Rare cell populations can be challenging to characterize using microfluidic single-cell RNA sequencing (scRNA-seq) platforms. Typically, the population of interest must be enriched and pooled from multiple biological specimens for efficient collection. However, these practices preclude the resolution of sample origin together with phenotypic data and are problematic in experiments in which biological or technical variation is expected to be high (e.g., disease models, genetic perturbation screens, or human samples). One solution is sample multiplexing whereby each sample is tagged with a unique sequence barcode that is resolved bioinformatically. We have established a scRNA-seq sample multiplexing pipeline for mouse retinal ganglion cells using cholesterol-modified oligos. We utilized the enhanced precision of this dataset to investigate cell type distribution and transcriptomic variance across retinal samples. Additionally, we demonstrate that our multiplexed dataset can be useful for the identification of multiplets in non-labeled samples, a common challenge in scRNA-seq analysis.
© 2024 The Author(s).

Atypical chemokine receptors (ACKRs) scavenge chemokines and can contribute to gradient formation by binding, internalizing, and delivering chemokines for lysosomal degradation. ACKRs do not couple to G-proteins and fail to induce typical signaling induced by chemokine receptors. ACKR3, which binds and scavenges CXCL12 and CXCL11, is known to be expressed in vascular endothelium, where it has immediate access to circulating chemokines. ACKR4, which binds and scavenges CCL19, CCL20, CCL21, CCL22, and CCL25, has also been detected in lymphatic and blood vessels of secondary lymphoid organs, where it clears chemokines to facilitate cell migration. Recently, GPR182, a novel ACKR-like scavenger receptor, has been identified and partially deorphanized. Multiple studies point towards the potential coexpression of these 3 ACKRs, which all interact with homeostatic chemokines, in defined cellular microenvironments of several organs. However, an extensive map of ACKR3, ACKR4, and GPR182 expression in mice has been missing. In order to reliably detect ACKR expression and coexpression, in the absence of specific anti-ACKR antibodies, we generated fluorescent reporter mice, ACKR3GFP/+, ACKR4GFP/+, GPR182mCherry/+, and engineered fluorescently labeled ACKR-selective chimeric chemokines for in vivo uptake. Our study on young healthy mice revealed unique and common expression patterns of ACKRs in primary and secondary lymphoid organs, small intestine, colon, liver, and kidney. Furthermore, using chimeric chemokines, we were able to detect distinct zonal expression and activity of ACKR4 and GPR182 in the liver, which suggests their cooperative relationship. This study provides a broad comparative view and a solid stepping stone for future functional explorations of ACKRs based on the microanatomical localization and distinct and cooperative roles of these powerful chemokine scavengers.
Copyright: © 2023 Melgrati et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

FOXD1+ cell-derived stromal cells give rise to pericytes and fibroblasts that support the kidney vasculature and interstitium but are also major precursors of myofibroblasts. ZEB2 is a SMAD-interacting transcription factor that is expressed in developing kidney stromal progenitors. Here we show that Zeb2 is essential for normal FOXD1+ stromal progenitor development. Specific conditional knockout of mouse Zeb2 in FOXD1+ stromal progenitors (Zeb2 cKO) leads to abnormal interstitial stromal cell development, differentiation, and kidney fibrosis. Immunofluorescent staining analyses revealed abnormal expression of interstitial stromal cell markers MEIS1/2/3, CDKN1C, and CSPG4 (NG2) in newborn and 3-week-old Zeb2-cKO mouse kidneys. Zeb2-deficient FOXD1+ stromal progenitors also took on a myofibroblast fate that led to kidney fibrosis and kidney failure. Cell marker studies further confirmed that these myofibroblasts expressed pericyte and resident fibroblast markers, including PDGFRβ, CSPG4, desmin, GLI1, and NT5E. Notably, increased interstitial collagen deposition associated with loss of Zeb2 in FOXD1+ stromal progenitors was accompanied by increased expression of activated SMAD1/5/8, SMAD2/3, SMAD4, and AXIN2. Thus, our study identifies a key role of ZEB2 in maintaining the cell fate of FOXD1+ stromal progenitors during kidney development, whereas loss of ZEB2 leads to differentiation of FOXD1+ stromal progenitors into myofibroblasts and kidney fibrosis.

Regulatory T cell (Treg) lymphatic migration is required for resolving inflammation and prolonging allograft survival. Focusing on Treg interactions with lymphatic endothelial cells (LECs), we dissect mechanisms and functional consequences of Treg transendothelial migration (TEM). Using three genetic mouse models of pancreatic islet transplantation, we show that Treg lymphotoxin (LT) αβ and LEC LTβ receptor (LTβR) signaling are required for efficient Treg migration and suppressive function to prolong allograft survival. Inhibition of LT signaling increases Treg conversion to Foxp3loCD25lo exTregs. In a transwell-based model of TEM across polarized LECs, non-migrated Tregs become exTregs. Such conversion is regulated by LTβR nuclear factor κB (NF-κB) signaling in LECs, which increases interleukin-6 (IL-6) production and drives exTreg conversion. Migrating Tregs are ectonucleotidase CD39hi and resist exTreg conversion in an adenosine-receptor-2A-dependent fashion. Human Tregs migrating across human LECs behave similarly. These molecular interactions can be targeted for therapeutic manipulation of immunity and suppression.
Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

Gene therapy using recombinant adeno-associated virus (rAAV) vectors to treat blinding retinal dystrophies has become clinical reality. Therapeutically impactful targeting of photoreceptors still relies on subretinal vector delivery, which detaches the retina and harbours substantial risks of collateral damage, often without achieving widespread photoreceptor transduction. Herein, we report the development of novel engineered rAAV vectors that enable efficient targeting of photoreceptors via less invasive intravitreal administration. A unique in vivo selection procedure was performed, where an AAV2-based peptide-display library was intravenously administered in mice, followed by isolation of vector DNA from target cells after only 24 h. This stringent selection yielded novel vectors, termed AAV2.GL and AAV2.NN, which mediate widespread and high-level retinal transduction after intravitreal injection in mice, dogs and non-human primates. Importantly, both vectors efficiently transduce photoreceptors in human retinal explant cultures. As proof-of-concept, intravitreal Cnga3 delivery using AAV2.GL lead to cone-specific expression of Cnga3 protein and rescued photopic cone responses in the Cnga3-/- mouse model of achromatopsia. These novel rAAV vectors expand the clinical applicability of gene therapy for blinding human retinal dystrophies.
© 2021 The Authors. Published under the terms of the CC BY 4.0 license.