MHC class II deletion mutant expresses normal levels of transgene encoded class II molecules that have abnormal conformation and impaired antigen presentation ability.

Successive transfers of HLA-DR alpha and beta genes restored expression of HLA-DR antigens to human B-lymphoblastoid cell line, LCL .174, from which all known expressible class II genes are deleted. While transferent cells displayed large amounts of DR on their surfaces, transgene-encoded DR3 molecules lacked a conformation-dependent epitope. DR1-restricted CTL lysis of DR1-expressing transferents pulsed with native influenza virus proteins was greatly reduced; the same cells were efficiently lysed in the presence of CTL-recognized influenza peptides. The properties of DR-expressing transferents of .174 suggest they are defective in producing peptides from exogenous proteins or in forming DR/peptide complexes. Comparison with other DR-expressing deletion mutants indicates that at least one gene in an approximately 230 kb DNA segment between the DQ1 and Ring 7 loci is needed for normal DR-mediated processing and presentation. Production of DR3 molecules having the conformation-dependent 16.23 epitope and efficient DR1-restricted presentation of influenza viral epitopes occurred in a B cell line that has a mutation specifically eliminating expression of the TAP1 transporter gene, which is in the approximately 230 kb interval and is needed for production of HLA class I/peptide complexes.     

Peptide presentation by MHC class I molecules

The presentation of peptides by class I histocompatibility molecules plays a central role in the cellular immune response to virally infected or transformed cells. The main steps in this process include the degradation of both self and 'foreign' proteins to short peptides in the cytosol, translocation of peptides into the lumen of the endoplasmic reticulum, binding of a subset of peptides to assembling class I molecules and expression of class-I-peptide complexes at the cell surface for examination by cytotoxic T cells. A molecular understanding of most of these steps is emerging, revealing a remarkable coordination between the processes of peptide translocation, delivery and binding to class I molecules.     

Rethinking peptide supply to MHC class I molecules

The notion that peptides bound to MHC class I molecules are derived mainly from newly synthesized proteins that are defective, and are therefore targeted for immediate degradation, has gained wide acceptance. This model, still entirely hypothetical, has strong intuitive appeal and is consistent with some experimental results, but it is strained by other findings, as well as by established and emerging concepts in protein quality control. While not discounting defectiveness as a driving force for the processing of some proteins, we propose that MHC-class-I-restricted epitopes are derived mainly from nascent proteins that are accessed by the degradation machinery prior to any assessment of fitness, and we outline one way in which this could be accomplished.     

Assembly of MHC class I molecules analyzed in vitro

Recent evidence suggests that peptide ligands take part in the assembly of class I molecules in living cells. We now describe a simple system for studying class I assembly in vitro. Detergent extracts of the mutant cells RMA-S and .174, in which class I assembly does not occur spontaneously, will support assembly in vitro when specific peptides are added. Peptides stabilize a conformational change in the class I heavy chain and association with beta 2-microglobulin, at concentrations approximately 100-fold lower than required in "peptide feeding" experiments with whole cells. We show that peptides bind class I molecules during assembly and demonstrate that the conformational change induced in the heavy chain is influenced by the concentrations of both peptide and beta 2-microglobulin.     

Presentation of antigenic peptides by MHC class I molecules

The basis for the immune response against intracellular pathogens is the recognition by cytotoxic T lymphocytes of antigenic peptides derived from cytosolic proteins, which are presented on the cell surface by major histocompatibility complex (MHC) class I molecules. The understanding of MHC class I-restricted peptide presentation has recently improved dramatically with the elucidation of the structural basis for the specificity of peptide binding to MHC class I molecules and the identification of proteins encoded in the class II region of the MHC that are putatively involved in the production of peptides and their transport into the endoplasmic reticulum, where they assemble with class I molecules.     

Signal transduction through class I MHC by a monoclonal antibody that detects multiple murine and human class I molecules.

We have generated a hamster anti-mouse class I reactive mAb that is capable of activating T cells in the presence of the cofactor PMA, as assayed by both IFN-gamma production and cellular proliferation. This mAb detects an epitope present on the majority of murine class I molecules, with the known exceptions of H-2Kk and H-2Kq, and is therefore not beta 2-microglobulin-specific. It also recognizes multiple human class I molecules. The epitope recognized by this antibody maps to the class I alpha 1 domain. The activation properties of this mAb are not mediated exclusively through the glycosylphosphatidylinositol-linked Qa-2 molecule, as the antibody activates spleen cells from Qa-2 negative strains. Although class I molecules are not usually considered as activation Ag, these data demonstrate their potential for involvement in signal transduction.     

Rhesus macaque MHC class I molecules show differential subcellular localizations

The MHC class I gene family of rhesus macaques is characterised by considerable gene duplications. While a HLA-C-orthologous gene is absent, the Mamu-A and in particular the Mamu-B genes have expanded, giving rise to plastic haplotypes with differential gene content. Although some of the rhesus macaque MHC class I genes are known to be associated with susceptibility/resistance to infectious diseases, the functional significance of duplicated Mamu-A and Mamu-B genes and the expression pattern of their encoded proteins are largely unknown. Here, we present data of the subcellular localization of AcGFP-tagged Mamu-A and Mamu-B molecules. We found strong cell surface and low intracellular expression for Mamu-A1, Mamu-A2 and Mamu-A3-encoded molecules as well as for Mamu-B*01704, Mamu-B*02101, Mamu-B*04801, Mamu-B*06002 and Mamu-B*13401. In contrast, weak cell surface and strong intracellular expression was seen for Mamu-A4*1403, Mamu-B*01202, Mamu-B*02804, Mamu-B*03002, Mamu-B*05704, Mamu-I*010201 and Mamu-I*0121. The different expression patterns were assigned to the antigen-binding α1 and α2 domains, suggesting failure of peptide binding is responsible for retaining ‘intracellular’ Mamu class I molecules in the endoplasmic reticulum. These findings indicate a diverse functional role of the duplicated rhesus macaque MHC class I genes.     

Marsupials and monotremes possess a novel family of MHC class I genes that is lost from the eutherian lineage

Background

Major histocompatibility complex (MHC) class I genes are found in the genomes of all jawed vertebrates. The evolution of this gene family is closely tied to the evolution of the vertebrate genome. Family members are frequently found in four paralogous regions, which were formed in two rounds of genome duplication in the early vertebrates, but in some species class Is have been subject to additional duplication or translocation, creating additional clusters. The gene family is traditionally grouped into two subtypes: classical MHC class I genes that are usually MHC-linked, highly polymorphic, expressed in a broad range of tissues and present endogenously-derived peptides to cytotoxic T-cells; and non-classical MHC class I genes generally have lower polymorphism, may have tissue-specific expression and have evolved to perform immune-related or non-immune functions. As immune genes can evolve rapidly and are subject to different selection pressure, we hypothesised that there may be divergent, as yet unannotated or uncharacterised class I genes.

Results

Application of a novel method of sensitive genome searching of available vertebrate genome sequences revealed a new, extensive sub-family of divergent MHC class I genes, denoted as UT, which has not previously been characterized. These class I genes are found in both American and Australian marsupials, and in monotremes, at an evolutionary chromosomal breakpoint, but are not present in non-mammalian genomes and have been lost from the eutherian lineage. We show that UT family members are expressed in the thymus of the gray short-tailed opossum and in other immune tissues of several Australian marsupials. Structural homology modelling shows that the proteins encoded by this family are predicted to have an open, though short, antigen-binding groove.

Conclusions

We have identified a novel sub-family of putatively non-classical MHC class I genes that are specific to marsupials and monotremes. This family was present in the ancestral mammal and is found in extant marsupials and monotremes, but has been lost from the eutherian lineage. The function of this family is as yet unknown, however, their predicted structure may be consistent with presentation of antigens to T-cells.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1745-4) contains supplementary material, which is available to authorized users.     

Antigen loading of MHC class I molecules in the endocytic tract

Major histocompatibility complex (MHC) class I molecules bind antigenic peptides that are translocated from the cytosol into the endoplasmic reticulum by the transporter associated with antigen processing. MHC class I loading independent of this transporter also exists and involves peptides derived from exogenously acquired antigens. Thus far, a detailed characterization of the intracellular compartments involved in this pathway is lacking. In the present study, we have used the model system in which peptides derived from measles virus protein F are presented to cytotoxic T cells by B-lymphoblastoid cells that lack the peptide transporter. Inhibition of T cell activation by the lysosomotropic drug ammoniumchloride indicated that endocytic compartments were involved in the class I presentation of this antigen. Using immunoelectron microscopy, we demonstrate that class I molecules and virus protein F co-localized in multivesicular endosomes and lysosomes. Surprisingly, these compartments expressed high levels of class II molecules, and further characterization identified them as MHC class II compartments. In addition, we show that class I molecules co-localized with class II molecules on purified exosomes, the internal vesicles of multivesicular endosomes that are secreted upon fusion of these endosomes with the plasma membrane. Finally, dendritic cells, crucial for the induction of primary immune responses, also displayed class I in endosomes and on exosomes.     

Peripheral pool of CD8+ T-cells contains lymphocytes with antigen-specific receptors that recognize syngeneic MHC class II molecules

The capacity of T-lymphocytes to recognize "nonself" and tolerating "self" is formed as a result of positive and negative selection in the thymus. While obtaining and testing specificity of T-hybridomas, we demonstrated that the major part of peripheral pool of CD8+ T-lymphocytes carried receptors specific to "self" MHC class II molecules. Such an unexpected specificity of receptors has been found in some T-cell hybridomas produced by fusion of activated peripheral CD8+ T-lymphocytes with a tumor partner transfected by the coreceptor CD4 gene. The reactivity to "self" is not an experimental artifact due to an increased avidity of interaction of the hybridoma cells with antigen-presenting cells. Also, it is not an expression of reactivity of T-cells to superantigens, products of endogenous viruses of mouse breast cancer. The formation of a pool of such T-cells involves both cells with double receptor specificity and cells coexpressing two alpha-chains of T-cell receptor. Their appearance in the periphery can be due to the capacity of thymocytes differentiating in the direction of CD4+ cells to avoid negative selection via change of expression of coreceptor CD4 to CD8.     

TAP association influences the conformation of nascent MHC class I molecules

The influence of TAP-MHC class I interactions on peptide binding to the class I heavy chain is assessed during TAP-dependent assembly using Kb-specific Abs that recognize conformational changes induced by assembly with beta2-microglobulin (beta2m) and by peptide binding. A significant portion (45%) of Kb molecules in TAP+, RMA-derived microsomes are associated with the TAP complex as measured by coimmunoisolation of Kb using anti-TAP1 Abs, while only 20% of the Kb heavy chain molecules are isolated as Kbbeta2m complexes with the alpha-Kb-specific Abs, Y-3 or K-10-56. The amount of Kb isolated with Y-3 and K-10-56 increases in proportion to transport and binding of peptide to the Kb molecules within the RMA microsomes. In contrast, less than 5% of the Kb within TAP2-RMA-S microsomes associated with the remaining TAP1 subunit. However, greater than 60% of Kb heavy chain is isolated as K-10-56- and Y-3-reactive Kbbeta2m complexes. We propose that a TAP-MHC class I interaction serves to stabilize the MHC class I:beta2m complex in an immature conformation (Y-3 and K-10-56 nonreactive) prior to high affinity peptide binding, preventing the export of class I molecules complexed with low affinity peptide ligands from the ER.     

ER quality control in the biogenesis of MHC class I molecules

Class I molecules of the major histocompatibility complex play a vital role in cellular immunity, reporting on the presence of viral or tumor-associated antigens by binding peptide fragments of these proteins and presenting them to cytotoxic T cells at the cell surface. The folding and assembly of class I molecules is assisted by molecular chaperones and folding catalysts that comprise the general ER quality control system which also monitors the integrity of the process, disposing of misfolded class I molecules through ER associated degradation (ERAD). Interwoven with general ER quality control are class I-specific components such as the peptide transporter TAP and the tapasin-ERp57 chaperone complex that supply peptides and monitor their loading onto class I molecules. This ensures that at the cell surface class I molecules will possess mainly optimal peptides with a long half-life. In this review we discuss these processes as well as a number of strategies that viruses have evolved to subvert normal class I assembly within the ER and thereby evade immune recognition by cytotoxic T cells.     

CLIP-region mediated interaction of Invariant chain with MHC class I molecules

An association between the MHC class II chaperone molecule Invariant chain (Ii) and MHC class I molecules is known to occur, but the basis of the interaction is undetermined. Evidence is presented here that the CLIP region of Ii is involved in this interaction. A peptide encoding residues 91-99 of CLIP (MRMATPLLM) stabilised multiple MHC class I alleles, with the methionine residue at position 99 having a crucial role in binding to H2-K(b), whereas methionine at position 91 also appeared important in binding to RT1-A(a). Ii can also be detected in the class I MHC peptide loading complex. These data provide an explanation for the association of Ii and MHC class I molecules.     

ER stress impairs MHC Class I surface expression and increases susceptibility of thyroid cells to NK-mediated cytotoxicity

We recently reported that, in thyroid cells, ER stress triggered by thapsigargin or tunicamycin, two well known ER stressing agents, induced dedifferentiation and loss of the epithelial phenotype in rat thyroid cells. In this study, we sought to evaluate if, in thyroid cells, ER stress could affect MHC class I expression and the possible implications of this effect in the alteration of function of natural killer cells, suggesting a role in thyroid pathology. In both, a human line of fetal thyroid cells (TAD-2 cells) and primary cultures of human thyroid cells, thapsigargin and tunicamicin triggered ER stress evaluated by BiP mRNA levels and XBP-1 splicing. In both cell types, TAD-2 cell line and primary cultures, major histocompatibility complex class I (MHC-I) plasmamembrane expression was significantly reduced by ER stress. This effect was accompanied by signs of natural killer activation. Thus, natural killer cells dramatically increased IFN-γ production and markedly increased their cytotoxicity against thyroid cells. Together, these data indicate that ER stress induces a decrease of MHC class I surface expression in thyroid cells, resulting in reduced natural killer-cell self-tolerance.     

Chaperones and folding of MHC class I molecules in the endoplasmic reticulum

In this review we discuss the influence of chaperones on the general phenomena of folding as well as on the specific folding of an individual protein, MHC class I. MHC class I maturation is a highly sophisticated process in which the folding machinery of the endoplasmic reticulum (ER) is heavily involved. Understanding the MHC class I maturation per se is important since peptides loaded onto MHC class I molecules are the base for antigen presentation generating immune responses against virus, intracellular bacteria as well as tumours. This review discusses the early stages of MHC class I maturation regarding BiP and calnexin association, and differences in MHC class I heavy chain (HC) interaction with calnexin and calreticulin are highlighted. Late stage MHC class I maturation with focus on the dedicated chaperone tapasin is also discussed.     

Activation of human T4 cells by cross-linking class I MHC molecules

These studies examined whether cross-linking class I MHC molecules results in functional or biochemical responses in human T4 cells. The initial studies demonstrated that cross-linking class I MHC molecules either by culturing highly purified T4 cells with immobilized mAb to class I MHC Ag or reacting the T4 cells with mAb to class I MHC Ag and then cross-linking the mAb with goat antimouse Ig (GaMIg) enhanced T4 cell proliferation induced by an immobilized mAb to CD3, OKT3. More-over, immobilized but not soluble mAb to class I MHC Ag enhanced T4 cell proliferation induced by the combination of two mAb to CD2, OKT11, and D66.2. Finally, T4 cells reacted with mAb to CD3 and class I MHC Ag proliferated in the presence of IL-2 when cross-linked with GaMIg more vigorously than T4 cells reacted with either mAb alone. Cross-linking class I MHC molecules was also found to stimulate T4 cells directly. T4 cells reacted with mAb to class I MHC Ag or beta 2 microglobulin and cross-linked with GaMIg proliferated vigorously in the presence of IL-2 or PMA. In addition, it was demonstrated that cross-linking class I MHC molecules by culturing T4 cells with immobilized mAb to class I MHC Ag induced T4 cell proliferation in the presence of IL-2. T4 cell proliferation in the presence of IL-2 and PMA could also be induced by reacting the cells with specific mAb to polymorphic determinants on class I MHC molecules and cross-linking with GaMIg. Cross-linking mAb to CD4 or CD11a did not have a similar functional effect on T4 cells. Finally it was demonstrated that adding GaMIg to T4 cells reacted with mAb to class I MHC Ag but not CD11a resulted in an increase in intracellular calcium concentration. The data demonstrate that cross-linking class I MHC molecules results in the generation of at least one activation signal, a rise in intracellular calcium concentration, and, thereby, stimulates human T4 cells.     

Specificity of amyloid precursor-like protein 2 interactions with MHC class I molecules

The ubiquitously expressed amyloid precursor-like protein 2 (APLP2) has been previously found to regulate cell surface expression of the major histocompatibility complex (MHC) class I molecule K(d) and bind strongly to K(d). In the study reported here, we demonstrated that APLP2 binds, in varied degrees, to several other mouse MHC class I allotypes and that the ability of APLP2 to affect cell surface expression of an MHC class I molecule is not limited to K(d). L(d), like K(d), was found associated with APLP2 in the Golgi, but K(d) was also associated with APLP2 within intracellular vesicular structures. We also investigated the effect of beta(2)m on APLP2/MHC interaction and found that human beta(2)m transfection increased the association of APLP2 with mouse MHC class I molecules, likely by affecting H2 class I heavy chain conformation. APLP2 was demonstrated to bind specifically to the conformation of L(d) having folded outer domains, consistent with our previous results with K(d) and indicating APLP2 interacts with the alpha1alpha2 region on each of these H2 class I molecules. Furthermore, we observed that binding to APLP2 involved the MHC alpha3/transmembrane/cytoplasmic region, suggesting that conserved as well as polymorphic regions of the H2 class I molecule may participate in interaction with APLP2. In summary, we demonstrated that APLP2's binding, co-localization pattern, and functional impact vary among H2 class I molecules and that APLP2/MHC association is influenced by multiple domains of the MHC class I heavy chain and by beta(2)m's effects on the conformation of the heavy chain.     

Mice lacking MHC class II molecules

We have produced mice that lack major histocompatibility complex class II antigens, permitting us to evaluate the role of these molecules in diverse aspects of T and B cell differentiation. The mutant mice show near-complete elimination of CD4+ T lymphocytes from the spleen and lymph nodes; the few remaining CD4-positive cells are preferentially localized to B cell follicles. Surprisingly, substantial numbers of CD4 single-positive cells reside in the thymus; however, these are not mature thymocytes as we currently recognize them. B lymphocytes occur in normal numbers and are capable of terminal differentiation to plasma cells. Nevertheless, several aberrations in the B cell compartment are demonstrable: a lack of germinal centers, fewer IgM+IgD+ cells in certain individuals, reduced production of serum IgG1, and complete inability to respond to T-dependent antigens. In short, the class II-negative mice have confirmed some old ideas about lymphocyte differentiation, but have provided some surprises.     

Novel MHC class I-related molecule MR1 affects MHC class I expression in 293T cells

Association with β₂-microglobulin and binding a ligand are necessary conditions for cell surface expression of the antigen presenting molecules. MHC class I-related protein, MR1, is suggested to have an antigen presentation function, nevertheless the physiological ligand(s) is (are) still to be determined. In the present study, by characterising the subcellular deportment of human MR1 transfectants, we have shown its differential mobilisation. Our results demonstrated a preferential association of MR1 with β₂-microglobulin in MHC class I-deficient B cell lines. Furthermore, we have evidenced diminished expression of classical MHC class I molecules in human MR1-transfected 293T cells, showing a possible interaction between MR1 and classical MHC class I molecules.