Ifn γ secretion assay

C-Kit-D816V-stimulated T cells were plated by limiting dilution (0.5–2.0 cells/well) and expanded in a 96-well U-bottom plate (Becton, Dickinson and Company) in AIM-V medium supplemented with 5% heat-inactivated human serum, 40 ng/mL anti-CD3 antibody (Clone UCHT 1; Becton, Dickinson and Company), and 120 IU/mL IL-2 (PeproTech, Inc.). The expanded clones were examined for IFNγ production after peptide stimulation, and a C-Kit-D816V-specific T cell clone was obtained. A single T cell from the obtained clone was sorted into a 96-well plate (4titude, Wotton, UK) by the FACSAria II cell sorter (Becton, Dickinson and Company). PIK3CA-H1047R specific T cells were enriched by IFNγ secretion assay (Miltenyi Biotec, Auburn, CA, USA), followed by sorting of a single T cell into a 96-well plate (4titude) by the FACSAria II cell sorter (Becton, Dickinson and Company).
The sequences of TCR genes of the sorted T cell clones were determined, as reported previously [33 (link)]. The cDNA of TCRα and TCRβ chain was amplified from single cells and sequenced at Eurofin Genomics K.K. (Tokyo, Japan).

Memory-like NK cells, control NK cells and resting NK cells were analyzed using a Miltenyi Biotec IFNγ secretion assay as per the manufacturer’s instructions. Briefly, the cells were incubated with an IFNγ specific catch reagent at 37 °C to allow cytokine secretion. The secreted IFNγ binds to the catch reagent on the positive secreting cells. Then, the cells were labeled with a second IFNγ-specific detection antibody conjugated to PE (R-phycoerythrin) for detection by flow cytometry.

PBMC were isolated from healthy adult donors (age range 25–50 years) with a previous history of bee or wasp sting, and from wasp and bee allergic individuals with a history of anaphylaxis under local ethics approval (CO2.291 and 09/H0606/71). T cells were isolated using CD3 MACs bead separation (Miltenyi Biotec, Germany) for ex‐vivo assays. Or, PBMCs were expanded by culturing with feeder cells for 14–20 days (1000 patient PBMCs plus 0.2 × 10⁶ irradiated donor PBMCs and 0.4 × 10⁶ transformed B‐cell line per well of a round‐bottom 96‐well plate in RPMI supplemented with 100 IU/mL penicillin, 100 μg/mL streptomycin, 2 mM L‐glutamine, and 5% human serum (Sigma‐Aldrich, MO, USA), nonessential amino acids, HEPES, sodium pyruvate, and 2‐mercaptoethanol (Life Technologies, CA, USA) in the presence of 1 nM IL‐2 (PeproTech, NJ, USA) and 50 ng/mL anti‐CD3 antibody (OKT3). After cell numbers had expanded, T cells were isolated using CD3 MACs beads (Miltenyi Biotec, Germany). T‐cell lines were generated using CD3+ cell isolated by MACs bead separation from healthy adult human peripheral blood. T cells were then cultured with wasp venom, bee venom, or PLA2 pulsed irradiated K562‐CD1a cells for 10–14 days. T‐cell lines were further enriched for PLA2 reactivity using the IFN‐γ secretion assay (Miltenyi Biotec, Germany) following manufacturer instructions.

Animal experiments were performed according to institutional and national guidelines and regulations following approval by the responsible authority (Landesamt fuer Gesundheit und Soziales, Berlin, Germany). Mice were primed with synthesized predicted HLA-A*02:01-restricted peptides on day 0 with 150 µg of peptide in a 1:1 solution of Incomplete Freund’s Adjuvant (IFA, Sigma-Aldrich, St. Louis; MO, USA) and 50 µg CpG1826 (Novus Biologicals) by subcutaneous injection. Mice were boosted three times, the earliest on day 21 after priming. Blood was collected 7 days after each boost, and mouse PBMCs were cultured with 1 × 10⁻⁶ M peptide overnight for Interferon-γ (IFN-γ) detection. Mice with IFN-γ-secreting CD8+ T cells in the periphery were sacrificed, and spleen and inguinal lymph nodes were collected. Splenocytes were isolated, and CD4+ cells were depleted. Splenocytes were expanded for 10 days in RPMI 1640 with 10% FBS, HEPES, NEAA, sodium pyruvate, 50 µM β-mercaptoethanol, 20 IU/mL IL-2, and 10⁻⁸ M peptide. After expansion, peptide-reactive cells were sorted in RLT buffer for RNA isolation following IFN-γ secretion assay (Miltenyi Biotech, Bergisch-Glattbach, Germany) and staining with antibodies against mouse CD3 (clone 17A2, APC-conjugated, BD Biosciences, Franklin Lakes, NJ, USA) and mouse CD8 (53-6.7, PerCP-conjugated, BD Biosciences, Franklin Lakes, NJ, USA).

PBMCs were diluted to 1 × 10⁷ cells/mL in RPMI 1640 GlutaMAX Supplement medium (Gibco, USA) with 5% autologous serum and stimulated with inactivated purified TBEV particles, Sofjin strain (18 (link)) (10 μg/mL of E protein determined using VectoTBEV-antigen kit [Vector-Best, Russia]). Stimulated cells were incubated in 24-well plates for 16 or 24 h at 37°C and 5−7% CO₂. After 16 h, half of the stimulated cells were subjected to the IFNγ Secretion Assay (Miltenyi Biotec, USA) according to the manufacturer’s protocol. After 24 h, the rest of the cells were washed and stained with PE-labeled anti-CD137 antibodies (Miltenyi Biotec, USA, clone REA765). In addition, cells were stained with eFluor450-labeled anti-CD3 antibodies (eBioscience, USA, clone UCHT1). Then, CD3⁺IFNγ⁺ and CD3⁺CD137⁺ cells were collected by fluorescence-activated cell sorting on Sony SH800S Cell Sorter with the Purity Mode allowing for at least 97% of purity (see Supplementary Figure 1 for the gating strategy and Supplementary Table 1 for the number of sorted cells) and lysed with Trizol reagent (Invitrogen, USA) immediately followed by total RNA isolation.