Live dead fixable yellow dye

We selected 13 commercially available vaccines that are delivered in soluble form intradermally or intramuscularly. The vaccines used are summarized in Table 1, including manufacturer, alum content and working concentration. To assess vaccine activation concentration, we cultured IL-4 DCs with three different concentrations of each vaccine for 24 h and assessed DC viability and activation status by flow cytometry. DC and monocyte purity were assessed with the following panel: FITC-CD16, PerCP-Cy5.5-CD14, PE-CD141, ECD-CD19, PE-Cy7-CD56, APC-CD1c, Alexa Fluor 700-HLA-DR, APC-H7-CD8 and V450-CD3. The purity analysis gating strategy is summarized in Supplementary Fig. 13a. Viability was assessed by the incorporation of Live/Dead Fixable Yellow dye (Life Technologies; Supplementary Fig. 13b,c). DC activation/maturation was assessed with the following panel: FITC-CD56, PerCP-Cy5.5-CD14, PE-CD1a/b/c, ECD-CD16, PE-Cy7-CD40, APC-CD83, Alexa Fluor 700-CD3/CD19/CD20, APC-H7-CD80, V450-CD86 (Supplementary Fig. 14). We selected the stimulation concentration that best activated IL-4 DCs while retaining elevated viability, and used these concentrations for CD1c+ DC maturation experiments. The stimulation concentrations are summarized in Table 1.

PBMCs from healthy human donors were obtained from Miltenyi Biotec, and naive CD4⁺ cells were negatively selected using the naive CD4⁺ T cell isolation kit II by MACS according to the manufacturer’s instructions. CD4⁺ cells were activated using anti-CD3 (clone OKT3, 5 μg/mL) and anti-CD28 (clone CD28.2, 10 μg/mL) mAb–precoated 96-well plates. Next, 2 × 10⁵ T cells and 4 × 10³ DLBCL cells (SU-DHL-4 or Karpas-422) were cocultured at a 50:1 ratio in AIM-V medium (Invitrogen) supplemented with HLA-DR/DP/DQ mAb (clone WR18, 10 μg/mL) for MHC II blockade for 3 days. T cell proliferation was assessed using CellTrace violet dye (Invitrogen). Prior to coculturing, CD4⁺ cells (1 × 10⁷ cells/mL) were stained with 5 μM CellTrace violet dye and incubated at 37°C for 15 minutes with gentle vortexing every 5 minutes. Flow cytometry was performed on the FACSSymphony flow cytometer (BD Biosciences) using the following Abs/stains: LIVE/DEAD Yellow Fixable dye 1:1000 (Invitrogen), CD4 BV786 clone SK3 1:200 (BD Biosciences), CD8 APC-H7 clone SK1 1:200 (BD Biosciences), and CD69 FITC clone FN50 1:50 (BioLegend).

Cells from the aorta, spleen and paLN were isolated as described before^{19 (link)}. Briefly, Apoe^−/−Il27ra^+/− or Apoe^−/−Il27ra^−/− mice were euthanized by CO₂ inhalation. The aortas were perfused with 30 ml of PBS containing 2% heparin and isolated under the dissection microscope. Collected aortas, spleens or paLNs were cut into small pieces followed by digesting in 2 ml of enzymatic cocktail, containing 450 U/ml collagenase type I, 250 U/ml collagenase type XI, 120 U/ml hyaluronidase type I, 120 U/ml DNAse I (all enzymes from Sigma) in 1x HBSS and incubated in a shaker at 37 °C for 55 min. Obtained cell suspension was stained with following antibodies: CD45-PerCP (30-F11; BioLegend), CD11b-eFluor 450 (M1/70; eBioscience), CD11c-APC (N418; eBioscience), MHCII-Alexa Fluor 700 (M5/114.15.2; eBioscience), TCRβ-eFluor 780 (H57-597; eBioscience), CD69-PE-Cy7 (H1.2F3; eBioscience), B220-FITC (RA3-6B2; eBioscience), CD4-APC (GK1.5; eBioscience), CD8-PE (53-6.7; eBioscience) and LIVE/DEAD Yellow fixable dye (Invitrogen) and analyzed by flow cytometry (LSRII, BD Biosciences). Obtained data were analyzed using FlowJo software.