling of engraftment capacity connects a critical biological function at a key developmental timepoint with its underlying molecular drivers, serving as a useful resource for the field.

cell biology↗

bioRxiv2020

Airway-associated macrophages in homeostasis and repair

There is an increasing appreciation for the heterogeneity of myeloid lineages in the respiratory system, but whether distinct populations associate with the conducting airways remains unknown. We use single cell RNA sequencing, flow cytometry and immunofluorescence to characterize myeloid cells of the mouse trachea during homeostasis and epithelial injury/repair. We identify submucosal macrophages that are similar to lung interstitial macrophages and intraepithelial macrophages, and find that repair of the tracheal epithelium is impaired in Ccr2-deficient mice. Following injury there are early increases in neutrophils and submucosal macrophages, including M2-like macrophages. Unexpectedly, intraepithelial macrophages are initially lost but later replaced from CCR2+ monocytes. Mast cells and group 2 innate lymphoid cells are sources of IL13 that polarizes macrophages and directly influences basal cell behaviors. Their proximity to the airway epithelium establishes these myeloid populations as potential therapeutic targets for airway disease.

cell biology↗

bioRxiv2018

Automated optimal parameters for T-distributed stochastic neighbor embedding improve visualization and allow analysis of large datasets

Accurate and comprehensive extraction of information from high-dimensional single cell datasets necessitates faithful visualizations to assess biological populations. A state-of-the-art algorithm for non-linear dimension reduction, t-SNE, requires multiple heuristics and fails to produce clear representations of datasets when millions of cells are projected. We developed opt-SNE, an automated toolkit for t-SNE parameter selection that utilizes Kullback-Liebler divergence evaluation in real time to tailor the early exaggeration and overall number of gradient descent iterations in a dataset-specific manner. The precise calibration of early exaggeration together with opt-SNE adjustment of gradient descent learning rate dramatically improves computation time and enables high-quality visualization of large cytometry and transcriptomics datasets, overcoming limitations of analysis tools with hard-coded parameters that often produce poorly resolved or misleading maps of fluorescent and mass cytometry data. In summary, opt-SNE enables superior data resolution in t-SNE space and thereby more accurate data interpretation.

bioinformatics↗

bioRxiv2017

HIV-1 Unspliced RNA Expression Induces Innate Immune Activation in Macrophages

Low-levels of type I interferons (IFN-I) are thought to be a driving force for immune activation and T cell exhaustion in HIV-1 infected individuals on highly active antiretroviral therapy (HAART), though the causative mechanisms for persistent IFN-I signaling have remained unclear. Here, we show Rev-CRM1 dependent nuclear export and peripheral membrane association of intron-containing HIV-1 unspliced RNA (usRNA), independent of primary viral sequence or viral protein expression, is subject to sensing, and results in IFN-I-dependent pro-inflammatory responses in macrophages. Our findings suggest that persistent expression of HIV-1 usRNA in macrophages contributes to chronic immune activation and that use of HIV RNA expression inhibitors as adjunct therapy might abrogate aberrant inflammation and restore immune function in HIV-infected individuals on HAART.

microbiology↗