We used male and female transgenic mice between 8-15 weeks of age with age and sex matched wildtype controls in all experiments. All mice were bred on a C57BL/6J background. All experiments were performed in accordance with local standards and the Animals (Scientific Procedures) Act 1986, licence PPL 70/7661 and were approved by the Babraham Institute Animal Welfare and Ethics Review Body.
Mice were bred and maintained at the Babraham Institute Biological Support Unit. Regular health monitoring confirmed that stock and holding rooms were free of primary pathogens and additional agents listed in the FELASA recommendations . Ambient temperature and lighting was kept between 19-21°C with a relative humidity of 52% and a 12-hour light: 12-hour dark cycle including 15 min ‘dawn’ and ‘dusk’ periods of subdued lighting. Mice were housed in individually ventilated cages with 1-5 animals per cage and were fed CRM (P) VP diet (Special Diet Services) ad libitum. Animals received sunflower or millet seeds at the time of cage-cleaning as part of their environmental enrichment.
• Isoflurane: IsoFlo 100% w/w Inhalation Vapour, liquid (Zoetis)
S. pneumoniae stock
Note: S. pneumoniae is a category 2 pathogen capable of causing disease in humans. Ensure that all work involving viable bacteria is completed in a containment level 2 laboratory and that appropriate risk assessments are in place.
We received S. pneumoniae TIGR4, serotype 4 as a kind gift from Professor Jeremy Brown, University College London. This strain is a clinical isolate and not genetically modified. Serotype 4 has been shown to be virulent in mice  and is included in the two clinically available vaccines: Prevenar-13 and Pneumovax. A variety of other serotypes and strains are available from ATCC and BEI Resources.
• S. pneumoniae culture media: Add 36.4g Todd-Hewitt broth powder (Oxoid CM0189) and 5g yeast extract (Oxoid LP0021) to 1L sterile milliQ water, heat and mix until dissolved, then autoclave.
• Blood-Agar plates: Prepare 500mL LB-Agar (ThermoFisher scientific 22700), autoclave and allow to cool to 50°C in a water-bath, then add 25mL defibrinated sheep’s blood (Oxoid SR0051). Mix well, pour into sterile 90mm petri dishes and allow to set under aseptic conditions. Add Optochin antibiotic disks (Sigma-Aldrich 74042-50DISCS-F) to selected plates. Plates can be stored under aseptic conditions at 4°C for up to 5 days.
• Phosphate buffered saline (PBS): Low endotoxin Dulbeccos’s phosphate buffered saline without Ca2+ or Mg2+ (Sigma-Aldrich D8537).
• Glycerol (Sigma-Aldrich G5516)
• Pneumovax II (pneumococcal polysaccharide vaccine, Sanofi Pasteur MSD). One 0.5mL dose contains 50µg/mL of each serotype (1-5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B. 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F). Dilute one 0.5mL dose 1:12.5 in sterile PBS for administration to mice.
• Prevenar 13 (pneumococcal 13-valent conjugate vaccine, Diphteria CRM197 protein, Pfizer). One 0.5mL dose contains 22µg of each serotype: 1-5, 6A, 7F, 9V, 14, 18C, 19A, 23F polysaccharides and 4.4µg of serotype 6B polysaccharide; 34µg CRM197 carrier protein, 100µg polysorbate 80, 295µg succinate buffer and 125µg aluminium phosphate adjuvant. Dilute one 0.5mL dose 1:6.4 in sterile PBS for administration to mice.
Immune cell isolation
• Red blood Cell lysis buffer: Red blood cell lysis buffer Hybri-Max (Sigma-Aldrich R7757)
• Mouse Lung dissociation kit (Miltenyi, 130-095-927) Prepared according to manufacturer’s instructions.
• Percoll (Sigma-Aldrich P1644): Prepare isotonic Percoll by adding 1 part sterile 10x concentrated PBS to 9 parts Percoll. Then prepare a 37.5% working solution of isotonic Percoll in sterile 1x PBS.
• Antibodies: see Table 1See figure in Figures section.
• Staining buffer: 0.5% BSA in PBS.
• Fixation buffer: 4% paraformaldehyde (Biolegend 420801)
• Transcription factor staining buffer set (eBioscience 00-5523-00)
• Non-fluorescent counting beads (AccuCount Blank Particles 5.3µm; Spherotech, USA)