A subscription to JoVE is required to view this content. Sign in or start your free trial.
We present improved protocols for retroviral transduction of trafficking receptors and competitive homing to study receptor-mediated organ- and microenvironment-specific lymphocyte positioning. This method offers valuable insights into immune cell trafficking mechanisms and has potential applications in future basic and therapeutic research.
Understanding how G-protein coupled receptor (GPCR) expression affects cell positioning within diverse tissue microenvironments is essential for elucidating immune cell trafficking mechanisms. We present a competitive homing assay designed to study GPCR-mediated T-cell localization to organs expressing their cognate chemoattractant ligands, applicable for both short-term and long-term studies. The approach involves an improved protocol for recombinant murine stem cell virus (MSCV) transduction of T cells to express the GPCR of interest or a control construct, followed by competitive homing in recipient mice. Cell distribution across different organs is analyzed using flow cytometry and/or confocal microscopy. In short-term experiments (10-12 h), confocal microscopy revealed distinct cell localization patterns, including to alveoli, bronchi submucosa, venous sites, and interstitium in the lung, as well as the epithelium lining the trachea, stomach, and uterine horn. In long-term studies (1-7 weeks), flow cytometry provided insights into preferential cell accumulation, revealing dynamic changes and potential maturation or repositioning within tissues over time. This competitive homing assay is a robust tool for studying GPCR-mediated cell positioning, offering valuable insights into tissue-specific distribution and potential applications in immunology and therapeutic research.
G-protein coupled receptors (GPCRs) are fundamental in regulating a variety of cellular processes, including signal transduction, neurotransmission, hormone regulation, and immune cell migration1. They play a crucial role in the spatiotemporal control of lymphocyte migration and localization2. During the priming phase of immune responses, the local microenvironment and cellular interactions prompt T lymphocytes to express a unique set of adhesion molecules and chemokine receptors known as homing receptors. This adaptation enables antigen-experienced T cells to engage with organ-specific endothelial cells (ECs) and migrat....
All mice in this study were maintained in specific pathogen-free (SPF) facilities at the Veterans Affairs Palo Alto Health Care System (VAPAHCS). B6/SJL Prprc Pep3BoyJ (CD45.1), C57B6/J (CD45.2), and Rag1-/- mice were purchased from Jackson Laboratories. While we used PepBoy to obtain CD45.1 cells, we recommend using JAXBoy (C57BL/6J-Ptprcem6Lutzy/J). JAXBoy is a fully coisogenic strain generated through CRISPR instead of traditional backcrossing, which improves genetic consistency. Historically, CD45 allotype-marked studies using PepBoy mice (CD45.1), which are not fully congenic, have included control homing and recirculation assays with wild-typ....
In this study, we present a detailed protocol for investigating the ability of specific receptors to direct T-cell localization in vivo. As a demonstration of this protocol, we used GPR2513. We are able to achieve 30%-40% transduction efficiency using this protocol, as assessed by Thy1.1 staining by flow cytometry. We performed in vitro transwell-based chemotaxis assays using GPR25-transduced cells alongside stuffer controls, testing their migration towards hCXCL17, mCXCL17, and CXCL12 as.......
The internally controlled homing assay outlined in this study is a comprehensive method for examining GPCR-mediated T cell trafficking and positioning within diverse organs and tissue microenvironments. This approach integrates several critical optimizations to enhance reproducibility, accuracy, and efficiency.
A critical aspect of this protocol is the efficient transduction of T cells using MSCV retroviral vectors, which is facilitated by the use of Plat-E cells for viral production. Key opti.......
The authors have nothing to disclose.
Supported by NIH grants R01 AI178113 and R01 AI047822, Grant 1903-03787 from The Leona M. & Harry B. Helmsley Charitable Trust, and Tobacco-Related Disease Research Program (TRDRP) grants T31IP1880 and T33IR6609 to E.C.B.; Y.B. was supported by a Research Fellows Award of the Crohn's and Colitis Foundation of America (835171). B.O. was supported by a postdoctoral fellowship of the Ramon Areces Foundation (Madrid, Spain) and a Research Fellows Award of the Crohn's and Colitis Foundation of America (574148). A.A. was supported by the California Institute for Regenerative Medicine (CIRM) - EDUC2-12677.
....Name | Company | Catalog Number | Comments |
AF647 anti mouse CD90.1-Thy1.1 (OX-7) | BiolegendΒ | 202507 | |
anti-CD31 (DyLight 633, clone 390) | InvivoMabΒ | BE0377 | |
anti-mouse CD28 37.51 | eBiosciencesΒ | ||
anti-mouse CD3 145-2c11 | eBiosciencesΒ | ||
APCCy7 anti mouse CD3 (145-2c11) | BiolegendΒ | 100329 | |
BV421 anti mouse CD8bΒ (Ly-3) | BiolegendΒ | 126629 | |
BV711 anti mouse CD4 (RM4-5)Β | BiolegendΒ | 100549 | |
CD90.1 microbeadsΒ | MiltenyiΒ | 130-121-273 | |
CFSEΒ | ThermoscientificΒ | C34554 | |
FITC anti mouse CD45.2 (104) | BD | AB_395041 | |
mouse IL2Β | PeprotechΒ | 200-02-50UG | |
mouse IL7Β | PeprotechΒ | Β 217-17-10UG | |
Mouse T CD4 isolation kitΒ | STEMCELL technologiesΒ | 18000 | |
MSCV-IRES- Thy1.1 GPR25 | VectorbuilderΒ | ||
MSCV-IRES- Thy1.1 Stuffer | VectorbuilderΒ | ||
PE-CD45 (30-F11) antibodyΒ | BiolegendΒ | 103105 | |
PECy7 anti mouse TCRbΒ (H57-597) | Tonbo | ||
PercpCy5.5 anti mouse CD45.1 (A20) | eBiosciencesΒ | ||
Platinum-E (Plat-E)Β | cell Biolabs. IncΒ | RV-101 | |
Yellow fluorescent dyeΒ | ThermoscientificΒ |
Request permission to reuse the text or figures of this JoVE article
Request PermissionExplore More Articles
This article has been published
Video Coming Soon
Copyright Β© 2025 MyJoVE Corporation. All rights reserved