Exposure of NLCs to rNAMPT enhances their immunosuppressive function. NLCs (n = 6) were generated in the presence of rNAMPT before assessing expression of CD163 (A) and CD206 (B) by flow cytometry and confocal microscopy. Box plots show cumulative analyses of pixel intensity (a.u.) in at least 4 fields for the different samples. (C) Conventionally obtained NLCs (n = 8) were incubated (15 minutes, 37°C) with FITC-dextran (1 mg/mL in PBS + 5% FCS) with or without rNAMPT. Phagocytosis was confirmed by costaining with caveolin-1 (in red, last 2 panels) and confocal microscopy analysis. Original magnification ×63. (D) Dot plots showing Annexin-FITC (AV) and propidium iodide (PI) staining of CD19⁺ cells cultured with NLC derived with or without NAMPT for 14 days. The graph represents cumulative data (n = 14). (E) CFSE-labeled, preactivated (anti-CD3/IL-2, 24 hours) autologous PBMCs were cocultured with predifferentiated NLCs (3-5 days with or without rNAMPT). Graph shows cumulative data (n = 8). (F) FACS analysis of basal Treg (CD4⁺/CD25^high/CD127^low) expression and after 5 days of coculture (with or without rNAMPT) with autologous NLCs. (G) FACS analysis of PD-1 expression on CD4⁺ T cells after 5 days of coculture (with or without rNAMPT) with autologous NLCs. The gray histogram represents the isotype control. Box plot represent the cumulative data (n = 9) showing the percentage of PD-1 expression. (H) PD-L1 expression on NLCs treated with rNAMPT for 24 hours was checked by flow cytometry and confirmed by quantitative PCR (n = 14).