Il 17a apc

Monoclonal antibodies specific to the following mouse antigens were purchased from Thermo Fisher Scientific, eBioscience, United States: CD4 Alexa488 (GK1.5), Foxp3 eFluor450 (FJK-16s), IL-17A APC (eBio17B7), CD8 PE (Ly-2), CD25 PE (PC61), and IFN gamma PE (XMG1.2).

For the detection of intracellular cytokines, cells were reactivated by PMA (50 ng/ml) and ionomycin (500 ng/ml) (Sigma‐Aldrich) with protein transport inhibitor (Thermo Fisher Scientific, Waltham, MA, USA) for 5 h. Cells were blocked by anti‐mouse CD16/32 antibodies before surface staining. Surface proteins expressed on cells were stained with fluorescence‐conjugated antibodies diluted in FACS buffer (1× phosphate‐buffered saline with 0.5% bovine serum albumin) for 30 min. Intracellular cytokine staining was performed using intracellular fixation and permeabilization buffer set (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's recommended protocol. The anti‐mouse CD4‐PerCP‐Cy7, IL‐17A‐APC, IL‐17A‐PE, IFNγ‐APC and IFNγ‐PE fluorescence‐conjugated antibodies for flow cytometry were purchased from Thermo Fisher Scientific (Waltham, MA, USA). After staining, cells were washed and acquired on FACS Canto II (BD Biosciences, Franklin Lakes, NJ, USA). To figure out cytokine‐producing CD4 T cells, lymphocytes were gated from FSC‐A and SSC‐A dot plot. Next, single cells were gated from FSC‐H and FSC‐A. CD4⁺ cells were gated from CD4‐Percp‐cy5.5 and FSC‐A dot plot. And then, intracellular cytokines in CD4 T cells were measured. Data were analysed by using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

At the beginning and conclusion of the study, peripheral blood samples were drawn into serum separator tubes (SST). LDL-C levels were quantified using the Cobas 6000 analyzer (Roche Diagnostics, Basel, Switzerland) through enzymatic colorimetric techniques.
For the analysis of PBMCs, we followed procedures based on a previously published article by our group (32 (link)). The study utilized several monoclonal antibodies, including anti-CD4-AF700, anti-CD8-PE, anti-CD8-APC, anti-CD28-APC, anti-CD57-FITC, anti-interferon (IFN)-γ-PE-Cy7, and anti-tumor necrosis factor (TNF)-α-APC, IL-17A-APC, Granzyme B-PE, perforin-APC, all sourced from eBioscience, San Diego, CA, USA. Following permeabilization, cells were stained for intracellular cytokines and cytotoxic molecules using anti-IFN-γ-PE-Cy7 and anti-TNF-α-APC. Multicolor flow cytometry was performed with the BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA). Data analysis was conducted using FlowJo V10 software (FlowJo, LLC, Ashland, OR, USA). Blood samples with a PBMC viability of 80% or more were used for FACS analysis, with 77 patients meeting this criterion at both baseline and follow-up visits.