7 aad viability dye

All patients were HLA class I genotyped to determine eligibility for CD8 T cell specific MCPyV-tetramer screening (Bloodworks Northwest, Seattle, WA). PBMC collected from patients with HLA class I (HLA-I) types that corresponded to available MCPyV-specific tetramers (A*02:01, A*24:02, B*07:02, B*35:02, or B*37:01; n = 17 patients) were analyzed without knowledge of patient viral status. PBMC was analyzed using a previously optimized and standardized HLA-I tetramer staining protocol as follows: PBMC (> 2 × 10^6) at baseline and 12 weeks after starting therapy were stained with anti-CD8-FITC antibody (Clone 3B5, Life Technologies), 7-AAD viability dye (BioLegend), and appropriate APC or PE-labeled tetramers (Immune Monitoring Lab, Fred Hutchinson Cancer Research Center) and data collected on a FACSAriaII (BD Biosciences). FlowJo version 10.0.8 (TreeStar) was used for analysis and determination of the percentage of live cells in the tetramer, CD8, double positive region. Samples with > 0.01% of CD8+ T cells co-staining with tetramers were considered positive. For patients with tetramer(+) T cells, all subsequent PBMC obtained on trial were also analyzed.

PBMC were thawed at 37°C, transferred into pre-warmed complete RPMI10 culture medium, pelleted by centrifugation, resuspended in RPMI10, and left to rest at 37˚C for 1.5 h. Cells were stained with a cocktail of cell surface antibodies in FACS buffer (PBS + 2% FCS) for 30min at room temperature (RT) in the dark. Cells were washed and resuspended in FACS buffer, 7AAD viability dye (Biolegend) was added, and cells were acquired on a BD FACSymphony A5 flow cytometer (BD Biosciences). Data were analysed by FlowJo V10 (BD Biosciences). The following anti-human flow cytometry antibodies were used: TCR-gd-FITC (B1, Biolegend), CD19-PerCP-Cy5.5 (HIB19, Biolegend), CD14-PE (M5E2, Biolegend), CD56-PE-CF594 (NCAM16.2, BD Biosciences), CCR7-PE-Cy7 (G043H7, Biolegend), PD-1-AF700 (EH12.2H7, Biolegend), CD45RO-APC-Cy7 (UCHL1, BD Biosciences), CD127-BV421 (A019D5, Biolegend), CD28-BV480 (CD28.2, BD Biosciences), CD20-BV605 (L27, BD Biosciences), CD16-BV650 (3G8, BD Biosciences), HLA-DR-BV711 (L243, Biolegend), CD4-BV786 (SK3, Biolegend), CD3-BUV395 (UCHT1, BD Biosciences), CD8-BUV496 (RPA-T8, BD Biosciences), CD25-BUV737 (2A3, BD Biosciences).

Processed cells were stained for flow cytometry analysis, as previously described (76 (link)). Briefly, isolated cells were washed in PBS, resuspended in staining buffer (PBS/1% FCS/0.1% NaN₃) and aliquots of 100 μL were dispensed into the wells of a 96-well round-bottom plate. Cells were incubated with anti-CD16/CD32–specific mAb (101320/93, BioLegend) for 30 minutes on ice to block FcγRΙΙΙ/ΙΙ receptors. The cells were then stained with 7-AAD viability dye (420404, BioLegend) to gate live cells. Cells were then stained with a panel of antibodies to lymphoid or myeloid cell surface markers (BioLegend) for 30 minutes on ice. The lymphoid panel included CD3-FITC (catalog/clone 100204/17A2), CD4-APC (catalog/clone 116014/RM4-4), CD8-APC-Cy7 (catalog/clone 100714/53-6.7), and CD19- PE-Dazzle 594 (catalog/clone 115554/6D5) antibodies. The myeloid panel included CD11b–Alexa Fluor 488 (catalog/clone 101217/M1-70), Ly6G-BV605 (catalog/clone 127639/1A8), NK1.1–PE-Dazzle 594 (catalog/clone 108748/PK136), CD11c–Alexa Fluor 700 (catalog/clone 117320/N418), and MHC class II–APC-Cy7 (catalog/clone 107628/M5/114.15.2). All antibodies were pretitrated in preliminary experiments and used at saturating concentrations. After washing, cells were analyzed on a BD FACSCanto II (BD Biosciences) and analyzed using BD FACSDiva software (BD Biosciences).

POP92 has been previously validated to contain a population of AC133 positive CICs^{25 (link)}. To isolate the AC133 positive CIC population, POP92 spheroids were mechanically and enzymatically dissociated with 0.25% trypsin, washed well and strained to single cell suspension, then stained with AC133 conjugated to Alexa-488 fluorophore (Miltenyi Cat# 130-105-225) and 7-AAD viability dye (Biolegend Cat# 420404). Live, singlet cells were FACS sorted using a BD FACSAria into the top 10% of AC133^high cells (CIC-enriched fraction), and the bottom 20% AC133^low cells (low CIC fraction).

M1 and M2 HMDMs and THP-1 macrophages were dissociated using enzyme-free cell dissociation buffer (13,151,014, Gibco) and scraping. Macrophages were stained with cell surface marker antibody or corresponding isotype control listed in Supplementary Table 1. Cells were resuspended in FACS buffer with 7-AAD viability dye (420,404, BioLegend) and incubated for 10 min at room temperature prior to data acquisition. Data were collected using an Attune NxT flow cytometer or Bio-Rad S3e flow cytometer and analysis was performed using Kaluza or FlowJo. Live macrophages were gated as 7-AAD negative cells. Delta median fluorescence intensity (MFI) was calculated as MFI of positive stained cells minus MFI of the isotype control stained cells.

Analysis of MLN, ILN, spleen, and tumor cells was carried out using multi-color flow cytometry, following our standard protocol (22 (link), 27 (link)). The following antibodies (all purchased from Biolegend, San Diego, CA, USA) were used in the current study: anti-CD45-APC (Cat# 103112), anti-CD19-PE (Cat# 115508), anti-CD19-PE-Texas Red (Cat# 115554), anti-CD3-BV785 (Cat# 100232), anti-CD4-FITC (Cat# 100509), anti-CD8-APC-Cy7 (Cat# 100714), anti-CD8-APC (Cat# 100712), anti-CD11b-Alexa Flour-488 (Cat# 101217), anti- CD11c-PE (Cat# 117308), anti- Ly6G-BV605 (Cat# 127639), Ly-6A/E (Sca-1)-PE-Texas Red (Cat# 108138), anti-MHC II (I-A/I-E)-BV785 (Cat# 107645), anti-MHC I H-2K^d -BV421 (Cat# 116623). Non-viable cells from tumors were excluded using 7-AAD viability dye (Biolegend) and non-viable cells from spleens, MLNs, and ILNs were excluded using Zombie Aqua dye (Biolegend). Data were collected on 10,000-50,000 cells (depending on the organ) using a FACSCelesta flow cytometer (BD Biosciences, Mountain View, CA, USA) and analyzed using FlowJo software (BD Biosciences).

M1 and M2 HMDMs and THP-1 macrophages were dissociated using enzyme-free cell dissociation buffer (13,151,014, Gibco) and scraping. Macrophages were stained with cell surface marker antibody or corresponding isotype control listed in Supplementary Table 1. Cells were resuspended in FACS buffer with 7-AAD viability dye (420,404, BioLegend) and incubated for 10 min at room temperature prior to data acquisition. Data were collected using an Attune NxT flow cytometer or Bio-Rad S3e flow cytometer and analysis was performed using Kaluza or FlowJo. Live macrophages were gated as 7-AAD negative cells. Delta median fluorescence intensity (MFI) was calculated as MFI of positive stained cells minus MFI of the isotype control stained cells.