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Mechanisms of Antigen Processing

Research Summary

Peter Cresswell is interested in the functions of major histocompatibility complex (MHC) molecules and CD1 molecules, which, respectively, bind peptides from foreign antigens and lipids, to form complexes recognized by T lymphocytes during immune responses. The mechanisms governing formation of MHC-peptide complexes are collectively known as antigen processing. Also of interest are the actions of a variety of proteins, induced by interferons, that play a role in immunity to infection.

MHC class I molecules are recognized by CD8-positive T cells, and MHC class II molecules by CD4-positive T cells. The mechanisms by which complexes of MHC molecules and peptides are generated, known collectively as antigen processing, are critical for successful adaptive immune responses. CD1 molecules bind lipids and present them to T cells, and we are interested in specific processing mechanisms that may play a role in this system. We are also interested in the mechanisms by which interferons eliminate viral infection during the innate immune response.

MHC Class I–Restricted Antigen Processing
Peptides generated in the cytosol by the proteasome bind to dimers of the MHC class I glycoprotein and β2-microglobulin (β2m) in the endoplasmic reticulum (ER). The peptides are translocated into the ER by the transporter associated with antigen processing (TAP). Tapasin, a transmembrane protein encoded by an MHC-linked gene, physically links peptide-free MHC class I molecules to TAP, forming the core of the "peptide-loading complex," which also includes the chaperone calreticulin and the thiol oxidoreductase ERp57. Translocation of a peptide followed by its successful interaction with an MHC class I molecule induces dissociation of the class I–peptide complex from the loading complex and transport to the cell surface.

Unfolded glycoproteins bind to calreticulin, or the related chaperone calnexin, via their N-linked glycans, specifically when they are in the monoglucosylated form, an intermediate in their maturation. ERp57 functions with calreticulin to ensure correct disulfide bond formation in many newly synthesized glycoproteins. We have found that within the loading complex, tapasin and ERp57 are connected by a disulfide bond. Formation of this bond occurs rapidly upon synthesis and is independent of MHC class I interaction with the loading complex. Our data suggest a peptide-loading cycle where newly assembled class I molecules bearing monoglucosylated N-linked glycans enter the loading complex with calreticulin and dissociate when peptides are loaded. We have used a cell-free system to show that the disulfide-linked conjugate of tapasin and ERp57 mediates binding of peptides to MHC class I molecules in solution. It can also "edit" the bound peptides, favoring the association of peptides with higher affinity at the expense of those with a lower affinity. This is a key step in ensuring the formation of stable class I–peptide complexes for recognition by CD8-positive T cells. Recently, in collaboration with Karin Reinisch's laboratory (Yale School of Medicine), we have determined the three-dimensional structure of the tapasin-ERp57 conjugate, which has allowed us to construct a rational model of the peptide-loading complex.

In dendritic cells (DCs), protein antigens internalized by phagocytosis or endocytosis can generate class I–associated peptides. This process is called cross-presentation, and the peptides are produced following protein translocation into the cytosol. Phagosomes in DCs appear to recruit some ER components into their limiting membrane, and we have shown that in DCs, proteins or protein fragments internalized by pinocytosis have access to the ER. In the ER there is a mechanism known as ERAD (ER-associated degradation) that allows misfolded proteins to be extruded into the cytosol for degradation by proteasomes. In DCs, therefore, the ER components accessible to extracellular soluble proteins and phagocytosed proteins provide the machinery for translocation into the cytosol, providing a likely explanation for the cross-presentation of extracellular antigens to CD8-positive T cells. We have shown that luciferase enzymes, used as model substrates, can enter the cytosol of DCs in a folded and functional form. The process appears to involve protein unfolding in the endocytic pathway and Hsp90-mediated refolding in the cytosol. We have also shown that the ability to cross-present antigens in immune complexes can be introduced into cells other than DCs by expressing Fc receptors that permit phagocytosis.

MHC Class II–Restricted Antigen Processing
Class II MHC molecules bind to the invariant chain immediately upon synthesis in the ER. The class II–invariant-chain complex consists of a core trimer of invariant chains associated with three class II molecules, each of which is a heterodimer. Association with the invariant chain prevents class II MHC molecules from binding unfolded proteins early in transport. The MHC class II–invariant-chain complex is transported from the ER to late endocytic structures, where the invariant chain is proteolytically degraded, releasing class II molecules capable of binding peptides after the HLA-DM–induced dissociation of an invariant-chain fragment called CLIP (class II–associated invariant-chain peptide).

In B cells and thymic epithelium, an additional class II–like heterodimer called HLA-DO associates with HLA-DM. HLA-DO is a DM inhibitor. It is believed that HLA-DO down-regulates the presentation of unrelated peptides by B cell class II molecules by restricting active DM to late endocytic compartments where the surface immunoglobulin of B cells delivers bound protein antigens.

Formation of CD1-Lipid Complexes
CD1 molecules are a family of MHC class I homologs that bind β2m and present lipids to certain T lymphocytes. Calreticulin, calnexin, and ERp57 are involved in CD1d assembly in the ER, but the order of events is different from MHC class I molecules. A complex consisting of partially oxidized CD1d heavy chain, calreticulin, calnexin, and ERp57 is formed prior to β2m association. After CD1d dissociates from the chaperones and disulfide bond oxidation is completed, β2m association occurs. Initial lipid binding occurs in the ER, but the complete complement of lipids on surface CD1d molecules, many likely to be acquired in the endocytic pathway, is still unknown. We recently identified two dominant components, the lipids phosphatidylcholine and sphingomyelin.

CD1d molecules are the target for natural killer T (NKT) cells, which recognize self-lipids. Degradation of sphingolipids in lysosomes requires lipid-extracting cofactors called saposins, which are small homologous proteins (saposins A–D) that are generated by lysosomal proteolysis from a precursor called prosaposin. We found that human CD1d expressed in fibroblasts lacking prosaposin cannot be lysosomally loaded with an exogenous lipid, α-galactosylceramide. Introduction of human prosaposin restores the ability of the cells to load the lipid. Using recombinant, purified saposins and prosaposin mutants lacking individual saposin species, we have found that saposin B is the major saposin involved.

NKT cells are involved in initial responses to viruses, and we showed that herpes simplex virus type 1 (HSV-1) escapes recognition by inducing the down-regulation of CD1d molecules from the surface of infected cells, including DCs. The virus prevents reexpression of CD1d molecules on the cell surface during the normal process of recycling through the endocytic pathway.

Functions of Interferon-Responsive Proteins
In pursuit of additional modulators of antigen processing, we looked for IFN-γ–responsive proteins that might play a role. We identified the enzyme gamma-interferon–inducible lysosomal thiolreductase (GILT), which is found in the late endocytic/lysosomal compartments where class II peptide loading occurs. We postulated that GILT is involved in the reduction of disulfide bonds within endocytosed protein antigens, unfolding them so that their class II epitopes are exposed. We showed that a GILT-knockout mouse is impaired in its ability to generate class II–restricted responses to protein antigens containing multiple internal disulfide bonds. Recently we have found that GILT is required for certain CD4-positive T cells to recognize murine melanoma antigens. We also found that GILT is secreted by macrophages in an active precursor form when they are stimulated by Gram-negative bacteria such as Escherichia coli or lipopolysaccharide from the bacteria. This results from the rerouting of precursor GILT and a number of lysosomal proteins rather than appropriate targeting to the lysosome. More recently we have shown that GILT is a host factor that facilitates infection of macrophages by Listeria monocytogenes. This organism uses a pore-forming toxin to facilitate its release from the phagosome into the cytosol, where it replicates, and GILT activates the toxin by reduction.

We also identified viperin, a novel protein that is induced by IFN-γ in macrophages and by type I interferons in most cell types. Viperin has antiviral activity for the human cytomegalovirus (CMV). Surprisingly, despite the antiviral activity of viperin, CMV actively induces its synthesis upon infection. During CMV infection, viperin moves from its normal intracellular site at the cytoplasmic face of the ER to sites of virus assembly. This may represent an immune evasion mechanism for CMV. We also found that viperin has antiviral activity for influenza A virus. The step in influenza virus replication affected by viperin expression is the release of the virus from infected cells, the last stage of the viral life cycle.

Grants from the National Institutes of Health provided partial support for the work on cross-presentation, MHC class II, GILT, and CD1d.

As of May 30, 2012

Scientist Profile

Yale University
Cell Biology, Immunology