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During necropsy spleens were collected in Iscove’s Dulbecco’s Medium (IMDM, Lonza) supplemented with 5% FBS and P/S/G for direct preparation of single cell suspensions using 100 µm strainers (Falcon). Erythrocytes were removed from single cell suspensions by treatment with red blood cell lysis buffer (Roche diagnostics). For intracellular cytokine staining, splenocytes were stimulated with 5 µM synthetic peptide (epitope NP_366–374: ASNENVEIM [Fig. S2] or ASNEMMETM [Fig. 4]) or 1 µg/250,000 cells recombinant HA protein from H5N1 influenza virus A/Vietnam/1203/04 or A/Indonesia/5/05 (Protein Sciences) in IMDM supplemented with GolgiStop and incubated for 6 h at 37 °C. Mock-treated splenocytes and splenocytes stimulated with 50 ng/ml PMA (Sigma-Aldrich) and 0.5 µg/ml ionomycin (Sigma-Aldrich) served as appropriate negative and positive controls. After stimulation, splenocytes were incubated with fluorochrome-labeled antibodies to CD3e^APC-Cy7 (BD Pharmingen), CD8b^FITC (BD Pharmingen), CD4^PerCP (BD Pharmingen) and viable cells were identified with Aqua LIVE/DEAD (Invitrogen). Subsequently, cells were fixed and permeabilized using BD Cytofix/Cytoperm^TM Plus (BD Biosciences), and incubated with anti-IFN-γ^PacificBlue (Biolegend). Samples were acquired on a FACS Canto II and data was analyzed as described previously^{55 (link),59} using FACS Diva software (BD Biosciences).

Single‐cell suspensions were obtained from cultured cells or tumor tissues. The tumors were enzymatically digested in 1 mg mL⁻¹ collagenase I and 1 mg mL⁻¹ collagenase IV. Fixable Viability Stain 700 (564997, BD) was used to exclude dead cells. The following antibodies were used for surface staining: CD3e‐APC‐Cy™7 (557596, BD), CD8a‐PerCP‐Cy™5.5 (551162, BD), CD335‐BV421 (562850, BD), CD11b‐FITC (557396, BD), CD80‐APC (ab95549, Abcam), CD86‐PerCP‐Cy5.5 (105027, Biolegend), Gr‐1‐APC (108411, Biolegend) and CD11c‐FITC (117306, Biolegend). After being stained at room temperature in the dark, the cells were fixed with 4% methanol, permeabilized with 0.5% Triton, and intracellularly stained with Foxp3‐Alexa Fluor 647 (560401, BD). A BD FACSymphony A5 instrument was utilized for flow cytometry, and NovoExpress Software (Tree Star) was employed to analyze the data. Cell populations were identified as follows: CD4⁺ T cells: live/CD45⁺/CD3⁺/CD4⁺/FoxP3⁻; CD8⁺ T cells: live/CD45⁺/CD3⁺/CD8⁺; Treg cells: live/CD45⁺/CD3⁺/CD4⁺/FoxP3⁺; natural killer (NK) cells: live/CD45⁺/CD3⁻/NKP46⁺; MDSC cells: live/ CD11b⁺/Gr1⁺ and DCs: live/CD45⁺/CD3⁻/CD11c⁺.