Efficient Retroviral Transduction and Competitive Homing for Investigating GPCR-Mediated T-Cell Localization in Diverse Tissue Microenvironments

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 migrate to distinct target tissues. The ability of T cells to acquire tissue tropism is vital for effective recall responses, particularly in the context of recurrent infections affecting the same organ^3,4.

GPCRs guide immune cells to specific tissues and organs where they perform critical functions -- such as directing CD8+ T and NK cells to tumor sites for cytotoxic action or aiding CD4+ T cells in orchestrating immune responses by supporting the activation of other immune cells. Understanding how GPCRs direct T cells to their precise locations is essential for advancing targeted immunotherapies^5,6. The challenge, however, lies in modeling these complex interactions in vitro, as replicating both spatially restricted cues and directional chemotactic signals simultaneously is difficult.

Elucidating the roles of specific leukocyte receptors is also often challenging due to their limited frequency of expression in endogenous populations and the fact that these receptors typically decorate distinct cell types. This complexity makes it difficult to isolate the role of a specific receptor from other cell subset-specific mechanisms. Ideally, methods should compare similar populations, differing only in the receptor of interest to provide clear insights.

To overcome these challenges, we have adopted a competitive homing assay that employs recombinant MSCV retroviral transduction for efficient GPCR expression in T cells. MSCV retroviral vectors, which combine elements from the myeloproliferative sarcoma virus (PCMV)-based MESV vectors and the Moloney murine leukemia virus (MMLV)-based LN vectors, incorporate an extended hybrid packaging signal derived from the LN vectors⁷. This modification enhances the efficiency of gene delivery, enabling both short-term and long-term studies of T-cell localization in vivo. By utilizing high-titer retroviral particles and confocal microscopy, the approach allows for precise visualization of T-cell positioning and interactions within complex tissue environments. We present detailed protocols for the retroviral transduction of trafficking receptors and the performance of internally controlled (so-called competitive) homing assays to study receptor-mediated organ- and microenvironment-specific lymphocyte positioning. The overall goal of this method is to provide valuable insights into immune cell trafficking mechanisms and to enable future applications in both basic research and therapeutic development.

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-Ptprc^em6Lutzy/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-type (WT/WT) comparisons to address potential variability. With JAXBoy mice now available as a fully isogenic alternative, these additional controls may no longer be necessary. Researchers should still consider that differences between CD45.1 and CD45.2 variants-such as their roles as protein tyrosine phosphatases-can influence cellular behavior and homing patterns. All protocols discussed in the text and below have been approved or meet the guidelines of the accredited Department of Laboratory Animal Medicine and the Administrative Panel on Laboratory Animal Care at the VA Palo Alto Health Care System (VAPAHCS). Animals were sacrificed using approved procedures. Mice of both sexes, aged 8-12 weeks, were included in the experiments.

1. MSCV vector preparation

1. Purchase MSCV-IRES-Thy1.1 retroviral vector with the coding region of the mouse gene of interest (GOI) or an ORF_Stuffer (negative ORF control)
   NOTE: This construct includes the Thy1.1 surface marker alongside the GPCR gene of interest. The Internal Ribosome Entry Site (IRES) linker allows for the co-expression of the GPCR with the Thy1.1, facilitating the identification of transduced cells by flow cytometry or purification and isolation by magnetic beads. This is particularly useful when antibodies specific to the GPCR are unavailable, as is often the case with poorly studied GPCRs.
2. Procure the MSCV plasmid in bacterial form and culture by inoculating Luria-Bertani (LB) broth supplemented with appropriate antibiotic selection based on the resistance gene encoded by the plasmid (e.g., ampicillin, kanamycin, or chloramphenicol). LB medium consists of tryptone (10 g/L), yeast extract (5 g/L), and NaCl (10 g/L), adjusted to pH 7.0 and sterilized by autoclaving. Incubate the culture at 37 °C in a shaking incubator (220 RPM) overnight.
3. Prepare plasmid stocks using standard molecular biology techniques using a DNA Preparation kit.
4. Following DNA isolation, measure the concentration of DNA with a spectrophotometer and prepare working plasmid solutions at a concentration of 1 µg/µL. Store plasmids at -20 °C for future use.

2. Establishing packaging cell line culture

NOTE: We used Platinum E (Plat-E) cells from Cell Biolabs. Plat-E cells are a 293T-based cell line with an EF1α promoter, which provides a stable and high-yield expression of retroviral structural proteins (gag, pol, and env genes), enabling retroviral packaging with a single plasmid transfection⁸. Although other cell lines, such as NIH-3T3 or 293T, might be used, we have not tested these alternatives.

1. Prepare Plat-E cell media by adding DMEM, 10% FBS, 1% Penicillin/Streptomycin, Blasticidin (10 µg/mL), and Puromycin (1 µg/mL). Use blasticidin and puromycin for cell maintenance but omit them during and after transfection.
2. Plate Plat-E cells shipped frozen in 1.0 mL, at >3 x 10⁶ cells/mL in DMEM, 20% FBS, and 10% DMSO. Thaw them rapidly in a 37 °C water bath. Transfer all of the thawed cells into a 15 mL conical tube containing Plat-E culture medium.
3. Centrifuge at 450 x g for 5 min at 4 °C. Resuspend the cell pellet in 1 mL of Plat-E medium by gently pipetting to create a single-cell suspension.
4. Add 9 mL of Plat-E medium to a 10 cm culture dish, then transfer the 1 mL of resuspended cells into the dish. Confirm that the thawed cells are viable by mixing an equal volume of cell suspension and Trypan Blue and counting the viable (unstained) and dead (blue-stained) cells using a hemocytometer or cell counter, aiming for >70% viability.
5. Incubate the cells at 37 °C in a humidified incubator with 5% CO₂. Do not change the medium for the first 3 days; it is normal to observe floating and dead cells upon the first thaw.
6. When the cells reach 85%-90% confluency, wash with DMEM/PBS Ca-/Mg-. Detach cells using 2 mL of 0.05% Trypsin/0.5 mM EDTA and incubate for 3 min at 37 °C. Add 8 mL of Plat-E medium, collect cells into a 15 mL conical tube, and centrifuge at 450 x g for 5 min at 4 °C.
7. Split at a 1:10 or 1:5 surface ratio (i.e., seed new plates with 1/10 or 1/5 of the total volume from the original dish). Resuspend in a final volume of 10 mL of Plat-E medium and incubate at 37 °C in a humidified incubator with 5% CO₂.
8. Aliquot the rest of the cells in 1 mL of FBS with 10% DMSO and freeze at -80 °C and subsequently in liquid nitrogen for long-term use.
   NOTE: Treat cells as described above when near 85% confluent. To maintain optimal cell health, avoid over-confluency and aim for 3-day splits with 1:10 dilution. If growth slows, use a fresh aliquot. Cell performance typically decreases with subsequent passages. We recommend using cells for transfection between passages 4 and 15 for best results.

3. Production of transduced cells

1. Day 1: Seed Plat E cells
   NOTE: Calculate the number of Plat E cells and plates required. We used 1 mL of viral supernatant per well in a 24-well plate to transfect cells 2x. We typically use approximately two 10 cm Petri dish transfected plates with Plate-E cells per 24-well plate with T cells in culture.
   1. Start by coating 10 cm tissue culture plates with 5 mL of 50 µg/mL poly-D-lysine in sterile water. Incubate at room temperature for 45 min. Wash 2x with sterile PBS Ca-/Mg -- to ensure no residue remains.
      NOTE: We recommend coating the plates as Plat-E cells tend to detach after transfection, leading to poor cell performance and reduced viral titer production.
   2. Detach Plat-E cells as described above in step 2.6 and perform a trypan blue exclusion assay to ensure viability. Mix an equal volume of cell suspension and Trypan Blue. Count the viable (unstained) and dead (blue-stained) cells using a hemocytometer/cell counter. Seed 3 x 10⁶live cells in a 10 cm tissue culture Petri dish with antibiotic-free Plat-E medium.
      NOTE: Seeded plates should reach 85%-90% confluency the next day. If this is not achieved, consider using a different cell aliquot. Adjust the seeding density based on the timing of plating; for evening plating, 3.5 x 10⁶ cells may be used.
2. Day 2: Transfection of Plat-E cells and coating plates for T cells with anti-CD3 and anti-CD28 antibodies
   1. Replace the medium on Plat-E cell cultures with 6.5 mL of reduced serum media.
   2. Prepare the transfection mix according to the manufacturer's instructions for the Lipofectamine reagent (Lipofectamine 2000 was used in this study).
   3. For each plate, mix 45 µL of Lipofectamine 2000 with 210 µL of reduced serum medium in one tube, and 15 µg of DNA with 235 µL of reduced serum medium in another tube. Combine the contents of the tubes by pipetting 3x-4x.
   4. Incubate the mixture for 5-20 min at room temperature to allow Lipofectamine/DNA complexes to form, then add dropwise to the plates. Incubate the plates for 16 h at 37 °C.
   5. T cell activation wells: Coat 24 well plates with 5 µg/mL of anti-mouse CD28 (37.51) and 10 µg/mL of anti-mouse CD3 (145-2c11) in 375 µL/well PBS overnight at 4 °C or for 3-4 h in the incubator at 37 °C on Day 3.
      ​NOTE: Wrap plates in transparent film if incubating overnight to prevent evaporation. Dynabeads mouse activator CD3/CD28 can be used as an alternative with comparable results.
3. Day 3: Change Plat-E media and isolate and activate T cells
   1. Change the Plat-E transfection media to 14 mL of DMEM complete medium in the morning. Prepare T Cell Media by adding RPMI-10: RPMI 1640 with L-glutamine, 10% FBS, 1 % Penicillin/Streptomycin, 1x MEM Non-essential amino acids, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, and 1 mM HEPES.
   2. Euthanize JAXBoy (CD45.1) and C57B6/J (CD45.2) mice using CO₂inhalation followed by cervical dislocation. Isolate spleens and lymph nodes under sterile conditions.
   3. Gently mash spleens on a 100 µm nylon mesh cell strainer with RPMI medium in a 6-well plate using a syringe plunger.
   4. Transfer the solution through a 40 µm nylon mesh cell strainer for a single-cell suspension into a 50 mL conical tube. Centrifuge at 450 x g for 5 min at 4 °C and wash them with PBS Ca-/Mg-.
   5. Perform magnetic negative selection using the Mouse T/T CD4 Isolation Kit following the manufacturer's instructions, or alternatively, sort the cells using sterile FACS
   6. Count the T cells using a cell counter and plate them in the T cell activation wells at 1-1.5 x 10⁶ per well in 1 mL of RPMI-10 medium per well. Incubate the cells at 37 °C with 5% CO₂in a humidified incubator. Allow 24-48 h for proper activation, as assessed under the microscope, as explained in the notes below.
      NOTE: The cross-linking of CD3 and CD28 effectively activates the T cells in this setup. One mouse spleen typically yields approximately 1 x 10⁸ splenocytes. CD4+ T cells generally constitute about 10% of the total splenocyte population. Therefore, to prepare a 24-well plate with 1-1.5 x 10⁶ CD4+ T cells per well, it is estimated that cells from 2-3 mice will be sufficient.
4. Day 4: Transduction
   1. Check the T cells after 24 h under a microscope. Ensure T cells are in a blasting state (forming clusters and appearing enlarged due to activation) before transduction. Blasting T cells ensure they are in a dividing state, which is crucial for MSCV transduction⁹.
   2. Collect ~10 mL of viral supernatant from the 10 cm plates in a conical tube. Replace the Plat-E culture plates with another 10 mL of Plat-E medium without antibiotics to ensure sufficient media to generate more media for a second transduction the next day. Adjust media volume in PLAT-E plates based on the number of T cell wells that need to be transfected the next day.
      NOTE: Transducing 2x significantly improves efficiency.
   3. Filter the viral supernatant by passing through a 0.45 µm syringe filter. Add 8 µg/mL of polybrene and 1:100 HEPES.
   4. Spin the 24-well T cell plate for 7 min at 950 x g. Carefully collect and save the supernatant without dislodging the cells. This supernatant contains cytokines and other factors secreted by the T cells after activation and replaced with this supernatant after the spinfection to maintain the cytokine and factors profile to support T cell proliferation and growth.
   5. Perform spinfection by adding 1 mL of viral supernatant to each well and spinning at 1,150 x g for 4 h at 32 °C. Seal the plates with plastic wrap during spinfection.
   6. After spinfection, carefully replace the media with the previously saved T cell supernatant.
5. Day 5: Repeat transduction
   1. Repeat the transfection as described on Day 4. Optionally, mix the saved supernatant in step 3.4.3 of initially activated T cells with fresh media in a 1:1 ratio if the media appears exhausted.
6. Day 6: Wash cells and expand
   1. Wash cells with PBS Ca+/Mg+. and transfer them to a new culture plate with 130 U/mL of mouse IL2 and 10 ng/mL of mouse IL7. Incubate the cells for at least 2 days or until they reach the desired expansion level, based on the number of cells needed for injection.
7. Day 8: Harvest and purification
   1. Harvest cells and purify with a Histopaque 1.077 density gradient.
      NOTE: This technique helps isolate viable cells (like lymphocytes or other immune cells) from dead cells or debris. Dead cells, which have a higher density, will settle at the bottom of the tube, while viable cells typically stay in the interface layer.
   2. Add 5 mL of Histopaque 1.077 to a 15 mL tube. Harvest cells by centrifuging at 450 x g for 5 min at 4 °C to pellet the cells.
   3. Resuspend the cell pellet in 5 mL of PBS Ca+/Mg+. Layer the cell suspension carefully on top of 5 mL of Histopaque 1.077 in the centrifuge tube.
   4. Centrifuge at 400-500 x g for 20 min at room temperature, ensuring that the centrifuge's break/acceleration is set to zero.
   5. After centrifugation, cells will separate into distinct layers based on density. The desired cells will typically be at the interface layer between the Histopaque and the upper medium, while dead cells will settle at the bottom. Carefully collect the cell layer at the interface layer using a pipette, avoiding contamination with other layers. The collected cells are now ready and clean for injections.
   6. Assess transduction efficiency by performing Thy1.1 staining and flow cytometry analysis. See the gating strategy in Figure 1.
      NOTE: After transduction, cells can be used in chemotaxis assays to assess their migration towards specific chemokines compared to control vector cells to functionally confirm their activity in vitro before proceeding with injections.
8. Long-term in vivo localization assay
   1. For long-term homing, use CD45.1 for the mouse GPCR of interest and CD45.2 for empty vector (or vice versa) transduced cells. Mix the two cell populations at a 1:1 ratio.
   2. Inject intravenously 20-30 x 10⁶ total cells for each Rag1-/- adult recipient mouse for 1 week tropism and 5 x 10⁶ for 7-week long tropism. We use lymphocyte-deficient Rag1-/- recipients to reduce competition with endogenous T cells.
   3. After 1 to 7 weeks (depending on the study), inject anti-CD45 antibody intravenously (i.v.) into the mice 5 min before harvest to label blood-borne cells.
   4. Euthanize the mice using CO₂ inhalation followed by cervical dislocation.
      NOTE: Studies of 1 week and 7 weeks have been conducted; longer studies may be possible but have yet to be tested.
   5. Harvest cells from different organs of interest and controls. Digest the tissues according to standard lymphocyte preparation protocols for each organ^10,11.
   6. Stain the collected cells with monoclonal antibodies (mAbs) for flow cytometric analysis.
9. Short-term competitive homing: T cell positioning and Imaging
   1. We recommend using C57B6/J mice as donors, given that we will be fluorescently labeling the cells. However, for very short homing assays up to a few hours, any strain could be used.
   2. On day 8 of culture, magnetically isolate transduced (Thy1.1+) cells using CD90.1 microbeads, according to manufacturer's instructions¹².
   3. Maintain only transduced cells (>95% purity) in culture after this point for 2 days under IL2 and IL7 as indicated above and allow to expand. The microbeads are biodegradable and after 48 h will not impair normal cell function.
   4. On Day 11, label the cells expressing the GPCR of interest with Carboxyfluorescein succinimidyl ester (CFSE is a fluorescent cell staining dye). For Stuffer cells, use a yellow fluorescent dye, or vice versa. You may use alternative dyes if preferred.
      1. Prepare a cell suspension at a concentration of 1 x 10⁶ cells/mL in RPMI with 2% FBS. Incubate the cells with the dye at a final concentration of 5 µM at 37 °C in a water bath with gentle agitation for 20 min. To ensure comparable results, alternate the dye assignments in separate experiments. Alternatively, use a single dye to label a common cell type as an internal standard, including experimental and control cells in different recipients.
      2. Wash the labeled cells and mix them at a 1:1 ratio. Estimate the cell concentration using a cell counter. Inject 15-30 x 10⁶ cells intravenously into recipient mice (WT or transgenic recipient mice, depending on the purpose of the experiment).
   5. Approximately 10-12 h later, inject the mice with anti-CD31 (e.g., DyLight 633 labeled, clone 390) antibody, which should be 10-15 min before sacrifice to delineate blood vessels and discriminate intravascular from extravasated cells.
   6. Euthanize the mice using CO₂ inhalation followed by cervical dislocation. Analyze cell localization by FACS of cell suspensions as described in step 3.8 above or by imaging tissue whole mounts as in step 3.9.8 or step 3.9.7 for organs of interest using confocal microscopy.
   7. For thin organs such as the trachea, uterine horn, or lymph nodes, prepare squash mounts. Place the tissue on a slide with double-sided tape on the sides, add a few drops of Fluoromount-G mounting solution, cover with a cover slip, and gently and evenly press to fix the coverslip to the tape using a separate slide or other flat object.
   8. For frozen sections, embed the tissue in the optimal cutting temperature (OCT) compound. For lungs, perfuse with 50% OCT/PBS before embedding.
   9. Use flow cytometry for control organs such as peripheral lymph node (PLN), spleen, or blood to assess transduction efficiency (% Thy1.1+) as shown in Figure 1 and normalize the results. Visualize PLN with squash mounts or sections using confocal microscopy. Count cells using confocal microscopy with Imaris software.
   10. Determine the ratio of GPCR transduced cells to control (Stuffer transduced) cells in either whole organs or specific organ compartments. Normalize to input ratios determined by FACS and/or to the ratio recovered from control organs where the GPCR is known or presumed to be irrelevant.
   11. For analysis of microenvironmental localization in the lungs, measure the distance of cells to histological landmarks (e.g., bronchial basement membranes or veins) using Imaris software.

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...

Materials

Name	Company	Catalog Number	Comments
AF647 anti mouse CD90.1-Thy1.1 (OX-7)	Biolegend	202507
anti-CD31 (DyLight 633, clone 390)	InvivoMab	BE0377
anti-mouse CD28 37.51	eBiosciences
anti-mouse CD3 145-2c11	eBiosciences
APCCy7 anti mouse CD3 (145-2c11)	Biolegend	100329
BV421 anti mouse CD8b (Ly-3)	Biolegend	126629
BV711 anti mouse CD4 (RM4-5)	Biolegend	100549
CD90.1 microbeads	Miltenyi	130-121-273
CFSE	Thermoscientific	C34554
FITC anti mouse CD45.2 (104)	BD	AB_395041
mouse IL2	Peprotech	200-02-50UG
mouse IL7	Peprotech	217-17-10UG
Mouse T CD4 isolation kit	STEMCELL technologies	18000
MSCV-IRES- Thy1.1 GPR25	Vectorbuilder
MSCV-IRES- Thy1.1 Stuffer	Vectorbuilder
PE-CD45 (30-F11) antibody	Biolegend	103105
PECy7 anti mouse TCRb (H57-597)	Tonbo
PercpCy5.5 anti mouse CD45.1 (A20)	eBiosciences
Platinum-E (Plat-E)	cell Biolabs. Inc	RV-101
Yellow fluorescent dye	Thermoscientific