Macsquant16

Tumors were harvested, mechanically cut into small pieces, and then enzymatically treated with 1 mg/mL collagenase D (IV type, Clostridium histolyticum, Sigma) and 0.02 mg/mL DNase I (from bovine pancreas grade 2, Roche) at 37 °C for 30 min. The enzymatic reaction was stopped by adding EDTA (Sigma). The ensuing single-cell suspension was filtered by means of a 70-µm cell strainer and prepared for cytofluorimetric analysis. After Fc blocking, single-cell suspensions were stained with the following antibodies from Miltenyi Biotec: CD45-VioGreen (clone: REA737); NK1.1-APC (clone: PK136); CD11b-Vioblue (clone: REA592); CD3-PE (clone: REA641); CD69-PEVio770 (clone: REA937); Ly6C-PE (clone: 1G7.G10); CD4-PEVio770 (clone: REA604); CD8-Vioblue (clone: 53.6.7); and F4/80-PercPVio700 (clone: REA126). Subsequently, cell viability was determined by LIVE/Dead-633 nm (L10120) staining according to the manufacturer’s instructions (Invitrogen); negative cells were considered viable. Doublet exclusion and gating on live CD45⁺ cells were performed. The following subpopulations of CD45⁺ were identified: CD11b⁺ Ly6C⁺ (monocytes), CD11b⁺ F4/80⁺ (macrophages), CD3⁺CD4⁺ and CD3⁺C8⁺ (T cells) and NK1.1⁺CD3⁻ (NK cells). Cells were analyzed on a MACSQuant16 (Miltenyi) and analyzed with FlowJo software (RRID:SCR_008520, Treestar) [28 (link)].

For characterization of infiltrating leukocyte populations two to three tumor slices were transferred onto a prewetted 70 µm cell strainer (Miltenyi Biotec) and mechanically dissociated with the rubberized part of a 3 ml syringe. After the cells have been rinsed through with 10 ml PBS, they were pelleted, and the supernatant was discarded. Single cell suspensions were stained with a live/dead discriminating dye (Zombie Aqua™ Fixable Viability Kit). After blocking human Fc receptors with Human TruStain FcX™, cells were stained extracellular with the following antibody cocktail: mouse α-human CD3 BV650 (clone OKT3), mouse α-human CD45 FITC (clone HI30), mouse α-human CD8α PerCP-Cy5.5 (clone RPA-T8), mouse α-human CD4 PE-Cy7 (clone OKT4), mouse α-human CD25 AlexaFlour700 (clone BC96) and mouse α-human CD69 APC/Fire700 (clone FN50). After fixation and permeabilization (True-Nuclear™ Transcription Factor Buffer Set) and another Fc receptors blocking step, cells were stained with the following antibody cocktail against intracellular proteins: rat α-human Granzyme B BV421 (clone QA18A28), mouse α-human Perforin PE (clone dG9) and mouse α-human FoxP3 AlexaFluor647 (clone 150D). All antibodies and reagents used for staining were purchased from Biolegend. Samples were measured on a MACSQuant16 (Miltenyi Biotec), analyzed using FlowJo (Tree Star Inc) and displayed with GraphPad Prism.

Donor cells were counted with the NucleoCounter NC-200 (Chemometec, Lillerod, Denmark). Dead cells were stained with Zombie Aqua for 10 min in the dark. Surface staining of cells was done at 4°C for 10 min with anti-human antibodies in PBS supplemented with 2% FBS and 0.01% sodium azide (FACS Buffer). Cells were then washed once with FACS buffer before analyzing. For the intracellular cytokine assay, cells were stained with anti-CD107a overnight during the period of activation. Then cells were washed once with PBS before staining with Zombie Aqua (BioLegend, San Diego, CA) and washed again with FACS buffer before surface staining as described above. Next cells were fixed and permeabilized with 1X eBiosciences Fixation/Permeabilization solution. Intracellular staining was performed with antibodies in 1X eBioscience Permeabilization Buffer. Cells were then washed twice with FACS Buffer and analyzed. For the flow-based killing assay, nuclear RFP expressed endogenously was used to detect tumor cells and CellTrace Far Red was used to detect PBMCs. Cells were analyzed on a MacsQuant10 or MacsQuant16 flow cytometer (Miltenyi Biotec). Data was analyzed with FlowLogic software (Inivai Technologies, Mentone, Australia).

All flow cytometry assays were carried out using the MACSQuant 16 analyzer (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany). The total number of events per sample was 20,000 in each experiment. Blue (488 nm), violet (405 nm), and red (640 nm) lasers were used. Cell cycle results were collected using a 579/34 PE B2 filter. The fluorescence results of Annexin V, MitoTrackerTM, and H₂-DCFDA were collected using a 525/50 FITC B1 filter. Fluorescence results of MitoSOXTM were collected using a V4 channel with 615/20 filter.

Pseudovirus production and neutralization assays were performed as described previously [17 (link)]. Briefly, either pseudovirus expressing coronavirus spike proteins variants of interest (B.1.1.7, B.1.351, P.1, B.1.617.2, BA.1, BA.5, SARS-CoV-1) (Elbe and Buckland-Merrett et al, 2017) [74 (link)], or full-length SARS-CoV-2 engineered to express eGFP [75 (link)], were applied for analysis. Plasma samples were heat inactivated at 56°C for 45min prior to analysis; Eight 5-fold dilutions in DMEM with 10%FBS and 50U/mL P/S (cDMDM) of plasma was mixed with virus at MOI = 0.01 and incubated at 37°C for 1 hour before addition of Vero TRMPSS2 SARS-CoV-2 [15 (link)] (Mycoplasma tested) permissive cells and further incubated for 24 hours at 37°C. Final total plasma dilutions rested ranged from 1:25–1:1953125. All antibodies were initially screened as supernatants at single concentration at 20ug/ml and 0.8ug/ml, after which full neutralization curves were performed on purified lead antibody candidates, in 5-fold dilutions in duplicates as described for plasma samples. The final total concentrations tested ranged from 517ug/ml– 0.256ng/ml. Cells were fixed in 1% PFA for at least 15 min at 4°C, prior to analysis of cell eGFP expression on a Miltenyi Biotec MACSquant16 flow cytometry.

After transduction, CAR T cells were activated with CD3/CD28 antibodies (ImmunoCult, StemCell, Vancouver BC, Canada) for 48 h. Then, cells were spinoculated with eGFP-HIV MOI 0.1 at 1250× g for 2 h and incubated with virus overnight. Infection was analyzed as eGFP expression by flow cytometry (MACSQuant 16, Miltenyi, Bergisch Gladbach, Germany).