Activated NKT cells enhance in vitro short-term clonogenic hematopoietic activity through production of GM-CSF. (A) Human cord-blood NKT cells (i) were expanded in vitro (ii), in the presence of αGalCer. Flow-sorted cells (iii) were fully activated as determined by CD69 and HLA-DR expression (iv), and almost all were CD4⁺ (v). (B) CD34⁺ cells were cocultured with activated NKT cells for 18 to 24 hours at different CD34/NKT cell ratios and plated in methylcellulose in the absence of exogenous cytokines (i). The clonogenic capacity of CD34⁺ cells was enhanced at CD34/NKT cell ratios of 1:2 or greater. Only myeloid colonies were formed (representative of 2 independent experiments). The enhancing effect of NKT cells on myelopoiesis was confirmed in an extended panel of CFU assays performed in the absence of exogenous cytokines (ii). Values are the mean and SEM of 5 independent experiments (*P < .05 by paired Student t test). (C) Effect of an anti–GM-CSF neutralizing antibody on the clonogenic capacity of CD34⁺ cells cocultured with activated CB NKT cells at 1:10 ratio. The clonogenic capacity was inhibited by anti–GM-CSF in a dose-dependent manner. For each condition, values are the mean and SEM of quadruplicate assays. Representative of 2 independent experiments.