Clone ucht1

In a subset of patients (n = 63), freshly isolated cells were lysed using the Immunoprep Reagent System (Beckman Coulter) and stained with CD45-FITC/CD56-PE/CD19-ECD/CD3-PC5 and CD45-FITC/CD4-PE/CD8-ECD/CD3-PC5 antibody mixes (all Tetrachrome, Beckman Coulter, Krefeld, Germany), according to manufacturer´s instructions. Flow-cytometry analysis was carried out and analyzed using NAVIOS cytometer and analysis software (Beckman Coulter).
In a small patient subpopulation, from which pretreatment PBMCs were available (n = 6, patient characteristics in Supplementary Table 1), expression of membrane bound LAG3 was quantified in T lymphocytes, key players of antitumoral response. For this instance, priorly isolated and cryopreserved PBMCs were thawed and stained for flow cytometry analysis using fluorescently labeled mAbs directed towards CD3, CD4, CD8 and LAG3, in order to quantify LAG3 positive Helper T lymphocytes (HTLs) (CD3 + CD4 + CD8-LAG3 + ) and cytotoxic T lymphocytes (CTLs) (CD3 + CD4-CD8 + LAG3 + ) (CD3: Clone UCHT1, Fluorophor APC-R700 ; CD4: Clone SK3, Fluorophor BUV737 ; CD8: Clone RPA-T8, Fluorophor BV786; LAG3: Clone T47-530, Fluorophor Alexa 647; all originating from BD Biosciences, San Jose, CA, USA). A LSR Fortessa (BD Biosciences, San Jose, CA, USA) was used for cytometry analysis and FlowJo 10.6 software (TreeStar, Ashland, OR, USA) for data analyses.

The following conjugated antibodies were used for cell-surface staining: CD3-Pacific Blue (BD Pharmingen; Clone UCHT1; Cat. No. 558117; San Diego, CA, USA), CD8-APC H7 (BD Pharmingen; Clone SK1; Cat. No. 641400), CD45RA-PE (BD Pharmingen; Clone HI100; Cat. No. 555489), CCR7-PE-Cy7 (BD Pharmingen; Clone 3D12; Cat. No. 557648), CD28-PerCP-Cy5.5 (BD Biosciences; Clone L293; Cat. No. 337181; San Jose, CA, USA), CD27-Alexa Fluor 700 (BD Pharmingen; Clone M-T271; Cat. No. 560611), CD95-APC (BD Pharmingen; Clone DX2; Cat. No. 558814) and CD127-FITC (BD Pharmingen; Clone HIL-7R-M21; Cat. No. 560549). Conjugated antibodies for intracellular staining included the following: IFN-γ-FITC (BD Pharmingen; Clone 4S.B3; Cat. No. 554551), IL-2-PerCP-Cy5.5 (BD Pharmingen; Clone MQ1-17H12; Cat. No. 560708) and TNF-α-AlexaFluor 700 (BD Pharmingen; Clone MAb11; Cat. No. 557996). To exclude dead cells, the Fixable Aqua Dead Cell Stain viability marker was used (Invitrogen; Cat. No. L34957; Eugene, OR, USA).

Fresh or cryopreserved single-cell suspensions from endometrial and decidual tissue, above, were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen, Cat# L34965) per manufacturer instructions. Cells were washed and treated with Human Fc Block (BD Biosciences, Cat# 564219, 1:200). For CD45+ cell purification for scRNAseq, cells were then washed and stained with CD45 (PerCP-Cy5.5, BioLegend, Clone 2D1, Cat# 368503, 1:100). All scRNAseq data presented, with the exception of Supplementary Figure 3, were generated from exclusively cryopreserved cells. For bulk NK (CD56+CD3-) and bulk macrophage (CD14+CD64+) cell purification for functional IOC experiments, cells were additionally stained with CD3 (BV785, BioLegend, Clone UCHT1, Cat# 300471, 1:100); CD14 (V450, BD Biosciences, Clone MφP9, Cat# 560349, 1:200); CD56 (PE, BD Biosciences, Clone B159, Cat# 555516, 1:50); and CD64 (PE-Cy7, BioLegend, Clone 10.1, Cat# 305022, 1:100). Antibody staining was performed at 4°C for 30 minutes in the dark. A BD FACSMelody sorter was used. All cells used for functional IOC experiments were isolated from cryopreserved single-cell suspensions. Cytospins were prepared in a cytocentrifuge at 600RPM for 3 minutes, after which cells were stained with hematoxylin and eosin.

A simplified VOA was performed as we previously described (55 (link)). Briefly, memory CD4⁺ T cells were cultured at 1 × 10⁶ cells/well in 1 mL media (RPMI, 10% FBS, 1% P/S) in a 48-well plate (Costar) coated with CD3 Abs (1 μg/mL; BD Biosciences, Clone UCHT1) and in the presence of soluble CD28 Abs (1 μg/mL; BD Biosciences, Clone CD28.2). At day 3 poststimulation, cells from each original replicate were individually washed with media, split into two new CD3 Abs-uncoated 48-well plates, and cultured in media containing IL-2 (5 ng/mL; R&D Systems) in the presence or in the absence of GGSK2691805A (5 μM). The cells from each well were further split into two new wells (without washing) at day 6 and 9 poststimulation, with 50% of the media being refreshed with IL-2 with/without GSK2691805A. The cells were kept in culture for a total of 12 d.

A subsequent staining procedure for phenotypic characterization of CD3^-CD8^dull GAD65 AA 114–122 pentamer reactive T cells was implemented by adding the following antibodies: mAb anti-human CD8 FITC (1:20, BD), mAb anti-human CD3 Alexa Fluor 700-A labelled (1:50, clone UCHT1, cat# 557943, BD), mouse mAb anti-human CD56 PE cyanine 7 (PECy7) labelled (clone B159, cat# 557747, BD) and mouse mAb anti-human ILT2/LIR1 (CD85J (Ig-like transcript (ILT)/leukocyte Ig-like receptor (LIR) (APC conjugated, clone HP-F1, cat# 17-5129-41, eBioscience) [29 (link)] both used at 1:50 dilution.

Treg were stimulated with rhIL-2 (25 U/mL) alone as control, or together with either soluble CD28 agonist mAb (1 μg/mL, Clone L293, Cat# 348040; BD Bioscience), plate-bound CD3 mAb (5 μg/mL, Clone UCHT1, BD Bioscience), plate-bound CD3 (5 μg/mL) plus soluble CD28 mAb (1μg/mL), CD28-superagonist ANC28.1 (1 μg/mL, Clone ANC28.1/5D10, Cat# 177–820, preservative free; Ancell, Bayport, USA), or anti-CD3/anti-CD28 mAb-coated microbeads (Invitrogen by Life technologies, Bleiswijk, The Netherlands) used in a 1:2 bead-to-cell ratio. Titration of soluble CD28-agonist and CD28-superagonist antibodies at a concentration range of 1–10 μg/mL led to similar FOXP3 expression levels as determined by flow cytometry. Cells were cultured in 96-well round bottom plates in RPMI 1640 (Invitrogen) as described previously44 (link). In selected experiments immunomodulating agents were analyzed; Treg were pretreated with wortmannin (5 μM), rapamycin (200 nM) (Sigma-Aldrich, St. Louis, USA)15 (link), or vehicle control for 30 min at 37 °C. Thereafter, stimulators as indicated were added to the culture mixture.