Anti cd44 alexa fluor 700

₅₃₃IYSTVASSL₅₄₁ (IVL_533-541)-tetramer was bought from MBL (cat. NO.TS-M520-1), anti-CD8α depletion antibody (clone: YTS 169.4). Rat IgG2b isotype antibody (clone: LTF-2) was purchased from Bio X Cell. Antibodies for flow cytometry included anti-CD3-APC-eFluor780 (clone: 17A2; eBioscience), anti-CD4-Pacific Blue (clone: GK1.5; BioLegend), anti–CD8α-PerCP/Cy5.5 (clone: 53–6.7; BioLegend), anti-CD11a-PE/Cy7 (clone: 2D7; BD Bioscience), anti–CD44-Alexa Fluor 700 (clone: IM7; BioLegend), anti-CD45-APC-eFluor780 (clone:30-F11;Biolegend), anti-CD62L-eVolve 605 (clone: MEL-14; eBioscience), anti-CD127-PE & PE/Cy7 (clone: A7R34; BioLegend), anti-KLRG1-FITC & BV510 (clone: 2F1/KLRG1; BioLegend), anti-CD103-APC (clone: 2E7; BioLegend), anti-CD69-FITC (clone: H1.2F3; BioLegend) and anti-IFNγ-APC (clone: XMG1.2; BioLegend), anti-Eomes-PE & PE/Cy7 (clone: Dan11mag; eBioscience).

CSC markers were evaluated by multi-color flow cytometric analysis (FACS) on a BD FACS LSRII (BD Biosciences, San Jose, CA, USA), after cell staining with 0.5 μg per 100 μl of anti-CD24-Pacific Blue (eBioscience, San Diego, CA, USA) and anti-CD44-Alexafluor 700 (Biolegend, San Diego, CA, USA), 0.5 μg per 100 μl of anti-human-CD133-AF700 (MACS, Auburn, CA, USA) or ALDEFLUOR reagent (0.5 μg per 100 μl; Stem Cell Technologies, Vancouver, BC, CA). Data are represented as mean±s.d. *P<0.05; **P<0.01 were calculated by two-tailed Student's t-test (n=6; replicated 2 ×). CD24^low/CD24^high sub-populations of NCI-H292 cells were sorted utilizing anti-CD24-Pacific Blue antibody, using a BD FACSAria high-speed cell sorter (BD Biosciences). Similarly, CD133+ cells were sorted and collected in high fetal bovine serum (20%)-containing RPMI-1640 cell media (Gibco, Grand Island, NY) supplemented with 5% penicillin/streptomycin. Cells were centrifuged, washed 2 × with 1 × PBS, and resuspended in StemPro mesenchymal stem cell growth media supplemented with StemPro MSC SFM supplement (Gibco) and placed in ultra-low attachment plates (Corning) and monitored over the course of 7 days.

Splenocytes were isolated for analysis via IFN-γ ELISpot, surface and intracellular cytokine staining (ICS) and flow cytometry as previously described^{69 (link)–71 (link)}. Splenocytes were restimulated with 2 μg/mL of the appropriate antigenic peptide pool (comprised of individual peptides that were 20 amino acids in length with 10 amino acids of overlap) spanning the full-length LASV GPC sequence (Mimotopes). Reference sequences for peptide synthesis are as follows: NP_694870.1 (Josiah), AIT17836.1 (Pinneo), AAF86703.1 (803213), CAA36645.1 (GA391). Cells were restimulated for 18–20 h or 6 h (at 37 °C and 5% CO₂) for IFN-γ ELISpot and ICS, respectively. Surface staining and ICS were carried out using the following antibodies: LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Thermo Fischer Scientific); anti-CD8a-PerCP/Cy5.5, anti-CD62L-PeCy7, anti-IFN-γ-eFluor 450, anti-TNF-α-Alexa Fluor 488, anti-IL-2-PE (eBioscience); anti-CD4-Brilliant Violet 650, anti-CD44-Alexa Fluor 700 (BioLegend); and anti-CD127-eFluor 660 (Invitrogen). Antigen-specific T-cell responses were quantified by subtracting the response (SFU for IFN-γ ELISpot and the percentage of cytokine-positive cells for ICS) measured without stimulation from that observed after restimulation.