delineating-the-metabolic-phenotype-of-biopsy-derived-kidney-cells

Delineating the Metabolic Phenotype of Biopsy-Derived Kidney Cells

Kidney transplantation is the optimal treatment for end-stage kidney disease; however, transplanted kidneys are often lost prematurely, with up to 50% ...

University Health Network

University of Toronto

Tissue-resident immune cells reside in distinct niches across organs, where they contribute to tissue homeostasis and rapidly respond to perturbations in the local microenvironment. Innate lymphoid cells (ILCs) are a family of innate immune cells that regulate immune and tissue homeostasis. Across anatomical locations throughout the body, ILCs adopt tissue-specific fates, differing from circulating ILC populations. Adaptations of ILCs to microenvironmental changes have been documented in several inflammatory contexts, including obesity, asthma, and inflammatory bowel disease. While our understanding of ILC functions within tissues have predominantly been based on mouse studies, development of advanced single cell platforms to study tissue-resident ILCs in humans and emerging patient-based data is providing new insights into this lymphocyte family. Within this review, we discuss current concepts of ILC fate and function, exploring tissue-specific functions of ILCs and their contribution to health and disease across organ systems.

Tissue-Dependent Adaptations and Functions of Innate Lymphoid Cells.

Knowledge of the transcriptional programs underpinning the functions of human kidney cell populations at homeostasis is limited. We present a single-cell perspective of healthy human kidney from 19 living donors, with equal contribution from males and females, profiling the transcriptome of 27677 cells to map human kidney at high resolution. Sex-based differences in gene expression within proximal tubular cells were observed, specifically, increased anti-oxidant metallothionein genes in females and aerobic metabolism-related genes in males. Functional differences in metabolism were confirmed in proximal tubular cells, with male cells exhibiting higher oxidative phosphorylation and higher levels of energy precursor metabolites. We identified kidney-specific lymphocyte populations with unique transcriptional profiles indicative of kidney-adapted functions. Significant heterogeneity in myeloid cells was observed, with a MRC1LYVE1FOLR2C1QC population representing a predominant population in healthy kidney. This study provides a detailed cellular map of healthy human kidney, and explores the complexity of parenchymal and kidney-resident immune cells.

Single-cell profiling of healthy human kidney reveals features of sex-based transcriptional programs and tissue-specific immunity.

Defining the immunological landscape of human tissue is an important area of research, but challenges include the impact of tissue disaggregation on cell phenotypes and the low abundance of immune cells in many tissues. Here, we describe methods to troubleshoot and standardize Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) for studies involving enzymatic digestion of human tissue. We tested epitope susceptibility of 92 antibodies commonly used to differentiate immune lineages and cell states on human peripheral blood mononuclear cells following treatment with an enzymatic digestion cocktail used to isolate islets. We observed CD4, CD8a, CD25, CD27, CD120b, CCR4, CCR6, and PD1 display significant sensitivity to enzymatic treatment, effects that often could not be overcome with alternate antibodies. Comparison of flow cytometry-based CITE-seq antibody titrations and sequencing data supports that for the majority of antibodies, flow cytometry accurately predicts optimal antibody concentrations for CITE-seq. Comparison by CITE-seq of immune cells in enzymatically digested islet tissue and donor-matched spleen not treated with enzymes revealed little digestion-induced epitope cleavage, suggesting increased sensitivity of CITE-seq and/or that the islet structure may protect resident immune cells from enzymes. Within islets, CITE-seq identified immune cells difficult to identify by transcriptional signatures alone, such as distinct tissue-resident T cell subsets, mast cells, and innate lymphoid cells (ILCs). Collectively this study identifies strategies for the rational design and testing of CITE-seq antibodies for single-cell studies of immune cells within islets and other tissues.

Strategies for optimizing CITE-seq for human islets and other tissues.

Natural killer (NK) cell activity is influenced by cytokines and microenvironment factors, resulting in remarkably diverse functions, by contributing to inflammatory responses or serving as rheostats of adaptive immunity. Using single cell RNA sequencing (scRNA-seq), we identified a  CD56NK cell population associated with hematopoietic stem cell transplant recipients protected from acute graft-versus-host disease (GVHD). We further define a role for the combination of interleukin-2 (IL-2) and transforming growth factor β1 (TGF-β1) in promoting a regulatory phenotype in NK cells. "Induced" regulatory NK cells produce high amounts of TGF-β1, inhibited T cells, could promote naive T cells differentiation into regulatory T cells, and exhibited a unique transcriptional program that includes expression of  (HELIOS) and  (HOBIT). This phenotype was not stable, and "induced" regulatory NK cells lost the ability to secrete TGF-β1 upon exposure to different cytokines. These findings define protective CD56NK cells post-hematopoietic stem cell transplantation, and demonstrate the combination of IL-2 and TGF-β1 promotes regulatory activity in NK cells.

Single cell profiling of hematopoietic stem cell transplant recipients reveals TGF-β1 and IL-2 confer immunoregulatory functions to NK cells.

Interleukin-10 (IL-10)-producing group 2 innate lymphoid cells (ILC2) regulate inflammatory immune responses, yet their therapeutic potential remains largely unexplored. Here, we demonstrate that cell therapy with human ILC2 inhibits pathogenic T cell responses in humanized mouse models of graft-versus-host disease (GVHD), resulting in reduced GVHD severity and improved overall survival without limiting the graft-versus-leukemia effect. ILC2 conferred superior protection from GVHD than IL-10 ILC2s, and blocking IL-10 and IL-4 abrogated ILC2 protective effects, indicating that these cytokines are important for the protective effects of ILC2. Notably, ILC2 provided comparable protection from GVHD to regulatory T cells without impairing T cell engraftment, instead decreasing intestinal T cell infiltration and suppressing CD4 Th1 and CD8 Tc1 cells. CITE-seq of expanded ILC2s revealed CD49d and CD86 are markers that allow for enrichment of ILC2 from conventional ILC2s and tracking of ILC2 in patient studies. Altogether, these findings demonstrate the potential of ILC2 in cell therapies for GVHD and other immune-mediated diseases.

Article	Year
Tissue-Dependent Adaptations and Functions of Innate Lymphoid Cells. Frontiers in immunology\| PubMed ID: 35359972	2022
Single-cell profiling of healthy human kidney reveals features of sex-based transcriptional programs and tissue-specific immunity. Nature communications\| PubMed ID: 36496458	2022
Strategies for optimizing CITE-seq for human islets and other tissues. Frontiers in immunology\| PubMed ID: 36936943	2023
Single cell profiling of hematopoietic stem cell transplant recipients reveals TGF-β1 and IL-2 confer immunoregulatory functions to NK cells. iScience\| PubMed ID: 39720529	2024
Cell therapy with human IL-10-producing ILC2s limits xenogeneic graft-versus-host disease by inhibiting pathogenic T cell responses. Cell reports\| PubMed ID: 39721022	2025

Julia M. Murphy

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