In Vivo T Cell Migration Assay

Introduction

In 2021, we published an article reporting the existence of a heteromeric receptor between the dopamine receptor D5 (D5DR) and the chemokine receptor CCR9 on CD4⁺ T-cells. The signalling of this heteromer, which is different from its monomers, promotes the migration to the gut and the infiltration to the colonic lamina propria, mediating intestinal inflammation. This work brought new perspectives into the neuroimmune regulation of the CD4⁺ T-cells, reporting the first ever heteromer in this subset and in the immune system, which can be pharmacologically disrupted by small peptides that alter the lymphocyte migration [1, 2]. Here, we detail our protocol to generate colitogenic (gut-tropic) CD4⁺ T-cells and in vivo migration assays, which were initially adapted from [3].

Obtention of splenocytes

Male or female 10-20 weeks-old D5DR-sufficient and insufficient mice were sacrificed by isoflurane (CP Pharma, Burgdorf, Germany) inhalation for 3 minutes. Then, spleens were collected and mechanically disaggregated in ice-cold MACs Running Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany; hereafter as MACs) using frosted microscopy slides. The suspension cells from spleens were treated with an Ammonium-Chloride-Potassium (ACK: Gibco^TM) lysis buffer for 3 minutes. After, MACs was added to each reaction to stop the erythrolysis and cells were washed by centrifugation at 1400 rpm for 7 minutes at 4ºC. Later, the cell pellets were resuspended in MACs, 70 μm-filtered, and the cellularity was extrapolated from the cell number of 10 mL cell suspension 1:1 diluted with Trypan Blue 0,4% (Gibco^TM) counted using a Neubauer’s chamber.

CD4+ T-cells purification and gut tropism induction

Total CD4⁺ T-cells were untouched-isolated by magnetic separation using CD4⁺ T Cell Isolation Kit mouse (Miltenyi Biotec) following the manufacturer’s guidelines, and flow cytometry checked purity was always >90%. Then, 10⁶cells/mL were cultured in 10% heat-inactivated 0,22 μm-filtered fetal bovine serum (Gibco^TM, Thermo Fisher Scientific) Glutamax-supplemented RPMI 1640 media (Gibco^TM) in the presence of 2-mercaptoethanol (50 μM; Gibco^TM), non-essential amino acids (NEEA, 1X, Gibco^TM), sodium pyruvate (1X; Gibco^TM) and Penicillin-Streptomycin (Pen/strep; 1X; Gibco^TM). To induce gut tropism, retinoic acid (RA; 100 nM; Merck, Darmstadt, Germany) was added together with recombinant mouse IL-2 (20 ng/mL; Biolegend San Diego, CA, USA) and low-endotoxin azide-free anti-CD28 (2 mg/mL; clone 37.51; BioLegend). CD4⁺ T-cells were plated in low-endotoxin azide-free anti-CD3e (2-4 mg/mL; clone 145-2C11; BioLegend) activated wells, generated by three hours incubation at 37ºC with 50 or 250 mL/well depending on the plate surface, and cultured in flat-bottom 96-well (200 mL/well) or 24-well (500 mL/well) plates (NUNC^TM). After two to three days, cells were harvested and washed by centrifugation at 1400 rpm for 7 minutes at 4ºC, and the cell number was determined as above. Using the same media conditions, cells were re-cultured for another two to three days in the presence of RA (100 nM) and rIL-2 (20 ng/mL) on wells previously activated with anti-CD3e/anti-CD28.

In vivo migration assay

Colitogenic D5DR sufficient (CD45.1 or CD45.1.2) and insufficient (CD45.2) CD4⁺ T-cells were harvested, washed and counted, as described above, and 2,5-5 x 10⁶ cells were resuspended in 50mL 1X of Dulbecco’s phosphate buffered saline (DPBS: Gibco^TM). Later, both genotypes were mixed in a ratio of 1:1 and 100mL were transferred to RAG1-deficient mice by the vain tail. An aliquot of the injected cells was immunostained with Zombie Aqua® (BioLegend), anti-a4b7/PE (clone DATK32; ;BioLegend), anti-TCRb/PerCP (clone H57-597; ; BioLegend) anti-CD4/PE-Cy7 (clone GK1.5; ;BioLegend), anti-CCR9/APC (clone eBioCW1.2; ; eBioscience), anti-CD45.2/APC-cy (clone 104; ; BioLegend) and anti-CD45.1 (clone A20; ; BioLegend) and the injected genotype ratio was analysed by flow cytometry as we exemplified in the original publication. After 16 hours, mice were sacrificed, and the spleen, mesenteric lymph nodes and distal large intestine (after cecum) were dissected. Later, suspension cells were obtained from all tissues and immunostained with the same antibody mix used to check the input ratio. The percentage and genotype of the CD4⁺ T-cells in each tissue were determined by flow cytometry, and the migration index was calculated in all of them, as published previously [4]. 

1.         Osorio-Barrios, F., et al., The Heteromeric Complex Formed by Dopamine Receptor D(5) and CCR9 Leads the Gut Homing of CD4(+) T Cells Upon Inflammation. Cell Mol Gastroenterol Hepatol, 2021. 12(2): p. 489-506.

2.         Adams, D.H., More Levels of Complexity in the Control of Intestinal Inflammation. Cell Mol Gastroenterol Hepatol, 2021. 12(2): p. 791-792.

3.         Kurmaeva, E., et al., Roles of T cell-associated L-selectin and beta7 integrins during induction and regulation of chronic colitis. Inflamm Bowel Dis, 2013. 19(12): p. 2547-59.

4.         Villablanca, E.J. and J.R. Mora, Competitive homing assays to study gut-tropic t cell migration. J Vis Exp, 2011(49).