Anti cd8α pe clone 53 6.7

The capacity of PDT and the NPs to induce antigen-specific T cells in the blood of TC-1 tumor-bearing mice was determined by analyzing (25 µL) blood obtained from the tail vein at day 8 after PDT. Red blood cells were removed using a lysis buffer, after which the cells were incubated with an APC-labeled, MHC class I (H-2Db) HPV16 E749-57 (RAHYNIVTF) (H-2Db) tetramer. Next, the cells were stained with anti-CD8α-PE (clone 53-6.7; eBioscience, Waltham, MA, USA), anti-CD3-eFluor 450 (clone 17A2; eBioscience, Waltham, MA, USA), and analyzed by flow cytometry on an LSR-II (BD Biosciences, San Jose, CA, USA). Gating of CD8⁺ T cells was based on CD3⁺CD8⁺ events after size/morphology and doublet exclusion was based on FSC/SCC patterns.

The presence of antigen-specific T cells in the blood of each mouse was determined by collecting 50 µL of blood via a puncture of the caudal vein at day 8 and day 16 after the first treatment. After removal of red blood cells by lysis, the cells were stained with anti-CD8α-PE (clone 53-6.7, eBioscience) and anti-CD3-eFluor 450 (clone 17A2, eBioscience). For mice bearing TC-1 tumors, the APC labeled HPV16 E7_49-57 (RAHYNIVTF) MHC class I (H-2D^b) tetramer was added to the staining mix. After thorough washing, the cells were subjected to flow cytometry measurements on an LSR-II laser flow cytometer controlled by CELLQuest software v. 3.0 (Becton Dickinson) and the data analyzed with FlowJo LLC v. 10 software (Tree Star).

Spleen cells were further seeded in triplicates at 5 × 10⁶ cells/well in a 24-well plate and stimulated with WH121 (10 μg/mL) in the presence of 1 μg/mL anti-CD28/CD49d (eBioscience CA, USA) for 20 h at 37°C under 5% CO₂. For the last 4 h of the stimulation, 3 μg/mL brefeldin A and 2 μM monensin solution (eBioscience CA, USA) were added. The cells were washed in FACS buffer (1% FCS-PBS) and stained for 30 min at RT with anti-CD4-PE-Cy7 (clone GK1.5, eBioscience), anti-CD8α-PE (clone53-6.7, eBioscience), anti-CD62L-FITC (clone MEL-14, BD Pharmingen), and anti-CD44-APC-Cy7 (clone IM7, BD Pharmingen) mAbs. After the cells had been washed and permeabilized with the Cytofix/Cytoperm kit (BD Pharmingen CA, USA), the intracellular cytokines were stained with anti-IFN-PerCP-Cy5.5 (clone XMG1.2; eBioscience CA, USA) and anti-IL-2-allophycocyanin (clone JES6-5H4; eBioscience CA, USA) mAbs for 30 min at RT. The cells were washed twice, resuspended in FACS buffer and analyzed using an LSRII multicolor flow cytometer (BD Biosciences CA, USA). The absolute number of IFN-γand/or IL-2 positive CD4⁺ and CD8⁺ T cells, T_CM (central memory T cells, CD62L^hiCD44^hi) and T_EM (effector memory T cells, CD62L^loCD44^hi) cells were analyzed with FlowJo software (Tree Star Inc., OH, USA) as previously described [25 (link)], respectively. The data are represented as the mean ± SD per group (n = 6).