Celltrace violet

Freshly thawed PBMC from CMV seropositive and seronegative donors were stained with CellTrace™ Violet and stimulated with CMV antigen in growth medium in the presence of neutralizing mouse monoclonal antibodies (mAb) anti-CTLA-4 (Biolegend; clone L3D10) and/or anti-PD-1 (Biolegend; clone A17788B). After 6 days of incubation, CellEvent™ Caspase-3/7 Green Detection Reagent was added to PBMC cultures 30 min, followed by Zombie Yellow™ viability dye, followed by anti-CD3 Alexa-Fluor 700 (eBiosciences) and anti-CD4 PC 5.5 (Beckman Coulter) for 30 minutes at room temperature. Lastly, cells were washed with Annexin V buffer diluted to 1X (eBiosciences) followed by Annexin V APC (eBiosciences). Cells were analyzed using a Gallios Flow Cytometer.

For in vivo suppression studies, 3×10⁵ Miltenyi (negative selection) enriched CD4⁺ and FACS sorted (>98% purity) CD45RB^high YFP⁻ cells from LNs and spleens of Foxp3^YFP-Cre/Cre mice were i.v. injected into the tail vein of Rag1 KO mice with or without 1×10⁵ Miltenyi (negative selection) enriched CD4⁺ and FCAS sorted (>98% purity) YFP^bright Treg cells from LNs and spleens of Foxp3^YFP-Cre/+ x CARMA1^{f/f (or f/+ or +/+)} x Rosa26^YFP mice.
For in vitro suppression studies, 1×10⁴ FACS sorted (>98% purity) CD4⁺ YFP⁻ conventional T cells from LNs and spleens of Foxp3^YFP-Cre/Cre mice were labeled with 5 μM CellTrace Violet and stimulated with 250 ng/ml of αCD3 mAb (145–2c11, Biolegend) in presence of 2.5 × 10⁴ T-cell depleted splenocytes and different concentrations (from 1:1 to 1:16) of Miltenyi (negative selection) enriched CD4⁺ and FACS sorted (>98% purity) YFP^bright Treg cells from LNs and spleens of Foxp3^YFP-Cre/+ x CARMA1^{f/f (or f/+ or +/+)} x Rosa26^{STOP f/f-YFP} mice. CD4⁺ YFP⁻ conventional T cell proliferation was read out after 72h, as previously described.^{41 (link)} Briefly, percentage of suppression was scaled from 0 (proliferation of conventional T cell in absence of Treg) to 100 (complete absence of proliferation).

MLNs were digested to generate single cell suspensions as described above and MLN DCs were isolated from each mouse using EasySep Mouse Pan-DC Enrichment Kit (Stem Cell Technologies #19763), counted by hemacytometer, normalized for cell concentration, and plated at 7.5e4 cells/well of a round-bottom TC plate. OT-II cells were isolated from pooled spleens and peripheral lymph nodes of OT-II mice using EasySep Mouse Naïve CD4⁺ T cell isolation kit (Stem Cell Tech #19765), labeled for 20min in 5μM CellTraceViolet (Biolegend #425101), and plated at 2e5 cells/well with 100ng/mL OVA. On day 3 of co-culture, T cell proliferation was assessed by flow cytometry.

Primary AML cells, HL-60 cells, and THP-1 cells were cultured in the presence or absence of 1 μM all-trans retinoic acid (Sigma Aldrich) overnight. The following day, targets were removed from culture, counted, and labeled with CellTrace Violet (Thermo Fisher) per the manufacturer’s instructions. Labeled targets were then co-cultured with expanded peripheral blood NK cells or iADAPT NK cells thawed immediately from cryopreservation at a 2:1 E:T ratio in the presence or absence of daratumumab (10 μg/ml) for 5 hours. Cells were then stained for Live/Dead Near IR (Thermo Fisher) and analyzed by flow cytometry. A fresh bone marrow aspirate was obtained from a relapsed MM patient, and cells were stained with CellTrace Violet and a fluorescently conjugated antibody against CD138 (281-2; Biolegend). Bone marrow aspirate cells were then co-cultured with expanded peripheral blood NK cells, non-transduced iNK cells, or iADAPT iNK cells in the presence or absence of daratumumab (10 μg/ml) overnight. The percentages of live CD138⁺ MM cells remaining in each culture was determined by flow cytometry. Percent specific killing was calculated with the formula (1-[% live tumor cells in the NK cell co-culture condition/% live tumor cells in the tumor alone condition]) x 100.