*2.6. T-Cell Responses to Influenza A Antigens*

T cell analysis was performed, as described here. Briefly, the splenocytes were isolated by physical disruption of the spleens, followed by Ficoll purification and washed twice with PBS. The depletion of CD8<sup>+</sup> T cells was performed while using Dynabeads (Dynal Biotech, Oslo, Norway), according to the manufacturer's instructions. The efficiency of CD8<sup>+</sup> cell depletion was confirmed by flow cytometry. On average, 98% <sup>±</sup> 2.4% of the CD8<sup>+</sup> cells were removed. Total and CD8<sup>+</sup> T cell depleted splenocytes from individual animals were suspended in RPMI 1640 (Sigma) supplemented with penicillin/streptomycin (Invitrogen) and 10% fetal calf serum (FCS, Sigma) and then subjected to anti-Interferon-γ (IFNγ) (Mabtech, Nacka, Sweden) antibody coated 96-well polyvinylidene fluoride (PVDF) bottomed plates (MAIPN 4510, Millipore Corporation, Bedford, MA, USA). WIV antigen restimulation was performed while using influenza A/H1N1/2006/SI or A/H3N2/1995/Wuhan at 100 TCID50. Peptide restimulations were performed using the H-2Kd binding NP peptides TYQRTRALV (147-156/aa), RLIQNSLTIERMVLS (55-69/aa) and the H-1Kb binding NP peptide ASNENMDAM (366-374/aa) at 1 μM final concentration (GenScript, Piscataway, NJ, USA). Concanavalin A (1 μg/well, Sigma) was used as a positive control to test cell activation and medium alone was used as the negative control. Spot-forming cells were quantified after 24 h incubation and then counted by an AID ELISPOT reader (AutoImmun Diagnostika, Strassberg, Germany). The results are given as cytokine-producing spot-forming cells (SFC) per million plated cells. The total and CD8<sup>+</sup> depleted splenocytes (106) were stained for 30 min. at 4 ◦C with FITC conjugated anti CD4 antibodies and with PerCP conjugated anti-CD8α antibodies (BD Pharmingen, Stockholm, Sweden). IL-5 production was determined by ELISA after restimulation of total splenocytes from individual mice with WIV A/H1N1/SI (1 μg total), as determined by the manufacturer (Omninvest, Budapest, Hungary) after 48 h from C57BL/6 samples and 72 h from the NMRI samples. The different time points chosen for the two mouse strains were

determined in an in vitro pre-study influenza/ConA stimulation of spleen cells, and the optimal time point for highest levels of IL-5 secretion in elderly animals was chosen. Furthermore, IL-5 secretion was shown to be secreted at higher amounts for a longer period than IL-4 in vitro (or possibly consumed less rapidly) in aged mice, thus making IL-5 easier to use as a Th2-biomarker than IL-4, as shown by McDonald et al. 2017.
