Evaluating Immune Checkpoint Inhibition in Leukemia

Bone marrow aspirates and peripheral blood samples were separated by Ficoll density gradient centrifugation. All experiments were performed using freshly isolated cells. Cells were labeled with CellTrace Violet (CTV, Thermo Fisher) and cultured in 96-well plates coated with 1 µg/mL anti-CD3 (BioLegend, Clone UCHT1) or control mIgG (BioLegend, Clone MOPC-21). Groups of wells were then treated with either 10 µg/mL control mIgG, 10 µg/mL anti-PD1 (EH12.2H7), 60 nM JQ1, or 60 nM JQ1 + 10 µg/mL anti-PD1. After 5 days, cells were stained for flow cytometry. Antibody clones and source are listed in Table 2. Viability was determined by Zombie Aqua staining and doublets were gated out of analysis by FSC-A vs. FSC-H. Flow cytometry data were acquired on a BD LSRFortessa or Cytek Aurora and analyzed using FlowJo v10 software. All human sample experiments are approved under IRB protocol #00004422, “Pathogenesis of Acute Leukemia, Lymphoproliferative Disorder and Myeloproliferative Disorders” (PI: Marc Loriaux, MD, PhD). Informed consent was obtained from all patients.Table 2

Antibodies used in this study for staining of patient samples.

Marker	Clone	Vendor
CD19	HIB19	BioLegend
CD45	HI30	BioLegend
CD8	RPA-T8	BioLegend
CTLA-4	BNI3	BioLegend
PDL1	29E.2A3	BioLegend
CD33	WM53	BioLegend
CD4	RPA-T4	BioLegend
PDL2	24F.10C12	BioLegend
CD56	HCD56	BioLegend
TIGIT	MBSA43	Thermo Fisher
TIM3	7D3	BD
PD1	EH12.2H7	BioLegend
CD3	HIT3a	BioLegend

List of specificity, clone and source of each antibody used in human flow cytometry experiments.