X vivo medium

The ZIKV (Zika virus) infection rate was determined in Vero E6 cells. Therefore, 12,000 cells were seeded into 96-well plates. The next day, medium was removed and 70 µl fresh x-vivo medium (Lonza, Basel, Switzerland) and 10 µl of peptide titration series were added. Then, cells were infected with 20 µL of ZIKV MR766. Two days later, infection rates were determined with a cell-based ZIKV immunodetection assay described previously [24 (link)]. HIV infection was quantified by the reporter activity of TZMbl cells. Therefore, 10,000 cells were seeded. The next day, medium was removed and 70 µl fresh x-vivo medium (Lonza) and 10 µl of peptide titration series were added. Then, cells were infected with 20 µL of HIV-1 NL4-3. Two days later, the infection rate was determined by quantification of β-galactosidase expression according to Müller et al. [24 (link)]. All infection values were corrected for the background signal derived from uninfected cells, and untreated controls were set to 100% infection.

Cells were thawed, and then rested overnight in x-vivo medium (Lonza, Basel, Switzerland) with 5% human serum in an incubator. The next day, cells were washed twice in FACS buffer, then stained with the following: LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Waltham, MA, USA), CD45RA-APC, CD62L-PE, and CD3-FITC (BD Biosciences, San José, CA, USA). Cells were stained for 30 min on ice, washed twice, and then resuspended in FACS buffer at 50 × 10⁶cells/mL. Next, cells were sorted on a FACS ARIA flow cytometer with appropriate application settings and compensation controls. Cell sorting was performed with a purity setting. After sorting, cells were allowed to rest in x-vivo medium (Lonza, Basel, Switzerland) in an incubator. After sorting of all the cells, samples of 10⁴ cells were analyzed to ensure proper sorting purity. Gating strategy for the identification of CD3⁺ T cells for the phenotypic analysis and subsequent sorting of different memory cell fractions is provided in Supplementary Material 2. Materials and methods for intracellular cytokine staining (ICS), establishment of T-cell cultures specific for mutant CALR epitopes and statistical analyses are provided in Supplementary Material 3.

C57BL/6 mice, at 6–8 weeks, were administered a lethal dose of Euthasol followed by transcardial perfusion with 0.9% ice cold heparinized saline. Brains were excised under aseptic conditions, and tissue dissociation was performed using the Neural Tissue Dissociation Kit, according to the manufacturer’s instructions. Following dissociation, myelin debris was separated and removed using Debris Removal Solution, according to the manufacturer’s instructions. CD11b-positive cells were enriched using anti-CD11b MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany), according to manufacturer’s instructions. The resulting cell:bead suspension was centrifuged at 400g for 10 min and resuspended in X-VIVO medium (Lonza Biosciences, Walkersville, MD), or X-VIVO medium supplemented with 1 ug/mL HMGB1 for immediate stimulation.