# Flow Cytometry

## Plots

Confirm that:

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

[x] 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).

[x] All plots are contour plots with outliers or pseudocolor plots.

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

## Methodology

**Sample preparation**

PBMC were isolated by density gradient centrifugation from whole blood samples and stored at -196 °C. Thawed PBMC were washed in PBS, and surface stained for 30 min with saturating concentrations of different combinations of antibodies in the presence of fixable live/dead stain. For ICS, cells were then fixed and permeabilized for detection of intracellular antigens.

**Instrument**

LSR Fortessa and LSRII (BD Biosciences)

**Software**

FlowJo v10.5 and v10.7

**Cell population abundance**

Sort of peripheral blood cells:

M37 410,000 Tn; 840,000 Tcm; 360,000 Ttm; 540,000 Tem

M80 230,000 CD4+ T cells; 550,000 Tn; 420,000 Tcm; 310,000 Ttm; 330,000 Tem

M94 520,000 CD4+ T cells

M102 340,000 CD4+ T cells

M111 380,000 CD4+ T cells

M114 2,100,000 CD4+ T cells

Purity of CD3+ CD4+ cells >95% as determined by flow cytometric analysis.

Sort of tissue cells:

M37 ileum 25,000 LPL; rectum 54,000 LPL

M51 lymph node 1,500 Tfh cells

M77 duodenum 215,000 LPL; ileum 105,000 LPL; rectum 83,000 LPL

No purity assessments performed.

See also Ext. Data Table 2 for cell population abundance (= cells used in the PCR assays).

**Gating strategy**

Cells were distinguished from debris based on FCS-A and SSC-A. Doublets were excluded based on FSC-A and FSC-H. Live cells were distinguished from dead cells by negative selection using LIVE/DEAD Fixable Aqua Dead Cell Stain kit (Thermo Fisher Scientific) or Zombie NIR fixable viability dye (BioLegend).

T cells and T-cell subsets were defined based on expression of CD3, CD4, CD8, CCR7, CD45RA, CD45RO, CD27, TCRγδ, CD161 and TCR Vα7.2. NK cells and NK cell subsets were defined based on expression of CD16 and CD56. See also supplementary data for gating strategy.

[x] Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.