Exciting applications of single chain trimers of MHC-I molecules.
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HLA-A2 Antigen↗
Biomedical subjects
Lonnie Lybarger
Publications and source records attributed to Lonnie Lybarger.
Licensing of natural killer cells by host major histocompatibility complex class I molecules.
Self versus non-self discrimination is a central theme in biology from plants to vertebrates, and is particularly relevant for lymphocytes that express receptors capable of recognizing self-tissues and foreign invaders. Comprising the third largest lymphocyte population, natural killer (NK) cells recognize and kill cellular targets and produce pro-inflammatory cytokines. These potentially self-destructive effector functions can be controlled by inhibitory receptors for the polymorphic major histocompatibility complex (MHC) class I molecules that are ubiquitously expressed on target cells. However, inhibitory receptors are not uniformly expressed on NK cells, and are germline-encoded by a set of polymorphic genes that segregate independently from MHC genes. Therefore, how NK-cell self-tolerance arises in vivo is poorly understood. Here we demonstrate that NK cells acquire functional competence through 'licensing' by self-MHC molecules. Licensing involves a positive role for MHC-specific inhibitory receptors and requires the cytoplasmic inhibitory motif originally identified in effector responses. This process results in two types of self-tolerant NK cells--licensed or unlicensed--and may provide new insights for exploiting NK cells in immunotherapy. This self-tolerance mechanism may be more broadly applicable within the vertebrate immune system because related germline-encoded inhibitory receptors are widely expressed on other immune cells.
Animals↗
Evidence for MR1 antigen presentation to mucosal-associated invariant T cells.
The novel class Ib molecule MR1 is highly conserved in mammals, particularly in its alpha1/alpha2 domains. Recent studies demonstrated that MR1 expression is required for development and expansion of a small population of T cells expressing an invariant T cell receptor (TCR) alpha chain called mucosal-associated invariant T (MAIT) cells. Despite these intriguing properties it has been difficult to determine whether MR1 expression and MAIT cell recognition is ligand-dependent. To address these outstanding questions, monoclonal antibodies were produced in MR1 knock-out mice immunized with recombinant MR1 protein, and a series of MR1 mutations were generated at sites previously shown to disrupt the ability of class Ia molecules to bind peptide or TCR. Here we show that 1) MR1 molecules are detected by monoclonal antibodies in either an open or folded conformation that correlates precisely with peptide-induced conformational changes in class Ia molecules, 2) only the folded MR1 conformer activated 2/2 MAIT hybridoma cells tested, 3) the pattern of MAIT cell activation by the MR1 mutants implies the MR1/TCR orientation is strikingly similar to published major histocompatibility complex/alphabetaTCR engagements, 4) all the MR1 mutations tested and found to severely reduce surface expression of folded molecules were located in the putative ligand binding groove, and 5) certain groove mutants of MR1 that are highly expressed on the cell surface disrupt MAIT cell activation. These combined data strongly support the conclusion that MR1 has an antigen presentation function.
Animals↗
Viral immune evasion molecules attack the ER peptide-loading complex and exploit ER-associated degradation pathways.
The CD8+ cytotoxic-T-cell response is a potent mechanism that controls intracellular pathogens, including many viruses. To facilitate transmission, viruses often counter this response by inhibiting the cell surface display of virus-derived peptides on MHC class I molecules. More specifically, recent studies have demonstrated that viruses have evolved remarkable mechanisms to inhibit MHC class I expression by interfering with the function of the MHC class I assembly machinery (the peptide-loading complex) in the endoplasmic reticulum and/or by exploiting endoplasmic-reticulum-associated degradation pathways. These viral molecules are proving invaluable as research tools to illuminate the novel features of physiological pathways that are central to normal cell biology. Furthermore, the detailed characterization of such pathways has yielded significant new insights into host-pathogen interplay.
Animals↗
Recognition of open conformers of classical MHC by chaperones and monoclonal antibodies.
There is considerable evidence that the conformation and stability of class I and class II major histocompatibility complex (MHC) proteins is dependent upon high-affinity peptide ligation, but structural data for an empty MHC protein unfortunately is lacking. However, several monoclonal antibodies (mAbs) that specifically detect open MHC conformers have been characterized, and they provide insights into the changes associated with peptide loading and unloading. Here, the structural changes make the argument that certain of these open conformer-specific mAbs recognize analogous MHC segments as the molecular chaperones tapasin and DM. MHC residues located in regions flanking the peptide-terminal anchoring pockets have been implicated in both chaperone and monoclonal antibody binding. Indeed, we propose these regions serve as peptide-binding hinges that are uniquely accessible in open MHC.
Animals↗
Requirements for the selective degradation of endoplasmic reticulum-resident major histocompatibility complex class I proteins by the viral immune evasion molecule mK3.
Recent studies suggest that certain viral proteins co-opt endoplasmic reticulum (ER) degradation pathways to prevent the surface display of major histocompatibility complex class I molecules to the immune system. A novel example of such a molecule is the mK3 protein of gammaherpesvirus 68. mK3 belongs to an extensive family of structurally similar viral and cellular proteins that function as ubiquitin ligases using a conserved RING-CH domain. In the specific case of mK3, it selectively targets the rapid degradation of nascent class I heavy chains in the ER while they are associated with the class I peptide-loading complex (PLC). We present here evidence that the PLC imposes a relative proximity and/or orientation on the RING-CH domain of mK3 that is required for it to specifically target class I molecules for degradation. Furthermore, we demonstrate that full assembly of class I molecules with peptide is not a prerequisite for mK3-mediated degradation. Surprisingly, although the cytosolic tail of class I is required for rapid mK3-mediated degradation, we observed that a class I mutant lacking lysine residues in its cytosolic tail was ubiquitinated and degraded in the presence of mK3 in a manner indistinguishable from wild-type class I molecules. These findings are consistent with a "partial dislocation" model for turnover of ER proteins and define some common features of ER degradation pathways initiated by structurally distinct herpesvirus proteins.
Animals↗
Applications of major histocompatibility complex class I molecules expressed as single chains.
Generation of CD8 T-cell responses to pathogens and tumors requires optimal expression of class I major histocompatibility complex/peptide complexes, which, in turn, is dependent on host cellular processing events and subject to interference by pathogens. To create a stable structure that is more immunogenic and resistant to immune evasion pathways, we have engineered class I molecules as single-chain trimers (SCTs), with flexible linkers connecting peptide, beta2m, and heavy chain. Herein we extend our earlier studies with SCTs to the K(b) ligand derived from vesicular stomatitis virus (VSV) to characterize further SCTs as probes of immune function as well as their potential in immunotherapy. The VSVp-beta2m-K(b) SCTs were remarkably stable at the cell surface, and immunization with DNA encoding SCTs elicited complex-specific antibody. In addition, SCTs were detected by cytotoxic T-lymphocytes specific for the native molecule, and the covalently bound peptide was highly resistant to displacement by exogenous peptide. SCTs can also prime CD8 T-cells in vivo that recognize the native molecule. Furthermore, SCTs were resistant to downregulation by the immune evasion protein mK3 of gamma herpesvirus 68. Moreover, owing to their preassembled nature, SCTs should be resistant to other immune evasion proteins that restrict peptide supply. Thus, SCTs possess therapeutic potential both for prophylactic treatment and for the treatment of ongoing infection.
Animals↗
Recognition of HLA-A2-restricted mammaglobin-A-derived epitopes by CD8+ cytotoxic T lymphocytes from breast cancer patients.
A breast cancer-associated antigen, mammaglobin-A, is specifically expressed in 80% of primary breast tumors. The definition of immune responses against this highly expressed breast cancer-specific antigen should be of great value in the development of new therapeutic strategies for breast cancer. Thus, the purpose of this study was to identify HLA-A2-restricted mammaglobin-A-derived epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL). We identified seven mammaglobin-A-derived candidate epitopes that bind the HLA-A2 molecule (Mam-A2.1-7) by means of a HLA class I-peptide binding computer algorithm from the Bioinformatics & Molecular Analysis Section of the National Institutes of Health. Subsequently, we determined that CD8+ CTLs from breast cancer patients reacted to the Mam-A2.1 (83-92, LIYDSSLCDL), Mam-A2.2 (2-10, KLLMVLMLA), Mam-A2.3 (4-12, LMVLMLAAL), Mam-A2.4 (66-74, FLNQTDETL), and Mam-A2.7 (32-40, TINPQVSKT) epitopes using an IFN-gamma ELISPOT assay. Interestingly, healthy individuals also showed high reactivity to the Mam-A2.2 epitope. Two CD8+ CTL lines generated in vitro against TAP-deficient T2 cells loaded with the candidate epitopes showed significant cytotoxic activity against the Mam-A2.1-4 epitopes. These CD8+CTL lines recognized a HLA-A2+breast cancer cell line expressing the Mam-A2.1 epitope. In addition, DNA vaccination of HLA-A2+/human CD8+ double-transgenic mice with a DNA construct encoding the Mam-A2.1 epitope and the HLA-A2 molecule induced a significant expansion of epitope-specific CD8+ CTLs that recognize the same HLA- A2+/Mam-A2.1+ breast cancer cell line. In conclusion, these results demonstrate the immunotherapeutic potential of mammaglobin-A for the treatment and prevention of breast cancer.
Adenocarcinoma↗
Model for the interaction of gammaherpesvirus 68 RING-CH finger protein mK3 with major histocompatibility complex class I and the peptide-loading complex.
The mK3 protein of gammaherpesvirus 68 and the kK5 protein of Kaposi's sarcoma-associated herpesvirus are members of a family of structurally related viral immune evasion molecules that all possess a RING-CH domain with ubiquitin ligase activity. These proteins modulate the expression of major histocompatibility complex class I molecules (mK3 and kK5) as well as other molecules like ICAM-1 and B7.2 (kK5). Previously, mK3 was shown to ubiquitinate nascent class I molecules, resulting in their rapid degradation, and this process was found to be dependent on TAP and tapasin, endoplasmic reticulum molecules involved in class I assembly. Here, we demonstrate that in murine cells, kK5 does not affect class I expression but does downregulate human B7.2 molecules in a TAP/tapasin-independent manner. These differences in substrate specificity and TAP/tapasin dependence between mK3 and kK5 permitted us, using chimeric molecules, to map the sites of mK3 interaction with TAP/tapasin and to determine the requirements for substrate recognition by mK3. Our findings indicate that mK3 interacts with TAP1 and -2 via their C-terminal domains and with class I molecules via their N-terminal domains. Furthermore, by orienting the RING-CH domain of mK3 appropriately with respect to class I, mK3 binding to TAP/tapasin, rather than the presence of unique sequences in class I, appears to be the primary determinant of substrate specificity.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Biochemical features of the MHC-related protein 1 consistent with an immunological function.
MHC-related protein (MR)1 is an MHC class I-related molecule encoded on chromosome 1 that is highly conserved among mammals and is more closely related to classical class I molecules than are other nonclassical class I family members. In this report, we show for the first time that both mouse and human MR1 molecules can associate with the peptide-loading complex and can be detected at low levels at the surface of transfected cells. We also report the production of recombinant human MR1 molecules in insect cells using highly supplemented media and provide evidence that the MR1 H chain can assume a folded conformation and is stoichiometrically associated with beta(2)-microglobulin, similar to class I molecules. Cumulatively, these findings demonstrate that surface expression of MR1 is possible but may be limited by a specific ligand or associated molecule.
Amino Acid Sequence↗
Enhanced immune presentation of a single-chain major histocompatibility complex class I molecule engineered to optimize linkage of a C-terminally extended peptide.
Major histocompatibility complex class I molecules can be expressed as single polypeptides wherein the antigenic peptide, beta2-microglobulin, and heavy chain are attached by flexible linkers. These molecules, single-chain trimers (SCTs), are remarkably stable at the cell surface compared with native (noncovalently attached) class I molecules. In this study, we used a structure-based approach to engineer an F pocket variant SCT of the murine class I molecule Kb that presents the SIINFEKL epitope of ovalbumin. Mutation of heavy chain residue Tyr84 (Y84A) in the SCT resulted in enhanced serological and cytolytic CD8 T cell recognition of the covalently linked peptide due to better accommodation of the linker extending from the C terminus of the peptide. These SCTs exhibit significant cell-surface stability, which we hypothesize is rendered by their ability to continuously and efficiently rebind the covalently attached peptide. In addition, we demonstrate that SCT technology can be applied to tetramer construction using recombinant SCTs expressed in Escherichia coli. SCT-based tetramers could have applications for the enumeration of T and natural killer cells that recognize peptide.class I complexes prone to dissociation.
Amino Acid Sequence↗
Virus subversion of the MHC class I peptide-loading complex.
Many viral proteins modulate class I expression, yet, in general, their mechanisms of specific class I recognition are poorly understood. The mK3 protein of gamma(2)-Herpesvirus 68 targets the degradation of nascent class I molecules via the ubiquitin/proteasome pathway. Here, we identify cellular components of the MHC class I assembly machinery, TAP and tapasin, that are required for mK3 function. mK3 failed to regulate class I in TAP- or tapasin-deficient cells, and mK3 interacted with TAP/tapasin, even in the absence of class I. Expression of mK3 resulted in the ubiquitination of TAP/tapasin-associated class I, and mutants of class I incapable of TAP/tapasin interaction were unaffected by mK3. Thus, mK3 subverts TAP/tapasin to specifically target class I molecules for destruction.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Cutting edge: single-chain trimers of MHC class I molecules form stable structures that potently stimulate antigen-specific T cells and B cells.
We report in this work the expression and characterization of class I molecules expressed as single-chain trimers consisting of an antigenic peptide-spacer-beta(2)-microglobulin-spacer H chain. Our results indicate that these single-chain constructs assemble efficiently, maintain their covalent structure, and are unusually stable at the cell surface. Consequently, these constructs are at least 1000-fold less accessible to exogenous peptide than class I molecules loaded with endogenous peptides, and they are potent simulators of peptide-specific CTL and Abs. Our combined findings suggest that single-chain trimers may have applications as DNA vaccines against virus infection or tumors.
Animals↗
Physical association of the K3 protein of gamma-2 herpesvirus 68 with major histocompatibility complex class I molecules with impaired peptide and beta(2)-microglobulin assembly.
To persist in the presence of an active immune system, viruses encode proteins that decrease expression of major histocompatibility complex class I molecules by using a variety of mechanisms. For example, murine gamma-2 herpesvirus 68 expresses the K3 protein, which causes the rapid turnover of nascent class I molecules. In this report we show that certain mouse class I alleles are more susceptible than others to K3-mediated down regulation. Prior to their rapid degradation, class I molecules in K3-expressing cells exhibit impaired assembly with beta(2)-microglobulin. Furthermore, K3 is detected predominantly in association with class I molecules lacking assembly with high-affinity peptides, including class I molecules associated with the peptide loading complex TAP/tapasin/calreticulin. The detection of K3 with class I assembly intermediates raises the possibility that molecular chaperones involved in class I assembly are involved in K3-mediated class I regulation.
Animals↗