Ghost dye

Sorted T cell subsets were stimulated with anti-CD3 and anti-CD28 antibodies conjugated to either Dynal beads or microbubbles for 30 min at 37°C with rotation, then placed into 12 well plates at 0.5 × 10⁶ cells/ml and incubated at 37°C. For a standard condition, stimulated cells were harvested at 48 h for hTERT mRNA and telomerase activity measurements. For the 15-day culture experiment, stimulated cells were collected every 3 days for cell count and other analyses including hTERT mRNA, telomerase activity, and cell viability by flow cytometry. Accumulated cell divisions were calculated based on cell counts as the sum of total cell doublings over the course of the culture. Cell viability was determined by dual staining with AnnexinV (Biolegend) and GhostDye (Tonbo Bioscience). Proliferation analysis by CFSE dilution was performed in ModFit (Verity Software House) to calculate proliferation index and cell viability analysis was performed using FlowJo software (FlowJo, LLC). Cell lifespan in culture was determined using cell viability data as follows: death of T cell subsets was defined as the day when the sum of all gates of AnnexinV and GhostDye positive cells reached ≥50%. Kaplan-Meier analysis of T cell subset lifespan was performed in Prism (GraphPad Software).

For macrophages staining, macrophages were pretreated with Fc Receptor Blocking Solution (1:20, BioLegend) for 10 min at room temperature and were stained with surface antibodies and Ghost Dye (1:1000, Tonbo Biosciences) at 4°C for 30 min in dark. Macrophages were also fixed and permeabilized with Fixation/Permeabilization Solution (BD Biosciences) and were stained with anti-CD68 (Y1/82A, BioLegend). For T cell intracellular staining, cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer (eBioscience), and intracellular cytokine/ nuclear transcription factor staining was performed according to the manufacturer’s protocol.
The following monoclonal antibodies (mAbs) were used: FITC anti-CD86 (BU63, BioLegend), PE anti-CD163 (GHI/61, BioLegend), APC anti-CD206 (15-2, BioLegend), and PerCP-Cy55 anti-CD68 (Y1/82A, BioLegend), FITC anti-CD4 (A161A1, BioLegend), PE-Cy7 anti-IFNγ (B27, BioLegend), PerCP-Cy55 anti-IL-17A (BL168, BioLegend), Alexa 647 anti-T-bet (O4-46, BD Biosciences), and PE anti-RORγt (AFKJS-9, BD Biosciences). Appropriately matched isotype control mAb to each antigen-specific mAb was used for control.
The stained cells were immediately analyzed on FACSAria II (BD Biosciences) flow cytometer, and data analysis was performed with the FlowJo software (Tree Star).

Cell surface and intracellular staining were performed using antibodies including CD3 (eFluor 450; eBioscience, Waltham, MA, United States; clone UCHT1), CD8a (FITC, eBioscience, clone RPA-T8; or APC-eFluor 780, eBioscience, clone RPA-T8), TRBV4-1 (PE; Miltenyi Biotec, Bergisch Gladbach, Germany; clone REA871), Ghost Dye (Violet 510; Tonbo Biosciences, San Diego, CA, United States), IFN-γ (PE-Cyanine7, eBioscience, clone 4S.B3), T-bet (BV421; BD Biosciences, San Jose, CA, United States; clone O4-46), Granzyme A (PE-Cyanine7, eBioscience, clone CB9), Granzyme B (FITC, BD eBioscience, clone GB11), Granzyme K (eFluor 660, eBioscience, clone G3H69), PRF1 (BV421, BD Biosciences, clone δG9), CCL4 (PerCP-eFluor 710, eBioscience, clone FL34Z3L), CCL5 (eFluor 660, eBioscience, clone VL1), and CXCR3 (PE-Cyanine7, eBioscience, clone CEW33D). Intracellular staining was performed on T cells stimulated with PMA (50 ng/mL; Sigma-Aldrich, St. Louis, MO, United States) and ionomycin (1 mM; Sigma-Aldrich) in the presence of GolgiStop (2/3 μL/mL, BD Biosciences) and Grid-plug (1 μL/mL, BD Biosciences) for 5 h. Flow cytometry was performed on a BD FACSCanto II flow cytometer using BD FACSDiva Software and FCS Express 5 software (De Novo Software, Los Angeles, CA, United States).

Spleen and lungs were processed and stained as previously described [55 (link),68 (link),69 (link),70 ]. Numbers of live cells were enumerated with Countess II (Thermo Fisher) with trypan blue stain. Two million live cells were seeded in each well, for staining (see Table 1, below). The acquisition of the data was performed by using BD-LSR II (Becton Dickinson), and the analysis was completed with FlowJo 10.0, following standard gating strategy. Statistical significance was calculated by using two-way ANOVA in GraphPrism.
We used fluorescence minus one (FMO), unstained control, and compensation beads controls in each one of our experiments. Dilutions were titrated prior to the assay, to guarantee no unspecific stains.
Our gating strategy included first selecting the single cells population, using Forward scatter height versus forward scatter area density plot for doublet exclusion (FSCH/FSC-A); then, with the selected population, we performed a doublet discrimination (SSCW/SSC-H); and, finally, we cleaned the sidescatter by using SSC-A/SSC-H. We then followed the manufacture’s recommendation to specifically select viable cells, using Ghost dye (Tonbo, 13-0871-T500). From there, we continued our grating strategy by following the criteria shown in Table 2.