CD1 proteins have been implicated as antigen-presenting molecules for T cell-mediated immune responses, but their intracellular localization and trafficking remain uncharacterized. CD1b, a member of this family that presents microbial lipid antigens of exogenous origin, was found to localize to endocytic compartments that included the same specialized subset of endosomes in which major histocompatibility complex (MHC) class II molecules are proposed to bind endocytosed antigens. Unlike MHC class II molecules, which traffic to antigen-loading endosomal compartments [MHC class II compartments (MIICs)] primarily as a consequence of their association with the invariant chain, localization of CD1b to these compartments was dependent on a tyrosine-based motif in its own cytoplasmic tail.

CD1b is an antigen-presenting molecule that mediates recognition of bacterial lipid and glycolipid antigens by specific T cells. We demonstrate that the nine-amino acid cytoplasmic tail of CD1b contains all of the signals required for its normal endosomal targeting, and that the single cytoplasmic tyrosine is a critical component of the targeting motif. Mutant forms of CD1b lacking the endosomal targeting motif are expressed at high levels on the cell surface but are unable to efficiently present lipid antigens acquired either exogenously or from live intracellular organisms. These results define the functional role of the CD1b targeting motif in a physiologic setting and demonstrate its importance in delivery of this antigen-presenting molecule to appropriate intracellular compartments.

CD1b and CD1c are antigen-presenting molecules that mediate recognition of bacterial lipids by T cells, but it is currently not known whether these two molecules are redundant or are specialized to perform different immunological functions. Here, we show that the distribution of CD1c in human dendritic cells was characterized by a high ratio of cell surface to intracellular molecules, whereas CD1b showed a reciprocal pattern of distribution. In contrast to the accumulation of CD1b in lysosomal major histocompatibility complex class II compartments, intracellular CD1c molecules accumulated in other endocytic compartments, most likely early and late endosomes. Deletion of the cytoplasmic tail of CD1c, containing a tyrosine-based internalization motif, abolished most of its intracellular localization. Functional studies using T cells specific for defined lipid antigens revealed that in contrast to CD1b-mediated antigen presentation, antigen presentation by CD1c was resistant to drugs inhibiting endosomal acidification and was independent of endosomal localization of CD1c. Taken together, these results support the hypothesis that CD1b and CD1c are specialized to survey the lipid content of different intracellular compartments.

The intracellular trafficking of major histocompatibility complex (MHC) class I and class II molecules has evolved to support their function in peptide antigen presentation optimally. We have analyzed the intracellular trafficking of newly synthesized human CD1b, a lipid antigen‐presenting molecule, to understand how this relates to its antigen‐presenting function. Nascent CD1b was transported rapidly to the cell surface after leaving the Golgi, and then entered the endocytic system by internalization via AP‐2‐dependent sorting at the plasma membrane. A second sorting event, possibly involving AP‐3 complexes, led to prominent accumulation of CD1b in MHC class II compartments (MIICs). Functional studies demonstrated the importance of nascent CD1b for the efficient presentation of a foreign lipid antigen. Therefore, the intracellular trafficking of nascent CD1b via the cell surface to reach MIICs may allow the efficient sampling of lipid antigens present in endocytic compartments.

A panel of murine monoclonal antibodies was generated against the extracellular domain of the human platelet-derived growth factor (PDGF) ␤ receptor (PDGFR␤). These antibodies were assayed for both the ability to inhibit binding of PDGF BB to PDGFR␤ ؉ cells as well as the capacity to inhibit PDGF BB-mediated mitogenesis. As expected, all antibodies that could prevent PDGF BB binding also inhibited mitogenesis. However one antibody (M4TS.11), with no detectable ability to inhibit PDGF BB binding, was a potent inhibitor of proliferation induced by PDGF BB. Further characterization indicated that M4TS.11 impaired PDGFR␤ dimerization, revealing the mechanism by which it prevented PDGF BB-mediated mitogenesis. Using domain deletion mutants of the extracellular portion of PDGFR␤, the determinant recognized by this antibody was localized to the fourth extracellular domain of PDGFR␤, indicating that this domain, which is not involved in ligand binding, actively participates in receptor dimerization and signal transduction. The M4TS.11 antibody could also inhibit PDGF BB-mediated proliferation of responsive cells from both the baboon and the rabbit, indicating the determinant recognized by the antibody is not limited to humans and making it possible to use this antibody to evaluate the therapeutic benefit of interfering with PDGF in animal models of human disease.

Human CD1 proteins present lipid and glycolipid Ags to T cells. Cellular trafficking patterns of CD1 proteins may determine the ability of differing isoforms of CD1 to acquire, bind, and present these Ags to T cells. To test this hypothesis, glycosyl-phosphatidylinositol (GPI)-modified variants of CD1b and CD1c were engineered by chimerization with a GPI modification signal sequence derived from decay-accelerating factor (DAF). GPI reanchoring was confirmed by demonstrating the phosphatidylinositol-specific phospholipase C sensitivity of the CD1b · DAF and CD1c · DAF fusion proteins expressed on transfectant cell surfaces. Using cytotoxicity and cytokine release assays as functional readouts, we demonstrated that CD1c · DAF is as efficient as native CD1c in presenting mycobacterial Ags to the human CD1c-restricted T cell line CD8-1. In contrast, CD1b · DAF, although also capable of presenting Ag (in this case to the CD1b-restricted T cell line LDN5), was less efficient than its native CD1b counterpart. The data support the idea that CD1c · DAF maintains the capacity to access CD1c Ag-loading compartment(s), whereas CD1b · DAF is diverted by its GPI anchor away from the optimal CD1b Ag-loading compartment(s). This constitutes the first GPI reanchoring of CD1 proteins and provides evidence that CD1b and CD1c have nonoverlapping Ag-presenting pathways, suggesting that these two Ag-presenting molecules may have distinct roles in lipid Ag presentation.