Differential effects of interleukin-10 on the expression of HLA class II and CD1 molecules induced by granulocyte/macrophage colony-stimulating factor/interleukin-4

Interleukin (IL)-10 down-regulates HLA class II molecules, whether constitutively expressed or up-regulated by interferon-γ or IL-4 on monocytes but not on B lymphocytes. In this study we show that IL-10 does not inhibit HLA class II expression induced by the combination granulocyte/macrophage colony-stimulating factor and IL-4 on monocytes, although it simultaneously abrogates the expression of CD1 molecules induced by the same combination of cytokines. CD1 molecules can act as element of genetic restriction for CD4^? CD8^? T lymphocytes, and the suppression of CD1 expression by IL-10 abolished antigen presentation to CD1-restricted CD4^? CD8^? T cell receptor-positive T cells. Although HLA class II expression was not down-regulated by IL-10, the antigen specific proliferative response of CD4⁺ T cells was nevertheless decreased. This was not caused by down-regulation of known co-stimulatory molecules such as B7.1, B7.2 and ICAM-1. IL-10 decreased the antigen specific proliferative response further by directly influencing the T lymphocytes. Our results indicate that IL-10 exerts some of its immunoregulatory functions by differential modulation of antigen presenting molecules, induced by the same combination of cytokines.     

Biochemical analysis of the rat MHC class I antigens RT1.Aa, RT1.Fa and Pa

In DA strain rats, there are two other MHC class I loci (Pa and RT1.Fa) in the vicinity of the classical class I locus RT1.Aa. The Pa antigen is the pregnancy-associated antigen, and it was detected by antibodies elicited in WF females pregnant by DA males without any other immunization. The Fa antigen was detected by a monoclonal antibody raised by alloimmunization. In the present work, the Aa, Fa and the Pa antigens have been compared by HPLC peptide mapping and by isoelectric focusing after their isolation by appropriate monoclonal antibodies. All the three antigens are identical in primary structure with respect to lysine, methionine, asparagine and the aromatic amino acid residues, but they differ from one another with respect to glutamic acid and/or aspartic acid residues. The pI values of the antigens differ slightly. All three antigens have two identical N-linked glycans, but the Fa antigen has an additional N-linked glycan. Based on the available amino acid sequence of the Pa antigen, it can be concluded that both Aa and Pa antigens are devoid of glycosylation in the second domain. This lack of glycosylation of the classical antigen Aa is unique for the rat, since classical class I antigens of the mouse show glycosylation in the first and second, and sometimes in the third domain, and those in the human, in the first domain only. The high degree of similarity among the Aa, Fa, and Pa molecules that this study indicates is also unique for the rat, since antigens encoded by different class I genes of the same haplotype are quite disparate in the mouse and human.     

Ligation of MHC class II molecules differentially upregulates TNF beta gene expression in B cell lines of different MHC class II haplotypes.

Although the production of selected cytokines by B cells is important for their regulation, little is known about MHC class II-induced cytokine expression in these cells. We designed the present studies to investigate MHC class II-mediated TNF-beta gene expression in 19 EBV-transformed homozygote B cell lines at similar stage of differentiation but presenting different MHC class II haplotypes. Our results demonstrate that in contrast to PMA, engagement of MHC class II with staphylococcal enterotoxin A (SEA), a natural ligand, or with anti-HLA-DR mAb L243, stimulates TNF-beta gene expression in some but not all B cell lines. The differential stimulation of TNF-beta gene expression via MHC class II was not due to the cells MHC class II expression level, nor to their capacity to bind the ligands as evidenced by SEA binding affinity studies. Together these results demonstrate that ligation of MHC class II molecules can stimulate TNF-beta gene expression in a B cell line-dependent manner. The differential cytokine gene expression might be due to an influence of MHC class II haplotype either by a linkage disequilibrium with TNF-beta gene or by a differential association with effector or cell surface molecules.     

Sheep MHC class II molecules. II. Identification and characterization of four distinct subsets of sheep MHC class II molecules

A panel of seven monoclonal antibodies, sequential immunoprecipitation and two-dimensional NEPHGE/SDS-PAGE analyses were used to identify and characterize subsets of sheep MHC class II molecules. Using sequential immunoprecipitation four distinct subsets of class II molecules were identified by the monoclonal antibodies SBU.II 28-1, 37-68, 38-27 and 42-20, while another monoclonal antibody, SBU.II 49-1, recognized all four subsets of class II molecules. These four subpopulations of sheep class II molecules displayed different two-dimensional gel profiles and, using splenocytes from four outbred sheep, the class II molecules recognized by SBU.II 28-1, 37-68 and 42-20 showed structurally detectable allelic polymorphism in their beta polypeptides, but no detectable variation in their alpha polypeptides. In contrast, the class II molecules recognized by SBU.II 38-27 showed allelic variation in both their alpha and beta polypeptides. Two-dimensional (2-D) gel analyses of non-glycosylated class II molecules immunoprecipitated by SBU.II 49-1 suggested that approximately 10-12 different class II molecules were expressed by a single sheep. The results of this study show that sheep express class II molecules that can be divided into four structurally and serologically distinct subsets, and provide additional evidence for the subdivision of the sheep MHC class II genetic region into at least three distinct subregions.     

CIITA M1-RNA抑制HeLa细胞表面MHC II类抗原的表达

探讨MHC II类转录激活因子(CIITA)的M1-RNA对细胞表面MHC II类分子表达的抑制。M1-RNA是核糖核酸酶P的催化活性单位,设计并克隆针对CIITA第452、629位点的M1-RNA(分别为M1-452-GS、M1-629-GS)及其相应的CIITA靶基因,分别插入pUC19、pGEM-7zf(+)载体,进行细胞外切割活性筛选。将细胞外切割作用明显的M1-629-GS亚克隆入psNAV载体(psNAV-M1-629-GS,pA629)并稳定转染HeLa细胞株,流式细胞术检测经典的MHC II类抗原(HLA-DR、-DP、-DQ)的表达,RT-PCR检测CIITA的mRNA水平。在重组人γ干扰素诱导下,pA629阳性HeLa细胞株表面HLA-DR、-DP抗原表达分别降低了83.03%及89.91%;同时CIITA的mRNA含量明显减少(P<0.05)。CIITA的M1-RNA抑制了自身mRNA含量,从而阻止其调控的MHC II类分子的表达,为移植物抗宿主病的研究提供了一种新方法。     

Impaired expression of MHC class II molecules in response to interferon-gamma (IFN-gamma) on human thymoma neoplastic epithelial cells.

A human thymoma is a neoplasm derived from the thymic epithelial cell, and is well known for its association with autoimmune diseases, especially myasthenia gravis. The neoplastic epithelial cells of thymoma clearly retain thymic epithelial functions, but the development of T cells in thymoma is somewhat impaired. In this study, we quantified by flow cytometry the in vitro expression of MHC molecules on neoplastic epithelial cells precultured with IFN-gamma. While MHC class I expression was comparable with that on normal thymic epithelial cells, the level of MHC class II molecules on neoplastic epithelial cells was lower than in controls, and also varied greatly from case to case. Additionally, there was a significant positive correlation between the expression level of MHC class II and the proportion of mature CD3+ cells in the CD4+CD8- subset. Thus, accumulation of CD3-CD4+CD8- cells in thymoma may result from impaired expression of the MHC class II molecules, suggesting that the function of the neoplastic epithelial cells might determine the maturation and the positively selected repertoire of T cells in thymomas.     

Identification of specific recognition molecules on murine mononuclear phagocytes and B lymphocytes for Vi capsular polysaccharide: modulation of MHC class II expression on stimulation with the polysaccharide.

Vi bacterial polysaccharide is a homopolymer of alpha 1-4 N-acetyl polygalacturonic acid with variable O-acetylation at position C-3 and forms a capsule around many bacteria. It has been referred to as the virulence factor of Salmonella typhi and is also a candidate vaccine against typhoid fever. The present study reports the interaction of this polysaccharide with murine mononuclear phagocytes and lymphocytes, and with human monocytes. Vi showed a dose-dependent binding to the murine monocyte cell lines WEHI-274.1 and J774. This binding was abrogated if the polysaccharide was deacetylated, suggesting involvement of acetyl groups in this interaction. Vi also bound to the murine B-cell lymphoma line A20, to peritoneal exudate cells and to a lesser degree to spleen cells and thymocytes from BALB/c mice. The polysaccharide also interacted with the human histiocytic lymphoma line U937 but not with the human monocyte cell line THP-1. Stimulation with Vi led to up-regulation of surface major histocompatibility complex (MHC) class II expression on A20 cells. Immunoprecipitation of Vi-bound molecules from cell surface biotinylated A20 and WEHI-274.1 revealed two bands with MW of about 32,000 and 36,000. The study demonstrates that Vi capsular polysaccharide can interact with mononuclear phagocytes and lymphocytes through specific cell surface molecules and modulate MHC class II expression.     

CD40 associates with the MHC class II molecules on human B cells

The MHC class II and CD40 molecules are two major components of the immune system that are involved in cell-cell interactions and signal transduction. Data obtained in the course of the present investigation show that these two molecules are physically associated on the surface of various human B cell lines and on normal tonsilar B cells. The CD40 / MHC class II complexes were not detected on the germinal center B cell line Ramos. However, stimulation of these cells via CD40 or MHC class II triggered their association, suggesting that the formation of the complex is related to the activation status of the cells. The formation of these complexes did not alter the interaction of MHC class II molecules with one of their natural ligands, the staphylococcal enterotoxin A (SEA), as evidenced by the ability of SEA to bind MHC class II / CD40 complexes. Cross-linking of MHC class II or CD40 molecules leads to the association as well as the co-association of both molecules to the NP-49-insoluble cellular matrix. Such association allowed us to demonstrate that only a fraction of these molecules can be physically associated on the cell surface. Based on previous observations and those presented here, it is highly possible that the CD40 / MHC class II complexes may have an important role in signal(s) induced via both molecules and during T / B cells interactions.     

Venom immunotherapy modulates interleukin-4 and interferon-gamma messenger RNA expression of peripheral T lymphocytes.

The mechanism by which specific immunotherapy exerts its beneficial effect remains unclear. In order to evaluate the influence of venom immunotherapy on the T-cell cytokine pattern of allergic reactions, we studied interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression of peripheral T lymphocytes from 12 patients undergoing rush venom desensitization, before treatment at Day 0 (D0), at Day 15 (D15) and Day 90 (D90) after treatment, and from seven controls. Antigen-specific T-cell proliferation was also determined. Cytokine mRNA expression was evaluated using in situ hybridization, 24 hr after culture of peripheral T cells with medium, venom, or an unrelated allergen. Allergen-induced T-cell proliferation decreased at D15 and D90 of rush immunotherapy (P < or = 0.02). In venom-stimulated cultures of the patient group, there was a decrease in IL-4 mRNA-positive cells at D15 and D90 (P < or = 0.001). Before desensitization, IFN-gamma mRNA expression was lower in patients than in controls and did not increase after in vitro allergen stimulation. In contrast, after immunotherapy, spontaneous IFN-gamma mRNA expression increased, but only at D90 (P < or = 0.001). The cytokine pattern observed at D90 after immunotherapy was similar to that observed in control subjects. In conclusion, venom immunotherapy induced an altered cytokine mRNA pattern in allergen-stimulated T cells which was dissociated from the early changes of allergen-induced T-cell responsiveness.     

Immunochemical evidence for multiple class I antigens coded by the MHC of the rat (RT1) and their differential expression on red blood cells and lymphocytes

Five monoclonal antibodies reacting with class I MHC antigens were produced by fusing lymphocytes from WF (RT1u) rats immunized against DA (RT1a) rats with P3-X63-Ag8.653 myeloma cells. Sequential immunoprecipitation studies with the mAb and the WF anti-DA alloantiserum demonstrated the presence of four different class I molecules: all four molecules were reactive with the alloantiserum; three of them contained the determinant for mAb 155; two of the latter three molecules shared the determinants for mAb 3, 56 and 60, and one of these two molecules also contained the determinant for mAb 118. The four molecules could be isolated from the antigen preparation by sequential immunodepletion first with 118, next with 3, then with 115 and finally with the alloantiserum or by sequential absorption with affinity columns of Sepharose 4B coupled to the antibodies. The three antigens which were sequentially isolated with the mAb 118, 3, and 155, respectively, were analysed by SDS-PAGE after digestion with Staphylococcus aureus V8 protease, and they showed differences in peptide patterns. The relative amounts of the antigens expressed on red blood cells and on lymphocytes were different based on the results of sequential isolation and indirect cellular radioimmunoassay: the antigen which reacted with both mAb 3 (and 56 or 60) and 155 was the major class I antigen on red blood cells, and the antigen which reacted with mAb 118, 3 (and 56 or 60) and 155 was the major class I antigen on lymphocytes.     

Adhesion of lymphocytes to cerebral microvascular cells: effects of interferon-gamma, tumour necrosis factor and interleukin-1.

Although lymphocyte traffic through the brain is normally low, this can increase dramatically in response to infection or an autoimmune reaction. We have studied the adhesion of lymphocytes to cerebral endothelium in vitro in an attempt to model the first step of the infiltration process--that is, the initial interaction between the lymphocytes and endothelial cells--by brief co-culture of lymph node cells with monolayers of cultured cerebral endothelium. In this system we find that the basal level of adhesion can be increased in a dose-dependent manner by pre-treatment of the endothelial cells with interferon-gamma (IFN-gamma) and/or tumour necrosis factor (TNF) but not with interleukin-1 (IL-1). This increased adhesion can be blocked by incubating the IFN-gamma-treated cells with an antibody that is thought to bind to the common beta-chain of the lymphocyte functional antigen-1 (LFA-1) family of molecules. This suggests that endothelial cells express either LFA-1 or a molecule sharing the beta-subunit (of which several have been described) and furthermore that this molecule is involved in the regulation of lymphocyte traffic into the brain.     

Intracellular transport and peptide loading of MHC class II molecules: regulation by chaperones and motors

Summary: MHC dass II molecules are important in the onset and modulation of cellular immune responses. Studies on the intracellular transport of these molecules has provided insight into the way pathogens are processed and presented at the cell surface and may result in future immunological intervention strategies. Recent reviews have extensively described structural properties and early events in the biosynthesis of MHC class II (1-3). In this review, the focus will be on the function of the dedicated chaperone proteins Ii, DM and DO in the class II assembly, transport and peptide loading as well on proteins involved in transport steps late in the intracellular transport of MHC class II.     

Differential MHC class II expression on human peripheral blood monocytes and dendritic cells

Both monocytes (MO) and dendritic cells (DC) in human peripheral blood are of a plastic-adherent nature. The expression of the MHC class II sublocus products HLA-DP, -DQ and -DR on human peripheral blood transiently adherent cells (TA) was examined by an immunocytochemical staining technique. While most TA showed strong expression of molecules of the HLA-DR subtype, only a small proportion of cells (2-6%) showed strong HLA-DP or -DQ positivity. This strong expression of the HLA-DP and HLA-DQ sublocus products by a subset of TA was seen only after short-term culture; freshly isolated cells expressed comparatively low levels of these molecules. Enrichment for Fc receptor-negative or low-density cells from TA produced populations with strong HLA-DQ and -DP expression. Such co-enrichment of the strongly HLA-DQ+ and strongly HLA-DP+ cells suggests that the same cells express high levels of both types of MHC class II molecule. Immunocytochemical analysis of TA indicated that the strongly HLA-DQ+ cells, at least, were only weakly or non-reactive with the MO-specific monoclonal antibodies OKM1, UCHM1, MO2 and EB11. In addition, strongly HLA-DQ- or -DP-positive cells were poorly phagocytic in comparison with the majority of adherent cells. The apparent FcR-negative, low-density and weakly phagocytic nature of the strongly HLA-DQ/DP+ cells, combined with their lack of reactivity with several MO-specific antibodies, suggests that they may represent the DC component of TA. Such strong HLA-DQ/DP expression by DC may aid their positive identification in human peripheral blood and may be of relevance to DC function in antigen presentation.     

CD4+ T cell responses in mice lacking MHC class II molecules specifically on B cells

The role of B lymphocytes in initiating and maintaining a CD4⁺ T cell response has been examined using a variety of strategies, but remains controversial because of weaknesses inherent to each of the approaches. Here, we address this issue by measuring CD4⁺ T cell priming both in mutant mice devoid of B cells and in chimeric animals lacking major histocompatibility complex class II molecules specifically on B cells. We find that peptide and some protein antigens do not require B cells expressing class II molecules, nor B cells themselves, to efficiently prime. This could be demonstrated by the usual lymph node proliferation assay, a rather indirect in vitro measure of priming, and by a direct ex vivo assay of population expansion and activation marker expression. Interestingly, one protein antigen, conalbumin, could not prime in the absence of B cells, but could in the presence of B cells devoid of class II molecules. This finding constrains the possible mechanisms whereby B lymphocytes contribute to the initiation of a CD4⁺ T cell response, arguing against the importance of surface immunoglobulin-mediated antigen presentation by B cells.