96 u bottom plate

Whole blood was taken in a single venepuncture into heparin tubes and 0.8 ml were added to each of the four QFT tubes (i.e., Nil, TB1, TB2, and PHA; Qiagen). The tubes were immediately incubated at 37°C for about 20 h. For the QFT_in-vitro assay, 100 μl whole blood was cultured in 100 μl RPMI supplemented with, Penicillin/Streptomycin (100 U/ml) and L-glutamine (2 mM) using a 96 U-bottom plate (Greiner). Samples were stimulated with recombinant ESAT6/CFP10 fusion protein (E6/C10: 2 μg/ml), purified protein derivative of Mtb (PPD: 10 μg/ml; Statens Serum Institute), phytohemagglutinin (PHA: 10 μg/ml; Sigma-Aldrich) or left unstimulated for 20 h at 37°C and 5% CO₂. The term QFT_in-vitro has been introduced since the results serve as reference between QFT and ICS-based in vitro assays (see below). In contrast to QFT, the QFT_in-vitro uses recombinant proteins (not optimized peptide mixtures) and blood dilution. However, for comparison of PPD stimulation, as well as PBMC and intracellular cytokine staining (ICS), this QFT comparable assay was needed.
Supernatants from both assays were harvested and stored at −80°C until further analysis.

Triplicates of 5x10⁴ cells were plated in a 96-U bottom plate (Greiner) and incubated with AF488 conjugated (glyco)-dendrimers or vehicle control (max. 0,2% DMSO) diluted in serum free IMDM for LCs and complete RPMI for moDCs. When indicated cells were pre-incubated for 1 hour at 4^oC, followed by incubation at 37^oC for indicated time-points. Next, cells were stained with a fixable viability dye eFluor780 (FVD; eBioscience), anti-human HLA-DR BV510, CD1a APC (clone HI149; BD) (moDC) and EpCAM BV421 (clone EBA-1; Biolegend) (LCs). Binding and uptake was analyzed using FACS.
For antibody blocking assays cells were pre-incubated with 20µg/ml mouse-anti-human IgG1 Langerin (10E2) or DC-SIGN (AZN-D1) for 30 minutes at 37^oC, followed by addition of (glyco)-dendrimers for 1 hour at 37^oC with blocking antibodies at a final concentration of 10µg/ml. For 3 hour pre-incubation cells were incubated with a 10x serial dilution starting at 10µg/ml AZN-D1, washed and cultured for 1 hour with (glyco)-dendrimers at 37^oC. Liposomes with LeY were taken along as positive control for AZN-D1 blocking assays. For DC-SIGN block using mannan moDC were pre-incubated for 30 minutes with a 10x serial dilution starting at 100µg/ml, followed by co-incubation with 0,01µM (glyco)-dendrimers.

For LETR1, 5 × 10⁵ LECs infected with pCDH-EV or pCDH-LETR1 were seeded into 10 cm dishes and cultured overnight in starvation medium (EBM supplemented with 1% FBS). The next day, infected LECs were transfected with scrambled control ASO or LETR1-ASO2 for 24 h as described above.
For KLF4, a consecutive transfection of ASOs and siRNAs was performed. siRNA sequences are listed in Supplementary Data 13. In all, 350,000 LECs were seeded into 10 cm dishes and cultured overnight in starvation medium. The next day, LECs were transfected with scrambled control ASO or LETR1-ASO2, as described above. 24 h post transfection with ASOs, the medium was changed, and LECs were treated for additional 24 h with 20 nM of high GC scrambled control siRNA or two siRNAs targeting KLF4 (Thermo Fisher Scientific) and 32 µL Lipofectamine RNAiMAX previously mixed in 800 µL Opti-MEM.
At the end of the experiment, LECs were detached and collected. In all, 1.5 × 10⁵ LECs per replicate were then transferred in a 96 U-bottom plate (Greiner bio-one). Aliquots of ~1 × 10⁵ LECs were lysed and subjected to qPCR, as described above. Cell cycle analysis using flow cytometry was performed as described above. To detect Ki-67, an eFluor 450-conjugated rat anti-human Ki-67 antibody (clone: SolA15, ebioscience) was used at a 1:200 dilution.