Lawrence J. Stern

Developmental plasticity of CNS microglia

Microglia arise from CD45+ bone marrow precursors that colonize the fetal brain and play a key role in central nervous system inflammatory conditions. We report that parenchymal microglia are uncommitted myeloid progenitors of immature dendritic cells and macrophages by several criteria, including surface expression of “empty” class II MHC protein and their cysteine protease (cathepsin) profile. Microglia express receptors for stem cell factor and can be skewed toward more dendritic cell or macrophage-like profiles in response to the lineage growth factors granulocyte/macrophage colony-stimulating factor or macrophage colony-stimulating factor. Thus, in contrast to other organs, where terminally differentiated populations of resident dendritic cells and/or macrophages outnumber colonizing precursors, the majority of microglia within the brain remain in an undifferentiated state.

The human class II MHC protein HLA-DR1 assembles as empty αβ heterodimers in the absence of antigenic peptide

We have produced the human class II histocompatibility protein, HLA-DR1, as a soluble, secreted glycoprotein in insect cells infected with baculoviruses carrying truncated alpha and beta subunit genes. The peptide-binding site is empty, and the empty molecules are fully competent to bind antigenic peptide. We used the empty molecules to measure an intrinsic rate for peptide association, and to investigate the role of peptide in stabilizing the class II structure. Peptide binding kinetics for the empty molecule are only 10-fold faster than for peptide exchange into an occupied site, suggesting that a conformational change may accompany peptide binding. The native alpha beta heterodimer assembles in the absence of antigenic peptide, but peptide binding stabilizes the empty heterodimer against aggregation and against SDS-induced denaturation.

Antigenic peptide binding by class I and class II histocompatibility proteins

The recent determination of the structure of a class II MHC molecule complexed to a specific peptide reveals both similarities and differences with peptide binding by class I MHC.

The Relationship of MHC-Peptide Binding and T Cell Activation Probed Using Chemically Defined MHC Class II Oligomers

A series of novel chemically defined soluble oligomers of the human MHC class II protein HLA-DR1 was constructed to probe the molecular requirements for initiation of T cell activation. MHC dimers, trimers, and tetramers stimulated T cells, as measured by upregulation of the activation markers CD69 and CD25, and by internalization of activated T cell receptor subunits. Monomeric MHC-peptide complexes engaged T cell receptors but did not induce activation. For a given amount of receptor engagement, the extent of activation was equivalent for each of the oligomers and correlated with the number of T cell receptor cross-links induced. These results suggest that formation or rearrangement of a T cell receptor dimer is necessary and sufficient for initiation of T cell signaling.

Phosphorylation of T cell receptor zeta is regulated by a lipid dependent folding transition.

The cytoplasmic domain of the T cell receptor ζ subunit (ζ cyt) is sufficient to couple receptor ligation to intracellular signaling cascades, but little is known about its structure or mechanism of signaling. In aqueous solution, ζcyt is unstructured. Here we report that in the presence of lipid vesicles ζcyt assumes a folded structure. The folding transition is reversible and dependent on the presence of acidic phospholipids. In the lipid-bound conformation, ζcyt is refractory to phosphorylation by src family tyrosine kinases, which are believed to play a key role in signal initiation in vivo. In the lipid-free, unstructured form, ζcyt is readily phosphorylated, and phospho-ζ cyt exhibits neither membrane association nor structure induction. The conformational change may provide a mechanism for coupling receptor clustering to cytoplasmic signaling events.

Proteomic Analysis of Microglia-Derived Exosomes: Metabolic Role of the Aminopeptidase CD13 in Neuropeptide Catabolism

Vesicle transport is a fundamental mechanism of communication in the CNS. In this study we characterized a novel type of vesicle released by murine brain microglial cells: microglial exosomes. Analysis of their protein content identified several enzymes, chaperones, tetraspanins, and membrane receptors previously reported in B cells and dendritic cell-derived exosomes. Additionally, microglia-derived exosomes expressed the aminopeptidase CD13 and the lactate transporter MCT-1. Exosomal CD13 was metabolically active in cleaving leucine- and methionine-enkephalins peptides by releasing the N-terminal tyrosine. Cleaved neuropeptides were unable to bind to the neuronal opioid receptor as assessed by cAMP response. Microglial exosomal vesicles may represent an important, previously unrecognized, cellular communication system in an organ in which cell motility is highly restricted.

Peptide immunotherapy in allergic asthma generates IL-10–dependent immunological tolerance associated with linked epitope suppression

Treatment of patients with allergic asthma using low doses of peptides containing T cell epitopes from Fel d 1, the major cat allergen, reduces allergic sensitization and improves surrogate markers of disease. Here, we demonstrate a key immunological mechanism, linked epitope suppression, associated with this therapeutic effect. Treatment with selected epitopes from a single allergen resulted in suppression of responses to other (“linked”) epitopes within the same molecule. This phenomenon was induced after peptide immunotherapy in human asthmatic subjects and in a novel HLA-DR1 transgenic mouse model of asthma. Tracking of allergen-specific T cells using DR1 tetramers determined that suppression was associated with the induction of interleukin (IL)-10+ T cells that were more abundant than T cells specific for the single-treatment peptide and was reversed by anti–IL-10 receptor administration. Resolution of airway pathophysiology in this model was associated with reduced recruitment, proliferation, and effector function of allergen-specific Th2 cells. Our results provide, for the first time, in vivo evidence of linked epitope suppression and IL-10 induction in both human allergic disease and a mouse model designed to closely mimic peptide therapy in humans.

Structural Basis For Antigenic Peptide Precursor Processing by the Endoplasmic Reticulum Aminopeptidase ERAP1

ERAP1 trims antigen precursors to fit into MHC class I proteins. To fulfill this function, ERAP1 has unique substrate preferences, trimming long peptides but sparing shorter ones. To identify the structural basis for ERAP1s unusual properties, we determined the X-ray crystal structure of human ERAP1 bound to bestatin. The structure reveals an open conformation with a large interior compartment. An extended groove originating from the enzymes catalytic center can accommodate long peptides and has features that explain ERAP1s broad specificity for antigenic peptide precursors. Structural and biochemical analyses suggest a mechanism for ERAP1s length-dependent trimming activity, whereby binding of long rather than short substrates induces a conformational change with reorientation of a key catalytic residue toward the active site. ERAP1s unique structural elements suggest how a generic aminopeptidase structure has been adapted for the specialized function of trimming antigenic precursors.

Self-Release of CLIP in Peptide Loading of HLA-DR Molecules

The assembly and transport of major histocompatibility complex (MHC) class II molecules require interaction with the invariant chain. A fragment of the invariant chain, CLIP, occupies the peptide-binding groove of the class II molecule. At endosomal pH, the binding of CLIP to human MHC class II HLA-DR molecules was counteracted by its amino-terminal segment (residues 81 to 89), which facilitated rapid release. The CLIP(81–89) fragment also catalyzed the release of CLIP(90–105) and a subset of other self-peptides, probably by transient interaction with an effector site outside the groove. Thus, CLIP may facilitate peptide loading through an allosteric release mechanism.

Class II major histocompatibility complex tetramer staining: progress, problems, and prospects

The use of major histocompatibility complex (MHC) tetramers in the detection and analysis of antigen‐specific T cells has become more widespread since its introduction 11 years ago. Early challenges in the application of tetramer staining to CD4+ T cells centred around difficulties in the expression of various class II MHC allelic variants and the detection of low‐frequency T cells in mixed populations. As many of the technical obstacles to class II MHC tetramer staining have been overcome, the focus has returned to uncertainties concerning how oligomer valency and T‐cell receptor/MHC affinity affect tetramer binding. Such issues have become more important with an increase in the number of studies relying on direct ex vivo analysis of antigen‐specific CD4+ T cells. In this review we discuss which problems in class II MHC tetramer staining have been solved to date, and which matters remain to be considered.