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. 2020 May 3;21(6):e48927. doi: 10.15252/embr.201948927

Figure 4. Fractionated neutral lipid from stimulated macrophages activates iNKT cells.

Figure 4

  1. Neutral (Fractions 1–3) and polar lipids (Fractions 4–6) were isolated from J774.2 macrophages stimulated with thapsigargin, tunicamycin or DMSO. Lipids were fractionated using solid‐phase extraction (SPE) cartridges. Bar graph represents NKT cell activation by CD1d protein‐bound neutral lipids fractions isolated from thapsigargin (black bars)‐ or tunicamycin (red bars)‐treated J774.2 cells. Polar lipid fractions induce very little IL‐2 secretion by 2C12. In total, n = 2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.
  2. Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin‐treated J774.2 at acidic pH and reduced mCD1d loading at neutral pH. Graphs show mean ± SEM from n = 2 biological replicates (two technical replicates per biological replicate).
  3. Neutral (Fractions 1–3) and polar lipids (Fractions 4–5), isolated from murine bone marrow‐derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid‐phase extraction (SPE) cartridges, were co‐cultured with purified NKT1 subsets. Bar graphs represent IFN‐γ release by NKT1 cells induced by CD1d protein‐bound neutral lipid fractions isolated from thapsigargin (black bars)‐ or DMSO (white bars)‐treated primary murine macrophages. Polar lipid fractions induce a minimal amount of IFN‐γ secretion by the NKT1 sublineage. In total, n = 2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.
  4. Neutral (Fractions 1–3) and polar lipids (Fractions 4–5) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid‐phase extraction (SPE) cartridges. Bar graph represents IFN‐γ release by NKT1 cells by CD1d protein‐bound neutral lipid fractions isolated from thapsigargin (black bars)‐ or DMSO (white bars)‐treated human primary macrophages. Polar lipid fractions induce only minimal amounts of IFN‐γ secretion by the NKT1 sublineage. In total, n = 2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.

Source data are available online for this figure.