Duarte C. Barral

CD1 antigen presentation: how it works

The classic concept of self–non-self discrimination by the immune system focused on the recognition of fragments from proteins presented by classical MHC molecules. However, the discovery of MHC-class-I-like CD1 antigen-presentation molecules now explains how the immune system also recognizes the abundant and diverse universe of lipid-containing antigens. The CD1 molecules bind and present amphipathic lipid antigens for recognition by T-cell receptors. Here, we outline the recent advances in our understanding of how the processes of CD1 assembly, trafficking, lipid-antigen binding and T-cell activation are achieved and the new insights into how lipid antigens differentially elicit CD1-restricted innate and adaptive T-cell responses.

Perlecan, the major proteoglycan of basement membranes, is altered in patients with Schwartz-Jampel syndrome (chondrodystrophic myotonia).

Schwartz-Jampel syndrome (SJS1) is a rare autosomal recessive disorder characterized by permanent myotonia (prolonged failure of muscle relaxation) and skeletal dysplasia, resulting in reduced stature, kyphoscoliosis, bowing of the diaphyses and irregular epiphyses. Electromyographic investigations reveal repetitive muscle discharges, which may originate from both neurogenic and myogenic alterations. We previously localized the SJS1 locus to chromosome 1p34–p36.1 and found no evidence of genetic heterogeneity. Here we describe mutations, including missense and splicing mutations, of the gene encoding perlecan (HSPG2) in three SJS1 families. In so doing, we have identified the first human mutations in HSPG2, which underscore the importance of perlecan not only in maintaining cartilage integrity but also in regulating muscle excitability.

Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli syndrome

Griscelli syndrome (GS) patients and the corresponding mouse model ashen exhibit defects mainly in two types of lysosome-related organelles, melanosomes in melanocytes and lytic granules in CTLs. This disease is caused by loss-of-function mutations in RAB27A, which encodes 1 of the 60 known Rab GTPases, critical regulators of vesicular transport. Here we present evidence that Rab27a function can be compensated by a closely related protein, Rab27b. Rab27b is expressed in platelets and other tissues but not in melanocytes or CTLs. Morphological and functional tests in platelets derived from ashen mice are all within normal limits. Both Rab27a and Rab27b are found associated with the limiting membrane of platelet-dense granules and to a lesser degree with alpha-granules. Ubiquitous transgenic expression of Rab27a or Rab27b rescues ashen coat color, and melanocytes derived from transgenic mice exhibit widespread peripheral distribution of melanosomes instead of the perinuclear clumping observed in ashen melanocytes. Finally, transient expression in ashen melanocytes of Rab27a or Rab27b, but not other Rabs, restores peripheral distribution of melanosomes. Our data suggest that Rab27b is functionally redundant with Rab27a and that the pathogenesis of GS is determined by the relative expression of Rab27a and Rab27b in specialized cell types.

The leaden Gene Product Is Required with Rab27a to Recruit Myosin Va to Melanosomes in Melanocytes

The function of lysosome‐related organelles such as melanosomes in melanocytes, and lytic granules in cytotoxic T lymphocytes is disrupted in Griscelli syndrome and related diseases. Griscelli syndrome results from loss of function mutations in either the RAB27A (type 1 Griscelli syndrome) or MYO5A (type 2 Griscelli syndrome) genes. Melanocytes from Griscelli syndrome patients and respective murine models ashen (Rab27a mutant), dilute (myosin Va mutant), and leaden exhibit perinuclear clustering of melanosomes. Recent work suggests that Rab27a is required to recruit myosin Va to melanosomes, thereby tethering melanosomes to the peripheral actin network and promoting melanosome retention at the tips of melanocytic dendrites. Here, we characterize the function of the leaden gene product. We show that Rab27a, but not myosin Va, can be localized to melanosomes in leaden melanocytes, suggesting that the leaden gene product acts downstream of, or in parallel to, Rab27a in melanocytes to promote recruitment of myosin Va to melanosomes. We also observed reduced levels of myosin Va protein in leaden and ashen melanocytes, suggesting that myosin Va stability is influenced by the leaden and ashen gene products. In leaden cytotoxic T lymphocytes, we observed that lytic granules polarize towards the immunological synapse and kill target cells normally. However, in contrast to melanocytes, we found that neither the leaden gene product (melanophilin) nor myosin Va was detectable in cytotoxic T lymphocytes. These results suggest that Rab27a interacts with different classes of effector proteins in melanocytes and cytotoxic T lymphocytes.

Primary deficiency of microsomal triglyceride transfer protein in human abetalipoproteinemia is associated with loss of CD1 function

Abetalipoproteinemia (ABL) is a rare Mendelian disorder of lipid metabolism due to genetic deficiency in microsomal triglyceride transfer protein (MTP). It is associated with defects in MTP-mediated lipid transfer onto apolipoprotein B (APOB) and impaired secretion of APOB-containing lipoproteins. Recently, MTP was shown to regulate the CD1 family of lipid antigen-presenting molecules, but little is known about immune function in ABL patients. Here, we have shown that ABL is characterized by immune defects affecting presentation of self and microbial lipid antigens by group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 molecules. In dendritic cells isolated from ABL patients, MTP deficiency was associated with increased proteasomal degradation of group 1 CD1 molecules. Although CD1d escaped degradation, it was unable to load antigens and exhibited functional defects similar to those affecting the group 1 CD1 molecules. The reduction in CD1 function resulted in impaired activation of CD1-restricted T and invariant natural killer T (iNKT) cells and reduced numbers and phenotypic alterations of iNKT cells consistent with central and peripheral CD1 defects in vivo. These data highlight MTP as a unique regulator of human metabolic and immune pathways and reveal that ABL is not only a disorder of lipid metabolism but also an immune disease involving CD1.

CD1a and MHC Class I Follow a Similar Endocytic Recycling Pathway

CD1 proteins are a family of major histocompatibility complex (MHC) class I‐like antigen‐presenting molecules that present lipids to T cells. The cytoplasmic tails (CTs) of all human CD1 isoforms, with the exception of CD1a, contain tyrosine‐based sorting motifs, responsible for the internalization of proteins by the clathrin‐mediated pathway. The role of the CD1a CT, which does not possess any sorting motifs, as well as its mode of internalization are not known. We investigated the internalization and recycling pathways followed by CD1a and the role of its CT. We found that CD1a can be internalized by a clathrin‐ and dynamin‐independent pathway and that it follows a Rab22a‐ and ADP ribosylation factor (ARF)6‐dependent recycling pathway, similar to other cargo internalized independent of clathrin. We also found that the CD1a CT is S‐acylated. However, this posttranslational modification does not determine the rate of internalization or recycling of the protein or its localization to detergent‐resistant membrane microdomains (DRMs) where we found CD1a to be enriched. We also show that plasma membrane DRMs are essential for efficient CD1a‐mediated antigen presentation. These findings place CD1a closer to MHC class I in its trafficking and potential antigen‐loading compartments among CD1 isoforms. Furthermore, we identify CD1a as a new marker for the clathrin‐ and dynamin‐independent and DRM‐dependent pathway of internalization as well as the Rab22a‐ and ARF6‐dependent recycling pathway.

Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.

The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

CD1c bypasses lysosomes to present a lipopeptide antigen with 12 amino acids

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.

The small GTPase Rab11 co-localizes with α-synuclein in intracellular inclusions and modulates its aggregation, secretion and toxicity

Alpha-synuclein (aSyn) misfolding and aggregation are pathological features common to several neurodegenerative diseases, including Parkinsons disease (PD). Mounting evidence suggests that aSyn can be secreted and transferred from cell to cell, participating in the propagation and spreading of pathological events. Rab11, a small GTPase, is an important regulator in both endocytic and secretory pathways. Here, we show that Rab11 is involved in regulating aSyn secretion. Rab11 knockdown or overexpression of either Rab11a wild-type (Rab11a WT) or Rab11a GDP-bound mutant (Rab11a S25N) increased secretion of aSyn. Furthermore, we demonstrate that Rab11 interacts with aSyn and is present in intracellular inclusions together with aSyn. Moreover, Rab11 reduces aSyn aggregation and toxicity. Our results suggest that Rab11 is involved in modulating the processes of aSyn secretion and aggregation, both of which are important mechanisms in the progression of aSyn pathology in PD and other synucleinopathies.

Gene therapy for choroideremia: in vitro rescue mediated by recombinant adenovirus

Choroideremia (CHM) is an X-linked retinal degenerative disease resulting from a lack of functional Rab Escort Protein-1 (REP-1). As a first step in developing gene-based therapies for this disease, we evaluated the feasibility of delivering functional REP-1 to defective lymphocytes and fibroblasts isolated from individuals with CHM. A recombinant adenovirus delivering the full-length human cDNA encoding REP-1 under the control of a cytomegalovirus promoter was generated. Adenovirus-mediated delivery of REP-1 rescued the defective cells as assessed through protein and enzymatic assays. Ultimately, it may be possible to use virus-mediated delivery of REP-1 to evaluate disease intervention in vivo.