Human invariant natural killer T (iNKT) cells have been shown to recognize lysophosphatidylcholine (LPC) as an antigen presented by CD1d.  However, the physiological sources of LPC that activate iNKT cells remain unclear, since LPC and related lysophospholipids are produced both intracellularly and extracellularly through the action of a variety of phospholipase A₂ enzymes.  The major extracellular repositories and transporters of lysophospholipids are apolipoproteins and serum albumin.  We found that pre-exposing human monocyte-derived DCs to medium containing human albumin, but not human apolipoproteins, resulted in an enhanced ability to activate iNKT cell IFN-g secretion.  Pre-exposing DCs to de-lipidated human albumin failed to improve their ability to stimulate the iNKT cells.  The activity of the de-lipidated albumin was recovered when it was pre-treated with synthetic LPC, but not when it was pre-treated with phosphatidylcholine (PC) or oxidized PC.  Mass spectrometric analysis identified a C18:1 form of LPC among the compounds in an organic extract of human serum albumin.  DCs exposed to LPC-loaded albumin showed upregulated expression of the adhesion ligand ICAM-1, and demonstrated increased conjugation with iNKT cells.  iNKT cell IFN-γ secretion via this pathway appeared to be partially dependent on CD1d recognition and TCR signaling, and was also dependent on IL-12 stimulation and JAK signaling.  Since addition of an anti-CD40 blocking mAb abrogated the iNKT cell IFN-γ secretion, we hypothesize that the iNKT cell response is a result of a feedback interaction in which the iNKT cells engage CD40 on the DCs, which stimulates the DCs to produce IL-12 that in turn co-stimulates iNKT cell IFN-γ secretion.  These results show for the first time that extracellular LPC is delivered to DCs via serum albumin, resulting in enhanced autoreactive activation of human iNKT cells.