Transferring lipid antigens from membranes into CD1 antigen-presenting proteins represents a major molecular hurdle necessary for T-cell recognition. Saposins facilitate this process, but the mechanisms used are not well understood. We found that saposin B forms soluble saposin protein–lipid complexes detected by native gel electrophoresis that can directly load CD1 proteins. Because saposin B must bind lipids directly to function, we found it could not accommodate long acyl chain containing lipids. In contrast, saposin C facilitates CD1 lipid loading in a different way. It uses a stable, membrane-associated topology and was capable of loading lipid antigens without forming soluble saposin–lipid antigen complexes. These findings reveal how saposins use different strategies to facilitate transfer of structurally diverse lipid antigens.

Fibroblasts are important participants in inflammation. Although not leukocytes, their capacity to produce cytokines, chemokines, and other inflammatory factors locally in tissues suggests that they can contribute to inflammatory diseases. For example, fibroblasts in a rheumatoid arthritis (RA) joint are a dominant source of IL-6 and RANKL in the synovium, both of which are therapeutic targets for inflammation and bone erosion. Previously, we found that fibroblasts can be targeted by mAb directed against cadherin-11 (cad-11), a mesenchymal cadherin that fibroblasts selectively express. Targeting cad-11 significantly reduced inflammation as assessed by joint swelling and clinical inflammation scores. However, the mechanism by which anti-cad-11 reduced inflammation was not known. Here, we show that cad-11 engagement induces synovial fibroblasts to secret proinflammatory cytokines including IL-6. Cad-11 engagement strongly synergized with TNF-α and IL-1β in the induction of IL-6. Importantly, cad-11 activated MAP kinases and NF-κB for IL-6 induction. IL-6 levels in ankles of inflamed joints were reduced in cad-11 mutant mice compared to wild-type mice with inflammatory arthritis. Thus, we suggest that cad-11 modulates synovial fibroblasts to evoke inflammatory factors that may contribute to the inflammatory process in RA.

Microbial lipids activate T cells by binding directly to CD1 and T cell receptors (TCRs) or by indirect effects on antigen-presenting cells involving induction of lipid autoantigens, CD1 transcription, or cytokine release. To distinguish among direct and indirect mechanisms, we developed fluorescent human CD1b tetramers and measured T cell staining. CD1b tetramer staining of T cells requires glucose monomycolate (GMM) antigens, is specific for TCR structure, and is blocked by a recombinant clonotypic TCR comprised of TRAV17 and TRBV4-1, proving that CD1b-glycolipid complexes bind the TCR. GMM-loaded tetramers brightly stain a small subpopulation of blood-derived cells from humans infected with Mycobacterium tuberculosis, providing direct detection of a CD1b-reactive T cell repertoire. Polyclonal T cells from patients sorted with tetramers are activated by GMM antigens presented by CD1b. Whereas prior studies emphasized CD8(+) and CD4(-)CD8(-) CD1b-restricted clones, CD1b tetramer-based studies show that nearly all cells express the CD4 co-receptor. These findings prove a cognate mechanism whereby CD1b-glycolipid complexes bind to TCRs. CD1b tetramers detect a natural CD1b-restricted T cell repertoire ex vivo with unexpected features, opening a new investigative path to study the human CD1 system.

The recent discovery of dideoxymycobactin (DDM) as a ligand for CD1a demonstrates how a nonribosomal lipopeptide antigen is presented to T cells. DDM contains an unusual acylation motif and a peptide sequence present only in mycobacteria, but its discovery raises the possibility that ribosomally produced viral or mammalian proteins that commonly undergo lipidation might also function as antigens. To test this, we measured T cell responses to synthetic acylpeptides that mimic lipoproteins produced by cells and viruses. CD1c presented an N-acyl glycine dodecamer peptide (lipo-12) to human T cells, and the response was specific for the acyl linkage as well as the peptide length and sequence. Thus, CD1c represents the second member of the CD1 family to present lipopeptides. lipo-12 was efficiently recognized when presented by intact cells, and unlike DDM, it was inactivated by proteases and augmented by protease inhibitors. Although lysosomes often promote antigen presentation by CD1, rerouting CD1c to lysosomes by mutating CD1 tail sequences caused reduction in lipo-12 presentation. Thus, although certain antigens require antigen processing in lysosomes, others are destroyed there, providing a hypothesis for the evolutionary conservation of large CD1 families containing isoforms that survey early endosomal pathways.

Dendritic cells (DC) present lipid and peptide antigens to T cells on CD1 and MHC Class II (MHCII), respectively. The relative contribution of these systems during the initiation of adaptive immunity after microbial infection is not characterized. MHCII molecules normally acquire antigen and rapidly traffic from phagolysosomes to the plasma membrane as part of DC maturation, whereas CD1 molecules instead continually recycle between these sites before, during, and after DC maturation. We find that in Mycobacterium tuberculosis (Mtb)-infected DCs, CD1 presents antigens quickly. Surprisingly, rapid DC maturation results in early failure and delay in MHCII presentation. Whereas both CD1b and MHCII localize to bacterial phagosomes early after phagocytosis, MHCII traffics from the phagosome to the plasma membrane with a rapid kinetic that can precede antigen availability and loading. Thus, rather than facilitating antigen presentation, a lack of coordination in timing may allow organisms to use DC maturation as a mechanism of immune evasion. In contrast, CD1 antigen presentation occurs in the face of Mtb infection and rapid DC maturation because a pool of CD1 molecules remains available on the phagolysosome membrane that is able to acquire lipid antigens and deliver them to the plasma membrane.

Immunity/lipids link An unexpected link between immunity and lipid metabolism has been found. Apolipoproteins critical in lipid trafficking during normal energy delivery and in atherosclerosis have been found to have a role in the immune system. Here they serve a special function in CD1-mediated presentation of lipid antigens for T-cell activation. This has implications for microbial immunity, autoimmunity and atherosclerosis.