Immunophenotyping

23-parameter flow cytometric analysis was perform on fresh PBMCs and mononuclear cells (10⁶ cells) derived from LN biopsies, BAL, and lung. Immunophenotyping was performed using antihuman monoclonal antibodies (mAbs), which we^62,87–89 and others, including databases maintained by the NHP Reagent Resource (MassBiologics), have shown as being cross-reactive in RMs. A panel of the following mAbs was used for longitudinal T-cell phenotyping in PBMCs: anti-CCR7-BB700 (clone 3D12; 2.5 μL; cat. # 566437), anti-CXCR3-BV421 (clone IC6; 2.5 μL; cat. # 562558), anti-Ki-67-BV480 (clone B56; 5 μL; cat. # 566109), anti-CCR4-BV750 (clone 1G1 1E5; 2.5 μL; cat. # 746980), anti-CD3-BUV395 (clone SP34–2; 2.5 μL; cat. # 564117), anti-CD8-BUV496 (clone RPA-T8; 2.5 μL; cat. # 612942), anti-CD45-BUV563 (clone D058–1283; 2.5 μL; cat. # 741414), anti-CD49a-BUV661 (clone SR84; 2.5 μL; cat. # 750628), anti-CD28-BUV737 (clone CD28.2; 5 μL; cat. # 612815), anti-CD69-BUV805 (clone FN50; 2.5 μL; cat. # 748763), and Fixable Viability Stain 700 (2 μL; cat. # 564997) all from BD Biosciences; anti-CD95-BV605 (clone DX2; 5 μL; cat. # 305628), anti-HLA-DR-BV650 (clone L243; 5 μL; cat. # 307650), anti-CD25-BV711 (clone BC96; 5 μL; cat. # 302636), anti-PD-1-BV785 (clone EH12.2H7; 5 μL; cat. # 329930), anti-CD101-PE-Cy7 (clone BB27; 2.5 μL; cat. # 331014), anti-FoxP3-AF647 (clone 150D; 5 μL; cat. # 320014), and anti-CD4-APC-Cy7 (clone OKT4; 2.5 μL; cat. # 317418) all from Biolegend; anti-CD38-FITC (clone AT1; 5 μL; cat. # 60131FI) from STEMCELL Technologies; and anti-CXCR5-PE (clone MU5UBEE; 5 μL; cat. # 12-9185-42), anti-GranzymeB-PE-TexasRed (clone GB11; 2.5 μL; cat. # GRB17), and anti-CD127-PE-Cy5 (clone eBioRDR5; 5 μL; cat. # 15-1278-42) all from Thermo Fisher. mAbs for chemokine receptors (i.e. CCR7) were incubate at 37°C for 15 min, and cells were fixed and permeabilized for 30 min at room temperature using a FoxP3 / Transcription Factor Staining Buffer Kit (Tonbo Biosciences; cat. # TNB-0607-KIT). A panel of the following mAbs was used for the longitudinal phenotyping of innate immune cells in whole blood (500 μL), as described in⁹⁰, and mononuclear cells (2×10⁶ cells) derived from LN biopsies, BAL, and lung: anti-CD20-BB700 (clone 2H7; 2.5 μL; cat. # 745889), anti-CD11b-BV421 (clone ICRFF44; 2.5 μL; cat. # 562632), anti-Ki-67-BV480 (clone B56; 5 μL; cat. # 566109), anti-CD14-BV605 (clone M5E2; 2.5 μL; cat. # 564054), anti-CD56-BV711 (clone B159; 2.5 μL; cat. # 740781), anti-CD163-BV750 (clone GHI/61; 2.5 μL; cat. # 747185), anti-CD3-BUV395 (clone SP34–2; 2.5 μL; cat. # 564117), anti-CD8-BUV496 (clone RPA-T8; 2.5 μL; cat. # 612942), anti-CD45-BUV563 (clone D058–1283; 2.5 μL; cat. # 741414), anti-CCR2-BUV661 (clone LS132.1D9; 2.5 μL; cat. # 750472), anti-CD16-BUV737 (clone 3G8; 2.5 μL; cat. # 564434), anti-CD101-BUV805 (clone V7.1; 2.5 μL; cat. # 749163), anti-CD169-PE (clone 7–239; 2.5 μL; cat. # 565248), and anti-CD206-PE-Cy5 (clone 19.2; 20 μL; cat. # 551136) and Fixable Viability Stain 700 (2 μL; cat. # 564997) all from BD Biosciences; anti-ACE2-AF488 (clone Polyclonal; 5 μL; cat. # FAB9332G-100UG) from R & D; anti-HLA-DR-BV650 (clone L243; 5 μL; cat. # 307650), anti-CD11c-BV785 (clone 3.9; 5 μL; cat. # 301644), and anti-CD123-APC-Fire750 (clone 315; 2.5 μL; cat. # 306042) all from Biolegend; anti-GranzymeB-PE-TexasRed (clone GB11; 2.5 μL; cat. # GRB17) from Thermo Fisher; anti-CD66abce-PE-Vio770 (clone TET2; 1 μL; cat. # 130-119-849) from Miltenyi Biotec; anti-NKG2A-APC (clone Z199; 5 μL; cat. # A60797) from Beckman Coulter. mAbs for chemokine receptors (i.e. CCR2) were incubated at 37°C for 15 min, and cells were fixed and permeabilized at room temperature for 15 min with Fixation/Permeabilization Solution Kit (BD Biosciences; cat. #554714). For each sample a minimum of 1.2×10⁵ stopping gate events (live CD3⁺ T-cells) were recorded. All samples were fixed with 4% paraformaldehyde and acquired within 24 hours of fixation. Acquisition of data was performed on a FACSymphony A5 (BD Biosciences) driven by FACS DiVa software and analyzed with FlowJo (version 10.7; Becton, Dickinson, and Company).