Biology subjects

Konnikova, L.

Publications and source records attributed to Konnikova, L..

15 recordsLinked to original sources

bioRxiv2026

IL1 blockade attenuates E. coli induced intrauterine inflammation and preterm labor in Rhesus macaques

Intrauterine infection and inflammation are major causes of preterm labor, yet antibiotics alone often fail to prevent preterm birth despite achieving microbial clearance. Interleukin1 (IL1) is a key mediator of intra-amniotic inflammation and functional progesterone withdrawal, processes linked to labor initiation. We tested whether IL1 blockade with the clinically approved IL1 receptor antagonist, anakinra, could reduce inflammation and preterm labor in a Rhesus macaque model of intra-amniotic Escherichia coli infection followed by delayed antibiotic treatment. Here we show that anakinra reduced the incidence of preterm labor by 40% (p=0.07), attenuated inflammation in the fetal membranes and cervix, and preserved progesterone receptor-B abundance in the fetal membranes. These effects were most pronounced in animals protected from preterm labor. Our findings in a relevant model support the translational potential of combining IL1 blockade with antibiotics for infection-associated preterm labor. One sentence summaryRecombinant human IL1 receptor antagonist (anakinra) decreased E. coli infection induced preterm labor and partially decreased intrauterine inflammation in a non-human primate model of inflammation mediated preterm labor.

immunology↗

bioRxiv2026

Early-life mucosal T cells direct intestinal stem cell fate via a coordinated developmental program

Although early-life immunity was once considered immature, the human fetal immune system is dynamic and compartmentalized by the second trimester. By 21 weeks of gestation, T lymphocytes become a major immune population in the fetal small intestine (SI), yet their functional roles within this tissue remain largely undefined. To explore their unique contributions to intestinal development, we established an ex vivo co-culture system in which mucosal T cells isolated from fetal, neonatal, or adult SI donors were cultured with tissue-derived 3D SI organoids derived from various ages. Homeostatic early-life (fetal and neonatal) SI T cells uniquely promoted organoid generation, a metric of stem cell renewal, by upregulating cell cycle-associated gene programs. These early-life T cells also directed intestinal stem cell differentiation toward the secretory lineage in both growth and differentiation phase, highlighting that T cells poise stem cells to adopt secretory fates. T cells from infants with necrotizing enterocolitis (NEC) - an inflammatory intestinal disease affecting predominantly preterm infants - failed to activate these same programs, suggesting a pathologic role for T cells in NEC. T cells from the adult SI similarly failed to support organoid growth or differentiation, revealing developmentally specialized, nonimmune functions for early-life T cells in the intestine. Similarly, T cells derived from cord blood did not enhance organoid generation, indicating that this function is not necessarily a generalized feature of early-life T cells but rather is restricted to mucosal T cells. Organoids derived from adults or NEC, however, could re-enter regenerative states when co-cultured with fetal T cells, indicating that fetal T cells can restore stem cell self-renewal across developmentally and disease-imposed states. We further identified that T cell-derived soluble factors alone were insufficient to modulate intestinal stem cell fate, implying the need for physical interactions. Concordant with this finding, we report that T cells heavily localize to the stem cell niche during prenatal development, where they express factors involved in Notch, Wnt, and growth factor signaling to support fetal stem cell function. Collectively, these findings reveal a coordinated developmental program in which fetal SI T cells balance stem cell self-renewal and differentiation, identifying a developmental immune-epithelial axis that can be harnessed to restore intestinal regeneration.

immunology↗

bioRxiv2025

Detection of serotonin and serotonin related gene reveals unique roles in human intestinal epithelial development

Although 95% of serotonin in the human body is produced in the gastrointestinal tract, most prior research work has focused on the brain. Despite its abundance in the gut, serotonins role in early intestinal development remains poorly understood. Here we show that serotonin is detected in both the small intestine and large intestine as early as 14-15 weeks gestational age. Using single-cell transcriptomics with histological validation, we generated a spatiotemporal atlas of serotonin and serotonin-related genes in the human gut from first trimester to late adulthood. The serotonin synthesis gene, TPH1, was exclusively expressed in enteroendocrine cells at all time points analyzed, with the highest expression in the second trimester. The serotonin reuptake gene, SLC6A4, was largely restricted to mature absorptive enterocytes and increased post-natally. Serotonin receptors also displayed distinct cellular and developmental expression patterns. HTR1E was confined to the first trimester during highly proliferative stage. HTR4 was enriched for in crypt regions, suggesting a role in proliferation for serotonin. Finally, HTR3E was associated with tuft cells throughout development. Together, these findings reveal the dynamic, cell-type-specific regulation of serotonin in the developing human gut, suggesting its importance in early intestinal development, and provide a framework to study its roles in intestinal development and diseases.

developmental biology↗

bioRxiv2025

CD8 T cells mediate immunosurveillance for neoantigen+ epithelial stem cells in the colon

Epithelial cells in the colon accumulate substantial numbers of somatic mutations, some of which can be recognised as neoantigens. The ability of CD8 T cells to survey for neoantigen+ cells in the healthy colon would provide an early detection mechanism to prevent cancer, but it is unclear whether neoantigen-specific CD8 T cells can mediate this process of immunosurveillance without becoming tolerant. To address this question, we used a genetically engineered mouse model to express a neoantigen in the epithelial cells of the adult proximal colon. Induction of neoantigen expression led to rapid elimination of neoantigen+ epithelial cells from the colon in a CD8 T cell-dependent manner. Neoantigen-specific CD8 T cells acquired cytolytic function within the colon tissue under steady-state conditions, which was required for elimination of the neoantigen+ epithelial cells. Despite the elimination of [~]25% of their epithelial cells over a two-day period, the colons looked histologically normal. Immunofluorescence and single-cell transcriptomic analyses revealed that neoantigen-specific CD8+ T cells specifically target neoantigen+ stem cells at the crypt base, which was associated with Ki67 in the crypt wall and abundance of neoantigen-negative stem cells. Infiltrating neoantigen-specific CD8 T cells made IFNg and expressed PD-1, raising the question of why PD-1-dependent suppression did not prevent the acquisition of effector functions by these neoantigen-specific CD8 T cells. Despite an increased signature of interferon-stimulated genes in colonic epithelial cells, PD-L1 expression was surprisingly absent. Moreover, we found that colonic epithelial stem cells also did not express PD-L1 under conditions of chronic inflammation, such as ulcerative colitis, immune checkpoint-induced colitis, and ageing, or when directly stimulated with IFN-{gamma} in vitro. Analyses of the PD-L1 gene promoter across humans and mice showed hypermethylation at sites associated with PD-L1 repression in cancer. Thus, our data support a model in which the acquisition of neoantigens by colonic epithelial cells triggers CD8 T cell-mediated immunosurveillance. This results in the elimination of PD-L1-negative neoantigen+ stem cells by effector CD8 T cells and simultaneous repair of the colon by neoantigen-negative epithelial cells to prevent immunopathology.

immunology↗

bioRxiv2025

Fetal and maternal immune cells exhibit tandem maturation across gestation and dysregulation in preterm birth

Appropriate regulation of maternal immune cells in the pregnant uterus is essential for successful pregnancy, yet the impact of immune cells in placental villi (PV) on placental health remain understudied. We enriched immune cells from term and preterm PV for transcriptomic and functional analyses. Maternal and fetal immune cells were abundant, with maternal chimerism increasing at term. Fetal lymphocytes displayed enhanced prostaglandin-D signatures, while maternal lymphocytes were enriched for antigen presentation pathways. Inflammatory cytokines and expanded maternal and fetal T cell clones peaked at term but did not induce cytotoxicity in placental organoids, whereas expanded T cells from idiopathic preterm cases impaired trophoblast growth and endocrine function ex vivo. These findings reveal significant immune chimerism within PV in both health and disease yet implicate PV T cells in idiopathic preterm birth. One Sentence SummaryFetal-maternal immune chimerism is characteristic of all placental villi, yet unchecked T cells and cytokines can hinder trophoblast growth and potentially precipitate preterm birth.

immunology↗

bioRxiv2025

Efficacy of DNA Methyltransferase Inhibitor Immune Priming Therapy in Combination with PD-1 Inhibitors to Treat High-Risk Pediatric Brain Tumors

BackgroundDespite intensive therapies, outcomes for high-risk pediatric brain tumors (PBTs) remain dismal, prompting the search for novel treatments. DNA methyltransferase inhibitors (DNMTi) have been shown to prime tumors to improve response to checkpoint inhibition. The aim of this study was to investigate the potential of decitabine (DAC), in combination with a PD-1 inhibitor, to improve survival in pediatric high-risk brain tumor models. MethodsAnalysis of human PBT datasets was performed to determine gene expression levels of immune cell associated markers. Tumor response to DAC, with or without a PD-1 inhibitor, was tested in murine models representing H3-wildtype diffuse intrinsic pontine glioma (DIPG), H3K27-mutant diffuse midline glioma (DMG), atypical teratoid rhabdoid tumor (ATRT), and medulloblastoma (MB). CyTOF analysis of allograft tumors was performed to characterize changes within the tumor microenvironment. ResultsAnalysis of PBT subtypes revealed heterogeneous expression of immune cell markers, checkpoint receptors, and MHC molecules. DAC treatment decreased DNA methylation and increased neoantigen expression in human and mouse tumor cells. DAC alone or in combination with a PD-1 inhibitor resulted in prolonged survival in syngeneic mouse models of DIPG and ATRT but not DMG and MB models. CyTOF analysis of mouse tumors revealed changes in local immune cell infiltration upon combination treatment. ConclusionsDAC in combination with a PD-1 inhibitor can alter the immune microenvironment in mouse tumor models. Changes were observed in H3-wildtype DIPG and ATRT models, suggesting that certain tumor subtypes may respond to checkpoint blockade after immune augmentation with DNMTi. Key PointsO_LIPBTs show heterogenous expression of immune cell infiltrates C_LIO_LIDAC or DAC plus a PD-1 inhibitor shows extension of survival in H3-wildtype DIPG and ATRT mouse models C_LIO_LIMyeloid-derived suppressor cell abundance could be a major contributing factor to treatment response C_LI Importance of the StudyChildren with high-risk PBTs face dismal outcomes. Immune checkpoint inhibitor (ICI) successes have been demonstrated in a variety of adult malignancies; however, such beneficial outcomes have not been realized in PBTs. Here we investigate single and combination treatment of DNMTi and PD-1 checkpoint inhibition in syngeneic mouse models of high-risk PBTs. Our results suggest that some H3-wildtype DIPG and ATRT tumor types may be responsive to checkpoint therapy post immunomodulation and warrant further investigation.

immunology↗

bioRxiv2024

BACH2-driven tissue resident memory programs promote HIV-1 persistence

Transcription repressor BACH2 redirects short-lived terminally differentiated effector into long-lived memory cells. We postulate that BACH2-mediated long-lived memory programs promote HIV-1 persistence in gut CD4+ T cells. We coupled single-cell DOGMA-seq and TREK-seq to capture chromatin accessibility, transcriptome, surface proteins, T cell receptor, HIV-1 DNA and HIV-1 RNA in 100,744 gut T cells from ten aviremic HIV-1+ individuals and five HIV-1- donors. BACH2 was the leading transcription factor that shaped gut tissue resident memory T cells (TRMs) into long-lived memory with restrained interferon-induced effector function. We found that HIV-1-infected cells were enriched in TRMs (80.8%). HIV-1-infected cells had increased BACH2 transcription factor accessibility, TRM (CD49a, CD69, CD103) and survival (IL7R) gene expression, and Th17 polarization (RORC, CCR6). In vitro gut CD4+ T cell infection revealed preferential infection and persistence of HIV-1 in CCR6+ TRMs. Overall, we found BACH2-driven TRM program promotes HIV-1 persistence and BACH2 as a new therapeutic target.

immunology↗

bioRxiv2024

Cataloguing the postnatal small intestinal transcriptome during the period of susceptibility to necrotizing enterocolitis

In the first postnatal month, the developing mouse intestine shifts from an immature to a mature intestine that will sustain the organism throughout the lifespan. Here, we surveyed the mouse intestine in C57Bl/6 mice by bulk RNA-Seq to evaluate the changes in gene expression over time from the day of birth through 1 month of age in both the duodenum and ileum. Using trends identified in the RNA-Seq analyses, we further evaluated expression of epithelial and mesenchymal cell markers, epithelial regulators, and immune cell markers. We confirmed key changes with qRT-PCR and immunofluorescence. In addition, we compared some findings to humans using human intestinal biopsies and organoids. This dataset can serve as a reference for other groups considering the role of single molecules or molecular families in early intestinal and postnatal development, expanding the limited literature on postnatal gene expression in the developing intestine.

developmental biology↗

bioRxiv2024

In utero human intestine contains maternally derived bacterial metabolites

Understanding when host-microbiome interactions are first established is crucial for comprehending normal development and identifying disease prevention strategies. Furthermore, bacterially derived metabolites play critical roles in shaping the intestinal immune system. Recent studies have demonstrated that memory T cells infiltrate human intestinal tissue early in the second trimester, suggesting that intestinal immune education begins in utero. Our previous study reported a unique fetal intestinal metabolomic profile with an abundance of several bacterially derived metabolites and aryl hydrocarbon receptor (AHR) ligands implicated in mucosal immune regulation. To follow up on this work, in the current study, we demonstrate that a number of microbial byproducts present in fetal intestines in utero are maternally derived and vertically transmitted to the fetus. Notably, these bacterially derived metabolites, particularly short chain fatty acids and secondary bile acids, are likely biologically active and functional in regulating the fetal immune system and preparing the gastrointestinal tract for postnatal microbial encounters, as the transcripts for their various receptors and carrier proteins are present in second trimester intestinal tissue through single-cell transcriptomic data.

microbiology↗

bioRxiv2024

Comprehensive evaluation and practical guideline of gating methods for high-dimensional cytometry data: manual gating, unsupervised clustering, and auto-gating

Cytometry is an advanced technique for simultaneously identifying and quantifying many cell surface and intracellular proteins at a single-cell resolution. Analyzing high-dimensional cytometry data involves identifying and quantifying cell populations based on their marker expressions. This study provided a quantitative review and comparison of various ways to phenotype cellular populations within the cytometry data, including manual gating, unsupervised clustering, and supervised auto-gating. Six datasets from diverse species and sample types were included in the study, and manual gating with two hierarchical layers was used as the truth for evaluation. For manual gating, results from five researchers were compared to illustrate the gating consistency among different raters. For unsupervised clustering, 22 tools were quantitatively compared in terms of accuracy with the truth and computing cost. While no method outperformed all others, several tools, including PAC-MAN, CCAST, FlowSOM, flowClust, and DEPECHE, generally demonstrated strong performance. For supervised auto-gating methods, four algorithms were evaluated, where DeepCyTOF and CyTOF Linear Classifier performed the best. We further provided practical recommendations on prioritizing gating methods based on different application scenarios. This study offers comprehensive insights for biologists to understand diverse gating methods and choose the best-suited ones for their applications.

bioinformatics↗

bioRxiv2022

Group 3 Innate Lymphoid Cells are regulated by WASP in a microbiota-dependent manner

Wiskott-Aldrich syndrome protein (WASP) is a cytoskeletal regulator that is largely restricted to hematopoietic cells. While WASP expression in both lymphocytes and macrophages play a critical role in maintaining intestinal homeostasis, the function of WASP in innate lymphoid cells is unknown. Here we analyzed the role of WASP in the differentiation and function of group 3 innate lymphoid cells (ILC3s). WASP-deficient mice (Was-/-) have a marked reduction in ILC3s. Moreover, antimicrobial peptide expression in response to ILC3-derived IL-22 was also reduced in the absence of WASP. In Was-/- mice, we observed a reduction in CCR6+ ILC3s, cells known to restrict immune responses to commensal bacteria. WASP-deficient mice were more susceptible to Citrobacter rodentium, an enteric infection controlled by ILC3s. Interestingly, there was no reduction in ILC3s in Was-/- germ-free mice when compared to WT germ-free mice. ILC3s lacking WASP expression also demonstrated microbially-dependent alterations in gene expression associated with cell migration. Finally, ILC3-like (Rorgt+CD3-) cells were reduced in the GI tract of WASP-deficient patients. In conclusion, ILC3-specific expression of WASP is critical for the generation and function of ILC3s in the presence of commensal microflora. Aberrant ILC3 function in the setting of WASP-deficiency may contribute to underlying disease pathogenesis.

immunology↗

bioRxiv2022

Single cell atlas of the neonatal small intestine with necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a gastrointestinal complication of premature infants with high rates of morbidity and mortality. A comprehensive view of the cellular changes and aberrant interactions that underlie this disease is lacking. Here, we combine single cell RNA sequencing, T Cell Receptor beta (TCR{beta}) analysis, bulk transcriptomics, and imaging to characterize cell identities, interactions and zonal changes in NEC. We find that inflammatory macrophages are abundant in NEC and that T cells exhibit increased expression of inflammatory genes and cytokines accompanied by an increase in TCR{beta} clonal expansion. Fibroblasts and endothelial cells increase in proportion and exhibit a switch to an activated pro-inflammatory state. Villus tip epithelial cell identity is substantially reduced in NEC and the remaining epithelial cells up-regulate pro-inflammatory genes. We establish a detailed map of aberrant epithelial-mesenchymal-immune interactions that may be driving inflammation in NEC mucosa. Our analyses highlight the cellular changes underlying NEC disease pathogenesis and identify potential targets for biomarker discovery and therapeutics.

immunology↗

bioRxiv2021

Human placental villi immune cells help maintain homeostasis in utero

Maintenance of healthy pregnancy is reliant on successful balance between the fetal and maternal immune systems. Although maternal mechanisms responsible have been well studied, those used by the fetal immune system remain poorly understood. Using suspension mass cytometry and various imaging modalities, we report a complex immune system within the mid-gestation (17-23 weeks) human placental villi (PV). Further, we identified immunosuppressive signatures in innate immune cells and antigen presenting cells that potentially maintain immune homeostasis in utero. Consistent with recent reports in other fetal organs, T cells with memory phenotypes were detected within the PV tissue and vasculature. Moreover, we determined PV T cells could be activated to upregulate CD69 and proliferate after T cell receptor (TCR) stimulation and when exposed to maternal uterine antigens. Finally, we report that cytokine production by PV T cells is sensitive to TCR stimulation and varies between mid-gestation, preterm (26-35 weeks) and term deliveries (37-40 weeks). Collectively, we elucidated the complexity and functional maturity of fetal immune cells within the PV and highlighted their immunosuppressive potential.

immunology↗

bioRxiv2021

Insulin is expressed by enteroendocrine cells during human fetal development

Generation of beta cells via transdifferentiation of other cell types is a promising avenue for the treatment of diabetes. Here, we reconstruct a single cell atlas of enteroendocrine cells in the human fetal and neonatal small intestine. We identify a subset of fetal enteroendocrine K/L cells that express high levels of insulin and other beta cell genes. Our findings highlight a potential extra-pancreatic source of beta cells and exposes its molecular blueprint.

developmental biology↗

bioRxiv2020

Artificial-Cell-Type Aware Cell Type Classification in CITE-seq

Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq), couples the measurement of surface marker proteins with simultaneous sequencing of mRNA at single cell level, which brings accurate cell surface phenotyping to single cell transcriptomics. Unfortunately, multiplets in CITE-seq datasets create artificial cell types and complicates the automation of cell surface phenotyping. We propose CITE-sort, an artificial-cell-type aware surface marker clustering method for CITE-seq. CITE-sort is aware of and is robust to multiplet-induced artificial cell types. We benchmarked CITE-sort with real and simulated CITE-seq datasets and compared CITE-sort against canonical clustering methods. We show that CITE-sort produces the best clustering performance across the board. CITE-sort not only accurately identifies real biological cell types but also consistently and reliably separates multiplet-induced artificial-cell-type droplet clusters from real biological-cell-type droplet clusters. In addition, CITE-sort organizes its clustering process with a binary tree, which facilitates easy interpretation and verification of its clustering result and simplifies cell type annotation with domain knowledge in CITE-seq.

bioinformatics↗