96 well cell culture plate

The mice in each group were killed at week 29 after M. tuberculosis infection, and the spleens were obtained to prepare splenocyte suspensions. A 100-μL volume of splenocytes at the concentration of 3 × 10⁶/mL was added to the well of a 96-well cell culture plate (Mabtech), followed by incubation with 50 μL of PBS (negative control), 50 μL of Th1 peptide (100 μg/mL), 50 μL of CTL peptide (100 μg/mL), 50 μL of peptide pool (100 μg/mL), or 50 μL of PHA (positive control, 50 μg/mL) at 37°C for 48 h. Then, after centrifugation at 500 × g for 10 min, the supernatant of each sample was collected to detect the levels of Th1/Th2/Th9/Th17/Th22/Treg cytokines (IFN-γ, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-5, IL-6, TNF-α, GM-CSF, IL-18, IL-10, L-17A, IL-22, IL-23, IL-27, and IL-9) by using a Th1/Th2/Th9/Th17/Th22/Treg cytokine 17-Plex mouse ProcartaPlex panel (catalog no. EPX170-26087-901; Thermo Fisher, China) according to the manufacturer’s instructions.

A 5 mL blood sample was collected from HCs (n = 7), LTBI individuals (n = 8), and ATB patients (n = 7). PBMCs were isolated from the blood samples as described above. Then, 2.5 × 10⁵ PBMCs in 100 μL AIM medium were added to one well of a 96-well cell culture plate (Mabtech AB, Nacka Strand, Sweden) and incubated with 50 μL PP19128R vaccine (100 μg/mL) in a CO₂ incubator at 37 °C for 48 h. The mixture of cells and AIM medium was aspirated and then centrifuged at 1000 rpm for 10 min to collect the supernatant. The supernatant was gently transferred to another tube. The levels of interleukin-2 (IL-2), IL-4, IL-6, IL-10, IFN-γ, tumor necrosis factor-α (TNF-α), and IL-17A were measured using a BD CBA Human Th1/Th2/Th17 Cytokine Kit (BD Bioscience, San Diego, CA, USA, Cat: 560484) according to the manufacturer’s instructions. In addition, another group of HCs (n = 10) was enrolled as a negative control. Approximately 2.5 × 10⁵ PBMCs in 100 μL of AIM were added to a 96-well cell culture plate and incubated with 50 μL of AIM medium in a CO₂ incubator at 37 °C for 48 h. Th1/Th2/Th17 cytokine levels were then detected as described above.

The mice were killed on the 91^st day after the first immunization, and the spleens were collected for the preparation of splenocytes suspension following the above methods. A volume of 100μl splenocytes (3×10⁶/ml) and 50μl MP3RT vaccine (60 μg/ml) were incubated in a well of 96-well cell culture plate (Mabtech AB, Nacka Strand, Sweden) at 37°C for 48h. Then, the culture solution was transferred into a new tube and centrifugated at 500g for 10 min. Finally, the supernatant was gently transferred into another new tube, and the levels of interleukin -2 (IL-2), IL-4, IL-6, IL-10, IFN-γ, tumor necrosis factor -α (TNF-α), and IL-17A were detected by a Mouse Th1/Th2/Th17 Cytokine Kit (Cat. No. 560485, BD Biosciences, San Jose, CA, USA) following the manufacturer’s instruction.