Analysis of IL-15 expression in human CD34⁺ cells from various sources. (A) Expression of mRNA transcripts for IL-15 in human CD34⁺ cells by RT-PCR. Two amplification products for the IL-15 gene (643 and 524 bp) were detected in hematopoietic progenitors isolated from G-CSF—mobilized peripheral blood (MPB), bone marrow (BM), and cord blood (CB). As positive controls, LPS-activated macrophages (Mφ) and dendritic cells (DCs) were used. The housekeeping gene GAPDH (240 bp) was used as an internal control. (B) Confocal microscopic analysis of intracellular IL-15 localization in BM (i), MPB (ii), and CB (iii-iv) CD34⁺ cells by using anti—IL-15 mAb M111 (red staining) and FITC-transferrin as markers of early endosomes (green staining). The pictures are overlay-compacted images of serial optical sections of 1-μm thickness from the outside of the cells toward the inner compartments. Yellow staining at the submembrane level shows the localization of some endogenous IL-15 inside the early cell endosomes. The effects of neutralizing anti—IL-15 mAb on intracellular IL-15 localization in early endosomes are shown in lower panels iii and iv. The loss of yellow staining confirms that neutralizing mAb inhibits the retrapping of endogenously secreted IL-15 in CB CD34⁺ cells (panel iv). These data are representative of 3 different experiments.