Apc conjugated anti cd3

Flow cytometry was used to determine the phenotype of the lymphocytes. Isolated lymphocytes were stained for 30 min in the dark at room temperature with 1 μL of monoclonal antibodies specific for T cell surface markers. These antibodies included APC-conjugated anti-CD3 (553,066, BD Biosciences, San Diego, USA), FITC-conjugated anti-CD4 (553,046, BD Biosciences, San Diego, USA) and PE-conjugated anti-CD8 (553,032, BD Biosciences, San Diego, USA). Then, the lymphocytes were washed with 0.01 M PBS (0.2 mL) and centrifuged at 500×g for 5 min; this step was repeated for a total of three washes. After the final wash, the cells were resuspended in 0.2 mL of PBS and subjected to flow cytometry analysis. The lymphocytes were analyzed on a FACSCalibur flow cytometer (BD Biosciences) using the CellQuest program (BD Biosciences).

PBMCs isolated from mobilized and non-mobilized donors were stimulated in 96 well plates at a concentration of 1×10⁷/ml for 24 hours with either CMVpp65 Peptivator or 1 µg/ml of Staphylococcal Enterotoxin B (SEB. Sigma-Aldrich, Gillingham, UK) or left untouched. Samples were taken at 1, 4, 6, 16 and 24 hours and stained with APC-conjugated anti-CD3, FITC-conjugated anti-CD4, PerCP-conjugated anti-CD8 and PE-conjugated anti-CD25 (BD Bioscience).

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).

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.

To assess the proliferation of the primed CD4⁺ and CD8⁺ T cells, murine splenocytes and cells from the dLNs and MLNs were stained with CFSE (Invitrogen) for 10 min at 37 °C and incubated with anti-CD28 and either NP and M1 peptide or anti-CD3 as a positive control in 96-well plates. After 72 h of incubation, the cells were labelled with APC-conjugated anti-CD3 and PE-conjugated anti-CD4 or anti-CD8 as described above (BD Biosciences). Finally, the samples were analysed using a BD FACSAria™ II flow cytometer, and the data were analysed using FlowJo software.

Single-cell suspensions were prepared as previously described (Dou et al., 2018 (link)). For detection of NK cells and their NKG2D expression, cells were stained with FITC-conjugated anti-NK1.1 (BD Bioscience), APC-conjugated anti-CD3 (BioLegend), PE-conjugated anti-NKG2D (BioLegend) and corresponding isotype controls. For detection of NK cell-produced IFN-γ, firstly, 1.0×10⁶ cells were activated in vitro with 2 μl Leukocyte Activation Cocktail (BD Bioscience) for 5 h at 37°C, followed by incubation with FITC-conjugated anti-NK1.1, APC-conjugated anti-CD3 for 30 min. Then, cells were permeabilized and fixed using Cytofix/Cytoperm Soln Kit (BD Bioscience) for 30 min, followed by incubation with PE-conjugated anti-IFN-γ (BD Bioscience). FACS was performed on BD FACS Calibur flow cytometer (BD Biosciences, NJ, United States) or CytoFLEX (BECKMAN COULTER, United States). Data were analyzed using FlowJo (version 10, United States). For analysis of NK cell infiltration, the lymphocyte population was gated from the general diagram of FSC-SSC. The negative control was then determined from cells stained with isotype controls. The location of CD3⁺ cells was determined by CD3-stained cells, and that of NK1.1⁺ cells was distinguished by NK1.1-stained cells. The percentage of NK1.1⁺CD3⁻ cells in the population was recorded.

Blood samples were immunostained with the following panel of antibodies: APC-conjugated anti CD3, FITC-conjugated anti CD4, PE-conjugated anti CD69, PECy5-conjugated anti CD8, PE-conjugated anti CD28, PerCP-conjugated anti CD3, FITC-conjugated anti CD16, PE-conjugated anti CD56, PerCP-conjugated anti CD19, FITC-conjugated anti CD25, PE-conjugated anti Foxp3 and APC-conjugated anti CD4 (all reagents from BD Pharmingen, San Diego, USA). Optimal antibody concentrations were previously defined by titration. For intracellular Foxp3 staining, samples were first stained for CD4 and CD25, then fixed and permeabilized with human Foxp3 buffer set (BD Pharmingen, San Diego, USA) according to manufacturer´s protocols. Briefly, cells were washed twice with permeabilization buffer and then incubated with anti-human Foxp3 at room temperature for 30 min in the dark, before being resuspended in PBS and analyzed.
Flow cytometry data was collected on a FACSCalibur flow cytometer equipped with two lasers (Becton–Dickinson, Oxford, UK). For data acquisition and analysis CellQuestPro software (Becton–Dickinson) was used.