Cd4 fitc

Conjugated antibodies and staining reagents included MIP-1β-PE, CD3-PB, CD3-PE-Cy7, CD3-PerCP-Cy5.5, CD3-APC-Cy7, CD3-Horizon V450, CD4-PerCP-Cy5.5, CD4-AmCyan, CD4-FITC, CD8α-APC, CD8α-APC-Cy7, CD8α-AlexaFluor700, CD8α-FITC, CD8α-APC-H7, CD69- ECD (Beckman Coulter), CD20-Horizon V450, CCR7-FITC (R&D Systems), CD95-APC, CCR7-PerCP-Cy5.5, CD28-PE-Cy7 (eBioscience), granzyme B-AlexaFluor700, perforin-FITC (MabTech), IFNγ-PE-Cy7, TNFα- AlexaFluor700, IL-2-APC, CD95-PE, CD95-APC, and Aqua LIVE/DEAD Fixable Dead Cell Stain (Invitrogen). All reagents are from BD Biosciences unless indicated otherwise. For construction of monomers and tetramers, the following peptides were synthesized and purified to >95% by HPLC by New England Peptide LLC: p11C (CTPYDINQM), p54AS (TVPWPNASL), p54E660 (TVPWPNETL), p68A (STPPLVRLV), and TL8 (TTPESANL). The monomers and tetramers were prepared as previously described [85] (link), [86] (link). Tetramers were prepared using either streptavidin-PE (Prozyme), -APC (Prozyme), -AlexaFluor488 (Invitrogen), or -Qdot655 (Invitrogen). Monomers used in surface plasmon resonance studies were further quantified using an RC DC protein kit (Bio-Rad).

The following antibodies were used in this study: CD4-APC (300514, Beckman Coulter, Brea, CA, USA), CD4-FITC (A07750, Beckman Coulter), CD25-PE (555432, BD Biosciences, San Jose, CA, USA), CD122-PE (IM1978, Beckman Coulter), CD132-APC (338608, BioLegend, San Diego, CA, USA), cyclin D (2936P, Cell Signaling, Danvers, MA, USA), cyclin E (551159, Cell Signaling, Danvers, MA, USA), JAK3 (ab45141, Abcam, Cambridge, UK), STAT5 (9363P, Cell Signaling, Danvers, MA, USA), pSTAT5 (9314S, Cell Signaling, Danvers, MA, USA), pAKT (4075S, Cell Signaling, Danvers, MA, USA), and pERK1/2 (4284S, Cell Signaling).

Blood samples were collected at W0, W8, W24 and W48 for immunophenotyping by flow cytometry in ethylenediaminetetraacetic acid (8.55 mg/tube). Peripheral blood samples (100 µL) were incubated with specific monoclonal antibodies (Beckman Coulter, Wycombe, UK) at room temperature (RT) for 15 min in the dark; afterward, the red cells were lysed, at RT in the dark, in a VersaLyse lysing solution (A09777, Beckman Coulter, Wycombe, UK) for 15 min and then analysed. LS were identified by the recognition of surface molecules belonging to the CD family. The samples successively underwent gating analysis (total lymphocytes and CD45+ vs. side scatter) (CD45-FITC, A07782, Beckman Coulter, Wycombe, UK), and the desired lymphocyte subpopulations were gated excluding doublets. The LS identification was based on monoclonal antibodies against Bmem CD19+/CD27+, Br1 CD19+/CD38+, Br2 CD19+/CD25+, Treg CD4+/CD25+, Th CD3+/CD4+ and Tc CD3+/CD8+ (CD19-PC7, IM3628; CD27-PE, IM2578; CD38-PE, AO7779; CD25-PE, A07774; CD4-FITC, A07750; CD3-PC5, A07749; CD8-PE, A07757; Beckman Coulter, UK). The samples were tested using a CytoFLEX flow cytometer and the CytExpert software (Beckman Coulter, Wycombe, UK).

At each time point, in addition to leukocyte count, we assessed immature neutrophils (CD16^low) and LOX-1⁺ MDSC (CD15⁺, CD45^dim, LOX-1⁺ polymorphonuclear cells) percentages as described by Coudereau et al. (2022) (link) and immune checkpoint inhibitor (PD-1 and TIM-3) expression on CD3, CD4 and CD8 T lymphocytes. Cell staining was performed on fresh whole blood sample within 4 h after sampling. We used the following antibodies: CD45-PB, CD3-APC-AF750, CD4-FITC, CD8-Kro, CD14-PB, CD16-APC from BeckmanCoulter (Brea, CA) and: PD1-APC, TIM-3-PE-Dazzle, CD15-AF700, LOX1-PE from BioLegend (San Diego, CA). Isotype control antibodies (BioLegend) were used to determine the percentages of positive cells for PD-1, TIM-3 and LOX-1. Samples were run on Navios flow cytometer (Beckman Coulter). T lymphocytes subsets’ absolute quantification was performed on Aquios flow cytometer (Beckman Coulter). Detailed protocols are presented in supplementary methods. Results were expressed as absolute counts for neutrophil subsets and T lymphocyte subsets (i.e., cells/mm3). Results were expressed as absolute cell counts for immature neutrophils and LOX-1⁺ MDSC. Immune checkpoint inhibitor expressions on T lymphocyte subsets were expressed as percentages of positive cells based on isotype controls.

PBMCs from UC patients and healthy controls were harvested and stained in duplicate for CD45-PC7, CD3-ECD, CD8-PE, CD4-FITC, CD19-PC5, CD38-FITC, CD27-PC7, CD86-PE (Beckman Coulter, Brea, CA), CXCR5-PC5 and ICOS-PE (eBioscience, San Diego, CA) at room temperature for 30 min. After being washed with PBS, the cells were characterized by flowcytometry analysis using the Beckman Coulter Cytomics FC500 (Beckman Coulter). Cells stained with separate antibodies were defined as certain T_FH cells (CD4⁺ CXCR5⁺ T_H cells and CD4⁺CXCR5⁺ICOS⁺ T_H cells) and subsets of B cells (CD38⁺CD19⁺ B cells, CD86⁺CD19⁺ B cells and CD27⁺CD19⁺ B cells). Data were analysed with a CXP Analysis Cytometer (Beckman Coulter).