Software version x

Immunophenotyping was performed on PBMCs before expansion and T cells on day 28. Cells were washed in complete medium, and 2 × 10⁵ cells were re-suspended in the 100 μl PBS containing 3% Bovine Serum Albumin (Sigma, USA). Then. The cells were incubated with 5 μl of Anti-Human CD3-PerCP (Miltenyi Biotec, Germany), Anti-Human CD4-FITC/CD8-PE (BD Biosciences, USA), Mouse IgG2a Isotype PerCP (Miltenyi Biotec, Germany), Mouse IgG1 Isotype-FITC (BD Biosciences, USA), and Mouse IgG1 Isotype-PE Biosciences, USA) at 4 °C in the dark. Cells were analyzed with BD FACSCalibur™ Flowcytometer (BD Biosciences, USA) for cell surface markers and the data was processed using FlowJo software version X (FlowJo LLC, USA).

The procedure to isolate RFP-labeled alveolar cells has been previously published in our lab [19 (link)]. In brief, after harvested lungs were dissociated in dispase and Collagenase Type IV at 37 °C for 40 min. with frequent agitation, single-cell suspensions were passed serially through 100-, 70- and 40-μm cell strainers (BD Biosciences). Red blood cells were eliminated using RBC lysis buffer (Sigma‐Aldrich), according to the manufacturer's protocol. Cells were then pelleted and resuspended in FACS buffer (0.1% sodium azide, 5% fetal calf serum (FCS), 0,05% in PBS) before being stained with antibodies: anti‐EpCAM (APC-Cy7‐conjugated, Biolegend,1:50), CD49F (APC‐conjugated, Biolegend,1:50), and anti‐PDPN (FITC‐conjugated, Biolegend, 1:20) for 20 min on ice in the dark, followed by washing. Next, cells were washed and stained with SYTOX (Invitrogen) according to the manufacturer’s instructions, to eliminate dead cells. Finally, flow cytometry and cell sorting were conducted using a FACSAria III cell sorter (BD Biosciences). Data were analyzed using FlowJo software version X (FlowJo, LLC).
Flow cytometry values were used in the figures. Significance was determined by unpaired two-tailed Student’s t tests. All data are presented as mean ± SEM. Values of p < 0.05 were considered significant. The number of independent samples (n) can be found in the figures.

The MACS^® Separator Kit was used to deplete CD45- and CD31-positive cells from lung single cell suspensions prepared above following recommended procedures by the manufacturers. Briefly, cell suspensions were centrifuged, with the supernatant completely aspirated before adding 100 μL of CD45 and/or CD31 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) per 10 million cells in MACS buffer. The cells were gently mixed and incubated for 15 min at 4 °C, then washed in 1 mL of buffer, and resuspended in 500 μL of MACS buffer before applying on the column. Next, flow-through unlabeled cells were collected and centrifuged to pellet the cells and resuspended in 500 μL of MACS buffer containing anti-EpCAM (APC-conjugated, 1:50; Biolegend, San Diego, CA, USA) for 45 min on ice in the dark. APC-conjugated rat IgG2a (Biolegend, 1:50) was used as the isotype control. Flow cytometry analysis and data acquisition were carried out using the ACEA NovoCyte flow cytometer. Data were analyzed using FlowJo software version X (FlowJo, LLC, Ashland, OR, USA).

Mouse blood and brain tissue were processed for flow cytometry as described [46 (link)]. Fc receptors were blocked by previous incubation for 10 min with CD16/CD32 (clone 2.4G2, BD Pharmingen) in FACS buffer (PBS, 2 mM EDTA, 2% FBS) at 4 °C. Live/dead Aqua cell stain (Molecular Probe, Invitrogen) was used to determine the viability of cells. Cells were incubated with the following mix of primary antibodies: CD11b (clone M1/70, APC-Cy7, BD Pharmingen), CD45 (clone 30-F11, Brilliant Violet 786, BD Horizon), Ly6G (clone 1A8, PE-Cy7, BD Pharmingen), F4/80 (clone BM8, Brilliant Violet 605, Biolegend), CD115 (clone AFS98, APC, Biolegend), CD3 (clone 17A2, violetFluor 450, Tonbo Biosciences), CD45R (clone RA3-6B2, Alexa fluor 488), Ly6C (clone HK1.4, eFluor 450, eBioScience), CD161 (NK1.1, clone PK136; PerCP/Cy5.5, Tonbo Biosciences) and CD335 (NKp46, clone 29A1.4, PerCP/Cy5.5, BD Pharmingen). Data was acquired in a BD LSRII cytometer using the FacsDiva software (BD Biosciences, San Jose, CA, USA). Data analyses were performed with FlowJo software (version X, FlowJo LLC, Ashland, OR, USA).