Transfer of yeast artificial chromosomes into mammalian cells and comparative study of their integrity.

Yeast artificial chromosomes (YACs) from the CEPH MegaYAC library (Paris, France) ranging in size from 350 to 1600 kb and mapping to the q22.1 and q22.2 regions of human chromosome 21 were transferred into mammalian cells by spheroplast fusion. The integrity of the YACs from two adjacent parts of the region was compared after retrofitting and stable transfer into mammalian cells. We found that large YACs could easily be manipulated to allow transfer of the YAC material into mammalian cells and that the size of the YAC did not appear to be limiting for fusion. However, we show that there was great variability in the integrity of the YACs from the two regions, which was not related to the size of the YACs. Four YACs in region I from sequence-tagged site (STS) G51E05 up to STS LL103 showed, in general, no loss of material and correct gene transfer into mammalian cells. In contrast, the three YACs in the more centromeric region II (from STS G51B09 up to G51E05) frequently showed a loss of human material during handling, retrofitting and transfer. As a YAC from another library covering region II was also found to be unstable, we propose that the integrity of the YACs is highly dependent on the incorporated human chromosomal DNA.

Transcriptional regulation of the MHC class Ib genes HLA-E, HLA-F, and HLA-G.

The restricted tissue expression of the MHC class Ib molecules HLA-E, HLA-F, and HLA-G has suggested specialized functions and tight transcriptional control of their genes. Transactivation of classical MHC class I genes is mediated by two groups of juxtaposed cis-acting elements, which can be viewed as regulatory modules. The most upstream module consists of the enhancer A and ISRE, and mediates constitutive and cytokine induced expression, whereas the SXY module is important for the constitutive and CIITA-mediated transactivation of MHC class I genes. Nucleotide sequence divergence in these regulatory elements in the promoters of HLA-E, HLA-F, or HLA-G determines their differential responsiveness to NF-kappaB, IRF1, and CIITA-mediated induction. HLA-E is not inducible by NF-kappaB or IRF1, but is responsive to IFN-gamma through an upstream STAT1 binding site. Furthermore, HLA-E is inducible by CIITA through the SXY regulatory module. HLA-F is inducible by NF-kappaB through the kappaB1 site of enhancer A, is responsive to IFN-gamma through the ISRE, and is inducible by CIITA. Both regulatory modules are divergent in HLA-G rendering this gene unresponsive to NF-kappaB, IRF1, and CIITA-mediated induction. This implies a unique regulation of HLA-G transcription amongst the MHC class Ib genes.

Lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells and is caused by methylation of the IFN-gamma inducible promoter (PIV) of CIITA.

Lack of MHC-mediated antigen presenting functions of fetal trophoblast cells is an important mechanism to evade maternal immune recognition. In this study we demonstrated that the deficiency in MHC expression and antigen presentation in the trophoblast cell lines JEG-3 and JAR is caused by lack of class II transactivator (CIITA) expression due to hypermethylation of its interferon-gamma (IFN-gamma)-responsive promoter (PIV). Circumvention of this lack of CIITA expression by introduction of exogenous CIITA induced cell surface expression of HLA-DR, -DP, and -DQ, leading to an acquired capacity to present antigen to antigen-specific T cells. Transfection of CIITA in JEG-3 cells also upregulated functional HLA-B and HLA-C expression. Noteworthy, this lack of IFN-gamma-mediated induction of CIITA was also found to exist in normal trophoblast cells expanded from chorionic villus biopsies. Together, these observations demonstrate that lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells.

Transcriptional control of MHC genes in fetal trophoblast cells.

Tight control of MHC expression is essential for the outcome of a successful pregnancy. The lack of MHC class II and class I mediated antigen presentation by fetal trophoblast cells is an important mechanism to evade maternal immune recognition. Interestingly, the deficient expression of MHC class II molecules (HLA-DR, -DQ and -DP) and of the classical MHC class I molecules HLA-A and HLA-B is also noted after IFN-gamma treatment in trophoblast-derived cell lines. Our studies show that in trophoblast cell lines the IFN-gamma induced transactivation of HLA-A and HLA-B promoters is repressed. Furthermore, it was found that trophoblast cells lacked IFN-gamma mediated induction of the class II transactivator (CIITA). This lack of CIITA expression in trophoblast cells is due to CIITA promoter hypermethylation. In addition to lack of CIITA expression, trophoblast cells also displayed a repressed expression of RFX5. Together, these observations reveal a silencing of multiple activation pathways that are critical to the transcriptional control of MHC class II and class I antigen presentation functions by trophoblast cells.

Transcriptional control of MHC genes and T cell development in MHC class II deficiency.

MHC class II deficiency has proven to be an excellent model to study transcription regulation of MHC genes and T cell development. Cell lines established from MHC class II deficient patients have been of great value for the identification of proteins necessary for MHC expression and their study has resulted in the identification of a common regulatory pathway for MHC class II and class I genes. The lack of MHC class II expression was found to have a profound effect on the development of the CD4+ T cell lineage, in particular on the composition of the T cell receptor repertoire, revealing aberrant thymic selection processes in these patients. Here, we will discuss several aspects of the transcriptional regulation of MHC genes and the impact of deficient MHC class II expression on T cell development.

Effect of oral and parenteral administration of B6 vitamers on the lymphopenia produced by feeding ammonia caramel or 2-acetyl-4(5)-(1,2,3,4-tetrahydroxy)butylimidazole to rats.

The ability of B6 vitamers to prevent the lymphopenic effects of ammonia caramel fed to rats has been evaluated. Diets containing 10 ppm pyridoxine or pyridoxal prevented the lymphopenia produced in rats consuming an 8% (w/v) solution of ammonia caramel, whereas the dietary content of pyridoxamine needed to be increased to 20 ppm to have the same effect. In contrast to the results of the enteral administration of the individual B6 vitamers, pyridoxamine was found to be the most effective vitamer in preventing the ammonia caramel-induced lymphopenia when administered parenterally. However, all the nutritionally active forms of vitamin B6 were able to prevent the depression of the peripheral blood lymphocyte count, which resulted from ingestion of ammonia caramel by rats. The proposal that oral administration of pyridoxine may prevent the intestinal absorption of the lymphopenic constituent of ammonia caramel, 2-acetyl-4(5)-(1,2,3,4-tetrahydroxy)butylimidazole (THI), is discredited, since THI was found to reduce the lymphocyte count after parenteral administration in rats fed 0.04 ppm pyridoxone in the diet and that increased amounts of dietary pyridoxine (10 ppm) could still prevent this effect. These findings further emphasise the important relationship between dietary vitamin B6 content and the lymphopenic effects of ammonia caramel/THI in the rat.

Immunosuppressive effects of 2-acetyl-4-tetrahydroxybutyl imidazole (THI) in the rat.

THI is a component of ammonia caramel, a widely used food colouring. The effect of THI on the immune system has been determined in the male F344 rat. THI was given in the drinking water at doses of 1, 10 and 50 mg/l (equivalent to 0.1, 1 and 5 mg/kg per day) to animals on a vitamin B6-deficient diet. After 1 week, the immune competence of the animals was assessed under continued THI treatment. No marked changes in thymus or spleen weight were observed after THI treatment, although there was an increased number of pyknotic cells in the thymic cortex, mainly engulfed by macrophages and there appeared to be a slight thinning of the cortex area. THI produced a significant loss in T and B lymphocytes in peripheral blood but not in the spleen. No change in natural killer (NK) cell activity against YAC-1 target cells was observed in the spleen. The observed increase in NK cell activity in peripheral blood was due to an increase in circulating large granular lymphocytes (LGL). Although the serum antibody titre against keyhole limpet haemocyanin (KLH) was not affected by THI treatment, B cells showed less proliferation when cultured with lipopolysaccharide. T cell function was impeded as measured in mitogen-induced proliferation assay, delayed-type hypersensitivity assay and host versus graft (popliteal lymph node) assay. The results indicate that THI is an immunosuppressor in the rat, in whom it can produce profound lymphopenia and suppression of cell-mediated immunity.

Effects of 2-acetyl-4-tetrahydroxybutyl imidazole (THI) on the thymus of rats.

2-Acetyl-4-tetrahydroxybutyl imidazole (THI), a component of the food colouring ammonia caramel, has been shown to produce suppression of cell-mediated immunity and a reduction in circulating TH and TC/S lymphocytes in rats. Accordingly in this study the effects of THI on the thymus has been investigated. THI (1 mg/kg/day) was given for up to 7 days in the drinking water to Fischer 344 rats on a vitamin B6 deficient diet. No marked change in thymus weight was found but the cellularity was marginally decreased and flow cytometric analysis of the lymphocyte subsets revealed an increase in the number of CD4+CD8- and CD4-CD8+ single positive (SP) cells and a reduction in the number of CD4+CD8+ double positive (DP) thymocytes. This reduction was in agreement with histological findings of increased numbers of pyknotic cells in the cortex, mainly engulfed by macrophages. Mitogen-induced proliferation of thymocytes prepared from THI-treated animals was increased, concordant with the gradual increase in the percentage of mature SP cells. No change in normal proliferation of thymocytes cultured in vitro, or, in proliferation in vivo, detected as 5-bromo-2'-deoxyuridine (BrdU) incorporation, was found. It is concluded that THI produced an increase in death of immature DP cells. However, THI did not affect thymocyte proliferation or their differentiation into mature SP cells in the thymus, but rather impairs their migration into the circulation. The mechanism of action of THI appears to be indirect, but THI does not act through increasing the release of adrenal corticosteroids to supra-physiological levels as the same histopathological changes in the thymus were found in adrenalectomized rats.

The role of lymphocyte production and migration in the lymphopenia caused by 2-acetyl-4-tetrahydroxybutyl imidazole.

2-Acetyl-4-tetrahydroxybutyl imidazole (THI), a component of the food colouring ammonia caramel, has been shown to produce a profound and rapid lymphopenia in peripheral blood in the rat. In order to investigate whether the cause of the lymphopenia was due to the reduced production and influx in the circulation, redistribution of lymphocytes into other lymphoid compartments or an increased cell death, THI (1 mg/kg/day) was given in the drinking water for up to 14 days to F344 rats. A profound depletion of lymphocytes after already 1 day was only found in the blood compartment, whereas no such marked and rapid changes were found in the cellularity of other lymphoid compartments. The proportion and absolute number of DNA-synthesizing cells in each lymphoid organ was quantified using an antibody directed against incorporated 5-bromo-2'-deoxyuridine (BrdU), 1 h after a single BrdU injection. Additionally, enumeration and localization of BrdU+ cells was determined at later time points after a single BrdU injection by flow cytometry and immunocytochemistry, in order to examine the distribution and localization of recently formed (BrdU+) lymphocytes. THI treatment had no effect on the proliferation rate and the distribution of newly formed (BrdU+) cells in the lymphoid organs. However, migration studies revealed that THI treatment resulted in an increased percentage of fluorescein-labelled peripheral blood lymphocytes found in the spleen and bone marrow and a decreased percentage in the cervical and mesenteric lymph nodes, 24 h after injection. Collectively these results indicate that the lymphopenia in the peripheral blood compartment after THI treatment, is caused by a rapid sequestration of lymphocytes into the spleen and bone marrow rather than by a reduced lymphocyte production and release into the periphery. The fact that THI also caused lymphopenia in splenectomized rats, indicates that the spleen does not play an active part in the change in migrational behaviour of lymphocytes after THI treatment. Finally, as there was no increase in the absolute number of lymphocytes found in the spleen or bone marrow it seems they are rapidly degraded.

The regulation of HLA class I expression: is HLA-G the odd one out?

The expression of HLA-G in extravillous cytotrophoblast cells coincides with a general lack of classical MHC class I expression in this tissue. This differential expression of HLA-G and classical HLA class I molecules in trophoblasts suggests a tight transcriptional control. Transactivation of classical MHC class I genes is mediated by two groups of juxtaposed cis -acting elements which can be viewed as regulatory modules. The most up-stream module consists of the enhancer A and ISRE, and mediates the constitutive and cytokine-induced expression. The recently identified S-X-Y module is important in the constitutive and CIITA mediated transactivation. Both modules are divergent in HLA-G rendering this gene unresponsive to NF-kappaB, IRF-1, and CIITA mediated induction pathways. However, other known regulatory sequences that could contribute to the tissue-specific expression of HLA-G have so far not been identified in the proximal promoter region (-1500 bp) and in the first five intronic sequences. This implies a unique regulation of HLA-G transcription. Here, the transcriptional control of HLA-G and classical class I molecules in trophoblast cells are discussed.

The effect of 2-acetyl-4-tetrahydroxybutylimidazole on lymphocyte subsets in peripheral blood of the rat.

A constituent of Ammonia Caramel, 2-acetyl-4-tetrahydroxybutylimidazole (THI), is known to cause a reduction in the number of circulating lymphocytes when fed to rats. In the present study the effect of giving THI 1 mg/kg by gavage daily for 7 days on the numbers of lymphocytes in subsets has been monitored in peripheral blood. Both immunoglobulin light chain-bearing B-cells (MARK-1+) and CD5 marker-bearing T-cells (OX-19+) were reduced in number within 1 day of treatment. Within the pan-T-cell population, Class II MHC reactive helper T-lymphocytes (W3/25-) were more reduced than the Class I MHC reactive cytotoxic/suppressor T cells (OX-8+). The number of null cells (MARK-1-, OX-19-) was not affected; the majority of these cells appeared to be large granular lymphocytes.

Site alpha is crucial for two routes of IFN gamma-induced MHC class I transactivation: the ISRE-mediated route and a novel pathway involving CIITA.

The constitutive and cytokine-induced levels of major histocompatibility (MHC) class I expression are tightly controlled at the transcriptional level. In this study, it is shown that the cis-acting regulatory element site alpha of the MHC class I promoter is essential for the IFN gamma-induced transactivation of MHC class I gene expression through the ISRE. Moreover, it was discovered that the class II transactivator (CIITA), which is itself under the control of the IFN gamma induction pathway, strongly transactivates MHC class I gene expression and exerts its activity through site alpha. Therefore, site alpha is a crucial regulatory element, mediating the classic route of IFN gamma induction via the ISRE as well as a novel route of MHC class I transactivation involving CIITA.

Antigen processing and presentation by human trophoblast-derived cell lines.

The trophoblast-derived choriocarcinoma cell lines JEG-3 and JAR express the nonclassical MHC class I molecules HLA-G (JEG-3) or, at a low level, HLA-E (JAR), but lack expression of the classical MHC class I molecules HLA-A and HLA-B. Expression of these nonclassical MHC class I genes was found to coincide with expression of the genes encoding the peptide transporter associated with Ag processing (TAP). In immunoprecipitation studies, a physical interaction between the TAP complex and HLA-G or HLA-E could be demonstrated. To investigate whether trophoblast-derived cell lines were capable of peptide processing, transport, and loading of MHC class I molecules, HLA-A*0201-expressing transfectants of JEG-3 and JAR were used for functional studies. These transfectants were recognized by both allospecific cytotoxic T cell clones and, after viral infection, by an influenza A matrix peptide-specific cytotoxic T cell clone, indicating that these trophoblast-derived cell lines were capable of presenting endogenously derived peptides in the context of HLA-A*0201. From these observations, it can be inferred that the TAP complex and other molecules involved in Ag processing and presentation by MHC class I molecules are functionally active in these trophoblast-derived cell lines. This implies that trophoblasts are able to provide antigenic peptides for presentation by nonclassical MHC class I molecules that are naturally expressed by this cell type.

Regulation of MHC class I and II gene transcription: differences and similarities.

Major histocompatibility complex (MHC) molecules serve as peptide receptors. These peptides are derived from processed cellular or extra-cellular antigens. The MHC gene complex encodes two major classes of molecules, MHC class I and class II, whose function is to present peptides to CD8+ (cytotoxic) and CD4+ (helper) T cells, respectively. The genes encoding both classes of MHC molecules seem to originate from a common ancestral gene. One of the hallmarks of the MHC is its extensive polymorphism which displays locus and allele-specific characteristics among the various MHC class I and class II genes. Because of its central role in immunosurveillance and in various disease states, the MHC is one of the best studied genetic systems. This review addresses several aspects of MHC class I and class II gene regulation in human and in particular, the contribution to the constitutive and cytokine-induced expression of MHC class I and II genes of MHC class-specific regulatory elements and regulatory elements which apparently are shared by the promoters of MHC class I and class II genes.

The role of enhancer A in the locus-specific transactivation of classical and nonclassical HLA class I genes by nuclear factor kappa B.

HLA class I expression is tightly controlled at the transcriptional level by several conserved regulatory elements in the proximal promoter region. In this study, the two putative kappa B motifs of enhancer A (kappa B1 and kappa B2) of the classical and nonclassical HLA class I genes were investigated for their binding properties of transcription factors and tested for their contribution to the NF-kappa B-induced route of transactivation. It was shown that NF-kappa B-induced transactivation through enhancer A is most important for the HLA-A locus, which contains two NF-kappa B binding sites. Although the enhancer A of HLA-B contains only one NF-kappa B binding site (kappa B1), there was still a moderate transactivation by NF-kappa B. Since HLA-F, which also possesses one NF-kappa B binding site but lacks protein binding to its KB2 site, was not transactivated by NF-kappa B, the NF-kappa B-mediated transactivation through the kappa B1 motif in HLA-B is most probably facilitated by binding of the transcription factor Spl to the upstream kappa B2 site. Thus, transcriptional regulation of HLA class I genes by NF-kappa B is restricted to the HLA-A and HLA-B loci.

Transactivation of classical and nonclassical HLA class I genes through the IFN-stimulated response element.

The IFN-stimulated response element (ISRE) is an important conserved cis-acting regulatory element in the promoter of MHC class I genes, but displays considerable locus-specific nucleotide variation. In this report, the putative ISREs of classical and nonclassical HLA class I genes were investigated for their contribution to MHC class I transactivation. It is shown that IFN-gamma induced MHC class I transactivation through the ISRE of HLA-A, HLA-B, HLA-C, and HLA-F. This is congruent with the binding of IFN regulatory factor-1 to the ISREs of these loci upon IFN-gamma treatment. Sp1 was shown to bind to the CG-rich sequences in the ISRE regions of HLA-B, HLA-C, and HLA-G. The putative E box 5' of the ISRE in most HLA-B alleles was shown to bind the upstream stimulatory factors (USF) 1 and 2. The Sp1 and USF binding sites did not influence IFN-gamma-induced transactivation. However, the USF binding site played a suppressive role in the constitutive expression of HLA-B. The locus-specific transcriptional control through the ISRE could be an important mechanism in the differential regulation of classical and nonclassical MHC class I expression, which determines adequate Ag presentation upon pathogenic challenge.

Novel mutations within the RFX-B gene and partial rescue of MHC and related genes through exogenous class II transactivator in RFX-B-deficient cells.

MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific regulatory factors. Fibroblasts derived from two recently identified bare lymphocyte syndrome patients, EBA and FZA, were found to contain novel mutations in the RFX-B gene. RFX-B encodes a component of the RFX transcription factor that functions in the assembly of multiple transcription factors on MHC class II promoters. Unlike RFX5- and RFXAP-deficient cells, transfection of exogenous class II transactivator (CIITA) into these RFX-B-deficient fibroblasts resulted in the induction of HLA-DR and HLA-DP and, to a lesser extent, HLA-DQ. Similarly, CIITA-mediated induction of MHC class I, beta2-microglobulin, and invariant chain genes was also found in these RFX-B-deficient fibroblasts. Expression of wild-type RFX-B completely reverted the noted deficiencies in these cells. Transfection of CIITA into Ramia cells, a B cell line that does not produce a stable RFX-B mRNA, resulted in induction of an MHC class II reporter, suggesting that CIITA overexpression may partially override the RFX-B defect.

Role of the interferon-stimulated response element in HLA-B downregulation in human melanoma cell lines.

Tumor cells are thought to escape immune surveillance from T cells by suppressing expression of major histocompatibility complex (MHC) class I molecules at their cell surface. Human MHC class I molecules are encoded by three different loci (HLA-A, -B, and -C). In primary human melanomas as well as melanoma cell lines, HLA class I expression is frequently downregulated in a B locus-specific manner. To study the involvement of promoter elements in HLA-B locus-specific downregulation, a series of reporter constructs containing 5'-flanking sequences of the HLA-A2 and -B7 genes were transfected into melanoma cell lines expressing high and low levels of HLA-B antigens. It is shown that enhancer A, which is generally believed to be a potent enhancer in HLA class I gene transcription, only weakly activates transcription in melanoma cell lines. In contrast, the interferon-stimulated response element (ISRE), known to induce MHC class I expression in response to IFNs, as well as a region comprising site alpha/enhancer B significantly stimulate constitutive transcription of HLA class I genes. Although none of the promoter elements tested could be demonstrated to mediate HLA-B locus-specific downregulation, high and low HLA-B melanoma cell lines do differ in ISRE activity as well as in ISRE-binding nuclear factors. The finding that high and low HLA-B melanoma cell lines contain different transcription factors binding to elements not actively involved in the process of HLA-B locus abrogation suggests that these cell lines originate from distinct types of melanocyte precursor cells expressing a different set of transcription factors.