Anti cd8a bv510

Tissues were digested using the Mouse Tumor Dissociation Kit (Miltenyi) on the gentleMACS device (Miltenyi) according to the manufacturer’s instructions. Red blood cell lysis buffer (BioLegend) was used to remove red blood cells. After washing with PBS, cells were incubated with TruStain fcX anti-mouse CD16/32 receptor blocking agent (BioLegend) diluted in Cell Staining Buffer (BioLegend) for 20 min at 4 °C. After washing, Zombie NIR cell viability dye (1:2,000, BioLegend) was added and incubated for 20 min at 4 °C. To assess immune cell composition, the following antibodies were added for 30 min at 4 °C: for lymphocytes, anti-CD45 PerCP Cy5.5 (30-F11, 1:100), anti-CD3 FITC (17A2, 1:100), anti-CD4 PB (RM4-5, 1:100), anti-CD8a BV 510 (53-6.7, 1:100) and anti-granzyme B AF 647 (GB11, 1:100); for macrophages, anti-CD45 PerCP Cy5.5 (30-F11, 1:100), anti-CD11B PB (M1/70, 1:100), anti-CD11C AF 488 (N418, 1:100), anti-Ly6C AF 647 (HK1.4, 1:100) and anti-Ly6G PE (1A8, 1:100), all from BioLegend. Granzyme B was added after surface staining was completed and after fixation–permeabilization (Fixation Buffer, BioLegend; 10× Intracellular Staining Perm Wash Buffer, BioLegend). Subsequently, samples were washed twice before data acquisition on the BD Aria III flow cytometer. The gating strategy is shown in Extended Data Fig. 3b.

Peripheral blood mononuclear cells (PBMCs) from SLE patients and HCs were isolated from the heparinized fresh blood by Lymphoprep (Stemcell Technologies, Vancouver, Canada). PBMCs obtained were pre-treated with FcR blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) and stained with the following fluorescence-conjugated anti-human antibodies: anti-CD3-PE-Cyanine (Cy) 7 (SK7), anti-CD4-Brilliant Violet (BV) 421 (RPA-T4), anti-CD8a-BV510 (RPA-T8), anti-CD19-APC (HIB19), anti-CD56-FITC (HCD56), anti-CD3-PerCP-Cy5.5 (UCHT1), anti-CD19-BV421 (HIB19), anti-CD20-PE-Cy7 (2H7), anti-CD27-BV510 (O323), anti-IgD-FITC (IA6-2), anti-CD38-APC-Cy7 (HIT2; all from BioLegend, San Diego, CA, USA), and anti-CD226-PE (DX11; Miltenyi Biotec). Isotype control antibodies (BD Biosciences, San Jose, CA, USA) were used to determine the level of background staining. Samples were analyzed with the FACS Aria II flow cytometer (BD Biosciences). Data analysis was performed using the FlowJo Software (Tree Star, Ashland, OR, USA). CD4⁺ T cells (CD3⁺CD4⁺), CD8⁺ T cells (CD3⁺CD8⁺), NK cells (CD3⁻CD56⁺), B cells (CD3⁻CD19⁺), naive B cells (CD3⁻CD19⁺IgD⁺CD27⁻), IgD⁺-memory B cells (CD3⁻CD19⁺IgD⁺CD27⁺), switched-memory (SM) B cells (CD3⁻CD19⁺IgD⁻CD27⁺), and plasmablasts (CD3⁻CD19⁺CD20⁻CD38⁺⁺) were all assessed by flow cytometry.