Anti cd8α

Tissues were processed as previously described⁵¹ . Briefly, tissues were fixed in paraformaldehyde (PFA), lysine, and sodium periodate buffer (PLP, 0.05M phosphate buffer, 0.1M L-lysine, pH 7.4, 2 mg/mL NaIO₄, and 10mg/mL PFA) overnight at 4°C followed by 30% sucrose overnight at 4^oC and subsequent embedding in OCT media. Frozen tissues were sectioned at 20μm using Leica CM3050S cryostat, and FcR blocked with anti-CD16/32 Fc-block (Clone 93, BioLegend) diluted in PBS containing 2% donkey serum, 2% fetal bovine serum (FBS), and 0.1% Triton-X for 1h at 25^oC. Sections were stained with anti-CD8α (Clone 53–6.7, BD Bioscience), anti-EpCAM (clone G8.8, eBioscience), anti-Influenza A virus (polyclonal, Abcam) and anti-CD11c (clone N418, BioLegend) diluted in PBS containing 2% goat serum, 2% FBS, 0.1% Triton-X, and 0.05% Fc block for 1h at 25^oC. Images were acquired using a Zeiss LSM 880 confocal microscope (Carl Zeiss) with the Zen Black software. The imaging data were processed and analyzed using Imaris software version 9.0.1 (Bitplane, Oxford Instruments).

For experiments analyzing P14 T cells, CD8 T cells were enriched from splenocytes from naïve P14 mice with the EasySep Mouse CD8 T Cell Isolation Kit (StemCell). 2×10³ P14 CD8 T cells were transferred i.v. into B6 recipient mice one day before infection with LCMV clone 13. In P14 cotransfer experiments, either Cd28^fl/fl CreERT2^neg or Cd28^wt/wt CreERT2⁺ cells were used as controls with similar results.
For Tpex transfer experiments, splenocytes were isolated from LCMV clone 13 infected mice at least 40 days post-infection (and 2–3 weeks post tamoxifen administration as indicated). Spleens were digested for 30 minutes at 37°C with 0.4 U/ml of collagenase D (Roche, # 11088882001). After CD8 enrichment (StemCell), 20×10⁶ cells were labeled with 5 μM cell trace violet (CTV, Thermo Fisher Scientific) during 20min at 37°C. CTV staining was quenched for 5 min with RPMI containing 10% FBS before proceeding with cell surface staining with anti-CD8α (53–6.7), -CD44 (IM7), -CD28 (E18), -CD39 (24DMS1) and -PD-1 (RMP1–30; non-blocking clone) from BD or Biolegend and Live/Dead fixable dead cell stain (Invitrogen). Live CD8⁺ PD-1⁺ CD39^neg Tpex were isolated by FACS (post-sort purity >96%) and 1×10⁵ cells were transferred i.v. to infection matched CD45.1⁺ mice. 2–5 weeks post-transfer, single cell suspensions were obtained from spleen and lung for staining and analysis.

For flow cytometric analysis, 5x10⁵ PPL, IEL and LPL were labelled with mouse anti-rat monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridininchlorophylla protein (PerCP) or allophycocyanin (APC). The antibodies used were anti-CD4, anti-CD8α, anti-CD8β, anti-TCRαβ, anti-TCRγδ, anti-NKR-P1A, anti-CD25 (BD Biosciences, Oxford, UK), anti-CD62L, anti-CD103 (Biolegend, San Diego, CA, USA) and anti-TLR4 (Novus Biologicals, Littleton, CO, USA). The cells were stained as previously described [19 (link)]. Briefly, lymphocytes were incubated with saturating amounts of antibodies in PBS-0.2% FBS-0.1% NaN₃ (darkness, 20 min, 4 °C). Consecutively, the cells were washed, and fixed with 0.5% p-formaldehyde (darkness, until analysis, 4 °C). A negative control staining was included in each cell sample. Analyses were performed with a Gallios Cytometer (Beckman Coulter, Miami, FL, USA) in the Scientific and Technological Centres of the University of Barcelona (CCiTUB).