Animals

VillinCreERT2 Ap1m2flox/flox mice backcrossed onto C57BL6/J genetic background were bred and maintained under specific pathogen-free conditions in the animal facility of Faculty of Pharmacy, Keio University. VillinCreERT2 Ap1m2flox/flox mice or Ap1m2flox/flox littermate mice were orally administrated 10 mg/ml tamoxifen at 100 µL (1 mg/mouse/day) for 5 consecutive days. After 2 days of interval, the mice were sacrificed by cervical dislocation under deep anesthesia. C57BL6/J mice were purchased from CLEA Japan and Sankyo Laboratory Services (SLC). Generation of Ap1m2 germline knockout mouse was previously described and defined as AP-1B KO mice in the text [8, 9]. All animal experiments were approved by the Animal Studies Committees of Keio University.

Cell preparation

Mouse intestinal tissues were treated with Hank’s Balanced Salt Solutions (HBSS) containing 1 mM dithiothreitol and 20 mM EDTA (all from Nacalai-Tesque, Kyoto, Japan) for 20 min in a shaking incubator (37 °C, 200 rpm) to isolate epithelial cells. Epithelial cells were collected into 50 ml tubes through 100 µm cell strainer and treated the remaining tissue once more with HBSS containing 20 mM EDTA for 20 min. After collecting the epithelial cells into 50 ml tubes through 100 µm cell strainer, we isolated IELs from epithelial cells by centrifuging in 40% percoll (Cytiva, Tokyo, Japan).

For isolation of lamina propria (LP) lymphocytes, the remaining tissues were minced and dissociated with RPMI1640 medium containing, 0.5 mg/ml Collagenase (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 0.5 mg/ml DNaseI (Sigma Aldrich, St. Louis, MO, USA), 2% new born calf serum (NBCS, Thermo Fisher Scientific, Waltham, MA, USA), 100 µg/ml penicillin, and streptomycin, and 20 mM HEPES (Nacalai Tesque) at 37 °C for 30 min. The cell suspensions were then filtered to obtain single-cell suspensions for flow cytometry [27].

Flow cytometry

The monoclonal antibodies used in flow cytometry were showed in Supplementary Table 1. These antibodies were used with Fixable Viability Stain 780 (BD Bioscience). Precision Count Beads (BioLegend) was used for cell counting. The stained samples were analyzed using a FACS LSRII or, a FACS Celesta (BD Bioscience). Data were analyzed with Flowjo software version 10 (BD Bioscience).

Iodixanol density gradient ultracentrifugation

From euthanized AP-1BΔIEC and control mice, a 10 cm long small intestine was cut from just below the stomach. After cutting open and washing off the luminal contents by ice-cold Hank’s Balanced Salt Solutions (HBSS), mouse intestinal tissues were treated with ice-cold HBSS containing 1 mM dithiothreitol and 30 mM EDTA (all from Nacalai-Tesque, Kyoto, Japan) for 10 min. Epithelium was carefully separated from the lamina propria by manipulation with fine needles under microscopic monitoring. The separated epithelium was washed with ice-cold HBSS one time and lysed by nitrogen cavitation, using nitrogen gas and cell disruption vessels (Parr Instrument Company, Moline, IL, USA) [28, 29]. Cell lysate was centrifuged at 3000 rpm for 5 min to remove nucleus and cell debris. Supernatant was ultracentrifuged at 160,000×g for 1 h to concentrate cell membrane. After discarding the supernatant, the pellet was suspended by 25 mM NaCl, 50 mM NaF, 20 mM HEPES 50% iodixanol solution. For iodixanol density gradient formation, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, and 30% iodixanol solutions were prepared by using 25 mM NaCl, 50 mM NaF, 20 mM HEPES [30]. Iodixanol gradient (10–30%) was made in 13 PA tube (Eppendorf Himac Technologies, Ibaraki, Japan) by gently adding the prepared solutions with micro syringe with sharp needle (both from Ito Corporation). The suspended sample was carefully added to the top of the gradient. The gradient was set to the P40ST rotor and himac Ultracentrifuge CP 80NX (Eppendorf Himac Technologies) and centrifuged at 64,000×g on average for 90 min without deceleration. After centrifuge, samples were slowly taken from the rotor, and dispensed the fractionated samples into protein low bind tubes (Eppendorf, Hamburg, Germany).

Western blotting

Separating gel for SDS-PAGE were made from, ×4 separating gel buffer solution, acrylamide/bis solution, TEMED, APS (all from Nacalai Tesque) and distilled water. Stacking gel was made from, ×4 stacking gel buffer solution, acrylamide/bis solution, TEMED, APS (all from Nacalai Tesque) and distilled water. Samples were boiled in ×2 sample buffer with 2-melcaptoethanol (Nacalai Tesque) at 95 °C for 5 min. Samples were separated in created polyacrylamide gels or precast gels (BIO-RAD) and transferred into PVDF membranes. The 5% skim milk in TBS-T or PVDF Blocking Buffer for Can Get Signal (TOYOBO) was used for PVDF membrane blocking, and Can Get Signal Immunoreaction Enhancer Solution (TOYOBO) was used for both primary and secondary antibodies listed in Supplementary Tables 2 and 3. Immunoreactivities were detected by Chemi-Lumi One series (Nacalai Tesque) and Amersham ImageQuant 600 and 800 (Cytiva). Densitometry of band was measured by ImageJ Fiji [31].

TMT-based quantitative proteome analysis

Fractionated samples were collected by trichloroacetic acid (TCA) precipitation. Briefly, 20% TCA was added to samples equivalently and mixed. After 30 min on ice, samples were centrifuged by 15,000×g, 4 °C for 10 min. Supernatants were discarded and pellets were suspended in cold acetone for wash. Pellets were dried and reduced with 10 mM DTT for 30 min, alkylated with 50 mM iodoacetamide for 30 min, and samples were digested with trypsin. Each sample was labeled with the tandem mass tag (TMT) reagents (6 plex, Thermo Fisher Scientific) [32]. After fractionation by strong cation exchange (SCX) chromatography, LC/MS/MS analysis was performed on an Orbitrap Fusion Lumos (Thermo Fisher Scientific) in MS3 mode [32]. Peptides and proteins were identified by means of automated database searching using MaxQuant (ver.1.6.17.0) against UniprotKB/Swissprot release 2017_04 with Trypsin/P specificity allowing for up to 2 missed cleavages. TMT6plex (N-Term), TMT6plex (K) and Carbamidomethyl (C) were set as fixed modifications. Acetyl (Protein N-term) and Oxidation (M) were allowed as variable modifications. 1% false discovery rate (FDR) cutoffs (peptide spectrum match (PSM) and protein) were employed for identifications. The search result was processed with the Perseus ver (1.6.14.0) software for the quantitative analysis. Subcellular location of the proteins obtained was annotated by UniProtR package (ver. 2.2.0) in R software (2022.02.3).

Immunofluorescence analysis

For frozen blocks, samples from mouse intestine were embedded into OCT compound (Sakura Finetech, Tokyo, Japan) without fixation and freeze by liquid nitrogen, and kept in −80 °C deep freezer. Samples were cut into 5 µm sections and dried for overnight. Samples were fixed by 4% paraformaldehyde (PFA) for 10 min or cold acetone in −30 °C for 10 min. Samples were blocked with 5% donkey serum (Merck, Darmstadt, Germany) and added primary antibody showed in Supplementary Table 4 were diluted in 0.2% BSA/PBS. After incubation overnight, samples were washed with PBS for 3 times, and added secondary antibodies diluted in 0.2% BSA/PBS. After 1 h incubation in room temperature, samples were washed with PBS for 3 times and added ProLong™ Gold antifade mountant (Thermo Fisher Scientific) and embedded on cover glass.

For whole mount staining, epithelium from mouse intestine was isolated by stripping with 30 mM EDTA in HBSS. Epithelial samples were fixed by 4% PFA for 30 min and blocked with 5% donkey serum. Samples were stained with antibodies in Supplementary Table 4 diluted in 0.2% saponin/0.2% BSA/PBS. After incubation overnight, samples were washed with PBS for 3 times, and added secondary antibodies diluted in 0.2% BSA/PBS. After 1 h incubation in room temperature, samples were washed with PBS for 3 times with ProLong™ gold antifade mountant (Thermo Fisher Scientific) and embedded on cover glass.

Samples were blocked with 5% donkey serum (Merck, Darmstadt, Germany) and added primary antibody were diluted in 0.2% BSA/PBS. After incubation overnight, samples were washed with PBS for 3 times, and added secondary antibodies diluted in 0.2% BSA/PBS. After one hour incubation in room temperature, samples were washed with PBS for 3 times and added ProLong™ Gold antifade mountant (Thermo Fisher Scientific) and embedded on slide glass. The samples were observed using fluorescence microscope BZ-X810 (Keyence, Osaka, Japan) with BZ-X810 Viewer application or confocal laser microscope FV3000 (Olympus, Tokyo, Japan) with FV31S-SW application. Obtained data were analyzed by ImageJ Fiji. Primary and secondary antibodies are showed in Supplementary Tables 4 and 5.

Preparation of recombinant Fc-fusion proteins

The cDNA was synthesized from total RNA of mouse thymus or intestinal epithelium by RT-PCR reaction (ReverTraAce qPCR master mix, TOYOBO, or SuperScript IV RT, Thermo Fisher Scientific). cDNAs encoding extracellular domain of CD166 and Btnl2 were amplified by PCR using primer pairs listed in Supplementary Table 6. PCR products were cleaved at the restriction enzyme recognition site added to the PCR primers and cloned into pFUSE-IgG1e2-Fc2, which was treated with the corresponding restriction enzyme (InvivoGen, San Diego, CA, USA). The DNA sequence of cloned genes was confirmed by Sanger DNA sequencing (Eurofins Japan). Plasmids were multiplied by E coli DH5α strain, purified by Midiprep system (Promega), and concentrated to 1 mg/mL by ethanol precipitation. Plasmid vector was transfected into Expi293F cell by using ExpiFectamine 293 transfection system (Thermo Fisher Scientific). Fc-fusion proteins were purified from culture supernatant by HiTrap rProtein A FF (Cytiva). Isolated IELs were incubated with purified Fc proteins (0.25 µg/106 cells) for 30 min at 4 °C. After washing, Fc proteins bound to the cell surface were detected by PE-conjugated anti-human IgG Fc antibodies.

Mouse small intestinal organoid culture

Small intestinal organoids were derived from C57BL/6N mice (Clea Japan) according to a previous report [33]. The duodenum was removed from mice and cut into 5-mm segments. The segments were incubated cold 2 mM EDTA in PBS for 5 min and washed by pipetting. The segments were incubated in cold 2 mM EDTA in PBS for 30 min, and crypts were isolated by pipetting with cold HBSS. Dissociated crypts were passed through a 70 µm cell strainer. The crypts were resuspended in DMEM/F12 medium (Themo Fisher Scientific); then, the number of crypts were counted, and the crypts were resuspended in Matrigel (Corning) or Geltrix (Thermo Fisher Scientific). Next, the crypts were plated in a 48-well plate with IntestiCult organoid growth medium (Stemcell Technologies). Media were changed every 2 days.

Lentiviral infection

The sequence for the shRNA was designed by using VectorBuilder. The shRNA target sequence is shown in Supplementary Table 7. The oligo DNA of the target sequence with linker and restriction enzyme recognition site shown in Supplementary Table 8 was first inserted to pENTR4-H1 and then transferred to CS-EF-Rfa-mRFP by using Gateway LR Clonase (Thermo Fisher Scientific). Lenti-X 293T cells were propagated in DMEM containing 10% (vol:vol) FBS, 1% GlutaMAX (Thermo Fisher Scientific), 100 U/ml penicillin, and 100 µg/ml streptomycin (Nacalai Tesque). Lentiviral supernatants were generated by transient transfection of shRNA containing SIN vector (17 µg) or control vector (CS2-EF-mRFP1, 17 µg), pCAG-HIVgp (10 µg), and pCMV-VSV-G-Rev (10 µg) into Lenti-X 293 T (5 × 106/10 ml; Clontech) using Polyethylenimine Max (Polysciences, Warrinton, PA, USA), according to the manufacturer’s protocol. All vectors were kindly provided by Dr. H. Miyoshi and RIKEN Bioresource Research Center. Transfected host cells were incubated for 24 h; after culturing for another 48 h, viruses were purified after removal of host cells and centrifugation. Dissociated organoids were infected by mixing with 250 µl of virus-including medium, followed by centrifugation at 600×g for 1 h at 32 °C. Then, cells were embedded into Matrigel and plated in 48 well plate.

Coculture of IELs and intestinal organoids

Isolated CD8β and CD4 IELs were positively selected by using biotin conjugated anti-mouse CD8b (YTS156.7.7; BioLegendd) and anti-CD4 (RM4-5; BioLegend) and Streptavidin particles. After IEL selection, organoids cultured more than a week were released from Matrigel by using Gentle dissociation buffer (Stem Cell Technologies). 100 organoids and 1 × 105 IELs were mixed in 400 µl of DMEM including 10 µM Y27632 [34]. After 30 min in 37 °C 5% CO2, samples were centrifuged for 1 min at 200 × g. Samples were resuspended in Matrigel or Geltrix. Next, the crypts were plated in a 48-well plate with organoid growth medium supplemented with 10 ng/ml IL-2, 10 ng/ml IL-7 and 10 ng/ml IL-15/Ra complex (all from BioLegend). Media were changed every 2 days.

After 4 days of co-culture, the samples were subjected to immunofluorescence analysis. The samples were fixed with 4% PFA for 20 min and then collected into protein low bind tubes. After three-times washing with PBS and incubation with 5% donkey serum for 15 min, samples were stained with Alexa Fluor 488 conjugated anti-CD45.2 (104; BioLegend) and Alexa Fluor 647 conjugated anti-EpCAM (CO17-1A; BioLegend) and Hoechst 33342 (Molecular Probes, Eugene, OR, USA) in the presence of 0.2% BSA in PBS for overnight at 4 °C. After wash with PBS, samples were suspended in antifade mountant (Thermo Fisher Scientific) and embedded on slide glass. The samples were observed using confocal laser microscope FV3000 (Olympus, Tokyo, Japan). Obtained data were opened with ImageJ software. Interactions between the IEL and the epithelium were quantified by analyzing each confocal image individually. Cells that appeared in the same location across consecutive images were identified as the same cells. The area of the organoids was determined from the projection images.

Quantitative RT-PCR

Total RNA was prepared using TRIzol (Life Technologies) from epithelium isolated from mice or organoids. First-strand cDNA synthesis was completed using the Revertra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). The qPCR reactions were conducted in CFX-connect (Bio-Rad, Hercules, CA, USA) using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). The specific primers used are shown in Supplementary Table 9.

Statistical analysis

Statistical significance between two groups was calculated by Student’s t test, t test Welch’s correction, or Mann–Whitney’s U test. Statistical significance between the control group and other multiple groups was calculated by Dunnet’s T3 multiple comparison test. P < 0.05 indicated statistical significance. Data were output by Prism version 9 (GraphPad, San Diego, CA, USA).

Data availability

The raw MS data and analysis files have been deposited with the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the jPOST partner repository (https://jpostdb.org) with the data set identifier PXD042900 [35].

Supplemental materials

Supplementary Tables 1–9 are primers and antibodies lists. Supplementary Table 10, associated with Fig. 7, is a list of proteins that were changed in AP-1B deficient epithelium. Supplementary Table 11, associated with Fig. 7D, lists basolateral proteins that decreased in AP-1B-deficient epithelium and increased in the apical region.