Human CD1b and CD1c isoforms survey different intracellular compartments for the presentation of microbial lipid antigens.

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.

Cytoplasmic tail-dependent localization of CD1b antigen-presenting molecules to MIICs.

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.

Mechanisms of lipid antigen presentation by CD1.

CD1 is a family of cell surface glycoproteins that are related in structure and evolutionary origin to the major histocompatibility complex (MHC)-encoded antigen-presenting molecules. In contrast to MHC-encoded antigen-presenting molecules, CD1 binds and presents lipid and glycolipid antigens for specific recognition by T cell antigen receptors. Recent work shows that several CD1 family members colocalize with MHC class II proteins within the endocytic system of antigen-presenting cells. Detailed studies of the intracellular trafficking of CD1 proteins reveal new mechanisms controlling delivery of antigen-presenting molecules to particular compartments within cells. The combination of overlapping yet distinct trafficking routes for the various CD1 family members, combined with emerging information on the heterogeneity of CD1-presented lipid antigens, suggest a model whereby different members of the CD1 family could present antigens that occur in various cellular compartments. Furthermore, the CD1 family as a group may present antigens from pathogens that are not normally accessible to or efficiently surveyed by the MHC Class I or II systems. The discovery of this third pathway for antigen presentation, together with the appreciation of a previously unrecognized universe of nonpeptide lipid antigens for T cell responses, are likely to have broad implications for our understanding of the cell-mediated immune response and its role in health and disease.

An antibody reactive with domain 4 of the platelet-derived growth factor beta receptor allows BB binding while inhibiting proliferation by impairing receptor dimerization.

A panel of murine monoclonal antibodies was generated against the extracellular domain of the human platelet-derived growth factor (PDGF) beta receptor (PDGFRbeta). These antibodies were assayed for both the ability to inhibit binding of PDGF BB to PDGFRbeta+ 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 PDGFRbeta dimerization, revealing the mechanism by which it prevented PDGF BB-mediated mitogenesis. Using domain deletion mutants of the extracellular portion of PDGFRbeta, the determinant recognized by this antibody was localized to the fourth extracellular domain of PDGFRbeta, 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.

Intracellular trafficking pathway of newly synthesized CD1b molecules.

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.

Glycosyl-phosphatidylinositol reanchoring unmasks distinct antigen-presenting pathways for CD1b and CD1c.

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.

The tyrosine-containing cytoplasmic tail of CD1b is essential for its efficient presentation of bacterial lipid antigens.

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.

CD1--a new paradigm for antigen presentation and T cell activation.

Despite identification of the CD1 family of molecules in the late 1970s, the function of CD1 was undetermined for more than a decade. Recent evidence has established that CD1 molecules comprise a novel lineage of antigen-presenting molecules, distinct from major histocompatibility complex (MHC) class I and class II molecules. Unlike the MHC molecules, which bind short peptides in their antigen-binding groove for presentation to either CD4+ or CD8+ T cells bearing alpha beta T cell receptors, the CD1 molecules appear to accommodate lipid and glycolipid antigens in their hydrophobic cavity for presentation to a wide variety of T cells, including double-negative alpha beta and gamma delta T cells and CD8+ alpha beta T cells. By using a unique cytoplasmic signal, some CD1 molecules traffic to endosomal compartments for sampling mycobacteria-derived lipid antigens, and subsequently lipid antigen-loaded CD1 molecules are expressed on the cell surface to activate specific T cells. These CD1-restricted T cells kill mycobacteria-infected cells and secrete interferon-gamma, indicating a potential role of CD1-mediated T cell responses in clearing mycobacterial infection. The identification of an MHC-independent antigen presentation pathway for nonpeptide antigens provides new insights into immunoregulation and host defense.