Disruption of Hydrogen Bonds between Major Histocompatibility Complex Class II and the Peptide N-Terminus Is Not Sufficient to Form a Human Leukocyte Antigen-DM Receptive State of Major Histocompatibility Complex Class II

Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306–318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue.     

Dynamics of Major Histocompatibility Complex Class I Association with the Human Peptide-loading Complex

Although the human peptide-loading complex (PLC) is required for optimal major histocompatibility complex class I (MHC I) antigen presentation, its composition is still incompletely understood. The ratio of the transporter associated with antigen processing (TAP) and MHC I to tapasin, which is responsible for MHC I recruitment and peptide binding optimization, is particularly critical for modeling of the PLC. Here, we characterized the stoichiometry of the human PLC using both biophysical and biochemical approaches. By means of single-molecule pulldown (SiMPull), we determined a TAP/tapasin ratio of 1:2, consistent with previous studies of insect-cell microsomes, rat-human chimeric cells, and HeLa cells expressing truncated TAP subunits. We also report that the tapasin/MHC I ratio varies, with the PLC population comprising both 2:1 and 2:2 complexes, based on mutational and co-precipitation studies. The MHC I-saturated PLC may be particularly prevalent among peptide-selective alleles, such as HLA-C4. Additionally, MHC I association with the PLC increases when its peptide supply is reduced by inhibiting the proteasome or by blocking TAP-mediated peptide transport using viral inhibitors. Taken together, our results indicate that the composition of the human PLC varies under normal conditions and dynamically adapts to alterations in peptide supply that may arise during viral infection. These findings improve our understanding of the quality control of MHC I peptide loading and may aid the structural and functional modeling of the human PLC.     

Class II Major Histocompatibility Complex Plays an Essential Role in Obesity-Induced Adipose Inflammation

1. Download : Download high-res image (274KB)
2. Download : Download full-size image

Highlights? Obesity enhances MHCII expression in primary human and mouse adipocytes ? Adipocytes activate CD4+ ARTs via MHCII and leptin to induce adipose inflammation ? Macrophage changes in adipose follow adipocyte and T cell interactions during HFD ? Adaptive immune mechanisms are essential to obesity-induced adipose inflammation     

Ii Chain Controls the Transport of Major Histocompatibility Complex Class II Molecules to and from Lysosomes

Major histocompatibility complex class II molecules are synthesized as a nonameric complex consisting of three αβ dimers associated with a trimer of invariant (Ii) chains. After exiting the TGN, a targeting signal in the Ii chain cytoplasmic domain directs the complex to endosomes where Ii chain is proteolytically processed and removed, allowing class II molecules to bind antigenic peptides before reaching the cell surface. Ii chain dissociation and peptide binding are thought to occur in one or more postendosomal sites related either to endosomes (designated CIIV) or to lysosomes (designated MIIC). We now find that in addition to initially targeting αβ dimers to endosomes, Ii chain regulates the subsequent transport of class II molecules. Under normal conditions, murine A20 B cells transport all of their newly synthesized class II I-A^b αβ dimers to the plasma membrane with little if any reaching lysosomal compartments. Inhibition of Ii processing by the cysteine/serine protease inhibitor leupeptin, however, blocked transport to the cell surface and caused a dramatic but selective accumulation of I-A^b class II molecules in lysosomes. In leupeptin, I-A^b dimers formed stable complexes with a 10-kD NH₂-terminal Ii chain fragment (Ii-p10), normally a transient intermediate in Ii chain processing. Upon removal of leupeptin, Ii-p10 was degraded and released, I-A^b dimers bound antigenic peptides, and the peptide-loaded dimers were transported slowly from lysosomes to the plasma membrane. Our results suggest that alterations in the rate or efficiency of Ii chain processing can alter the postendosomal sorting of class II molecules, resulting in the increased accumulation of αβ dimers in lysosome-like MIIC. Thus, simple differences in Ii chain processing may account for the highly variable amounts of class II found in lysosomal compartments of different cell types or at different developmental stages.The initiation of most immune responses requires antigen recognition by helper T lymphocytes. The antigen receptors on T cells can only recognize antigens as small peptides bound to major histocompatibility complex (MHC)¹ class II molecules at the surface of antigen presenting cells (Cresswell, 1994; Germain, 1994). The complexes between class II molecules and antigenic peptides are formed intracellularly somewhere along the endocytic pathway (Germain, 1994; Wolf and Ploegh, 1995). This process requires the internalization of protein antigen and its delivery to a site suitable for the generation of antigenic peptides. In addition, the peptides must be generated within, or transferred to, a site to which newly synthesized MHC class II molecules are delivered and rendered competent for peptide binding (Davidson et al., 1991).Invariant (Ii) chain plays a central role in controlling the intracellular transport of MHC class II (Cresswell, 1996). In the ER, Ii chain is synthesized as a trimer that complexes with three αβ dimers of MHC class II (Roche et al., 1991). Its NH₂-terminal cytoplasmic domain contains a wellknown targeting signal that directs class II–Ii chain complexes to endosomes after exit from the TGN (Bakke and Dobberstein, 1990; Lotteau et al., 1990; Neefjes et al., 1990; Odorizzi et al., 1994; Pieters et al., 1993). Once in endosomes, Ii chain is subjected to proteolysis by acid hydrolases (Roche and Cresswell, 1991). Degradation occurs in a stepwise fashion, resulting in the appearance of class II– bound NH₂-terminal intermediates containing the Ii chain cytoplasmic domain, membrane anchor, and parts of its luminal domain (Newcomb and Cresswell, 1993). The intermediates accumulate in the presence of protease inhibitors that interfere with Ii chain processing such as the serinecysteine protease inhibitor leupeptin, treatment with which can also block the transport of at least some class II haplotypes to the cell surface (Amigorena et al., 1995; Blum and Cresswell, 1988; Neefjes and Ploegh, 1992). How leupeptin inhibits surface appearance is unknown.In human cells, Ii chain degradation intermediates include a 21–22-kD fragment (designated LIP [leupeptininducible peptide]) and a 10–12-kD fragment (designated SLIP [small leupeptin-inducible peptide]) (Blum and Cresswell, 1988; Maric et al., 1994). In murine cells, only a 10– 12-kD fragment has been identified (Ii-p10) (Amigorena et al., 1995). Ii-p10 remains as a trimer associated with three αβ dimers and blocks the binding of antigenic peptides (Amigorena et al., 1995; Morton et al., 1995). It is thus likely that Ii-p10 includes a luminal region of Ii chain (designated CLIP) known to occupy the peptide binding groove of αβ dimers. Cleavage of Ii-p10 by a leupeptinsensitive protease causes its dissociation from αβ dimers, while leaving CLIP in the peptide binding groove. The removal of CLIP is favored at acidic pH but is additionally catalyzed by a second MHC gene product, HLA-DM (Sloan et al., 1995; Denzin and Cresswell, 1995; Karlsson et al., 1994; Roche, 1995). In mutant cells lacking HLA-DM, there is defective loading of antigenic peptides and the appearance of CLIP-αβ dimers on the plasma membrane (Mellins et al., 1994; Riberdy et al., 1992).The precise site(s) where these events occur remains unclear. In A20 B cells, a specialized population of endosome-like vesicles designated CIIV (for class II vesicles) represents a site through which a majority of newly synthesized class II molecules pass en route to the cell surface and a place where antigenic peptides bind αβ dimers of the I-A^d haplotype (Amigorena et al., 1994, 1995; Barnes and Mitchell, 1995). CIIV are physically distinct from the bulk of endosomes and lysosomes and contain at least some HLA-DM (Pierre et al., 1996). Despite the fact that most of the αβ dimers reaching CIIV are newly synthesized, CIIV contain little or no intact Ii chain (Amigorena et al., 1995). Thus, Ii chain–αβ complexes first may be delivered to endosomes where Ii chain is cleaved before being delivered to CIIV. That peptide loading can occur in CIIV has been demonstrated by experiments showing that leupeptin causes CIIV to transiently accumulate Ii-p10– containing complexes, which can then bind peptide (Amigorena et al., 1995).In human Epstein-Barr virus–transformed B lymphoblasts, most class II molecules have been localized to structures collectively designated MIIC (for MHC class II compartment) (Peters et al., 1991; Tulp et al., 1994; West et al., 1994). MIICs differ from CIIVs in that the latter contain endosomal but not lysosomal markers, while MIICs have most or all of the features of lysosomes (Peters et al., 1991, 1995; Pierre et al., 1996). Interestingly, the distribution of class II between endosomal (CIIV) and lysosomal (MIIC) compartments varies widely among cell types. Since lysosomes are classically defined as terminal degradative organelles (Kornfeld and Mellman, 1989), such variations may reflect differences in the rates at which class II is turned over in different cell types. On the other hand, MIICs also contain the bulk of HLA-DM and can host the loading of antigenic peptides onto class II molecules (Sanderson et al., 1994). The extent to which these complexes escape degradation and reach the cell surface is unclear. Nor is it at all clear how different cell types regulate the intracellular distribution of class II molecules between early and late endocytic compartments.We now show that murine A20 cells expressing endogenous I-A^d and transfected I-A^b normally localize little class II in lysosomes. Selective lysosomal accumulation of I-A^b αβ dimers can be induced after leupeptin treatment. Interestingly, I-A^b dimers, but not I-A^d dimers, are induced by leupeptin to form stable complexes with Ii-p10. Upon removal of the inhibitor, the Ii-p10 was removed and class II molecules were slowly transported from lysosomes to the cell surface. Thus, the rate of dissociation of Ii chain intermediates can regulate whether newly synthesized class II molecules are transported to the plasma membrane or to lysosomes.     

Differential Transmembrane Domain GXXXG Motif Pairing Impacts Major Histocompatibility Complex (MHC) Class II Structure

Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II αβ heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2⁺ class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2⁺ I-A^k conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2⁺ versus Ia.2⁻ I-A^k class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response.     

Mycobacterium tuberculosis Promotes HIV trans-Infection and Suppresses Major Histocompatibility Complex Class II Antigen Processing by Dendritic Cells

Mycobacterium tuberculosis is a leading killer of HIV-infected individuals worldwide, particularly in sub-Saharan Africa, where it is responsible for up to 50% of HIV-related deaths. Infection by HIV predisposes individuals to M. tuberculosis infection, and coinfection accelerates the progression of both diseases. In contrast to most other opportunistic infections associated with HIV, an increased risk of M. tuberculosis infection occurs during early-stage HIV disease, long before CD4 T cell counts fall below critical levels. We hypothesized that M. tuberculosis infection contributes to HIV pathogenesis by interfering with dendritic cell (DC)-mediated immune control. DCs carry pathogens like M. tuberculosis and HIV from sites of infection into lymphoid tissues, where they process and present antigenic peptides to CD4 T cells. Paradoxically, DCs can also deliver infectious HIV to T cells without first becoming infected, a process known as trans-infection. Lipopolysaccharide (LPS)-activated DCs sequester HIV in pocketlike membrane invaginations that remain open to the cell surface, and individual virions are delivered from the pocket into T cells at the site of contact during trans-infection. Here we report that M. tuberculosis exposure increases HIV trans-infection and induces viral sequestration within surface-accessible compartments identical to those seen in LPS-stimulated DCs. At the same time, M. tuberculosis dramatically decreases the degradative processing and major histocompatibility complex class II (MHC-II) presentation of HIV antigens to CD4 T cells. Our data suggest that M. tuberculosis infection promotes a shift in the dynamic balance between antigen processing and intact virion presentation, favoring DC-mediated amplification of HIV infections.Dendritic cells (DCs) comprise a diverse family of cell types whose primary function is to initiate and drive immune responses. Myeloid DCs (myDCs) are essential antigen-presenting cells that monitor peripheral tissues for invading pathogens. myDCs bind and internalize bacteria and viruses using a variety of surface receptors. When stimulated by pathogenic or inflammatory signals, peripheral-tissue DCs migrate to lymphoid tissues and undergo maturation, degrading stored antigens into peptides that are loaded onto major histocompatibility complex class II (MHC-II) molecules and expressed on the cell surface for presentation to CD4 T cells (reviewed in reference 4). In addition to presentation of processed peptide antigens, DCs carry intact, unprocessed proteins and pathogens from peripheral tissues to lymph nodes, where they can be passed to other antigen-presenting cells to increase the breadth of the immune response (reviewed in reference 10).HIV can exploit the natural trafficking of DCs to establish and amplify infection of CD4 T cells. DCs efficiently transfer intact, infectious HIV to T cells during immune interactions through a process known as trans-infection (14). DCs trans-infect HIV by binding and concentrating the intact virus at the cellular interface, forming an “infectious synapse” that concentrates HIV receptors on the T cell to the same site (24). Importantly, trans-infection does not require productive infection of the DCs, which are not infected efficiently by HIV in vitro or in vivo (14). Immature DCs significantly enhance infection of T cells through trans-infection, and prior activation by cytokine or bacterial stimuli markedly increases infectious synapse formation and concomitant trans-infection (2, 24, 33).Worldwide, nearly one-third of HIV-infected people are coinfected with Mycobacterium tuberculosis, and active tuberculosis disease (TB) is the number one cause of death in HIV-infected people. Coinfected individuals are 30 times more likely to progress to active TB, which can in turn increase HIV replication and accelerate the progression to AIDS (35). The mechanisms by which coinfection with M. tuberculosis and HIV accelerates the progression of both diseases are poorly understood.Lung macrophages are the primary target of M. tuberculosis infection, and active disease is characterized by unconstrained replication in these cells. Dendritic cells can also be infected by M. tuberculosis, but M. tuberculosis growth is restricted due to a lack of nutrient access in the DC phagolysosomal structure in which it resides (20). Importantly, M. tuberculosis-infected DCs traffic between the infected lung and draining lymph nodes, bringing bacterial antigens into lymphoid tissues to initiate CD4 T cell responses essential for disease control (39).Others have established that M. tuberculosis binds to and is internalized by DCs via an interaction between the mycobacterial cell wall component mannosylated lipoarabinomannan (ManLAM) and the cell surface receptor DC-SIGN on dendritic cells (15). After ManLAM stimulation, DCs begin to secrete interleukin-10 (IL-10) and show defects in immunostimulatory functions (15). However, a more recent study suggests that ManLAM may not be solely responsible for these outcomes (1).Previously, it has been shown that lipopolysaccharide (LPS) potently stimulates HIV trans-infection of CD4 T cells by DCs (24, 33). Therefore, we reasoned that M. tuberculosis and its products might similarly stimulate DC trans-infection during active M. tuberculosis infections. Further, we hypothesized that DC activation by M. tuberculosis would result in downmodulation of processing and MHC-II presentation of newly bound HIV particles, shifting the balance away from immune control in favor of viral dissemination and pathogenesis.Here, we demonstrate that M. tuberculosis infection of DCs enhances HIV trans-infection mediated through surface-accessible, pocketlike invaginations of the plasma membrane. Increased HIV trans-infection is accompanied by decreased MHC-II processing and presentation of HIV antigens to CD4 T cells. Our results suggest one mechanism whereby M. tuberculosis infection can fuel HIV dissemination in coinfected individuals and at the same time decrease immune control of both HIV and M. tuberculosis infections.     

Genetic Diversity in Exon 2 of the Major Histocompatibility Complex Class II DQB1 Locus in Nigerian Goats

The DQB1 locus is located in the major histocompatibility complex (MHC) class II region and involved in immune response. We identified 20 polymorphic sites in a 228 bp fragment of exon 2, one of the most critical regions of the MHC DQB1 gene, in 60 Nigerian goats. Four sites are located in the peptide binding region, and 10 amino acid substitutions are peculiar to Nigerian goats, compared with published sequences. A significantly higher ratio of nonsynonymous/synonymous substitutions (d _N/d _S) suggests that allelic sequence evolution is driven by balancing selection (P < 0.01). In silico functional analysis using PANTHER predicted that substitution P56R, with a subPSEC score of ?4.00629 (P_deleterious = 0.73229), is harmful to protein function. The phylogenetic tree from consensus sequences placed the two northern breeds closer to each other than either was to the southern goats. This first report of sequence diversity at the DQB1 locus for any African goat breed may be useful in the search for disease-resistant genotypes.     

Target HCV NS3 CD4+ Th1 Epitope to Major Histocompatibility Complex Class II Pathway

A hepatitis C virus (HCV) plasmid vaccine was constructed, based on class II-associated invariant chain peptide (CLIP) substitution which endogenously targets HCV non-structure protein 3 (NS3) CD4⁺ T helper 1(Th1) epitope (1248AA-1261AA) to major histocompatibility complex (MHC) class II antigen. The in vitro expression results demonstrated that the vaccine was expressed efficiently in COS-7 cell line. The expressed protein could co-localize in endo-membrane system with BALB/c mouse MHC class II molecule I-A^d. The recombinant invariant chain molecule could aggregate with BALB/c mouse I-A^d molecule and form the theoretical nonomer structure in the COS-7 cell line. The assembled molecules migrate to the cell surface by exocytosis. This has implications for HCV vaccine development.     

Mixed-Haplotype and Mixed-Isotype Major Histocompatibility Complex Class II Molecules Detected by Murine T-Cell Clones and Their Possible Involvement in Autoimmunity

The recognition of antigen by T lymphocytes (T cells) is restricted by Class I or Class II major histocompatibility complex (MHC) gene products, the phenomenon called “MHC restriction.” MHC restriction is speculated to be one of the major elements for the association of disease susceptibility to MHC haplotypes. Clones of T cells have been shown to be powerful tools for the analysis of such restriction specificity. In this report, I describe unique mixed-isotype Aβ^d/Eα^drestriction molecules detected by T-cell clones in (B6Eα^d× BALB/c)F1 transgenic mice. The restriction specificity of these clones was confirmed by anti-Class II mAb blocking experiments. The ability of spleen cells from Aβ^dand Eα^ddouble transgenic B6 (B6Aβ^dEα^d) mice that express Aβ^d/Eα^dmolecules to present KLH to these clones supported the existence of such unique specificity. I also describe autoreactive as well as KLH-reactive T-cell clones restricted by mixed-haplotype Aβz/Aα^dClass II molecules derived from (NZB × NZW)F1 (B/WF1) mice. The restriction specificity was demonstrated by mAb blocking experiments and by experiments using Class II gene-transfected antigen-presenting cells. It is possible that such unique mixed-isotype and mixed-haplotype Class II molecules are critically involved in autoimmunity. In addition, the detailed methodology for establishing T-cell clones currently employed in my laboratory is described.     

Human Herpesvirus 7 U21 Downregulates Classical and Nonclassical Class I Major Histocompatibility Complex Molecules from the Cell Surface

Herpesviruses have evolved numerous strategies to evade detection by the immune system. Notably, most of the herpesviruses interfere with viral antigen presentation to cytotoxic T lymphocytes (CTLs) by removing class I major histocompatibility complex (MHC) molecules from the infected cell surface. Clearly, since the herpesviruses have evolved an extensive array of mechanisms to remove class I MHC molecules from the cell surface, this strategy serves them well. However, class I MHC molecules often serve as inhibitory ligands for NK cells, so viral downregulation of all class I MHC molecules should leave the infected cell open to NK cell attack. Some viruses solve this problem by selectively downregulating certain class I MHC products, leaving other class I products at the cell surface to serve as inhibitory NK cell ligands. Here, we show that human herpesvirus 7 (HHV-7) U21 binds to and downregulates all of the human class I MHC gene products, as well as the murine class I molecule H-2K^b. HHV-7-infected cells must therefore possess other means of escaping NK cell detection.Human herpesvirus-7 (HHV-7) is a betaherpesvirus that infects over 90% of the population by the age of 3 (for a review, see reference 58). Like all other herpesviruses, HHV-7 establishes a latent or persistent infection, lasting for the lifetime of its host. Primary infection is usually accompanied by febrile illness, but long-term infection with the virus is asymptomatic (3, 53). HHV-7 is T-lymphotrophic, but it has also been found in salivary epithelial cells (30, 62).As viruses that remain latent or persistent throughout the life of their hosts, the herpesviruses must interact continually with the host immune system. In so doing, all herpesviruses have evolved mechanisms to interfere with viral antigen presentation by class I major histocompatibility complex (MHC) molecules as a means to escape detection by cytotoxic T lymphocytes (CTLs). Some herpesvirus gene products interfere with proteolysis of antigens or peptide transport into the endoplasmic reticulum (ER) (1, 20, 56, 61). Others retain or destroy class I molecules (2, 26, 59, 64), enhance the internalization of class I molecules, or divert class I molecules to lysosomes for degradation (11, 23, 25, 44). Judging from the number and molecular diversity of these strategies, the removal of MHC class I-peptide complexes from the cell surface must be evolutionarily advantageous to these viruses as a means of escaping immune detection. We have described one such immunoevasin, U21, from HHV-7. HHV-7 U21 binds to class I MHC molecules in the ER and diverts them to a lysosomal compartment, where they are degraded, effectively removing them from the cell surface (23). The mechanism of U21-mediated diversion of class I molecules to lysosomes is not known, but the relocalization of class I MHC molecules is specific—U21 does not cause the rerouting of either the transferrin receptor or CD4 to lysosomes (22, 23).Since the herpesviruses have evolved such an extensive array of mechanisms to remove class I MHC molecules from the cell surface of infected cells, this strategy must serve them well. However, when natural killer (NK) cells detect an absence of class I MHC molecules on the surface of a cell (i.e., “missing self”), they become activated to kill that cell. NK cells detect the absence of class I MHC molecules through interaction of NK cell receptors with NK cell receptor ligands present on the surface of the target cell (for a review, see references 6 and 7). When an NK cell surveys a potential target, it integrates the number and strength of the activating and inhibitory signals it receives; after weighing the balance, it either remains indifferent to the target or becomes activated to kill it.Class I MHC molecules are ligands for inhibitory NK cell receptors. Thus, when a virus removes class I MHC molecules from the cell surface to escape detection by CTLs, it simultaneously renders the cell vulnerable to NK cell attack. Not surprisingly, viruses have evolved counterstrategies to protect their host cells from NK cell-mediated attack. The class I MHC locus contains three classical class I gene products, HLA-A, -B, and -C, as well as other “nonclassical” products, including HLA-E and HLA-G. As a strategy to avoid both CTL and NK cell attack, some viral immunoevasins selectively downregulate HLA-A and HLA-B locus products, while leaving HLA-C, -E, and other inhibitory class I-like molecules at the plasma membrane (10, 16, 35). It has therefore been speculated that HLA-A and -B may be more effective at antigen presentation to CTLs than HLA-C (15, 40). The nonclassical class I molecule HLA-E, on the other hand, functions primarily to inhibit NK cell activation and does not present foreign antigen to CTLs (33). As such, its expression at the cell surface is even promoted by at least one immunoevasin, UL40 from human cytomegalovirus (HCMV) (54, 57).We do not know how HHV-7 responds to the selective pressures exerted by NK cells. We have shown previously that U21 can associate with and downregulate HLA-A and -B, but we do not yet know the full extent of its promiscuity (23). For this reason, we now examine the ability of U21 to bind to and downregulate the various classical and nonclassical class I MHC gene products. We find that, unlike many other viral immunoevasins, HHV-7 U21 can associate with and downregulate HLA-C, -E, and -G and even murine class I MHC molecules. In an infection, this would shift the balance of inhibitory NK cell ligands on the cell surface to favor NK cell attack, suggesting that HHV-7 might compensate for such an imbalance through other means of NK cell evasion.U21 is 55-kDa type I membrane protein with a short (50-amino-acid [aa]) cytoplasmic tail. We have shown that its transmembrane domain and cytoplasmic tail are not involved in its association with the lumenal domain of the class I molecule (22). In addition to gaining information about U21''s potential influence on CTL and NK cell detection of HHV-7-infected cells, we also hoped that a survey of its ability to associate with various class I MHC gene products might help to illuminate regions of the class I molecule important for association with U21.     

Modes of Calreticulin Recruitment to the Major Histocompatibility Complex Class I Assembly Pathway

Major histocompatibility complex (MHC) class I molecules are ligands for T-cell receptors of CD8⁺ T cells and inhibitory receptors of natural killer cells. Assembly of the heavy chain, light chain, and peptide components of MHC class I molecules occurs in the endoplasmic reticulum (ER). Specific assembly factors and generic ER chaperones, collectively called the MHC class I peptide loading complex (PLC), are required for MHC class I assembly. Calreticulin has an important role within the PLC and induces MHC class I cell surface expression, but the interactions and mechanisms involved are incompletely understood. We show that interactions with the thiol oxidoreductase ERp57 and substrate glycans are important for the recruitment of calreticulin into the PLC and for its functional activities in MHC class I assembly. The glycan and ERp57 binding sites of calreticulin contribute directly or indirectly to complexes between calreticulin and the MHC class I assembly factor tapasin and are important for maintaining steady-state levels of both tapasin and MHC class I heavy chains. A number of destabilizing conditions and mutations induce generic polypeptide binding sites on calreticulin and contribute to calreticulin-mediated suppression of misfolded protein aggregation in vitro. We show that generic polypeptide binding sites per se are insufficient for stable recruitment of calreticulin to PLC substrates in cells. However, such binding sites could contribute to substrate stabilization in a step that follows the glycan and ERp57-dependent recruitment of calreticulin to the PLC.     

Giant Panda Genomic Data Provide Insight into the Birth-and-Death Process of Mammalian Major Histocompatibility Complex Class II Genes

To gain an understanding of the genomic structure and evolutionary history of the giant panda major histocompatibility complex (MHC) genes, we determined a 636,503-bp nucleotide sequence spanning the MHC class II region. Analysis revealed that the MHC class II region from this rare species contained 26 loci (17 predicted to be expressed), of which 10 are classical class II genes (1 DRA, 2 DRB, 2 DQA, 3 DQB, 1 DYB, 1 DPA, and 2 DPB) and 4 are non-classical class II genes (1 DOA, 1 DOB, 1 DMA, and 1 DMB). The presence of DYB, a gene specific to ruminants, prompted a comparison of the giant panda class II sequence with those of humans, cats, dogs, cattle, pigs, and mice. The results indicated that birth and death events within the DQ and DRB-DY regions led to major lineage differences, with absence of these regions in the cat and in humans and mice respectively. The phylogenetic trees constructed using all expressed alpha and beta genes from marsupials and placental mammals showed that: (1) because marsupials carry loci corresponding to DR, DP, DO and DM genes, those subregions most likely developed before the divergence of marsupials and placental mammals, approximately 150 million years ago (MYA); (2) conversely, the DQ and DY regions must have evolved later, but before the radiation of placental mammals (100 MYA). As a result, the typical genomic structure of MHC class II genes for the giant panda is similar to that of the other placental mammals and corresponds to BTNL2∼DR1∼DQ∼DR2∼DY∼DO_box∼DP∼COL11A2. Over the past 100 million years, there has been birth and death of mammalian DR, DQ, DY, and DP genes, an evolutionary process that has brought about the current species-specific genomic structure of the MHC class II region. Furthermore, facing certain similar pathogens, mammals have adopted intra-subregion (DR and DQ) and inter-subregion (between DQ and DP) convergent evolutionary strategies for their alpha and beta genes, respectively.     

Structure and Polymorphism of the Major Histocompatibility Complex Class II Region in the Japanese Crested Ibis,Nipponia nippon

The major histocompatibility complex (MHC) is a highly polymorphic genomic region that plays a central role in the immune system. Despite its functional consistency, the genomic structure of the MHC differs substantially among organisms. In birds, the MHC-B structures of Galliformes, including chickens, have been well characterized, but information about other avian MHCs remains sparse. The Japanese Crested Ibis (Nipponia nippon, Pelecaniformes) is an internationally conserved, critically threatened species. The current Japanese population of N. nippon originates from only five founders; thus, understanding the genetic diversity among these founders is critical for effective population management. Because of its high polymorphism and importance for disease resistance and other functions, the MHC has been an important focus in the conservation of endangered species. Here, we report the structure and polymorphism of the Japanese Crested Ibis MHC class II region. Screening of genomic libraries allowed the construction of three contigs representing different haplotypes of MHC class II regions. Characterization of genomic clones revealed that the MHC class II genomic structure of N. nippon was largely different from that of chicken. A pair of MHC-IIA and -IIB genes was arranged head-to-head between the COL11A2 and BRD2 genes. Gene order in N. nippon was more similar to that in humans than to that in chicken. The three haplotypes contained one to three copies of MHC-IIA/IIB gene pairs. Genotyping of the MHC class II region detected only three haplotypes among the five founders, suggesting that the genetic diversity of the current Japanese Crested Ibis population is extremely low. The structure of the MHC class II region presented here provides valuable insight for future studies on the evolution of the avian MHC and for conservation of the Japanese Crested Ibis.     

Redox-regulated Export of the Major Histocompatibility Complex Class I-Peptide Complexes from the Endoplasmic Reticulum

In contrast to the fairly well-characterized mechanism of assembly of MHC class I-peptide complexes, the disassembly mechanism by which peptide-loaded MHC class I molecules are released from the peptide-loading complex and exit the endoplasmic reticulum (ER) is poorly understood. Optimal peptide binding by MHC class I molecules is assumed to be sufficient for triggering exit of peptide-filled MHC class I molecules from the ER. We now show that protein disulfide isomerase (PDI) controls MHC class I disassembly by regulating dissociation of the tapasin-ERp57 disulfide conjugate. PDI acts as a peptide-dependent molecular switch; in the peptide-bound state, it binds to tapasin and ERp57 and induces dissociation of the tapasin-ERp57 conjugate. In the peptide-free state, PDI is incompetent to bind to tapasin or ERp57 and fails to dissociate the tapasin-ERp57 conjugates, resulting in ER retention of MHC class I molecules. Thus, our results indicate that even after optimal peptide loading, MHC class I disassembly does not occur by default but, rather, is a regulated process involving PDI-mediated interactions within the peptide-loading complex.