Nature - USA (2020-09-24)

3

nature research | reporting summary

October 2018

clone L243), anti-rat CD3 (no.201414, Biolegend, APC conjugate, clone 201414), anti-rat CD4 (no.203405, Biolegend, FITC

conjugate, clone 203405).

For Western blotting, the following antibodies were used in this study. anti-mouse phospho-mTOR (Ser2448) (#2971, Cell

signaling), anti-mouse mTOR antibody (#2972, Cell signaling)

This information is summarised in Supplementary Table 1.

Validation All the antibodies used in this study were commercial antibodies and were only used for applications, with validation procedures

described on the following sites of the manufacturers:

https://www.thermofisher.com; https://www.biolegend.com; https://www.cellsignal.com; https://www.sigmaaldrich.com;

https://www.abcam.com; https://www.bdbiosciences.com.

Animals and other organisms

Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals C57BL/6J mice, Balb/C and Jcl:Wistar were purchased from CLEA Japan, INC. (Tokyo, Japan). Ly5.1 mice, Cx3cr1GFP/GFP

transgenic (Cx3cr1gfp) mice, Rag2 knockout (Rag2-/-) mice, Myd88 knockout (Myd88-/-) mice and Foxp3eGFP-Cre-ERT2 mice

were obtained from The Jackson Laboratory (Maine, USA). B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J were provided by

Dr. Hongkui Zeng, Allen Institute for Brain Science. Chrm1/Chrm2/Chrm4 triple-knockout (mAChR TKO) mice were obtained from

Center for animal resources and development (Kumamoto, Japan). Wnt1 promoter/enhancer (Wnt1-Cre) were mated with EGFP

reporter mice (CAG-CATloxP/loxP-EGFP) to obtain Wnt1-Cre/Floxed-EGFP double-transgenic mice.Foxp3eGFP-Cre-ERT2 were

mated with CAG-tdTomato mice to obtain Foxp3eGFP-Cre-ERT2/CAG-tdTomato double-transgenic mice. Experimental animals

were maintained under specific payhogen free (SPF) conditions. Male GF mice (C57BL/6 background strain, 6-weeks old) were

purchased from Sankyo Lab Service Corporation (Tokyo, Japan) and were kept in the GF Facility of Keio University School of

Medicine. Male mice, aged 8 to 12 weeks and weighing 20 to 25 g, were used in all experiments.

Wild animals This study did not involve wild animals.

Field-collected samples This study did not involve samples collected from the field

Ethics oversight Keio University School of Medicine

Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants

Policy information about studies involving human research participants

Population characteristics The study enrolled biomaterials from 3 patients who underwent colon resection.

Recruitment Normal intestinal mucosa was obtained from unaffected areas of patients with colon cancer. All experiments were approved by

the institutional review board of Keio University School of Medicine and written informed consent was obtained from all

patients.

Ethics oversight Keio University School of Medicine

Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry

Plots

Confirm that:

The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).

The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).

All plots are contour plots with outliers or pseudocolor plots.

A numerical value for number of cells or percentage (with statistics) is provided.

Methodology

Sample preparation Isolation of colonic lamina propria mononuclear cells in mice

Lamina propria mononuclear cells (LPMC) isolation was performed as previously described6. Dissected colon mucosa was cut

into 5-mm pieces. Tissue was incubated with Ca2+, Mg2+-free HBSS containing 1 mM DTT (Sigma-Aldrich) and 5 ђM EDTA

(Thermo Fisher Scientific, MA, USA) at 37°C for 30 min, followed by further digestion with collagenase (FUJIFILM Wako

Chemicals, Osaka, Japan) and DNase (Sigma-Aldrich) for 45min. Cells were then separated with a Percoll density gradient (GE

Healthcare, IL, USA). The numbers of live cells were determined by Countess II (Thermo Fisher Scientific).

Isolation of human colonic LP cells

Large intestines were dissected and cleaned in situ of mesenteric fat and connective tissue. The entire large intestine was cut

into 0.5 cm pieces for digestion. These pieces were first washed in HBSS before incubation at 37°C for 20 min in PBS containing 1

mM DTT, and 5 mM EDTA. The supernatant, containing the IEL fraction, was discarded. The remaining LP fraction was then