CD3+CD4+ T cells were sorted from single-cell suspensions of mLNs stained with a viability dye (Tonbo) and antibodies to CD45.2, CD3, and CD4 (positive stain) and CD19 and CD11b (negative stain) (BioLegend), using a BD FACSAria Flow Cytometer (BD Biosciences). Freshly isolated CD4+ T cells were then shipped to SingulOmics (Bronx, NY), where they were subjected to scRNAseq using the 10X Genomics (Pleasanton, CA, USA) Chromium platform. Data were analyzed by Mayo Clinic biostatisticians; the 10X Genomics Cell Ranger Single Cell Software Suite (v2.0.2) was used to perform FASTQ reads alignment to the mm10 mouse genome, filtering, barcode counting, and unique molecular identifier (UMI) counting. For subsequent analyses, we followed the standard integrated analysis workflow in the Seurat package (v2.3.4).21 (link), 22 (link) Genes expressed in fewer than three cells and cells expressing fewer than 200 genes and >35% mitochondrial genes were excluded from downstream analysis in each sample. Each dataset was preprocessed using log-normalization and scaled for each gene across all cells. All datasets were integrated, scaled, and clustered on the low-dimensional space. Enriched gene markers in a cluster, conserved across two experimental conditions, were identified, and differentially expressed genes within each cluster between the two conditions were detected.
Viability dye
Manufactured by Cytek Biosciences
The Viability dye is a laboratory reagent used to assess the viability of cells in a sample. It is a key tool for flow cytometry applications, providing a simple and accurate method to differentiate between live and dead cells.
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Lab products found in correlation
Cd19 car detection reagent, by Miltenyi Biotec (2 mentions)
Anti human cd8 bv785, by BioLegend (2 mentions)
Anti human cd62l bv421, by BioLegend (2 mentions)
Anti human cd25 viobright fitc, by Miltenyi Biotec (2 mentions)
Anti human tigit pe, by BioLegend (2 mentions)
Anti human cd3 apc h7, by BD (2 mentions)
Facsaria fusion, by BD (2 mentions)
Anti human cd27 pe cf594, by BD (2 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Ionomycin, by Cytek Biosciences (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Brefeldin a, by Cytek Biosciences (1 mentions)
70 m strainer, by Thermo Fisher Scientific (1 mentions)
Efluor 506, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Collagenase a, by Roche (1 mentions)
Celltrace cfse, by Thermo Fisher Scientific (1 mentions)
6 protocols using viability dye
1
Transcriptional Profiling of CD4+ T Cells
2
CD8+ CAR T Cell Sorting and TCR Profiling
3
Lung Cell Isolation and Characterization
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Lungs were perfused with 5mL PBS via right ventricular puncture, extracted and maintained on ice in PBS with 0.7 mg/mL Collagenase A (Roche, Cambridge MA), 30 μg/mL DNase I (Sigma-Aldrich) and 2% FBS. Lung tissue was dissociated into a single cell suspension using a gentleMACS dissociator (Miltenyi Biotec, Somerville, MA) according to the manufacturer’s instructions. Lung cells were filtered through a 70 µm strainer (Thermo Fisher Scientific) and counted in Trypan Blue (Sigma-Aldrich) via hemocytometer (eFluor 506, Thermo Fisher Scientific). Cells were stained with viability dye (eFluor 506, Thermo Fisher Scientific), fixed and permeabilized using the FoxP3 kit (eBioscience San Diego, CA) according to the manufacturer’s instructions prior to staining for flow cytometry. In some cases, cells were stimulated in a commercial cocktail of phorbol 12-myristate 13-acetate, ionomycin, brefeldin A and monensin (Tonbo Biosciences, San Diego CA) for 4 hours prior to viability dye, fixing, permeabilization, and staining.
4
T Cell Suppression Assay Protocol
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nT reg cells were FACS sorted from pooled spleens and LNs of 3–4-wk-old WT GFP and ΔCNS0,3 male littermates or mixed bone marrow chimeric mice of WT GFP and ΔCNS0,3 described in Fig. 5 C . CD4 Tn cells were sorted from the spleens of CD45.1 mice and stained with CellTrace CFSE according to the manufacturer’s manuals (Thermo Fisher Scientific). Antigen-presenting cells were prepared from the splenocytes of male CD45.1 mice by depleting CD90.2+ T cells and TER-119+ red blood cells followed by lethal irradiation (20 Gy). T reg cell suppression assay was conducted in 96-well U-bottom plates, each well containing 200 µl complete RPMI1640 supplemented with 1 µg/ml anti-CD3 antibody, 105 antigen-presenting cells, and 4 × 104 CD4 Tn cells. T reg cells were added at different ratios to CD4 Tn cells. Cells were harvested 3 d later for flow cytometric analysis after being stained with viability dye (Tonbo Biosciences) and antibodies against CD4 and CD45.1.
5
Treg Cell Suppression Assay
6
Sorting of Effector and Precursor CAR T Cells
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Cryopreserved GMP product cells were thawed at 37°C and washed. Cells were then stained with the CD19-CAR detection reagent (Miltenyi; cat. #130-115-965) for 10 minutes at room temperature followed by staining with an antibiotin antibody conjugated to APC (Miltenyi; cat. #130-110-952), a cocktail of antibodies (CD3, CD8, CD27, CD62L, CD25, and TIGIT), and a viability dye (Tonbo Biosciences; cat. #13-0870-T100). After staining for 10 minutes at room temperature, the cells were washed twice and resuspended in FACS buffer for sorting on the FACSAria Fusion (BD Biosciences). CD8+ CAR T cells with either the predicted effector surface profile (TIGIT+, CD62Llo, and CD27−) or the opposite, noneffector precursor profile (TIGIT−, CD62L+, and CD27+) were sorted into complete RPMI media. Cells were lysed with TRIzol for bulk TCR repertoire sequencing.
Antibodies used were anti-human CD3-APC-H7 (BD Pharmingen; cat. #560176), anti-human CD8-BV785 (BioLegend; cat. #344740), anti-human CD27-PE-CF594 (BD Horizon; cat. #562297), anti-human CD62L-BV421 (BioLegend; cat. #304828), anti-human CD25-VioBright FITC (Miltenyi Biotec; cat. #130-113-283), and anti-human TIGIT-PE (BioLegend; cat. #372703).
Antibodies used were anti-human CD3-APC-H7 (BD Pharmingen; cat. #560176), anti-human CD8-BV785 (BioLegend; cat. #344740), anti-human CD27-PE-CF594 (BD Horizon; cat. #562297), anti-human CD62L-BV421 (BioLegend; cat. #304828), anti-human CD25-VioBright FITC (Miltenyi Biotec; cat. #130-113-283), and anti-human TIGIT-PE (BioLegend; cat. #372703).
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