Flow cytometry
For Cohort 1 GMNC and Cohort 3 thawed PBMC, the cellular subset analysis
and MAIT cell count was performed using a near infra-red live/dead stain
(Invitrogen) and the following mAb: CD3 PE-CF594(UCHT1), CD4-BV650(SK3),
CD8-AF700(SK1), CD38-FITC(HIT2), CD161-PE(HP-3G10), CCR6-BV421(G034E3),
FOXP3(236A) (all BD Biosciences), CD45RO-BV510(UCHL1),
granzyme-B-BV421(QA1828), HLA-DR-BV711(L243) (Biolegend) and TCR
Vα7.2-PE-VIO770(REA175) (Miltenyi Biotec). Granzyme B and FOXP3 staining
was performed using an intracellular and intranuclear permeabilization
kit (ThermoFisher). Isotype control stains were performed using mouse
IgG2aκ, IgG1λ (BD Biosciences), IgG2b (Biolegend), IgG2aκ and REA
control S antibody (Miltenyi Biotec).
For Cohort 2, leukocyte subsets were identified on thawed PBMC using
multiparameter flow cytometry, a near infra-red live/dead stain
(Invitrogen, Carlsbad, CA, USA) and the following mAb:
CD3-PerCPCy5.5(UCHT1), CD4-BUV496(SK3), CD8-BUV-395(G42-8),
CD19-BV785(SJ25C1), CD25-PE-Cy5(M-A251), CD27-APC-R700(M-T271),
CD27-PE-CF594(M-T271), CD38-PECy7(HIT2), CD45RA-BUV-737(HI100),
CD56-APC(VNK75), CD127-BV785(HIL-7R-M21), CD161-APC(DX12),
HLA-DR-BV711(G46-6), HLA-DR-FITC(G46-6), IgD-BV421(IA6-2),
IgM-PE(G20-127) (BD Biosciences, San Jose, CA, USA), IgA-APC(IS11-8E10),
CD161-APC(191B8) (both Miltenyi Biotec) CD45-AF700(HI30), and
CCR6-BV421(29-2L17) (Biolegend, San Diego, CA,USA).
Flow cytometry was performed on a three-laser LSR Fortessa X-20 in the
UK and a five-laser LSR Fortessa X-20 in Sydney (BD Biosciences) using
FACSDIVA v8.0.1 software (BD Biosciences). Compensation was calculated
before each experiment. Approximately 150 000 lymphocytes (50 000 live T
cells) were analysed per colon biopsies and 30 000 lymphocytes (10
000-15 000 live T cells) were analysed per PBMC sample. Gating
strategies for both peripheral blood and gut-derived mononuclear cells
are shown in Supplementary Figure 1. Lymphocyte populations are reported
as a proportion of parent populations. Specifically, live
CD3+ T cells are reported as percentage of total live
lymphocytes (as defined by side-scatter vs forward scatter,
CD45+ and exclusion of viability dye). MAIT cells are
reported as a percentage of live T cells. Plasmablasts are reported as a
percentage of live CD19+ lymphocytes.
CD4+ and CD8+ T cells are reported
as a percentage of live T cells. Activated
(HLA-DR+CD38+) cells are reported as
percentage of memory CD45RO+CD4+ or
CD45RO+CD8+ T cells. Treg are
reported as a percentage of CD4+ T cells.