Cd69 apc

Peripheral blood mononuclear cells (PBMC) of patients and control subjects were isolated by Ficoll-Hypaque (GE Healthcare, Pittsburgh, PA) density-gradient centrifugation, and cellular viability was evaluated by trypan blue staining and it was always higher than 95%. Mononuclear cells were stained for 30 minutes in darkness at 4°C with the following monoclonal antibodies (mAbs): CD4-FITC (eBioscience, San Diego, CA) or CD4-APC/Cy7 (BioLegend, San Diego, CA), CD25-APC/Cy7 (Becton-Dickinson, Franklin Lakes, NJ), NKG2D-FITC (eBioscience), antilatency-associated peptide (LAP, a surrogate marker for TGF-β)-PerCp/Cy5.5 (BioLegend), and CD69-APC (eBioscience). Then, cells were washed and fixed and permeabilized with the Foxp3 Fix/Perm kit (eBioscience) for 30 minutes. Subsequently, mononuclear cells were stained with mAbs against IL-10 (PE) (BioLegend) and Foxp3 (PE/Cy7) (eBioscience). Doublet discrimination was performed by analyzing FSC-A versus FSC-W dot plots from the lymphocyte gate. In all cases, at least 1 × 10⁶ events were analyzed, and gates were set up by using fluorescence minus one controls and labeled mAb isotype controls. CD4⁺CD69⁺ and CD4⁺NKG2D⁺ cells were analyzed separately, and data were acquired in FACSCanto II flow cytometer (Becton Dickinson) and analyzed using the Flow Jo software v10 (Tree Star Inc., Ashland, OR).

Spleen, MLN, and small intestine lamina propria cells were resuspended in PBS/1% bovine serum albumin and incubated with anti-mouse CD16/CD32 (Mouse BD Fc Block; BD Pharmingen, San Jose, CA, USA) to block non-specific binding sites. For surface staining, cells were incubated with CD4-PerCp-Cy5.5, CD69-APC, CXCR3-PE, CD25-AlexaFluor488, CD69-PE-Cy7, CD11c-PerCp-Cy5.5, CD8α-PE, CD11b-PE-Cy7, CD103-APC, CD11b-PE, CD83-FITC, CD86-APC (eBiosciences, San Diego, CA, USA), T1ST2-FITC (MD Biosciences, St. Paul, MN, USA), or CX3CR1-AlexaFluor488 (R&D Systems, Oxon, UK). Viable cells were distinguished by means of a fixable viability dye eFluor^® 780 (eBioscience). For detecting transcription factors, cells were first fixed and permeabilized with Foxp3 Staining Buffer Set (eBioscience) according to manufacturer’s protocol and then stained with Foxp3-APC and RorγT-PE antibodies (eBioscience). Results were collected with BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed with FlowLogic software (Inivai Technologies, Mentone, VIC, Australia).

The flow cytometry analysis was used to identify ERCs and detect the population of T cell subsets in this study. The staining process was the same as we previously mentioned [20 (link)]. Briefly, after being separated into single-cell suspension, the collected cells (100 µL/test) were stained with Zombie NIR™ (BioLegend, USA) to identify dead/live cells. And then stained these cells with fluorescent-labeled antibodies, which were purchased from BioLegend and eBioscience, including anti-human: CD45-FITC (Clone: HI30), HLA-DR-FITC (Clone: L243), CD73-FITC (Clone: AD2), CD105-PE-Cyanine 7 (Clone: SN6), and anti-mouse: CD45-PE-Cyanine7 (Clone: 30-F11), CD45-Percp-Cyanine 5.5 (Clone: 30-F11), CD3-FITC (Clone: 145-2C11), CD4-PE (Clone: GK1.5), CD4-FITC (Clone: RM4-5), CD8a-Percp-Cyanine 5.5 (Clone: 53-6.7), CD69-APC (Clone: H1.2F3), CD154-PE (Clone: SA047C3), IFN-γ-PE (Clone: XMG1.2), IL-17A-Percp-Cyanine 5.5 (Clone: eBio17B7), CD25-PE (Clone: PC61.5), Foxp3-APC (Clone: FJK-16s).

Stimulated PBMCs or γδ cells were stained with the following antibodies: Live-dead, (L34966, Invitrogen), CD3(clone UCHT1), CD19-PerCP (clone HIB19), γδ TCR-PE (clone 5A6.E9), αβ TCR-PE-Cy7(clone IP26), CD69-APC (clone FN50), CD137-BV650 (clone4B4-1). After staining, cells were washed twice and stored on ice until acquisition and sorting on a FACS Aria Fusion cell sorter (BD Biosciences). CD3⁺CD19^-αβ^-γδ⁺CD69⁺CD137⁺were single cells sorted directly into individual wells in a 96-well plate (Eppendorf 951020303) containing RT-PCR buffer.

Antibodies used were against HSA (CD24): M1/69 fluorescein or phycoerythrin (BD Pharmingen, Erembodegem, Belgium) or allophycocyanin (Biolegend, San Diego, CA), CD69 (APC, Invitrogen Life Technologies, Darmstadt, Germany), HIV-1 Vpr (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, Germantown, MD, USA); HIV-1 Vpr (1–46) antiserum from Dr Jeffrey Kopp (catalogue #3951) or mouse anti-HIV-1 Vpr serum [25 (link)] (kindly provided by Ulrich Schubert, Erlangen), HIV-1 p24 (AIDS Research and Reference Reagent Program; Monoclonal Antibody to HIV-1 p24 (AG3.0) from Dr Jonathan Allan) [60 (link)] or HIV-1 p24 clone KC57-RD1 (PE or FITC conjugated, Beckman Coulter, Krefeld, Germany), mouse anti-tubulin (Sigma-Aldrich, Munich, Germany), rabbit anti-NFAT (CellSignaling Merck-Millipore, Schwalbach, Germany). Secondary antibodies were IRdye 800CW Goat anti-Rabbit IgG and IRdye 680LT Goat anti-mouse IgG (Li-Cor, Lincoln, USA), Alexa-555 donkey anti-rabbit and Alexa-488 goat anti-mouse (Invitrogen) and goat anti-mouse as well as goat anti-rabbit HRP (Dianova, Hamburg, Germany). Insulin and SB213763 were from Sigma-Aldrich (Munich, Germany) and FK506 from Invitrogen.