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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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....

Results

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.......

Discussion

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.......

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
AF647 anti mouse CD90.1-Thy1.1 (OX-7)BiolegendΒ 202507
anti-CD31 (DyLight 633, clone 390)InvivoMabΒ BE0377
anti-mouse CD28 37.51eBiosciencesΒ 
anti-mouse CD3 145-2c11eBiosciencesΒ 
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)BDAB_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 GPR25VectorbuilderΒ 
MSCV-IRES- Thy1.1 StufferVectorbuilderΒ 
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 

References

  1. Cheng, L., et al. Structure, function and drug discovery of GPCR signaling. MolBiomed. 4 (1), 46 (2023).
  2. Lammermann, T., Kastenmuller, W. Concepts of GPCR-controlled navigation in the immune system. Im....

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