Rosettesep human cd8 t cell enrichment cocktail

CD8⁺ T cells were isolated from apheresis leukoreduction collars using RosetteSep Human CD8⁺ T Cell Enrichment Cocktail (STEMCELL Technologies, 15063). CD38⁺CD8⁺ T cells were removed by using EasySep Human Biotin Positive Selection Kit II (STEMCELL Technologies, 17663) and Biotin anti-CD38 monoclonal antibody (clone HIT2, BioLegend, 303518). The remaining CD38⁻CD8⁺ T cells were cultured overnight in six-well plates coated with plate-bound anti-CD3 (1 μg/ml; clone OKT3, BioLegend, 317326) and anti-CD28 (2 μg/ml; clone CD28.2, BioLegend, 302934). After overnight stimulation, activated CD8⁺ T cells were transfected with control vector or CD38 overexpression vector with C-GFPSpark tag (Sino Biological, HG10818-ACG) by using the Human T Cell Nucleofector Kit (Lonza, VPA-1002). For MitoTracker and immunofluorescence staining, control OFPSpark Vector (Sino Biological, CV025) and CD38 overexpression vector with OFPSpark (Sino Biological, HG10818-ACR) were used because of the consideration of fluorescence colors. Transfected cells were rested for 6 hours and then cultured overnight with control dimethyl sulfoxide (DMSO) (0.01%), 0.5 μM SRT1720, 20 μM 78c, or 20 μM MK-0159 as indicated in 48-well plates coated with plate-bound anti-CD3 and anti-CD28.

6 mL EDTA-anticoagulant-treated peripheral blood samples were collected on the day before the first dose of radiation and camrelizumab and during treatment (after delivery of 40 Gy radiation and 2 rounds of camrelizumab). Within 2 hours after the fresh whole-blood collection, CD8⁺ T cells were isolated from the whole-blood samples by negative selection by using the RosetteSep™ Human CD8⁺ T Cell Enrichment Cocktail (StemCell, 15063) according to the manufacturer’s instruction. After being washed twice with cold phosphate-buffered saline (PBS), purified CD8⁺ T cells were resuspended in RNAlater (ThermoFisher, AM7020) and preserved at ‒80°C for further TCR analysis. The purification of CD8⁺ T cells was immediately confirmed by flow cytometry analysis after isolation. All purification of isolated CD8⁺ T cells was above 93%. We also isolated the peripheral blood monocyte cells (PBMCs) from 5 mL peripheral blood both at baseline and during treatment by Ficoll-Paque® density gradient centrifugation. The fresh PBMCs were tested by using flow cytometry.^{16 (link)} The baseline peripheral blood granulocytes on the upper layer of erythrocytes after Ficoll-Paque® density gradient centrifugation were preserved directly at ‒80°C, which were used as normal tissue control in TMB analysis.

Primary human T cells were isolated from leukoreduction chamber residuals following Trima Apheresis (Blood Centers of the Pacific) using established protocols (35 (link)). Briefly, peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll separation in SepMate tubes (STEMCELL Technologies) in accordance with the manufacturer’s instructions. CD8⁺ T cells were isolated from PBMCs using either the EasySep Human CD8⁺ T cell Isolation Kit or the RosetteSep Human CD8⁺ T Cell Enrichment Cocktail (STEMCELL), following the manufacturer’s protocol. CD4⁺CD25^- conventional T cells and CD4⁺CD127^lowCD25⁺ Tregs were isolated from PBMCs with the EasySep Human CD4⁺CD127^lowCD25⁺ Regulatory T Cell Isolation Kit (STEMCELL). Isolated cell populations were analyzed for purity by flow cytometry on a Beckman Coulter CytoFlex flow cytometer using a panel of antibodies (anti-CD3 [UCHT1], anti-CD4 [OKT4], anti-CD8a [RPA-T8], anti-CD25 [M-A251], anti-CD45RA [HI100], and anti-CD127 [A019D5], all from BioLegend). Pilot experiments comparing FACS sorting and magnetic bead separation showed similar results with respect to cell purity, so magnetic bead separation was used.

Naive CD8⁺ T-cells were isolated isolated from blood using RosetteSep human CD8⁺ T cell enrichment cocktail (Stemcell) and Ficoll (GE Healthcare Life Sciences) gradient centrifugation. Naive CD8⁺ T-cells were stimulated by anti-CD3⁺CD28 Dynabeads (ThermoFisher) in Biotarget medium (Biological Industry) supplemented with 4% human platelet lysate (Ultra-GRO-Advanced, AventaCell) with 100U/mL IL-2 (R&D System) for 48hrs. Activated CD8⁺ T-cells were maintained in medium containing 100 U/mL IL-2, 10 ng/mL IL15 and 10 ng/mL IL7 (R&D System). T-cells were restimulated every 14-20 days by feeder cells, which were freshly isolated peripheral blood mononuclear cells (PBMC) and irradiated at 30 Gy. PBMC and T-cells were resuspended at a ratio of 2:1 in the medium with concentrations of 100U/mL IL-2, 10 ng/mL IL7, 10 ng/mL IL15 and 1.5 μg/mL of Lectin from Phaseolus vulgaris (Sigma-Aldrich). Blood collection protocol was approved by NUS IRB. Informed consent was obtained from all donors.

Blood samples were collected at the Blood Centers of the Pacific in San Francisco, CA. All donors were kept anonymous and provided written informed consent. Total human CD8⁺ T cells were enriched via negative selection using the RosetteSep human CD8⁺ T cell enrichment cocktail (15063; Stemcell). The following antibodies were used to stain peripheral blood mononuclear cells or enriched CD8⁺ naive, CD8⁺CD28⁺ memory, and CD8⁺CD28⁻ T cells: CD3-PECy5 (555334; BD Biosciences) and CD3-APC-H7 (560176; BD Biosciences), CD8-V450 (560347; BD Biosciences), CD28-PE (12–0289; eBioscience), CD45RA-APC (17–0458; eBioscience), and CCR7-PECy7 (557648; BD Biosciences). Flow cytometry was performed on an LSRII or Calibur DxP8 flow cytometer (both BD Biosciences) and was analyzed with FlowJo software. Sorting was performed on an ARIA II (BD Biosciences).