Cd154

Intracellular cytokine staining (ICS) was performed as previously described [14 (link)]. PBMC were thawed and rested overnight. Cells (1 x 10⁶ cells/well) were stimulated in 96-well round bottom cell culture plates with Ag85A, Ag85B, and TB10.4 peptide pools (1 μg/peptide/mL), peptides were pre-diluted in R10 medium containing CD107a-Alexa488 (Biolegend, USA), GolgiStop and GolgiPlug (BD Biosciences, USA). Dimethyl sulfoxide (Sigma) was used as a negative control and SEB (0.5 μg/mL) as the positive control. Cells were stimulated for 6–7 hours at 37°C and 5% CO2. After incubation, the cells were washed and stained with viability dye (Invitrogen, USA) before staining with fluorochrome-conjugated antibodies to surface markers CD4 (eBioscience, USA), CD8 (Biolegend), CD14 and CD19 (BD), then permeabilized and stained for CD3 (Beckman Coulter, USA), IFN-γ, IL-2, CD154, TNF-α (all four from BD), IL-17A (Biolegend), and IL-22 (eBioscience). Cells were acquired on BD LSR flow cytometers (BD Biosciences). All sample analysis was performed with FlowJo software (TreeStar Inc., USA).

ATCs were analyzed by flow cytometry for the surface markers including CD69, HLA-DR, CD45RO, CD154 and CD137 at day 5 and 12 in addition to CD3, CD4 and CD8. Early activation markers were noted to be highest on day 5 and maximal T cell growth was noted on day 12. Additionally, sterility testing for endotoxin, mycoplasma, gram stain and environmental culture was performed to ensure quality of the cell products for future clinical trials. Antibodies (BD Bioscience) used were anti-human CD3 (Cat. 564,810), CD4 (Cat. 555,346), CD8 (Cat. 560,662), CD69 (Cat. 562,883), HLA-DR (Cat. 564,231), CD45RO (Cat: 561,136), CD154 (Cat. 557,299) and CD137 (Cat. 561,702). Growth of ATCs was also measured along with the activation marker analysis. Maximal growth of T cells was noted to be at day 12 (data not shown).