Mhm 88

PBMCs were first stained with fluorophore-labeled antibodies against cell surface markers CD3 (OKT3, BioLegend), CD19 (SJ25-C1, Thermo Fisher), CD20 (SI Appendix, Table S2), and CD27 (O323, BioLegend), fixed (Lysing Solution, BD Biosciences), permeabilized (Permeabilizing Solution 2, BD Biosciences), and stained with fluorophore-labeled antibodies against IgG (G18-145, BD Biosciences), IgA (8E10, Miltenyi Biotec), IgD (IA6-2, BioLegend), and IgM (MHM-88, BioLegend). The stained cells were acquired on a FACS Canto II flow cytometer (BD Biosciences) and analyzed in FlowJo software v9.9.6 (BD Biosciences).

Lungs were perfused with 20 ml cold PBS, cut into small pieces and incubated with 1 mg ml^–1 collagenase (Thermo Fisher) and 50 U ml^–1 DNase I (Life Technologies) in PBS for 30 min at 37 °C. Samples were filtered through 70-μm nylon strainers, and red blood cells were lysed using 0.83% ammonium chloride before resuspension in FACS buffer (2% FCS and 0.05% sodium azide in PBS). Samples were stained for 30 min at room temperature with fluorescently labelled antibodies to CD45 (BioLegend, 30-F11), B220 (BioLegend, RA3-6B2), GL7 (BioLegend, GL7), CD95 (BioLegend, SA362F7), CXCR4 (BioLegend, L276F12), CD86 (BioLegend, GL-1), TCRβ (BioLegend, H57-597), CD4 (BioLegend, GK1.5), PD-1 (BioLegend, 29F.1A12) or CXCR5 (BioLegend, L138D7) or unlabelled anti-eMLV Env (83A25, in house), anti-mouse IgG (BioLegend, Poly4060), anti-mouse IgA (Southern Biotech, 11-44-2), anti-mouse IgM (BioLegend, RMM-1), anti-human IgG (BioLegend, M1310G05), anti-human IgA (Miltenyi Biotec, 130-114-002) or anti-human IgM (BioLegend, MHM-88), all at a 1:200 dilution in FACS buffer along with Near-IR Live/Dead stain (Thermo Fisher). Samples were run on an LSR Fortessa running BD FACSDiva v.8.0 or a Ze5 analyser running Bio-Rad Everest v.2.4 and analysed with FlowJo v.10. Gating strategies used for the identification of different cell types are shown in Extended Data Fig. 12a.

The stool samples from eight study participants were washed in 1 ml filtered sterile PBS (226 g, 5 min), prior to resuspension of the bacterial pellet in 1% BSA/PBS. Bacteria were then incubated with 10 μl of rat serum (Sigma, USA) at room temperature for 15 min prior to incubation with an anti-human IgM antibody (MHM-88; BioLegend, USA) at 4°C for 30 min. Bacteria were washed twice with filtered sterile PBS prior to resuspension in PBS. IgM-bound (IgM⁺) and unbound bacteria from each sample were sorted using FACSAria (BD Biosciences, USA).