Anti cd27

Peripheral blood lymphocytes (PBLs) from HSCT recipients were cocultured with pamidronate-pretreated autologous or allogeneic (refers to third-party healthy subjects) DCs at the ratio of 2:1, at a final of 3 × 10⁵ cells/well in 96-well plates. In some experiments, the neutralization antibody anti-NKG2D (10 μg/ml, BioLegend, USA) was used for blocking NKG2D during coculture. After 7 and 14 days of coculture, the percentages and differentiation and activation phenotypes of Vδ2⁺ T cells were detected by flow cytometry. Different combinations of monoclonal antibodies allowed identifying the differentiation profile of Vδ2⁺ T cells by the expression of CD45RO and CD27 (Naive: CD45RO⁻CD27⁺, Central Memory (CM): CD45RO⁺CD27⁺, Effector Memory (EM): CD45RO⁺CD27⁻ and terminal differentiation (TD): CD45RO⁻CD27⁻). For cell differentiation and activation assay, the cultured cells were stained with fluorochrome-labeled anti-CD27, anti-CD45RO, anti-HLA-DR, anti-CD38, and anti-NKG2D antibodies (BioLegend, USA).

All lymphocytes, pre- and post-selection, were analyzed using flow cytometry and the following antibodies: anti-CD45 (AF700; clone HI300), anti-CD3 (PE, FITC or PE-Cy7; clone OKT3), anti-CD8 (PE or BV510; clone HIT8a), anti-CD4 (BV421 or BV785; clone OKT4), anti-CD27 (BV785; clone 323), anti-CD14 (PC7; clone HCD14), anti-CD56 (APC; clone MEM-188), anti-EGFR (PE; clone AY13), TCRαβ (APC-Fire750; clone IP26) and anti-CD19 (BV421; clone HIB19) (all from Biolegend). To determine T cell activation state, T cells were stained with CD69 antibody (APC-Fire750; clone FN50) and CD25 antibody (BV650; clone BC96) (Biolegend) at indicated time points after initial stimulation. For live/dead discrimination propidium iodide (Sigma) was used. CAR expression has been monitored using an anti-idiotype antibody specific for the CD19-directed CAR, an in-house developed antibody. Cell associated fluorescence was analyzed by flow cytometry using either CytoFLEX or CytoFLEX LX flow cytometers (Beckman Coulter), unless indicated otherwise.
Live nucleated cells were enumerated pre- and post-selection (including fractions and downstream culture) using either NuceloCounter NC-3000 (Chemotec) or XN-350 Cell Counter (Sysmex) according to manufacturers’ instructions.

The following fluorescently conjugated antibodies were used for phenotypic analysis of NK cells: anti-LFA-1 (363404); anti-CXCR3 (353720); anti-PD-1 (329906); anti-CD107a (328612); anti-NKP44 (325116); anti-CD158e1 (312706); anti-CD158d (347006); anti-CD27 (356412); anti-CCR4 (359412); anti-DNAM-1 (338316); anti-CD16 (302040); anti-Granzyme B (515406); anti-CD62L (304806); anti-CD69 (310910); anti-NKP80 (346706); anti-NKP30 (325210); anti-CD158f (341304); anti-CD158b (312612); anti-Tim-3 (345012); anti-CD94 (305504); anti-TIGIT (372706); anti-TRAIL (308206); anti-CD57 (322306); anti-CX3CR1 (341610); anti-NKG2D (320808); anti-Perforin (353310); anti-Ki67 (350504); anti-IFN-γ (506518); anti-CD94 (305506); anti-NKp46 (331916); anti-CTLA4 (369614); anti-CD96 (338416); anti-41BB (309818); anti-CD25 (356108) were purchased from Biolegend. anti-CD159a/NKG2A (FAB1059P) and anti-NKG2C (FAB138G) were purchased from R&D.
The following fluorescently conjugated antibodies were used to identify immune cell types in eNK or PBMC: anti-human Lineage Cocktail (348803); anti-CD56 (362550); anti-CD3 (300430); anti-CD33 (366620); anti-HLA-DR (307606); anti-CD14 (301836); anti-CD19 (302242); anti-CD11b (301322); anti-CD25 (356108); and anti-FOXP3 (320108) were purchased from Biolegend, San Diego, CA, USA.

ASTRLs and PBMCs were immunophenotyped for various cell surface markers by flow cytometry with fluorophore conjugated human anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD127, anti-CD39 and anti-CD73 anti-CTLA4, anti-GITR, anti-ICOS, anti-CD45RA, anti-CD226, anti-LAP, anti-GARP, anti-CD56, anti-CD16, anti-CD19, anti-CD11b, anti-CD38, anti-CD27, and anti-CD24 (Biolegend). The data were acquired using a Canto II cytometer (BD Biosciences) and analyzed using Flowjo. The gating strategy for phenotyping included initial gating of a live PBMC population followed by the CD3⁺CD4⁺ population. The expression of CD25, CD127, CD39, and CD73 were expressed as % of the CD3⁺CD4⁺ population.
T cell proliferation and suppression was determined by CFSE dye dilution of the responder cells. Analysis of CFSE distribution was performed on Flowjo Proliferation platform and data are represented by Replication Index (RI). RI, determines the fold-expansion of only the responding cells, and it is the average number of divisions that all cells have undergone after they had been stained by a cell proliferation dye (13 (link)). The percentage of suppression was calculated from proliferation and suppression values.

The cells were incubated with Fc blockade reagent for 20 min at room temperature and stained with fluorochrome-conjugated antibodies in serum and sodium azide-containing buffer. Flow cytometry was performed as previously described (16 (link)). Antibodies in the present study were listed as follows: FITC anti-CD56 (Cat#362546, BioLegend, USA), PE anti-NKp30 (Cat#325208, BioLegend, USA), anti-CD56 (Cat#362508, BioLegend, USA), anti-NKp44 (Cat#325108, BioLegend, USA), anti-CD27 (Cat#356406, BioLegend, USA), PerCP-Cy5.5 anti-CD3 (Cat#317336, BioLegend, USA), anti-NKG2D (Cat#320818, BioLegend, USA), APC anti-NKp46 (Cat#331918, BioLegend, USA), anti-CD11b (Cat#301310, BioLegend, USA), anti-CXCR6 (Cat#356006, BioLegend, USA), anti-CD3 (Cat#317318, BioLegend, USA), PE-Cy7 anti-NKp46 (Cat#331916, BioLegend, USA), anti-CD3 (Cat#317334, BioLegend, USA), APC-Cy7 anti-CD3 (Cat#317342, BioLegend, USA), and anti-CD45 (Cat#304014, BioLegend, USA). FlowJo software was used for data analyzing (Tree Star, Inc., Ashland).