Fusion cell sorter

Stained dermal single-cell suspensions were prepared as described above. CD45⁺ and CD45^–EpCAM^– cells were FACS sorted separately in a BD Fusion cell sorter to enrich for the less abundant CD45⁺ cells following the sorting strategy shown in Supplementary Fig. 1a. Cells were collected in PBS + 0.5% bovine serum albumin at 4 °C in LoBind tubes (Eppendorf) and processed immediately with the microfluidics Chromium platform (10X Genomics). For the adult and aged experiment, three technical replicates were performed for CD45⁺ sorting. Replicate 1 included skin dermal cells of three adult and one aged mice, replicate 2 had two adult and one aged mice and replicate 3 comprised two adult and two aged mice. For CD45^–EpCAM^– cell sorting two replicates were carried out, both comprising one mouse per condition. For aged/IL-17-blocked and aged/IgG control 10X scRNA-seq of dermal skin cells, both CD45⁺ and CD45^–EpCAM^– conditions included four replicates in total. In both conditions, replicate 1 consisted of two aged/IL-17-blocked and two aged/IgG control mice. Replicates 2, 3 and 4 consisted in all cases of one aged/IL-17-blocked and one aged/IgG control mouse.

For cell sorting of LSK cells and myeloid progenitors (GMP + MDP), BM cells were collected from individual mice by flushing bones from both femurs, tibias and the pelvis and stained as described above. For sorting myeloids and lymphocytes, blood leukocytes staining was performed as described above. Adding anti-CD61 antibody (clone 2C9.G2 (HMβ3-1)) to the lineage allowed platelet and megakaryocyte exclusion. FACS-sorting was performed by FACS Aria III and Fusion cell sorter (BD Pharmingen).

Healthy donor PBMCs were collected via leukapheresis and stored in liquid nitrogen until use. CD8⁺ T cells were enriched from PBMCs using STEMCELL Technologies EasySep kits. Cells were then stained with fluorochrome-conjugated antibodies against CD8 (SK1), CD45RA (HI100), and CCR7 (G043H7) (all BioLegend, clones in parentheses). Naive CD8⁺CD45RA⁺CCR7⁺ T cells were sorted using a FACSAria IIIu or Fusion cell sorter (BD Biosciences). Sorted naive CD8⁺ T cells were resuspended in T cell culture media (RPMI, 10% FBS, l-glutamine, penicillin-streptomycin) with 10 IU/mL rhIL-2 (Prometheus) and equilibrated overnight to 2% O₂ in a hypoxic chamber (Coy Laboratory Products) or in a standard cell culture incubator (~20% O₂, Thermo Fisher Scientific). Cells were then activated with anti-CD3/anti-CD28 beads (Gibco, Thermo Fisher Scientific) at 3 cells per bead for 4 days. On day 4, 1.25 ng/mL rhTGF-β1 (BioLegend) was added, and cells were cultured for an additional 2 days. For phenotype stability assays i-T_RM were generated as described, sorted, and resuspended in media pre-equilibrated to 20% O₂ or 2% O₂ with 20 IU/mL rhIL-2 (Prometheus) or 10 ng/mL rhIL-15 (R&D Systems, Bio-Techne), with or without 1.25 ng/mL rhTGF-β1 (BioLegend).