Il 4 apc

Cells in BAL fluid were stained with CD3 FITC, CD11c PercP, Siglec F Alexa 647, CD11b PE-Cy7, viability dye APC Cy7 (all BD Biosciences, San Jose, CA, USA), Ly6G Alexa700 (Biolegend, San Diego, CA, USA), MHCII PE, and CD45 PE-eFluor610 (eBiosciences, San Diego, CA, USA) in the presence of Fc blocker (CD16/CD32, eBiosciences). Single cell suspensions from lungs were stained with CD4 FITC, CD45 PerCP-Cy5.5 (eBiosciences), GATA-3 Alexa 647, and viability dye APC-Cy7 (BD Biosciences). The following markers were used for the analysis of ILCs in lung tissue: Lineage (Lin) markers including CD3e, CD19, GR1, B220, Ter119, FcaR1 (all FITC, Biolegend), CD45 Alexa700, CD90 PE, ST2 Brilliant Violet 421 (Biolegend), CD49b PE-Cy7 (eBiosciences) and CD3 Percp-Cy5.5 (BD biosciences). Mediastinal lymph nodes (mLN) cells were stained with CD45 PerCP-Cy5.5, CD4 FITC, GATA-3 Alexa 647 (BD Biosciences) and IL-4 APC (Biolegend). For intracellular/intranuclear staining, cells were permeabilized and fixed using a FOXp3 Staining Buffer set (eBioscience) and subsequently stained with the appropriated markers. All appropriate Fluorescence Minus One (FMO) controls were used. Data were collected on a BD Biosciences Canto II flow cytometer or BD FACSAria™ III and analyzed using FlowJo software (Treestar, Palo Alto, CA, USA).

Cell surface and intracellular molecular expressions were evaluated by flow cytometry using CytoFLEX (BeckmanCoulter, U.S.A.). Fluorescein-conjugated mouse anti-human antibodies were used, including CD3-Alexa Fluor 700, CD3-BV650, CD8-BV786, CD8-PerCP/Cy5.5, CD45RA-APC/CY7, CCR7-PE/CY7,CD27-PE, CD28-BV421, CD69-APC/CY7, CD103-BV605, CXCR3-BV510, HLA-DR-APC, CD39-BV421, PD-1-PE, CD127-PE/CY7, CD62L-PE/CY7, Perforin-APC, Granzyme B-PE, IFN-γ-PE, and IL-4-APC (Biolegend, UK). For cell-surface staining, single-cell suspensions were stained on ice for 30 min in PBS with 1% fetal bovine serum (FBS). For intracellular staining, cells were fixed and permeabilized using the Fix/Perm kit (BD Biosciences, U.S.A.). To detect intracellular cytokines, CD8⁺T cells were stimulated for 6 h with phorbol 12-myristate 13-acetate (PMA; 1 μg/mL; Sigma) and ionomycin (1 μg/mL; Sigma), and 4 h with GolgiStop (1 μL/mL; BD Biosciences) in a round-bottom 96-well plate. Thereafter, cells were harvested, stained for surface expression, and then fixed and permeabilized for intracellular staining. Flow cytometry data was analyzed using FlowJo software (BD, UK) and CytoExpert software (Beckman Coulter, U.S.A.).

Anti-mouse CD16/32 (clone: 93), CD3-PE-Cy7 (clone: 17A2), CD4-BV510 (clone: RM4-5), CD8α-Percp/Cy5. 5 (clone: 53-6. 7), B220-FITC (clone: RA3-6B2), B220-APC (clone: RA3-6B2), IgE-FITC (clone: RME-1), CD11b-APC (clone: M1/70), Ly-6G-PE (clone 1A8), Ly-6C- Percp/Cy5. 5 (clone: HK1. 4), CD11c-BV421 (clone: N418), Siglec-F-FITC (clone: S17007L), CD25-PE (clone: PC61. 5), CD44-APC-Cy7 (clone: IM7), PD1-FITC (clone: 29F. 1A12), CXCR5-BV421 (clone: L138D7), and IL4-APC (clone: 11B11) antibodies were purchased from BioLegend. Anti-STING (clone: 41) antibody, DNAse I from bovine pancreas, and collagenase D were purchased from Sigma-Aldrich (Tokyo, Japan).

On day 8 after transplantation, the spleens of the aGvHD mouse model were crushed through 70-μm screens and the erythrocytes lysed. Single-cell suspensions were incubated in PBS containing the following fluorescently labeled antibodies: CD3 (PE), CD4 (APC/Cy7), and CD8 (BV510) (all from BioLegend, San Diego, CA, USA) at 4°C for 20 min. Apoptosis was assessed using PE Annexin V Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ, USA). Chimerism was assessed by H2Kb (FITC; BioLegend) positive population. For intracellular cytokine staining, the cells were stimulated with cell stimulation cocktail plus protein transport inhibitors (BioLegend) for 4 h. Cells were then harvested and washed. After surface staining of CD3, CD4, and CD8, the cells were fixed and permeabilized using a fixation and permeabilization kit (BD IntraSure Kit) and stained for the intracellular cytokines IFNγ (PerCP) and IL4 (APC) (both from BioLegend). The CD4⁺CD25⁺Foxp3⁺ population assay was carried out using a mouse regulatory T cell (Treg) staining kit (eBioscience, San Diego, CA, USA). Data were acquired on a FACS Canto II (BD) system and were analyzed using FlowJo software.

For functional assays on cytokine production by T cells, thawed isolated PBMCs were stimulated for 16 h at 37 °C in a 5% CO2 atmosphere with anti-CD3/CD28 (1 μg/mL) in complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum and 1% each of l-glutamine, sodium pyruvate, nonessential amino acids, antibiotics, 0.1 M HEPES, and 55 μM β-mercaptoethanol). For each sample, at least 2 million cells were left unstimulated as negative control, and 2 million cells were stimulated. All samples were incubated with a protein-transport inhibitor containing brefeldin A (Golgi Plug, Becton Dickinson) and previously titrated concentration of CD107a-PE. After stimulation, cells were stained with LIVE-DEAD Aqua (ThermoFisher Scientific) and surface mAbs recognizing CD4 AF700, and CD8 APC-Cy7 (Biolegend, San Diego, CA, USA). Cells were washed with stain buffer and fixed and permeabilized with the cytofix/cytoperm buffer set (Becton Dickinson) for cytokine detection. Cells were next stained with previously titrated mAbs recognizing CD3 PE-Cy5, IL-17 BV421, TNF BV605, IFN-γ FITC, IL-4 APC, or granzyme-B BV421 (all mAbs from Biolegend). Then, a minimum of 100,000 cells per sample were acquired on a Attune NxT acoustic cytometer (ThermoFisher)^{53 (link)}. FCS data were acquired in list mode by Attune nxT Software v4.2. mAbs used are listed in Sup Data 6.