Cryopreserved peripheral blood mononuclear cell (PBMC) samples from baseline and post-treatment (cycles 1–12) were thawed and rested overnight at 37°C. Then cells were stained with a viability dye (eBioscience
Fixable Viability Dye eFluor 450) and a master mix of antibodies for surface stains including
CD4-BV605 (BioLegend Cat# 317438, RRID:AB_11218995), CD3-PE-Cyanine5.5 (Invitrogen Cat# 35-0036-42, RRID: AB_11220085), CD8a-PE-Cyanine7 (Invitrogen Cat# 25-0088-42, RRID: AB_1659702),
PD1-APC (BioLegend Cat# 329908, RRID: AB_940475),
CD152-PE-Cy5 (BD Biosciences Cat# 555854, RRID:AB_396177). Cells were next fixed and permeabilized with the
eBioscience Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher) and subsequently stained intracellularly with
Alexa Fluor 700 antihuman Ki67 antibody (BioLegend Cat# 350530, RRID: AB_2564040). Stained cells were acquired on a BD Canto RUO and analyzed with FlowJo software (FlowJo, RRID:SCR_008520).
Liao J.B., Gwin W.R., Urban R.R., Hitchcock-Bernhardt K.M., Coveler A.L., Higgins D.M., Childs J.S., Shakalia H.N., Swensen R.E., Stanton S.E., Tinker A.V., Wahl T.A., Ancheta R.G., McGonigle K.F., Dai J.Y., Disis M.L, & Goff B.A. (2021). Pembrolizumab with low-dose carboplatin for recurrent platinum-resistant ovarian, fallopian tube, and primary peritoneal cancer: survival and immune correlates. Journal for Immunotherapy of Cancer, 9(9), e003122.
