Flow cytometry

Stainings of myeloid and lymphoid cells were performed separately. In isolated spleen cells, stainings were performed in 5x10⁶ cells. Stainings of cells isolated from the gonadal AT were performed with the highest number of cells possible, by diving the cell suspension equally between each staining (never exceeding 5x10⁶ cells).

For myeloid staining of surface determinants, cells were incubated for 45 minutes at 25°C in cRPMI, in the presence of 5% normal mouse serum (NMS), with the following antibodies: F4/80-FITC (clone BM8, BioLegend), Ly6G-PerCP/Cy5.5 (clone 1A8, BioLegend), CD274-PE/Cy7 (PD-L1, clone 10F.9G2, BioLegend), CD11b-APC/Cy7 (clone M1/70, BioLegend), CD45-Brilliant Violet (BV) 510 (clone 30-F11, BioLegend) and Ly6C-BV605 (clone HK1.4, BioLegend). To stain non-viable cells, Zombie Violet Fixable Viability Kit (BioLegend) was used, incubating cells in PBS with 5% NMS, for 15 minutes at 25°C. Finally, stained cells were resuspended in cRPMI for flow cytometry acquisition.

For lymphoid staining, cells were first incubated in cRPMI with phorbol 12-myristate 13-acetate (20ng/mL, PMA) and ionomycin (1μg/mL) to stimulate cytokine production by T cells, for 1h45 at 37°C. Next, to block cytokine secretion, brefeldin A (10μg/mL) and monensin (5μM), were added for the final 1h at 37°C of incubation. Staining of surface determinants was performed by incubating cells for 45 minutes at 25°C in cRPMI with 5% NMS and the following antibodies: CD3-FITC (clone 17A2, BioLegend), CD279-PE (PD-1, clone J43, eBioscience), CD45-BV510 (clone 30-F11, BioLegend), CD4-BV605 (clone RM4-5, BioLegend) and CD8-BV711 (clone 53–6.7, BioLegend). To stain non-viable cells, LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Invitrogen) was used, incubating cells in PBS with 5% NMS, for 15 minutes at 4°C. For staining of intracellular antigens, fixation and permeabilization of cells was performed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and the following antibodies: IFN-γ-PerCP/Cy5.5 (clone XMG1.2, BioLegend), Foxp3-APC (clone FJK-16s, eBioscience) and TNF-α-eFluor450 (clone MP6-XT22, eBioscience). Finally, stained cells were resuspended in cRPMI for flow cytometry acquisition.

For staining of B cell surface determinants, cells were incubated for 45 minutes at 25°C in cRPMI, in the presence of 5% normal mouse serum (NMS), with the following antibodies: CD45-BV510 (clone 30-F11, BioLegend), IgD-APC (clone 12-26c, eBioscience) and CD19-APC/Cy7 (clone 6D5, Biolegend). For staining of intracellular antigens, fixation and permeabilization of cells was performed using Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and the following antibody Ki67-PE (clone 16A8, eBioscience). Samples were analysed on a BD LSRFortessa flow cytometer with FACSDiva 6.2 Software. All data were analysed using FlowJo software version 10.0.7r2. A schematic of the gating strategy used is represented in S2 Fig. Immune cell populations normalized to tissue mass and corresponding organ masses are available in the S3 and S4 Files.