Madcam 1

Adhesion assay protocol was adapted from Strazza et al. [28 (link)]. Non-cancer control primary NK cells were isolated from PBMC by magnetic cell sorting using human NK cell isolation kit (Stemcell, Vancouver, British Columbia, Canada) according to manufacturer’s instructions. NK cells were resuspended at a density of 1 × 10⁵/100µL of NK MACS media (Miltenyi Biotec) supplemented with 100 IU IL-2 (Peprotech) and treated with 100 ng IL-15 (Immunotools) and/or 100 ng/mL fractalkine (Peprotech) for 2 and 24 h. NK cells were also treated with OAC patient-derived TCM or ACM for 2 and 24 h with and without pre-treatment with 5 nM CX3CR1 antagonist E6130 (MedChemExpress, Monmouth Junction, NJ, USA) for 1 h prior to exposure to ACM. A 96 well plate was coated with goat-anti-human IgG (Fc specific) in PBS and incubated overnight at 4 °C. Plates were subsequently coated with 2.5 µg/mL MAdCAM-1 (R&D, Minneapolis, MN, USA) for 1 h at 37 °C. NK cells were stained with CFSE and allowed to adhere for 20 min at 37 °C. Unbound cells were washed away. Unwashed wells were used as controls. The percentage of adherent cells was determined by a fluorescent plate reader and calculated as follows: $$\frac{\mathrm{Avg} \mathrm{intensity} \mathrm{in} \mathrm{well}}{\mathrm{Avg} \mathrm{intensity} \mathrm{in} \mathrm{unwashed} \mathrm{well}} \times \frac{100}{1}$$

T cells were magnetically sorted from host mice transplanted from an allogeneic donor. A total of 10⁶ T cells were suspended in 200 μl RPMI and placed in the upper compartment of a 24‐well sterile Transwell device (Corning Life Sciences) that was separated from the bottom well by a 5‐μm filter. The filters of the upper chamber were coated with recombinant mucosal vascular addressin cell adhesion molecule‐1 (MAdCAM‐1; 10 μg/ml; R&D Systems) before incubation. To promote migration, recombinant mouse CCL25 (BioLege) was added to the bottom well (100ng/ml, 1 ml final volume), and incubated at 37°C. After 6 h, the migrated T cells were counted by flow cytometry analysis within the bottom compartment and calculated as the number of T cells that migrated in response to the recombinant mouse MAdCAM‐1.

Plates (96-wells) were pre-coated with murine recombinant MadCAM-1 (R&D Systems, Minneapolis, MN.), fibronectin, collagen, or 5% BSA overnight at 4°C. Then, plates were blocked with 5% BSA in PBS for 1 h a 37°C. Next, 2 x 10⁵ γδT IELs or PMA activated-γδT IEL were seeded and incubated at 37°C for 1 h. After that, non-adhered cells were removed, and plates were washed once with PBS. Then cells were fixed with PFA 4% for 10 min at room temperature. Next, plates were washed and stained with crystal violet for 30 min; plates were washed 5 times and then incubated with 10% SDS for 30 minutes. Finally, plates were read in a 680-microplate reader (Bio-Rad, Hercules CA.) equipment.

For the quantification of dynamic adhesion in the presence of a combination of MAdCAM-1 and VCAM-1, an established protocol was adapted^{34 (link)}. In short, FACS-purified β7⁺ CD4 and CD8 memory T cells were rested overnight in RPMI 1640 with 10% FCS and 1% P/S and subsequently incubated with Cell Trace CFSE (Invitrogen) for 15 min at 37 °C. Rectangle miniature capillaries (CM Scientific) were coated with a combination of 5 µg/ml of each MAdCAM-1- (R&D Systems) and VCAM-1-Fc Chimeras (Biolegend) in capillary coating buffer (150 mM NaCl, 1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) for 1 h and then blocked with 10% FCS in phosphate buffered saline (PBS). Labeled cells were resuspended in adhesion buffer (150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂) at a concentration of 0.75 * 10⁶/ml. Subsequently, 1 mM MnCl₂ was added to the cells and the cell solutions were perfused through the coated capillaries at 10 µl/min using a peristaltic pump (Shenchen). Afterwards, capillaries were rinsed at 50 µl/min to remove non-adherent cells and, finally, capillaries were imaged using an inverted microscope (Leica). Twelve images were acquired per capillary and adhering cells were analyzed using Fiji (National Institute of Health).

Sterile tissue culture plates were coated overnight at 4 °C with 5 μg/mL recombinant P-selectin (R&D Systems, #137-PS-050) or MAdCAM-1 (R&D Systems, #6056-MC-050) proteins. The following day the wells were washed 3X with PBS before being blocked with 1% BSA in PBS at room temperature (RT) for 1 h. Following blocking, the wells were washed as before, and virus stocks were added to the wells for 2 h at RT. For IMC viruses generated through transfection the viral input was normalized by p24 while PBMC and T cell line viruses were used at their undiluted titre. After viral incubation, the wells were washed to remove unbound virus and were either treated with 0.5% triton X-100 in PBS to lyse captured virus or 15,000 TZM-bl cells were added to the wells to detect the ability of captured virus to be transferred to permissive cells. The viral lysates were assayed for p24 and TZM-bl cell luminescence was read 1–4 days later depending on the starting concentration of virus used. In brief, T cell line viruses that were of higher titre were incubated for 1–2 days, whereas viruses produced through transfection or infection of PBMC were incubated for 2–4 days to allow more time for replication.