Viral and bacterial minigene products are presented by MHC class I molecules with similar efficiencies

MHC class I molecules present short peptides, usually 8-10 amino acids in length, to CD8⁺ T cells. These peptides are typically generated from full-length endogenously synthesized proteins degraded by the antigen processing machinery of the target cell. However, exogenous proteins, whether originating from intracellular bacteria or parasites or via phagocytosis during cross-presentation, can also be processed for presentation by MHC class I molecules. It is currently not known whether endogenously synthesized proteins and proteins acquired from exogenous sources follow the same presentation pathway. One clue that the processing pathways followed by endogenous and exogenous proteins may not be identical is the vastly different presentation efficiencies reported for viral versus bacterial antigens. Because class I antigen processing involves multiple steps, we sought to determine where in the processing pathway these differences in efficiency occur. To accomplish this, we expressed identical minimal peptide determinants from viral and bacterial vectors using a minigene expression system and determined the rate of peptide-MHC generation per molecule of minigene product synthesized. We found that peptides expressed from either the viral or bacterial vector were presented with virtually identical efficiencies. These results suggest that differences in the processing pathways followed by endogenous versus exogenous proteins most likely occur at a point prior to where free peptide is liberated from full-length protein.     

Major histocompatibility complex class II (DR) antigen and costimulatory molecules on in vitro and in vivo activated human polymorphonuclear neutrophils

We have previously shown that normal human peripheral blood polymorphonuclear neutrophils (PMNs) contain cytoplasmic 'stores' of three key molecules normally associated with antigen presentation and T-cell costimulation, i.e. major histocompatibility complex class II (DR) antigen, CD80 (B7-1) and CD86 (B7-2). These cytoplasmic molecules were found to translocate to the cell surface within a few minutes following cross-linking (X-L) of Mac-1: an early neutrophil activation signal. In this study we have compared X-L of Mac -1 in parallel with four other well documented in vitro neutrophil activators: phorbol myristate acetate, N-formyl methionyl leucyl phenylalanine, lipopolysaccharide, and phagocytosis of immunoglobulin G-Latex particles. In addition, we have used paired samples of neutrophils obtained from peripheral blood (as a control) and synovial fluid from patients with rheumatoid arthritis as a source of in vivo activated cells. With the exception of phagocytosis, all activators resulted in the rapid (within 30 min) generation of two populations of activated neutrophils (designated P1 and P2) based on flow-cytometry measurements of size, granularity and phenotype. Significant up-regulation of DR and costimulatory molecules was observed, predominantly on P2 cells, with all activators except phagocytosis. CD80 and CD86 were noted to respond to the various activation signals in a different pattern suggesting that their intracellular granule location may be different. Dual-staining confocal laser microscopy studies showed that CD80 is largely confined to secretory vesicles (SVs) while CD86 appears to have a much wider distribution being found in SVs and within secondary (specific) and primary (azurophilic) granules. Increased surface expression of these antigens was also observed on P2 synovial fluid neutrophils appearing as large heterogeneous clusters on the cell surface when visualized by confocal laser microscopy.     

Aggregation of MHC class I molecules on a CD8+ alphabeta T cell clone specifically inhibits non-antigen-specific lysis of target cells

MHC class I molecules are target molecules recognized by TCR or NK receptors encoded in the NK gene cluster or leukocyte receptor cluster. We show that aggregation of MHC class I molecules by specific monoclonal antibodies on cytotoxic T cells, inhibits the anti-CD94 redirected lysis of P815. This inhibition is not the consequence of apoptosis or anergy of the cytotoxic T lymphocytes. In contrast, aggregation of MHC class I molecules does not inhibit either the anti-CD3 redirected cytotoxicity or the CD94-triggered up-regulation of CD25 molecules of the same T cell clone. MHC class I ligand molecules expressed by antigen presenting cells and/or T lymphocytes could therefore be able to modulate nonspecific cytotoxicity upon interaction with MHC class I molecules expressed by effector cytotoxic T lymphocytes.     

Mechanistic understanding and significance of small peptides interaction with MHC class II molecules for therapeutic applications

Major histocompatibility complex (MHC) class II molecules are expressed by antigen-presenting cells and stimulate CD4⁺ T cells, which initiate humoral immune responses. Over the past decade, interest has developed to therapeutically impact the peptides to be exposed to CD4⁺ T cells. Structurally diverse small molecules have been discovered that act on the endogenous peptide exchanger HLA-DM by different mechanisms. Exogenously delivered peptides are highly susceptible to proteolytic cleavage in vivo; however, it is only when successfully incorporated into stable MHC II–peptide complexes that these peptides can induce an immune response. Many of the small molecules so far discovered have highlighted the molecular interactions mediating the formation of MHC II–peptide complexes. As potential drugs, these small molecules open new therapeutic approaches to modulate MHC II antigen presentation pathways and influence the quality and specificity of immune responses. This review briefly introduces how CD4⁺ T cells recognize antigen when displayed by MHC class II molecules, as well as MHC class II–peptide-loading pathways, structural basis of peptide binding and stabilization of the peptide–MHC complexes. We discuss the concept of MHC-loading enhancers, how they could modulate immune responses and how these molecules have been identified. Finally, we suggest mechanisms whereby MHC-loading enhancers could act upon MHC class II molecules.     

Determinants of the endoplasmic reticulum (ER) lumenal-domain of the adenovirus serotype 2 E3-19K protein for association with and ER-retention of major histocompatibility complex class I molecules

The E3-19K immunomodulatory protein from adenoviruses (Ads) inhibits antigen presentation by major histocompatibility complex (MHC) class I molecules. As a result, the ability of Ad-specific cytotoxic T lymphocytes (CTLs) to lyse infected cells is suppressed. The ER-lumenal domain of E3-19K is subdivided into a variable (residues 1 to ∼78/81) and conserved (residues ∼79/82 to 98) region followed by a linker (residues 99-107). Using molecular and cellular approaches, we characterized in detail the properties of the ER-lumenal domain of E3-19K that enable it to target MHC class I molecules. Proteolysis of recombinant serotype 2 E3-19K (residues 1-100) (with six His residues) generated a large N-terminal (residues 1-88) and a small C-terminal fragment (residues 94-100) in solution. Neither of these fragments associates with HLA-A*1101 as shown by a native gel band-shift assay. In contrast, the N-terminal 1-93 residues of Ad2 E3-19K exhibited the same binding affinity to HLA-A*1101 as E3-19K. Using a site-directed mutational analysis and flow cytometry, we show that Tyr⁹³, but not Tyr⁸⁸, critically modulates the cell-surface expression of MHC class I molecules. Taken together, these results indicate that the sequence comprising residues 89-93 (M⁸⁹SKQY⁹³), and in particular Tyr⁹³, in the conserved region of E3-19K is critical for its immunomodulatory function. Residues 89-93 likely form a linker or loop in E3-19K. Overall, our data provide novel insights into the structure of E3-19K and identify key determinants for association with and ER-retention of its cellular target protein. This knowledge is important for our understanding of the molecular basis of Ad pathogenesis.     

ADAM metallopeptidase domain 17 (ADAM17) is naturally processed through major histocompatibility complex (MHC) class I molecules and is a potential immunotherapeutic target in breast, ovarian and prostate cancers

Selection of suitable antigens is critical for the development of cancer vaccines. Most desirable are over-expressed cell surface proteins that may serve as targets for both antibodies and T cells, thus maximizing a concerted immune response. Towards this goal, we characterized the relevance of tumour necrosis factor-α-converting enzyme (ADAM17) for such targeted therapeutics. ADAM17 is one of the several metalloproteinases that play a key role in epidermal growth factor receptor (EGFR) signalling and has recently emerged as a new therapeutic target in several tumour types. In the present study, we analysed the expression profile of ADAM17 in a variety of normal and cancer cells of human origin and found that this protein is over-expressed on the surface of several types of cancer cells compared to the normal counterparts. Furthermore, we analysed the presentation of a human leucocyte antigen (HLA)-A2-restricted epitope from ADAM17 protein to specific T cells established from normal donors as well as ovarian cancer patients. Our analysis revealed that the HLA-A2-restricted epitope is processed efficiently and presented by various cancer cells and not by normal cells. Tumour-specific T cell activation results in the secretion of both interferon-γ and granzyme B that can be blocked by HLA-A2 specific antibodies. Collectively, our data present evidence that ADAM17 can be a potential target antigen to devise novel immunotherapeutic strategies against ovarian, breast and prostate cancer.     

抗MHC I类单抗对NK细胞杀伤能力的影响

探讨ＮＫ细胞杀务与靶细胞表面ＭＨＣＩ类分子的关系。方法用ＨＬＡ－ＡＢＣ单抗培养上清或腹水封闭靶细胞Ｋ５６２表面的ＨＬＡ－ＡＢＣ分子后,分别观察了外周血新鲜ＮＫ细胞、纯化ＮＫ细胞、ＬＡＫ细胞和ＣＤ３ＡＫ细胞对Ｋ５６２细胞杀伤能力的变化。     

中华鳖MHC Iα2链基因克隆及序列分析

目的：为了探明中华鳖ＭＨＣ的结构与功能和寻找该种群的分子标记，对中华鳖ＭＨＣｃｌａｓＩα２基因中由两个半胱氨酸形成二硫键的区域进行了克隆和序列分析。方法：根据人和动物的ＭＨＣＩａα２链中两个半胱氨酸侧翼的保守序列设计了混合型引物，采用ＰＣＲ法从中华鳖基因组ＤＮＡ中克隆了ＭＨＣＩα２链（ＴｒｓｉＢＸ１）。结果：经氨基酸序列的同源性分析，ＴｒｓｉＢＸ１存在抗原多肽结合位点（Ｔ１４３、Ｋ１４６、Ｗ１４７、Ｙ１５９）以及β２微球蛋白结合位点（Ｑ１１５、Ａ１１７、Ｄ１１９、Ｇ１２０、Ｄ１２２），并与人和动物的ＭＨＣＩａα２存在４１１％～６３９％同源性。结论：ＴｒｓｉＢＸ１属ＭＨＣｃｌａｓｓＩａ类基因，可作为中华鳖种群的分子标记。     

Polymorphism of human CD1 genes

Human CD1 genes have been reported to be invariant or to show limited polymorphism. Recently, certain functions of CD1 antigens have been described to include the presentation lipid and glycolipid antigens. These observations prompted a thorough survey of the genetic polymorphism in the five human CD1 genes (CD1a-CD1e). Using polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) combined with sequence analyses, exons 2 and 3 from CD1a-CD1e were characterized from a total of 110 unrelated healthy donors. Results showed that all five genes (CD1a-CD1e) are polymorphic in exon 2. Substitutions in CD1b and CD1c are silent, whereas, substitutions in CD1a, CD1d and CD1e result in amino acid replacements in the deduced protein products. CD1a and CD1e polymorphisms are prevalent in the population. The substitutions in CD1a have characteristics that may influence interactions with beta2-microglobulin beta2-m) or accessory molecules. The substitution in CD1e is located in the region predicted to interact with ligands and may differentially impact the ability of CD1e alleles to bind antigen.     

Internalization of MHC class I molecules is a prerequisite for endocytosis of endorphin by lymphocytes

The nature of the interaction between gamma-type endorphins and the HLA class I molecules was studied by immunoelectronmicroscopy. The HLA molecules were not involved in the actual binding of endorphin to the cell. In contrast, for the endocytosis of gamma-endorphin, co-internalization of the HLA class I molecules is essential. The internalization process starts with clustering of gamma-endorphin and HLA class I molecules in coated pits. Cells that do not carry HLA class I molecules (Daudi) or do not internalize HLA class I molecules (EBV-transformed B cells) bind but do not internalize gamma-endorphin. On the basis of these observations, we suggest that the MHC class I molecules may function as transport molecules. Whether it is a general phenomenon that non-immunological ligands use the HLA class I molecules to get into the cell and immunological ligands (viral proteins) to reach the cell surface, remains to be established.     

Expression of MHC class I molecules together with antigenic peptides on filamentous phages

The availability of combinatorial T cell epitope libraries made using a phage display system would be useful for identifying the antigens recognized by T cells of unknown specificity. To this end, we have investigated here whether single chain-MHC class I molecules (scMHC-I) could be expressed together with antigenic peptides on filamentous phages. The results show that filamentous phages can express scMHC-I. Moreover, the expressed scMHC-I was able to bind antigenic peptide. These data support the use of combinatorial scMHC/T cell epitope libraries for screening potential T cell antigens.     

Spontaneous Autoimmune Disease in (NZB × NZW)F1 Mice is Ameliorated by Treatment with Methimazole

(NZB × NZW)F₁ mice spontaneously develop with age an autoimmune disease that resembles the human disease, systemic lupus erythematosus (SLE). The present study demonstrates that methimazole (MMI), an agent used in the treatment of autoimmune thyroid disease, is effective in mitigating the development of this SLE-like autoimmune disease in (NZB × NZW)F₁ mice. MMI significantly reduces the incidence and severity of proteinuria and deposition of immune complexes in the kidney. Previous studies have demonstrated that development of an experimentally induced SLE, which was prevented by MMI treatment, depended on the expression of MHC class I molecules. We now report that class I levels on both T cells and B cells from old (NZB × NZW)F₁MHC class I are markedly elevated relative to those from young F₁ mice. Furthermore, treatment of (NZB × NZW)F₁ mice with MMI reduced MHC class I expression on their PBL concomitant with amelioration of disease, raising the possibility that class I molecules may play a role in the generation of spontaneous autoimmune disease in these mice.     

MHC class II molecules activate NFAT and the ERK group of MAPK through distinct signaling pathways in B cells

MHC class II (MHC‐II) molecules are capable of transducing signals with the help of associated molecules. Although the search to find associated molecules over the past few years has been fruitful, it remains clear that not all signaling components and their mechanisms of action have been identified. In this study, we investigated calcium and MAPK signaling pathways using the BJAB and Raji human B cell lines. We demonstrate that calcium mobilization is an isotype‐independent event that triggers the dephosphorylation of NFAT. We also show that BCR activation followed by MHC‐II ligation increases the activation of NFAT. This signaling pathway differs from MHC‐II‐mediated MAP activation, where MEK1/2 and ERK1/2 phosphorylation are isotype‐specific events, which correspond to the induction of c‐Fos and formation of AP‐1. Future studies should elucidate the intertwined, intricate signaling cascades triggered by BCR and MHC‐II leading to humoral immune responses.     

Innate mucosal‐associated invariant T (MAIT) cells are activated in inflammatory bowel diseases

Inflammatory bowel diseases are characterized by a deregulated immune response targeting the gut bacterial flora. Mucosal‐associated invariant T (MAIT) cells are major histocompatibility complex (MHC) class Ib‐restricted innate‐like lymphocytes with anti‐bacterial functions. They display an effector/memory phenotype and are found in large numbers in the blood, mucosae and liver. They have also been implicated in inflammatory diseases such as multiple sclerosis. Therefore, we aimed to analyse the possible involvement of MAIT cells in Crohn's disease (CD) and ulcerative colitis (UC). To this end, a phenotypical and functional analysis of MAIT cells isolated from the blood of healthy subjects, CD and UC patients was undertaken. MAIT cells were also quantified in ileal biopsies of CD patients. The frequency of blood MAIT cells was specifically reduced in IBD patients compared with healthy donors, whereas it was dramatically greater in the inflamed versus healthy tissue. MAIT cells were activated as they expressed significantly more the Ki67 antigen, and this was accompanied by phenotypical changes such as increased expression of natural killer (NK)G2D and B and T lymphocyte attenuator (BTLA). Finally, in‐vitro‐activated MAIT cells from CD and UC patients secreted significantly more interleukin (IL)‐17, together with a decreased interferon (IFN)‐γ in CD but an increased IL‐22 in UC. These data show that MAIT cells are activated in IBD, which results in an increased recruitment towards the inflamed tissues, an altered phenotype and a switch in the pattern of cytokine secretion. This is the first demonstration that MAIT cells are immune players in IBD, whose precise functions in this context need to be addressed.     

A novel HLA-A determinant recognized by a cytotoxic human hybridoma IgG1 monoclonal antibody (TrJ14)

Abstract: TrJ14 is a cytotoxic human IgG1Λ hybridoma mAb that recognized a novel HLA-A epitope expressed by lymphoblastoid B cells that are homo- or heterozygous for A2, A3, A11, A30, A31, A33, A68 and A69. Based on these results, the HLA type of cell line TEM (10w9057) was retyped as A66. When peripheral blood T cells isolated freshly from 265 HLA-typed normal individuals served as targets, TrJ14 killed cells expressing two TrJ14-positive HLA-A alleles, as well as the majority of cells having one TrJ14-positive and one TrJ14-negative HLA-A antigen. However, TrJ14 failed to recognize or reacted weakly with most HLA-A2 and -A3 heterozygous normal T cells when A2 or A3 was coexpressed together with a TrJ14-negative antigen. The serological reactivity of TrJ14 correlated with the amino acid valine and aspartic acid at positions 76 and 77 of the αl-domain helix. These amino acids were shared exclusively by all the identified TrJ14⁺ alleles.     

Molecular genetic and biochemical analysis of woodchuck (Marmota monax) MHC class I polymorphism

The woodchuck (Marmota monax) is an animal model that is used in the study of human hepatitis B virus ( HBV ) infection. A knowledge of woodchuck MHC class I (Mamo-I) genes and gene products is therefore essential for understanding the antigen-specific T-cell responses in this animal model. A number of Mamo-I genes have been identified by molecular cloning and sequencing. However, the allelic nature of these genes has not been proven by classical genetics like the segregation analysis in families. In this study, we analyzed the allelic diversity of Mamo-I in two three-generation woodchuck families including 15 members by sequencing of Mamo-I genes and immunoblotting of Mamo-I proteins after one-dimensional isoelectric focusing (1D-IEF). In addition to four published Mamo-I alleles, six new alleles that belonged to the same locus as the known Mamo-I alleles (Mamo-A) were found within the two woodchuck families. A typical Mendelian segregation of Mamo-I gene and antigens was observed in the families studied. For simple and rapid detection of allelic variability of Mamo-I gene, a typing method based on the detection of PCR products amplified by sequence specific primers (SSP) has been developed and tested in 41 unrelated animals. The most prevalent allele was Mamo-A*01 with a frequency of 21.9% followed by Mamo-A*07 (12.2%). Our study established Mamo-A as a classical MHC class I locus by the polymorphic and allelic nature of Mamo-I gene in the woodchuck.