Lyse fix

To evaluate the in vivo CAR T cells, whole blood and mononuclear cell preparations from tissue biopsies were stained with antibodies labeled with fluorochromes for cytometric analysis. All samples (100 μL whole blood or 10⁶ small lymphocytes from tissue samples or cultured CAR T cells) were initially stained with anti-EGFR (Hu1: Biotin, R&D Systems), and Live/Dead Fixable Aqua Dead Cell Stain Kit (Thermo Fisher) for 30 min. After washing, the samples were stained with surface antibodies 30 min: CD45 (D058-1283: BUV395, BD Biosciences), anti-CD8a (SK1: BUV737, BD Biosciences), streptavidin (BV421, BD Biosciences), anti-CXCR5 (MU5UBEE: Super Bright 600, eBioscience), anti-CCR7 (150503: BV711, BD Biosciences), anti-CD4 (L200: BV786, BD Biosciences), anti-CD95 (DX2: PE, BioLegend), anti-CD28 (CD28.2: PE-DAZZ, BioLegend), anti-PD-1 (J105, PerCP-eFluor710, eBioscience), anti-CCR5 (3A9: APC, BD Biosciences), anti-CD3 (SP34-2, Alexa Fluor700, BD Biosciences), and anti-CD20 (2H7: APC/Fire750, BioLegend). Intracellular staining with anti-Ki67 (B56: FITC, BD Biosciences) was performed for 45 min after lyse/Fix (BD Biosciences) and permeabilizations. Polychromatic (8–14 parameter) flow-cytometric analysis was performed on a LSR II BD instrument as previously described.⁵¹ (link) List mode multiparameter data files were analyzed using the FlowJo software program (Tree Star, v.9.9.6).

Flow cytometry was carried out using a BD Accuri C6 (BD, Franklin Lakes, NJ). Murine BALF cells were incubated with Fixable Viability Stain 510 (FVS510) (BD cat. #564406) to differentiate live and dead cells. This was followed by washing with Stain buffer (BD cat. #554656) and incubation with Lyse Fix (BD cat. #558049). Fixed cells were washed with Stain buffer and aliquoted into two parts. One was incubated with CD11b (BD cat. #553312), CD11c (BD cat. #558079), Ly6G (BD cat. #551461) antibodies and the other aliquot was incubated with CD11c, MHC (BD cat. #558593), and SiglecF (BD cat. #562680) antibodies.

Bronchoscopy was performed under general anesthesia, and BAL fluid (BALF) collected by rinsing the right middle lobe with three aliquots of normal saline. The second and third aliquots were pooled and used for research purposes, as they provided the most reliable yield in volume and BALF cells (2 (link)). Samples were placed on ice immediately after collection. BALF culture was performed as part of routine clinical care in accordance with local guidelines, and results extracted from subjects’ patient records. Remaining BALF was homogenized with a 19G needle, and centrifuged 10 minutes at 800xg to pellet cells. Cells were washed with PBS containing 2.5 mM EDTA, centrifuged, and counted. For flow cytometry, 50.000-200.000 BALF cells were used per staining panel, and fixed with LyseFix (BD Biosciences) prior to acquisition. Antibody panels are listed in Supplementary Table 1.

For each subject, blood was collected by venipuncture into 2 mL sodium heparin tubes at 0, 1, 6, and 12 h post upadacitinib or tofacitinib dose. Recombinant human IL-6 (400 ng/ml), or IL-7 (400 ng/ml), (R&D Systems, Minneapolis, MN) was added to blood and incubated for 10 min at 37°C. Surface antibodies were added (CD14-APC, CD3-fluorescein isothiocyanate [FITC]; BD Biosciences, San Jose, CA) and incubated on ice for an additional 20 min. Samples were lysed (Lyse/Fix, BD Biosciences, San Jose, CA) and incubated for 10 min at 37°C. Samples were washed and stored at − 70°C. For intracellular staining, samples were thawed, washed, and resuspended in BD Perm buffer III (BD Biosciences, San Jose, CA) on ice for 30 min. Samples were washed and stained with pSTAT5-PE or pSTAT3-PE (BD Biosciences, San Jose, CA) for 60 min at room temperature and then analyzed immediately on a FACSCalibur. Geometric means were determined using FlowJo analysis software. Percent inhibition of relevant STAT phosphorylation was calculated as follows: (1-(Induction of pSTAT at 1 h – baseline pSTAT at 0 h) / (Induction of pSTAT at 0 h – baseline pSTAT at 0 h)*100.