Anti cd14 fitc

NPM1^mut-specific CTL products were characterized for phenotype by monoclonal antibody staining and flow cytometry. Anti-CD3 FITC, anti-CD4 PE, anti-HLA-DR PE, anti-CD8 APC, CDγδ FITC, anti-CD56 Pc5.5, anti-CD14 FITC, anti-CD56 PE, anti-CD3 Pc5.5, anti-CD19+ CD20 APC, anti-CCR7 FITC, and anti-CD45RA PE (Becton Dickinson, Franklin Lakes, NJ, USA) were employed.

Eight weeks after starting biweekly intradermal vaccination (i.e., after 4 vaccines) into the upper arms near the axilla, a DTH skin test was performed on the upper back with single intradermal injections of the vaccine or its components, including KLH alone. Two days later skin reactions were measured and skin biopsies were taken. DTH skin test-infiltrating lymphocytes (DILs) were collected and allowed to expand for 2-3 weeks in medium with 100 IU/mL of IL-2. After harvest, RHAMM-specific CD8⁺ T cells were quantified in DILs through flow cytometry following staining with R3 or R5 peptide-loaded (p)HLA-A*0201 tetramers (Fred Hutchinson Cancer Research Centre Seattle). For each condition, 1 × 10⁶ viable thawed DILs were washed and stained for 30 min at 4°C with allophycocyanin-labeled pHLA-A*0201 tetramers. Samples were then stained with LIVE/DEAD® Fixable Aqua Dead Cell Stain (cat# L34957, Thermo Fisher Scientific), anti-CD3-PerCP-Cy5.5 (cat# 560835, BD Biosciences), anti-CD14-FITC (cat# 345784 BD Biosciences), anti-CD19-FITC (cat# 345776, BD Biosciences) and anti-CD8-Pacific Blue (cat# MHCD0828, Thermo Fisher Scientific) for another 30 min at 4°C, followed by detection on a Cyflow ML flow cytometer (Partec). Dead (LIVE/DEAD⁺) cells and monocytes and B cells (FITC⁺) were excluded from analysis.

For analysis of the frequencies of different cell subsets among the PBMCs and for analysis of the purity of CD4 PBMCs and isolated CD4⁺ and CD14⁺ cells, cells were stained with combinations of the following antibodies: anti-CD14 FITC, anti-CD4 PerCP, anti-CD19 PE, anti-CD3 APC, anti-CD45RO PE and anti-CD8 FITC (BD). Cells were analyzed using FACSCalibur, equipped with blue and red lasers (BD) and the flow cytometric data was analyzed with FlowJo software (Tree Star Inc.). Isolated CD4⁺ cells contained >95% CD3⁺CD4⁺ cells. Isolated CD14⁺ T cells contained >95% CD14⁺ cells. The frequency of CD4⁺ in PBMCs after depletion of CD4⁺ cells was <5%.

The expanded NK cells were stained with the following monoclonal Abs; anti-CD3- fluorescein isothiocyanate (FITC), anti-CD14-FITC, anti-CD16- phycoerythrin (PE), anti-CD19-PE, anti-DNAM-1-PE, anti-CD56-PE-cyanine (Cy)5, anti-CXCR3-PE, anti-NKp30-PE, anti-NKp44-PE, anti-NKp46-PE (all from BD Biosciences, San Jose, CA, USA), anti-NKG2A-PE, anti-NKG2C-PE, and anti-NKG2D-PE (all from R&D systems, Minneapolis, MN, USA). Stained NK cells were acquired on LSR Fortessa and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). To confirm phenotypic changes in NK cell receptors in co-culture of tumor cells with NK cells, cryopreserved NK cells were thawed and co-cultured with MIA PaCa-2 at an E:T ratio of 1:1. On 1, 2, or 3 days after co-culture, the harvested NK cells were immunostained with anti-CD16-PE, anti-NKp30-PE, anti-NKp44-PE, anti-NKp46-PE, anti-DNAM-1-PE, anti-CXCR3-PE (all from BD Biosciences, San Jose, CA, USA), anti-NKG2D-PE (R&D systems, Minneapolis, MN, USA), anti-CD96-PE, and anti-CD161-PE (all from eBioscience, San Diego, CA, USA) then analyzed by flow cytometry, as described above.

Undiluted whole blood samples were washed by addition of 3 ml of 0.9% NaCl in polypropylene round-bottom tubes (BD Biosciences) and centrifuged (1000 × g). Then, blood sample was put into a TruCOUNT^™ Tube (BD Biosciences, San Jose, USA) and stained on ice for 30 min with: anti-CD45-APC, anti- HLA-DR-PerCP, anti-CD14-FITC, anti-CD16-PE (BD Biosciences) and anti-PD1-PE-Cy7 (eBioscience) monoclonal antibodies (mAbs). The samples were then treated with FACS Lysing Solution (BD) until erythrocytes were lysed, and the cells were immediately processed in the FACSCanto flow cytometer (Becton Dickinson, San Jose, USA) along with 10.000 of beads per tube. The absolute numbers of CD14⁺⁺CD16^-, CD14⁺⁺CD16⁺ and CD14⁺CD16⁺⁺ monocytes were calculated with reference to the bead count. The percentage and the absolute numbers of PD-1- positive cells were estimated in each of the monocyte subset. Flow cytometric data were analyzed using FlowJo software (Tree Star, Inc, Ashland, OR). The gating strategy and analysis of MO subsets was previously described by us [21 (link)] and others [22 (link)] and is presented on Fig 1.