Anti cd44

Spleens were sieved (70 µm), before washing [phosphate-buffered saline (PBS); 350 g, 5
min], re-sieving (40 µm), RBC lysis (eBioscience), washing, and resuspension in FACS
buffer (1% BSA, 0.05% NaN₃, in PBS) or full RPMI 1640. Cells in FACS buffer
were Fc blocked (1:100; BioLegend; 10 min, RT) before staining for T-cell activation with:
anti-CD4 (1:800; eBioscience), anti-CD8 (1:100), anti-CD62L (1:80), anti-CD44 (1:400; all
BioLegend; 20 min, RT); or for Tregs with: anti-CD4 (1:800), anti-CD25 (1:80; BioLegend;
20 min, RT), before washing, fixation, permeabilization (FOXP3 Fix/Perm; BioLegend), then
anti-FOXP3 (1:20; BioLegend; 30 min, RT). Splenocytes in RPMI were treated
±ionomycin/phorbol 12-myristate 13-acetate/brefeldin A (1:500; BioLegend), incubated (5 h,
37°C), washed, resuspended in FACS buffer, Fc blocked, stained with anti-CD4 or anti-CD8
(20 min, RT), washed, fixed, permeabilized (BioLegend), then anti-IL-10, anti-IL-17, and
anti-interferon γ (IFNγ; all 1:100; BioLegend; 30 min, RT). For neutrophil, monocyte or
Ly6C⁺ cells, whole blood (ethylenediaminetetraacetic acid) was stained with
anti-CD115 (1:100; eBioscience), anti-CD11b (1:800), anti-Ly6G (1:80; both BioLegend),
anti-Ly6C (1:400; AbD Serotec, Hercules, CA), at RT for 30 min, before RBC lysis, washing,
and analysis by flow cytometry (Accuri C6).

We used the following antibodies: anti-CD4 (clone: GK1.5)-FITC, -PerCP-Cy5, or -APC, anti-CD8 (clone: 53-6.7)-FITC, -PerCP-Cy5, or -APC, anti-CD44 (clone: IM7)-APC, anti-BrdU (clone: Bu20a)-PE, 7AAD, anti-IFN-γ (clone: XMG1.2)-APC, anti-IL-17 (clone: TC11-18H10.1)-PE, anti-IL-4 (clone: 11B11)-PE, anti-IL-6 (clone: MPS-20F3)-PE, anti-IL-12 (clone: C15.6)-PE, anti-IL-22 (clone: Poly5164)-PE, anti-IL-10 (clone: JES5-16E3)-PE, anti-IL-9 (clone: MH9A4)-PE, anti-TNF-α (clone: MP6-XT22)-PE (all from Biolegend, USA), anti-Active Caspase-3 (clone: C92-605)-FITC or -PE (from BD Pharmingen™, USA), and anti-CD69 (clone: H1.2F3)-PE (from eBiosciences, USA).

MSCs were isolated from human umbilical cords following previously described methods after informed consent was obtained according to institutional guidelines under the approved protocol^{17 (link)}. MSCs were cultured in DMEM/F-12 (#11320033, GIBCO BRL, Grand Island, NY, USA) containing 10% foetal bovine serum (#10099141C, GIBCO BRL) and 1% penicillin/streptomycin (#15240062, GIBCO BRL). MSCs between passages 5–8 were used for subsequent experiments. To confirm the identity of the cells, the phenotypic profile of MSCs was evaluated by flow cytometry analysis using PE-labeled human anti-CD29 (#102216, Biolegend, San Diego, CA, USA), anti-CD44 (#338804, Biolegend), anti-CD90 (#328109, Biolegend), anti-CD11b (#101210, Biolegend) or anti-CD45 antibody (#103106, Biolegend). The morphology was observed by inverted microscopy. MSCs were cultured in MSC osteogenic differentiation medium for differentiation, and differentiated cells were identified by Alizarin Red staining (#G8550, Solarbio, Beijing, China) and Oil Red O staining (#O8010, Solarbio).

Single-cell suspensions from spleen and lymph nodes were surface stained for 30 min at 4 °C. Mouse CD4⁺ naive T cells (CD4⁺B220⁻CD44^loCD25⁻) were sorted using mouse anti-CD4 1:400 (BioLegend cat#100422 ), anti-CD44 1:200 (BioLegend cat#103006), CD25 1:200 (BioLegend cat#101910) and B220 1:100 (BioLegend cat#103208). For bone marrow chimeras, CD45.1 1:200 (BioLegend cat#110722 )and CD45.2 1:200 (BioLegend cat no#109824) congenic markers were used. Total RNA from T cells from four wt and four mutant mice was extracted using mirVana microRNA isolation kit (Ambion) and miRNA microarrays were performed using Agilent mouse miRNA-v1_95_May07 chips at the Ramaciotti Center for Genomics (UNSW) and analysed using the Genespring software (Agilent Technologies, Inc.)

Single cell suspensions were obtained from PBMCs, and various tissues as described previously. Dead cells were gated out using Live/Dead fixable dead cell stain (Invitrogen). SARS-CoV-2 spike and SF162 peptide pools used for intracellular cytokine staining (ICS) and these were obtained from BEI Resources. Biotinylated MHC class I monomers (K^bVL8, sequence VNFNFNGL; D^bGP33, sequence KAVYNFATC; D^bGP276, sequence SGVENPGGYCL; OVA, sequence SIINFEKL; K^bB8R, sequence TSYKFESV) were used for detecting virus-specific CD8 T cells, and were obtained from the NIH tetramer facility at Emory University. Cells were stained with fluorescently-labeled antibodies against CD8α (53–6.7 on PerCP-Cy5.5), CD44 (IM7 on FITC), CD62L (MEL-14 on PE-Cy7), CD127 (A7R34 on Pacific Blue), and tetramers (APC). TNFα (MP6-XT22 on PE-Cy7), IFNγ (XMG1.2 on APC), or Ki67 (SolA15 on PE-Cy7). Fluorescently-labeled antibodies were purchased from BD Pharmingen, except for antiCD127 and anti-CD44 (which were from Biolegend). Flow cytometry samples were acquired with a Becton Dickinson Canto II or an LSRII and analyzed using FlowJo v10 (Treestar).