CD1b-mycolic acid tetramers demonstrate T-cell fine specificity for mycobacterial lipid tails

Mycobacterium tuberculosis synthesizes a thick cell wall comprised of mycolic acids (MA), which are foreign antigens for human T cells. T-cell clones from multiple donors were used to determine the fine specificity of MA recognition by human αβ T cells. Most CD1-presented lipid antigens contain large hydrophilic head groups comprised of carbohydrates or peptides that dominate patterns of T-cell specificity. MA diverges from the consensus antigen motif in that it lacks a head group. Using multiple forms of natural and synthetic MA and MA-specific T-cells with different T-cell receptors, we found that, unlike antigens with larger head groups, lipid length strongly controlled T-cell responses to MA. In addition, the three forms of MA that naturally occur in M. tuberculosis that differ in modifications on the lipid tail, differ in their potency for activating MA-specific T-cell clones. Thus, naturally occurring MA forms should be considered as separate, partly cross-reactive antigens. Two of the three forms of MA could be loaded onto human CD1b proteins, creating working CD1b-MA tetramers. The creation of CD1b-MA tetramers represents a new tool for future studies that track the effector functions and kinetics of MA-specific T-cells ex vivo.