Tnf mab11

SIV-specific CD8⁺ T cells were quantified in mononuclear cells isolated from blood and LN by flow cytometric intracellular cytokine analysis, as previously described in detail^{45 (link)}. Briefly, mixes of sequential (11 amino acid overlapping) 15-mer peptides (AnaSpec) spanning the SIVmac239 Gag, Env, Pol, Nef, Rev/Tat, Vif/Vpr/Vpx proteins were used as antigens in conjunction with anti-CD28 (BD Biosciences) and anti-CD49d co-stimulatory mAb (BD Biosciences). Cells were incubated at 37 °C with peptide mixes/pools and co-stimulatory antibodies for 1 hr, followed by an additional 8 hr. incubation in the presence of Brefeldin A (5 μg/ml, Sigma-Aldrich). Co-stimulation in the absence of peptides served as background control. Cells were stained with fluorochrome-conjugated monoclonal antibodies [CD3 (SP34-2), CD4 (L200), CD8a (SK1), IFN-γ (B27)(BD Biosciences), TNF (MAB11, eBioscience), CD69 (FN50, BD Biosciences)], and data were collected on an LSRII (BD Biosciences) and analyzed using the FlowJo software program (version 8.8.7; Tree Star, Inc.). Response frequencies to each peptide mix were defined by the intracellular expression of TNF and/or IFN-γ by CD3⁺, CD4⁻, CD8⁺ T cells after subtraction of background. Response frequencies to each peptide mix were added to obtain total SIV-specific response frequencies.

CD19 CAR transduced T cells (1×10⁶ cells) were incubated at an effector target ratio of 1:1 with either CD19⁺ K562, NGFR⁺ K562 or autologous B cells (CLL cells or EBV-transformed B cells), in AIM-V medium supplemented with 5% heat-inactivated human AB serum in presence of 10 μg/mL brefeldin A (Sigma-Aldrich), GolgiStop (BD Biosciences) and the anti-CD107a (BD Biosciences) antibody for 6 hours at 37°C and 5% CO₂. After incubation, monoclonal antibodies directed against CD3 (SK7), CD4 (RPA-T4), and CD8 (SK1) (all from BD Biosciences) were used for cell surface staining, and after fixation and permeabilization with the BD Cytofix/Cytoperm kit (BD Biosciences), antibodies against IL-2 (MQ1–17H12) (Biolegend), TNF (Mab11) (eBioscience), IFN-γ (4S.B3), and IL-17 (N49–653) (BD Biosciences), or in other experiments granzyme B (GB11) (BD Biosciences) were used for intracellular staining. For maximal stimulation controls, PMA and ionomycin (Sigma-Aldrich) were used at 25 ng/mL and 1 μg/mL, respectively, in presence of brefeldin A at 10 μg/mL and GolgiStop for 6 hours at 37°C and 5% CO₂. Cells were analyzed by flow cytometry as described above.