Lymphoprep

Peripheral blood mononucleated cells (PBMCs) were isolated from mouse blood using Lymphoprep (ProGen, Heidelberg, Germany). For analysis, PBMCs suspended in PBS were stained with a mixture of FITC-conjugated K^d (SF1-1.1), I-A^b (KH74), I-A^k (11-5.2), I-E^k (14-4-45), D^b (27-11-13S)-specific mAbs (BioLegend, St. Louis, MO) and PE-conjugated K^b (AF6-88.5), L^d (28-14-8)-specific mAbs (BioLegend). Two-color or one-color analysis was conducted by FACS (FACSCalibur, Nippon Becton Dickinson, Japan).

The PBMCs were isolated from 2 × 6 mL of blood using Lymphoprep (Progen, Heidelberg, Germany) and washed in RMPI-1640 (25 mM HEPES). The purified PBMCs were resuspended in autologous plasma and preserved in aliquots of approximately 5 × 10⁶ cells per vial in 40% RPMI-1640 (10% DMSO) by gradually decreasing the temperature by 1°C/min to −80°C and then transferred to liquid nitrogen until further analysis.

For cell preparation, mononuclear cells (MNC) were isolated from BM aspirates using density gradient centrifugation with LymphoPrep™ (PROGEN Biotechnik, cat. #1,114,547, Heidelberg, Germany).
To expand the mesenchymal stromal cell fraction, isolated MNCs were cultured in the Mesencult medium [Mesencult™ Proliferation Kit (human), STEMCELL Technologies Inc, cat. #05411, Vancouver, BC, Canada], with an addition of 10% AB human serum and 1% penicillin–streptomycin solution. BM stromal cells were generated up to passage (P) 3. To enrich and expand the MSC fraction, the isolated MNCs were cultured in the starvation medium RPMI (Biological Industries, cat. #1640, Beit-Haemek, Israel), with an addition of 10% FBS/human serum and 1% penicillin–streptomycin solution.
To induce MSC differentiation into adipocytes/osteocytes, BM-MSCs were stimulated using adipogenic or osteogenic differentiation inducing factors. In brief, P0 MSCs were cultured for 21 days at 37⁰C in either adipogenic or osteogenic medium (basal medium: MEM-α Biological Industries, cat. #01–042-1A; adipogenic supplement: R&D Systems, cat. #CCM011, Minneapolis, MN, USA; osteogenic supplement: R&D Systems, cat. #CCM008). The obtained cells are further referred to as “induced” MSCs, adipocytes or osteocytes. The MSCs that have not undergone such stimulation are further termed “non-induced”.

Heparinized blood samples were collected from participants prior to vaccination (P0), 28 days post completion of the primary series (P28), prior to booster vaccination (B0) and 28 days post booster vaccination (B28) (Figure 1A). From all blood samples, peripheral blood mononuclear cells (PBMC) were isolated using Lymphoprep (Progen) and cryopreserved at −135°C.

Fresh peripheral blood was collected from healthy donors into lithium heparin collection tubes after informed consent under a protocol that was approved by the Trinity College Dublin Faculty of Science, Technology, Engineering, and Mathematics research ethics committee. Neutrophils were isolated by dextran sedimentation and gradient separation using Lymphoprep (Axis shield, Progen). Red blood cells were lysed using ammonium–chloride–potassium (ACK) lysis buffer (Gibco), and the resulting hPMN was resuspended in Dulbecco’s Modified Eagle’s Medium with Glutamax (Gibco) supplemented with 10% fetal bovine serum (Sigma) and HEPES (10 µM, Sigma). Unless otherwise stated, the final concentration of hPMN was 2 × 10⁶ cells per replicate. Cell purity was assessed by flow cytometry using the neutrophil markers CD15 and CD16 which was confirmed to be >90%.