Apc cy7 conjugated anti cd4

Flow cytometry was used to analyze surface phenotype and transduction efficiency. The following monoclonal antibodies that detect surface proteins were used: PerCP-conjugated anti-CD3 antibody (BD Biosciences, San Jose, CA), APCcy7-conjugated anti-CD4 (BD Biosciences), FITC-or APCcy7-conjugated anti-CD8 (BD Biosciences), RD1-conjugated anti-CD45RA (Beckman Coulter, Fullerton, CA), and FITC- or APC-conjugated CCR7 (R&D Systems, Minneapolis, MN). To analyze the gene transfer efficiency of TCR-modified T cells, we double-stained the transduced PBMCs with FITC-conjugated anti-CD8 and PE-conjugated MAGE-A4_143-151/HLA-A*2402 tetramers (Ludwig Institute for Cancer Research, New York, NY). Samples were run through a FACS CantoII flow cytometer (BD Biosciences). Data were analyzed using FACS Diva software (BD Biosciences).

PBMCs were initially stimulated with IL-2 (100 U/mL) for six days. Cells were then washed and restimulated with plate-bound anti-CD3/CD28 antibodies (anti-CD3, 0.2 μg/mL; anti-CD28, 0.4 μg/mL), IL-2 (100 U/mL), or HIV gag peptide (10 μg/mL), with or without anti-Tim-3/PD-1 antibodies (25 μg/mL). For the detection of IL-2 and IFN-γ production, GolgiStop was added 1 hour after restimulation, and 4 hours later cells were stained for PerCP-conjugated anti-CD3 and APC-cy7-conjugated anti-CD4 and intracellular APC-conjugated anti-IL-2 or APC-conjugated anti-IFN-γ, according to the manufacturer's recommendations (BD Biosciences). Intracellular expression of IL-2 or IFN-γ within CD4+ and CD8+ (CD3+CD4−) T cells was detected using an LSRII instrument and analyzed with FlowJo software.

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll centrifugation. The following monoclonal antibodies (mAbs) and reagents were used in this study: PE-Cy7-conjugated anti-CD3, APC-conjugated anti-CD3, APC-Cy7-conjugated anti-CD4, PE-conjugated anti-CD38, APC-conjugated anti-HLA-DR, APC-Cy7-conjugated anti-HLA-DR, FITC-conjugated anti-CD45RA, PerCP-Cy5.5-conjugated anti-CCR7 (BD Biosciences, USA); Violet-conjugated anti-CD38, FITC-conjugated anti-CD38, PE-Cy7 conjugated anti-CD25, APC conjugated anti-CD69, APC conjugated anti-CD127, Amcyan-conjugated anti-CD45RA (BioLegend, San Diego, CA, USA). For the expression of all markers, flow cytometric gating was defined using fluorescence minus one (FMO) controls. CD4⁺ T cell subsets were identified in terms of CD45RA and CCR7 expression. CD38 and HLA-DR were measured on gated CD4⁺ T cell subsets: naive CD4⁺ T cells (Tn, CD3⁺CD4⁺CD45RA⁺CCR7⁺), central memory CD4⁺ T cells (Tcm, CD3⁺CD4⁺CD45RA⁻CCR7⁺), and effector memory CD4⁺ T cells (Tem, CD3⁺CD4⁺CD45RA⁻CCR7⁻). The expression of CD25, CD69, and CD127 were measured on gated CD38⁺ Tcm, HLA-DR⁻ Tcm, and CD4⁺HLA-DR⁺ cells. Data were collected using a BD LSRII flow cytometer (BD Biosciences) and analyzed using Flowjo software (TreeStar, USA).

PBMCs were plated in round-bottom 96-microtiter plates at 500,000 cells/well in 200 μL complete RPMI 1640 containing anti-CD3 (5 μg/mL) and anti-CD28 (2 μg/mL) antibodies (BD Biosciences) or HIV gag peptide (10 μg/mL, XiAn Meilian Company), with or without anti-Tim-3 and anti-PD-1 (25 μg/mL) antibodies (Biolegend). Cells were cultured for three days, and GolgiStop was added during the last five hours of the culture. The cells were incubated with conjugated antibodies PerCP-conjugated anti-CD3, APC-cy7-conjugated anti-CD4, PE-conjugated anti-Tim-3, and FITC-conjugated anti-PD-1, and intracellular cytokine staining for APC-conjugated anti-IFN-γ (BD Biosciences Pharmingen) or APC-conjugated IL-2 (Biolegend) was carried out using the Cytofix/Cytoperm Fixation/Permeabilization Kit according to the manufacturer's instructions (BD Biosciences). Intracellular expression of IL-2 or IFN-γ within CD4+ and CD8+ (CD3+CD4−) T cells was detected using an LSRII instrument and analyzed with FlowJo software.

At the time of study enrollment, the patient characteristics were assessed; additionally, peripheral venous blood samples were taken and available for all study participants. All patients were enrolled in a stable condition free of any signs of congestion of acute cardiac ischemia. In patients presenting with ischemic HFrEF, the definition of heart failure was made at least 6 weeks after the acute ischemic event to overcome selection bias based on postinfarction myocardial stunning as recommended by the European Society of Cardiology [12 (link), 13 (link)]. Routine laboratory parameters were analyzed and processed according to the local standards of the Department of Laboratory Medicine of the Medical University of Vienna. In addition, cells from fresh EDTA blood samples were stained with APC-Cy7-conjugated Anti-CD4 (BD Biosciences, San Jose, CA, USA) and FITC-conjugated Anti-CD8. Regulatory T cells were identified via their intracellular forkhead-box protein P3 (Fox-P3) and CD25 expression using PE-conjugated Anti-Fox-P3 (BioLegend, San Diego, CA, USA) as well as APC-conjugated Anti-CD25 (BioLegend, San Diego, CA, USA) in a second FACS panel. Stained cells were analyzed using a BD FACS Canto II Flow Cytometer System and FACSDiva software.

At day 45 p.t.i. mice with residual primary lymphoma from CHOP×2 group (i.e. animals with partial response to chemotherapy) as well as PBS control, were sacrificed and tumors were removed and prepared as a single-cell suspension. Cells were immunostained at 4 °C in the dark for 30 min with the following panel of antibodies: FITC-conjugated anti-CD49b, PECy7-conjugated anti-CD8, APC-conjugated anti-CD3, APCCy7-conjugated anti-CD4, PerCPCy5.5-conjugated anti-CD19, FITC-conjugated anti-CD4, PE-conjugated anti-FoxP3, PECy7-conjugated anti-CD3, APC-conjugated anti-CD25, FITC-conjugated anti-Ly6C, PE-conjugated anti-Ly6G, (all reagents from BD Pharmingen, San Diego, CA). Optimal antibody concentration was previously defined by titration. For intracellular FoxP3 staining, cells were first stained with anti-CD4 and anti-CD25 antibodies, then fixed and permeabilized with a mouse FoxP3 buffer set (BD Pharmingen) according to the manufacturer’s protocols. Cells were washed twice with permeabilization buffer and then incubated with anti-FoxP3 at 4 °C for 30 min in the dark. Flow cytometry data were collected on a FACS Canto II flow cytometer equipped with two lasers (Becton–Dickinson, Oxford, UK). For data acquisition and analysis, FACSDiva (Becton–Dickinson) and Infinicyt (Cytognos, Spain) software were used.

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient (Sigma) and were washed once with FACS buffer (PBS, with 1% bovine serum albumin). For staining, 1 × 10⁶ cells were incubated with conjugated antibodies APC-cy7-conjugated anti-CD3, PerCP-conjugated anti-CD8 (BD Biosciences Pharmingen), PE-conjugated anti-Tim-3, and FITC-conjugated anti-PD-1 (Biolegend) for 30 min at 4°C. Analysis was performed using an LSRII instrument (BD Biosciences), and at least 10,000 events were collected. Tim-3 and PD-1 expression on CD4+ (defined as CD3+CD8− cells) and CD8+ T cells were analyzed with FACSDiva software (BD Biosciences). For the detection of cytokine-induced Tim-3 and PD-1 expression, freshly isolated PBMCs were plated in round-bottom 96-microtiter plates at 0.5 million cells/well in 200 μL complete RPMI 1640 containing anti-CD3 (5 μg/mL) and anti-CD28 (2 μg/mL) antibodies (BD Biosciences) or recombinant IL-2, IL-7, IL-15, and IL-21 (25 ng/mL) (IL-2, IL-7, and IL-15: R&D Systems; IL-21: Biosource). Cells were cultured for five days and were incubated with conjugated antibodies against PerCP-conjugated anti-CD3, APC-cy7-conjugated anti-CD4 (BD Biosciences Pharmingen), PE-conjugated anti-Tim-3, and FITC-conjugated anti-PD-1 for 30 min at 4°C. Analysis was performed using an LSRII instrument and analyzed with FlowJo software.