Cd3 efluor 605

Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation with Lymphoprep (Axis-Shield). PBMC or whole blood samples were stained with the following fluorochrome-conjugated monoclonal antibodies: CD3-efluor605, CD4-efluor450, CD27-APC-efluor780, HLA-DR-efluor780, CD45RA-efluor605, FOXP3-PE (eBioscience), CD4-APC-H7, CD8-Percp, CD8-PE-Cy7, CD31-AF647, CD45RO-FITC, CD45RO-PE-Cy7, CCR7-PE-Cy7, Ki-67-Percp-cy5.5, CTLA-4-BV421 (BD Biosciences), PD-1-PE, CD28-AF700 (Biolegend), and CD161-PE (Miltenyi Biotec). Intracellular staining for FOXP3, Helios, Ki-67, and CTLA-4 was performed after cells were permeabilized with a FOXP3 staining buffer set according to instructions of the manufacturer (eBioscience). Whole blood samples were treated with BD lysing solution according to the instructions of the manufacturer (BD Biosciences). Stained samples were analyzed on a LSR-II flow cytometer (BD Biosciences). Analysis was performed with Kaluza Flow Analysis Software (Beckman Coulter). Absolute numbers of CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells were determined according to the MultiTest TruCount method (BD Biosciences), as described by the manufacturer. TruCount samples were measured on a FACSCanto-II (BD Biosciences) and analyzed with FACSCanto Clinical Software (BD Biosciences).

Human cervical tissue was obtained from two sets of five healthy women (age range 42–47 years old) undergoing hysterectomy for benign indication at HUGTP. After confirmation of healthy tissue status by the Pathological Anatomy Service, a piece from ecto and endocervix separated by anatomical localization was delivered to the laboratory in refrigerated RPMI 1640 medium (Cellgro, Manassas, VA) containing 10% FBS (Lonza, Basel, Switzerland), 500U/mL penicillin, 500μg/mL streptomycin, 5μg/mL fungizone and 1μg/mL gentamycin (Life Technologies). Tissue was processed within the next 12 h after surgery, and 8-mm³ block-dissection was performed as described [26 (link)]. Tissue digestion of 5–9 pieces of ecto or endocervix with collagenase IV (Invitrogen) was immediately executed as described [26 (link)]. Tissue blocks were then dissociated manually with a disposable pellet pestle and filtered through a 70μm-cell strainer (BD Biosciences). After centrifugation, pellet was suspended in staining buffer (1% mouse serum, 1% goat serum in PBS) and stained with different combinations of CD3-eFluor 605 (eBiosciences), CD14-V450, CD11c-PE-Cy7, CD8-V500, HLA-DR-PerCP-Cy5.5, CD69-Horizon PE-CF594 (BD Biosciences), NK1.1-PE, γδTCR-FITC, Vα7.2-APC-H7 (Miltenyi Biotec), CD103-FITC and Vα24-APC (BioLegend). Data were acquired and analyzed as described for blood.

Blood samples (EDTA) were stained with the following monoclonal antibodies: CD3-eFluor605, CD4-eFluor450, TCRγδ-PE (eBioscience), CD45RO-FITC, CCR7-PE-Cy7, CD4-PerCP, CD8-PerCP, CD8-APC-H7, DNAX accessory molecule-1 (DNAM-1)-FITC (BD), CD161-PE, CD161-APC (Miltenyi), 2B4-PE, NKG2D-PE-Cy7, TCR-Vα7.2-FITC, TCR-Vα24-Jα18-FITC (Biolegend), TCR-Vβ11-PE (Beckman Coulter), and KLRG1-FITC (generous gift from H. Pirchner). An overview of antibody panels is shown in Table S1 in Supplementary Material. Samples were subsequently treated with BD Lysing Solution (BD Biosciences) according to instructions of the manufacturer. Samples were measured on a LSR-II flow cytometer (BD) and analyzed with Kaluza Analysis Software (Beckman Coulter). In addition, the absolute numbers of circulating lymphocyte subsets were determined according to the MultiTest TruCount method (BD), as described by the manufacturer. Data were acquired on a FACSCanto-II flow cytometer (BD) and analyzed with FACSCanto Clinical Software (BD). The number of events for a particular T cell population needed to be more than 100 to allow for subsequent analysis of cellular markers, cytokines, and cytotoxic molecules.