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. 2026 Sep 15;17:1878920. doi: 10.3389/fimmu.2026.1878920

Figure 2.

Panel A shows a table listing concentrations of asparagine, aspartate, and glutamine in IMDM, DMEM, and DMEM(-Gln) media. Panel B displays an experimental schematic for culturing sorted LI ILC2 cells in different media for one week plus 48 hours. Panel C presents representative FACS histograms showing live/dead staining with viability percentages for DMEM(-Gln), DMEM, and IMDM. Panel D shows a bar graph quantifying cell viability for the three media. Panel E contains flow cytometry dot plots analyzing IL-5 and IL-13 production in IMDM, DMEM, and DMEM(-Gln). Panel F provides a bar graph quantifying IL-5+ IL-13+ ILC2 percentages. Panel G displays bar graphs of secreted IL-5 and IL-13 concentrations. Panel H provides a diagram of the asparagine synthetase (ASNS) pathway converting glutamine and aspartate to asparagine. Panel I gives a schematic for a supplemental experiment adding asparagine and aspartate to DMEM(-Gln) and culturing LI ILC2 as before. Panel J offers FACS histograms for cell viability in IMDM, DMEM(-Gln+Asn), DMEM(-Gln+Asp), and DMEM(-Gln). Panel K bar graph quantifies cell viability across these conditions. Panel L shows IL-5 and IL-13 flow cytometry for the same four conditions. Panel M quantifies IL-5+ IL-13+ ILC2 percentages as a bar graph. Panel N provides bar graphs of secreted IL-5 and IL-13 for each condition. Statistical significance and replicate markers are visible in relevant graphs.

Asn deprivation renders ILC2s dependent on de novo Asn synthesis. (A) The concentrations of L-Asn, L-aspartate (Asp), and L-glutamine (Gln) in IMDM, DMEM, and glutamine-deficient DMEM (DMEM(-Gln)) medium. (B–G) LI lamina propria ILC2s were sorted from C57BL/6 mice, expanded in IMDM for 1 week, and then cultured for 48 h in IMDM, DMEM, or DMEM(-Gln), followed by flow cytometric analysis or ELISA. Experimental workflow (B). Representative histograms showing the viability of ILC2s (C). Quantification of the frequency of live cells (without PAM/Ionomycin/Brefeldin A stimulation) (D). Representative flow cytometry plots showing IL-5 and IL-13 production by ILC2s (E). Quantification of the frequencies of IL-5+ IL-13+ ILC2s (F). Concentrations of IL-5 and IL-13 in ILC2 culture supernatants measured by ELISA (G). Each dot represents an individual mouse. (H) Schematic illustration of the metabolic pathway linking Gln, Glu, Asp, and Asn. (I–N) LI lamina propria ILC2s were sorted from C57BL/6 mice, expanded in IMDM for 1 week, and then cultured for 48 h in IMDM, DMEM(-Gln) and DMEM(-Gln) supplemented with Asn (28.4 mg/L, DMEM(-Gln+Asn)) or Asp (30 mg/L, DMEM(-Gln+Asp)), followed by flow cytometric analysis or ELISA. Experimental workflow (I). Representative histograms showing the viability of ILC2s (J). Quantification of the frequency of live cells (without PAM/Ionomycin/Brefeldin A stimulation) (K). Representative flow cytometry plots showing IL-5 and IL-13 production by ILC2s (L). Quantification of the frequencies of IL-5+ IL-13+ ILC2s (M). Concentrations of IL-5 and IL-13 in ILC2 culture supernatants measured by ELISA (N). Each dot represents an individual mouse. Data are presented as mean ± SD. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s multiple comparisons test; ns, not significant; **p < 0.01; ***p < 0.001; ****p < 0.0001.