Activation state and intracellular trafficking contribute to the repertoire of endogenous glycosphinogolipids presented by CD1d (130.22)

Abstract CD1d molecules present lipid antigens that activate CD1d-restricted natural killer T (NKT) cells. NKT cell activation leads to potent downstream activation of other immune cells through cell-cell interactions and rapid production of large quantities of cytokines. Endogenous lipids bound to CD1d are sufficient for activation of NKT cells in the setting of TLR induced cytokines. The most potent NKT cell antigens identified are glycosphingolipids (GSL). The repertoire of GSL bound to CD1d expressed in myeloid antigen presenting cells at steady state and in the setting of activation has not been delineated. We have identified the range of GSL bound to soluble CD1d molecules that sample the ER/secretory routes and cell surface cleaved CD1d that also samples the endocytic system. CD1d bound specific GSL preferentially as the CD1d GSL profile did not solely reflect cellular GSL abundance. GM1a and GD1a are prominent CD1d ligands for molecules following both the ER/secretory and lysosomal trafficking routes, while GM2 was eluted from soluble CD1d but not lysosomal trafficking CD1d. Further, after LPS activation the quantities of soluble CD1d bound GM3 and GM1a markedly increased. A unique a-galactose-terminating GSL was also found to be preferentially bound to CD1d at steady state and increased with APC activation. Our studies identify the range of GSL presented by CD1d and how presentation varies based on CD1d intracellular trafficking and microbial activation. In press PNAS