Pd1 fitc

Single-cell suspensions were stained according to standard protocols and subsequently stained for 30 min at 4°C with the following antibodies (BD Biosciences): TIGIT-FITC, CD226-FITC, PD1-FITC, CD155-PE, CD33-APC, CD34-FITC, CD3-PerCP, CD4-APC, CD8-PE, CD16-APC, CD56-BV421, and CD56-PB450. CD56⁺ NK cells were divided into CD56⁺CD16^– NK (CD56^bright NK) cells and CD56⁺CD16⁺ NK (CD56^dim NK) cells using an anti-human CD16 antibody (24 (link)). After washing the suspension twice, the cells were analyzed by FCM. The fluorescence compensation between channels was adjusted to circle the target cell group, and the FCM data were subsequently analyzed using Cell Quest^TM Pro 4.0.2 software (BD Biosciences).

Cells dissociated from granuloma were collected with manual pipette, incubated with 0.05% Trypsine-0.53 mM EDTA for 15 min at 37 °C to dissociate cells. Cells from five similar wells were pooled, washed and re-suspended in FACS buffer (PBS, 0.5% BSA, and 2 mM EDTA). Cells were then labeled with a mixture of the following fluorescent antibodies: CD45-PerCp CD8-APC-Cy7, CD137-Alexa700 and PD-1-FITC(all from BD bioscience. San Jose, CA, USA). Cells were analyzed on a NovoCyte Quanteon and the data were analyzed using FlowJo Software (TreeStar Ashland, OR, USA, Ver10.7.2).

Different immune cell subpopulations were immunophenotyped with flow cytometry from fresh PB samples with 6 panels of different cell-surface markers, including the following immune checkpoint receptors and cytotoxicity and migration markers: CD3-PerCP-Cy5.5 (BD, catalog 332771), CD4-PE-Cy7 (BD, catalog 560649), CD45-APC-H7 (BD, catalog 560178), CD8-BV510 (BD, 563919), CD56-BV421 (BD, 562751), CXCR1-FITC (BioLegend, catalog 341606), CD16-PE (BD, 561313), TCR γδ-APC (BD, catalog 555718), PD1-FITC (BD, catalog 557860), LAG3-PE (BD, catalog 125209), ICOS-PE-Cy7 (eBioscience, catalog 25-9948-42), CTLA-4–APC (BD, catalog 560938), HLA-DR-BB515 (BD, catalog 560938), CD27-PE (BD, catalog 555441), CD25-PE-Cy7 (BD, catalog 561405), CD11b-APC (BD, catalog 550019), NKG2C-AF488 (R&D Systems, catalog FAB138G), CD161-PE (BD, catalog 556081), NKG2D-PE-Cy7 (BD, catalog 562365), NKG2A-APC (R&D Systems, catalog FAB1059A), DNAM-BB515 (BD, catalog 565152), CD57-PE (BD, catalog 560844), NKp46-PE-Cy7 (BD, catalog 562101), NKp30-AF647 (BD, 558408), CXCR3-AF488 (BD, catalog 561730), CCR7-PE (R&D Systems, catalog FAB197P), CD45RO-PE-Cy7 (BD, catalog 560608), and CXCR4-APC (BD, catalog 560936). CD45⁺ lymphocytes were acquired with the BD FACS Verse, and the data were analyzed with FlowJo, version 10.4. The results are shown in Supplemental Table 1.

PBMCs from patients with SLE or RA and HC were collected and analyzed immediately for the molecular phenotypes of lymphocytes. The antibodies used for the surface marker analysis include anti-human CD3-ECD (Beckman Coulter, USA), CD4-Percp-Cy5.5, CD8-PE-Cy7, PD1-FITC, TIGIT-APC, ICOS-PE, TIM3-BV421, CD19-V500, CD3-FITC, CD8-V500, CD25-APC, HLA-DR- PE-Cy7, CD69-V450, and CD127-PE (BD Biosciences, USA). Briefly, 50 μl of cells was incubated with appropriate antibodies on ice in the dark for 30 min, followed by washing in PBS. All the samples were analyzed with a Navios flow cytometer (Beckman Coulter) and Kaluza analysis software (Beckman Coulter).