Immune senescence: significance of the stromal microenvironment.

The immune system undergoes age-associated changes known as immunosenescence, resulting in increased susceptibility to infections, cancers and autoimmunity in the aged. The basis of our understanding of immunosenescence has been derived primarily from studies examining intrinsic defects within many of the cells of the immune system. While these studies have provided insight into the mechanisms of immunosenescence, a picture is now emerging that the stromal microenvironment within lymphoid organs also contributes significantly to the age-associated decline of immune function. These extrinsic defects appear to impact the functional activity of immune cells and may offer a potential target to recover immune activity. Indeed, rejuvenation studies which have targeted the stromal niche have restored immune function in aged successfully, highlighting the impact of the microenvironment towards the aetiology of immunosenescence.

The effect of age on the phenotype and function of developing thymocytes.

The immune system declines with age leading to a progressive deterioration in the ability to respond to infection and vaccination. Age-associated thymic involution is one of the most recognized changes in the ageing immune system and is believed to be a major contributor towards immunosenescence; however, the precise mechanisms involved in age-associated thymic involution remain unclear. In order to gain further insight into the effect of ageing on T-cell development, steady-state thymopoiesis was studied in mice ranging from 1 to 18 months of age. There was a decrease in thymic cellularity with age, but the most dramatic loss occurred early in life. Although there were no alterations in the proportion of the major thymocyte subsets, there was a significant decline in the expression of other key molecules including CD3 and CD24. There was a decline in the ability of thymocytes from older mice to respond to mitogens, which was demonstrated by a failure to up-regulate expression of the activation marker CD69 and to enter the G(2)--M phase of the cell cycle. This was concurrent with an increased resistance to apoptosis in thymocytes from aged animals. Together, these results suggest that T cells may be flawed even before exiting to the periphery and that this could contribute to the age-associated decline in immune function.

Truncation of the Mll gene in exon 5 by gene targeting leads to early preimplantation lethality of homozygous embryos.

The mixed lineage leukemia gene (MLL) was originally identified through its involvement in reciprocal translocations in leukemias. MLL codes for a large multidomain protein and bears homology to the Drosophila developmental control gene trithorax in two small domains in the amino terminal region, the central zinc finger domain and the carboxy SET domain. Like the Drosophila trx, MLL has also been shown to be a positive regulator of Hox gene expression. We have targeted Mll (the murine homologue of MLL) in exon 5 causing expression of three truncated in-frame Mll transcripts. These transcripts retain all or some of the AT hook motifs and the DMT domain. This mutant allele causes early in vivo preimplantation lethality of homozygous embryos prior to the 2-cell stage. Embryos cultured in vitro progress to the 2-cell stage, but further development is arrested. The heterozygotes exhibit mild skeletal defects as well as defects in some neuroectodermal derivatives.

Phage display technology : protocols.

Over the past few years, considerable effort has gone into genetic engineering and manipulation of antibody molecules. One consequence of this research has been the use of filamentous phage as a vehicle on which to display antibody fragments. It is possible to express a variety of molecules on the surface of phage, including peptides and antibody fragments. With regard to antibodies, libraries of both sFv and Fab fragments have been successfully cloned onto the surface of bacteriophages, usually via the N terminal of the gene III product, although expression on the gene VIII product has also been employed to isolate low-affinity antibodies.

Intrathymic function of the human cortical epithelial cell surface antigen gp200-MR6: single-chain antibodies to evolutionarily conserved determinants disrupt mouse thymus development.

The mouse monoclonal antibody MR6 recognizes a 200 000 MW protein (gp200-MR6), which is expressed highly on human thymic cortical epithelial cells. The antigen is also expressed on some epithelial tumours and we have previously shown that MR6 inhibits the proliferation of the colon carcinoma cell lines HT29. However, the role of this molecule in the thymus is not known. In order to generate reagents that could be used in murine thymic functional studies we isolated antibodies specific to human gp200-MR6, using a phage display library expressing single-chain (sFv) antibodies. Three independent clones were isolated by panning with purified protein and their specificity was confirmed by immunohistochemistry, Western blotting and flow cytometry. In addition to human thymus, these phage antibodies also recognized the homologous antigen in mouse, pig and other species. Expressed as soluble sFv one of these clones inhibited the proliferation of HT29 cells and a mouse thymic epithelial cell line, suggesting that this antibody exhibits similar functional activity to MR6. In fetal thymic organ culture, thymocytes recovered from thymic lobes cultured in the presence of this sFv, were reduced in number fivefold compared with the control and the majority remained at the double-negative stage of development. These data indicate that gp200-MR6 plays an important role in thymocyte development. In addition, this is the first report to demonstrate that specific sFv can be used to study, and alter, thymic development. This work also highlights the advantage of phage antibody technology in selecting such reagents for functional assays.

The human thymic microenvironment: new approaches to functional analysis.

Although immunohistological techniques have revealed molecular niches within the human thymic microenvironment, functional analysis is limited by the assay systems available. However, our recent studies using antibody phage display have revealed a high degree of molecular conservation between the microenvironmental molecules of different species. We have therefore isolated single-chain antibodies specific for evolutionarily conserved epitopes, shared by human and mouse, and used these in murine thymic functional assays. The detection of such shared human/rodent molecules makes it possible to functionally 'walk' from mouse to man, providing us with an experimental approach to functional analysis of the human thymus.

The gp200-MR6 molecule which is functionally associated with the IL-4 receptor modulates B cell phenotype and is a novel member of the human macrophage mannose receptor family.

The human gp200-MR6 molecule has previously been shown to have either an antagonistic or agonistic effect on IL-4 function, demonstrated by inhibition of IL-4-induced proliferation of T cells or mimicking of IL-4-induced maturation of epithelium, respectively. We now show that gp200-MR6 ligation can also mimic IL-4 and have an anti-proliferative pro-maturational influence within the immune system, causing up-regulation of co-stimulatory molecules on B lymphocytes. Biochemical analysis and cDNA cloning reveal that gp200-MR6 belongs to the human macrophage mannose receptor family of multidomain molecules. It comprises 1722 amino acids in toto (mature protein, 1695 amino acids; signal sequence, 27 amino acids) organized into 12 external domains (an N-terminal cysteine-rich domain, a fibronectin type II domain and 10 C-type carbohydrate recognition domains), a transmembrane region and a small cytoplasmic C terminus (31 amino acids) containing a single tyrosine residue (Y1679), but no obvious kinase domain. Strong amino acid sequence identity (77%) suggests that gp200-MR6 is the human homologue of the murine DEC-205, indicating that this molecule has much wider functional activity than its classical endocytic role. We also show that the gp200-MR6 molecule is closely associated with tyrosine kinase activity; the link between gp200-MR6 and the IL-4 receptor may therefore be via intracellular signaling pathways, with multifunctionality residing in its extracellular multidomain structure.

Characterization of a recent retroposon insertion on mouse chromosome 2 and localization of the cognate parental gene to chromosome 11.

The genomic sequence on mouse Chromosome (Chr) 2 corresponding to a previously identified novel cDNA has been characterized. The genomic organization of this locus, adjacent to the beta 2 microglobulin gene, has the properties of a processed gene or retroposon including the presence of a short flanking direct repeat, a polyadenylation signal/poly A tract, and the absence of introns. Analysis of inbred and wild-derived Mus DNAs reveals the retroposon to be a feature only of M. m. domesticus subspecies, suggesting that the insertion event is relatively recent. This notion is supported by the presence of an open reading frame and the lack of sequence divergence in the flanking direct repeats. The complex chromatin configuration characteristic of this region in mouse and human is not, therefore, related to this cDNA. The cognate parental gene encoding the cDNA was mapped to Chr 11. A further, more ancient retroposon present in many Mus species localizes to Chr 17.

Selection of antibodies to cell surface determinants on mouse thymic epithelial cells using a phage display library.

The network of thymic epithelium contributes significantly to the thymic stromal cell environment, which plays a vital role in the generation and maturation of thymocytes. Monoclonal antibodies (mAb) have revealed considerable heterogeneity within this epithelial component of the mouse thymic microenvironment, but many of these antibodies recognize epitopes that are located inside the cell and so cannot be used in functional studies. As an alternative approach to isolate antibodies specific to thymic epithelium, we used a phage display library expressing single chain Fv antibodies. For selection, a thymic cell suspension was incubated with the phage display library, and major histocompatibility complex class II positive cells, the majority of which are epithelial, were then specifically selected. Phage bound to these cells were eluted and the selection procedure was repeated for a further five rounds. Immunohistochemical analysis revealed that these phage antibodies show differential staining of thymic epithelial subsets. Flow cytometric analysis of a thymic epithelial cell line using a panel of these antibodies demonstrated that they recognize epitopes on the cell surface. Furthermore, some of these antibodies also labelled human thymic epithelium, suggesting that the epitopes recognized by these antibodies are conserved between human and rodent thymus. Our approach therefore provides a rapid method to select antibodies specific for thymic epithelial cell surface determinants in their native configuration.

Analysis of T cell repertoire and function in mice transgenic for the human V beta 3 TCR.

We have constructed mice containing the human V beta 3 TCR gene from the influenza virus haemagglutinin specific human CD4+ T cell clone HA1.7. Similar cell yields were obtained from transgenic and non-transgenic lymphoid tissue, with normal levels of T cells and with no unusual bias of the CD4 or CD8 subpopulations. Immunostaining and FACS analysis of transgenic thymocytes, spleen, and mesenteric lymph nodes revealed that the majority of T cells expressed the human V beta 3 TCR on the cell surface. Small numbers of cells expressing murine TCR beta chain were also detected. Polymerase chain reaction analysis revealed that an extensive V alpha TCR repertoire was used in the human V beta 3 transgenic mice. Lymphocytes from the spleen and mesenteric lymph nodes of transgenic mice were assessed for functional activity in vitro. Isolated cells were stimulated with mitogen or superantigen, as well as directly through the TCR-CD3 complex, and their ability to proliferate and secrete lymphokines analysed. Cells from transgenic mice responded well after stimulation with phytohaemagglutinin, concanavalin A, anti-CD3 antibody, anti-CD3 antibody with phorbol ester, and Staphylococcus aureus enterotoxin B, and also showed alloreactivity in a mixed lymphocyte reaction. Minimal levels of response were detected after stimulation with murine TCR beta antibody. Together, these data suggest that a human TCR beta chain is able to associate with a murine TCR alpha chain, to form a fully functional surface TCR-CD3 complex.

Is TCR beta expression an essential event in early thymocyte development?

The evolution of a T cell through the thymus is characterized by several distinct morphological changes. The underlying mechanisms allowing a double negative thymocyte to mature to a double positive one expressing both CD4 and CD8 are still hazy. Here, Donald Palmer, Adrian Hayday and Michael Owen discuss the latest development in the field and data that underscore the crucial role of TCR components in the transition of double negative to double positive thymocytes.

Expression of the alpha beta T-cell receptor is necessary for the generation of the thymic medulla.

The architecture of the thymus of mice that congenitally fail to express the alpha beta T-cell receptor (TCR alpha beta) has been examined by immunohistology. In these mice, a defined mutation was introduced into the TCR alpha gene by homologous recombination. By using antibodies specific for cortical or medullary epithelium and for major histocompatibility complex antigens, the network of cortical epithelium in these mice was shown to be essentially unaltered in comparison with that of normal mice. In contrast, the thymic medulla was considerably reduced in size. This analysis shows that expression of the alpha beta TCR but not the gamma delta TCR is obligatory for establishing the thymic medulla and suggests that the growth of medullary epithelial cells may require contact with TCR alpha beta-expressing cells.

The chromatin structure of the mouse beta-2-microglobulin locus.

Within the promoter regions of both major histocompatibility complex (MHC) class I genes and the beta 2-microglobulin (beta 2m) gene, there are a number of common regulatory elements suggesting co-ordinate control. However, there is also evidence to suggest that beta 2m and class I are differentially regulated, indicating that these genes may have distinct regulatory elements. We sought to explore this question by analysing DNase I hypersensitive (DH) sites flanking the beta 2m gene. Five DH sites have been found within the vicinity of the beta 2m gene. One of these sites (DH1) located within the promoter region, correlates with the transcriptional activity of beta 2m since it is weak in embryonal (beta 2m negative) cell lines. The remaining DH sites (2-5) are located downstream of the beta 2m gene. The most proximal downstream site, (DH2) located 5.5 kb from the last exon, was observed only in embryonal cell lines, indicating possible involvement in the downregulation of beta 2m. Furthermore, this site is markedly diminished in differentiated F9 cells. Possible roles for the remaining sites are discussed, in particular relationship to a second transcriptional unit identified in the vicinity. In addition, a similar analysis reveals a cluster of DH sites located downstream from the last exon of the human beta 2m gene.

Evidence that the intracellular effects of adenosine in the guinea-pig aorta are mediated by inosine.

Previous studies have demonstrated that high concentrations of adenosine interact with both a cell surface receptor and with an intracellular site to evoke relaxation of the guinea-pig aorta. The intracellular action of adenosine was investigated in the present study. The purine sensitive 'P-site' did not appear to be involved since other P-site agonists did not consistently evoke relaxation. A major interaction with intracellular S-adenosylhomocysteine hydrolase also appeared unlikely since 1-homocysteine had only minor effects on adenosine-evoked responses. Inhibition of adenosine deaminase attenuated responses evoked by high concentrations of adenosine. The deaminated metabolite of adenosine, inosine, also evoked aortic relaxation. These responses were mediated solely via an intracellular site since they were blocked by an inhibitor of nucleoside-facilitated diffusion but were unaffected by an adenosine receptor antagonist. These results indicate that a major part of the intracellular effect of adenosine is mediated by its deaminated metabolite inosine.

Comparison of the potency of 8-phenyltheophylline as an antagonist at A1 and A2 adenosine receptors in atria and aorta from the guinea-pig.

The potency of 8-phenyltheophylline as an antagonist at A1 adenosine receptors in guinea-pig atria and at A2 adenosine receptors in the guinea-pig aorta has been investigated. 8-Phenyltheophylline was an apparently competitive antagonist of the negative chronotropic effect of adenosine, 2-chloroadenosine, L-N6-phenyl-isopropyl adenosine (L-PIA) and 5'-N-ethylcarboxamide adenosine (NECA) on atria and of the relaxant effect of adenosine, 2-chloroadenosine and NECA on the aorta. The pA2 values for 8-phenyltheophylline ranged from 6.4 to 6.6 and were not significantly different, irrespective of the agonist or tissue used. These results indicate that 8-phenyltheophylline is a relatively potent antagonist at adenosine receptors but does not exhibit selectivity for either of the putative sub-types in isolated tissues.

Hyperphagocytosis and the effect of lipopolysaccharide injection in tumour-bearing mice.

(AxT6)F1 hybrid mice received s.c. transplants from (AxT6)F1 mammary carcinomas. At 1, 2 or 4 weeks after tumour transplantation, the mice were bled to obtain plasma and then challenged with 25 micron E. coli lipopolysaccharide (LPS) endotoxin i.v. The mice were killed 24 hr later, further plasma was obtained and their liver ratios and spleen ratios were determined. A similar procedure was carried out on non-tumour-bearing mice. Progressive tumour growth was associated with an increase in the liver ratio. In parallel, mice with 4-week tumour transplant showed increased uptake of colloidal carbon particles and 51Cr-labelled sheep red blood cells in the liver. The plasma amino aspartate transaminase (AST) and the ornithine carbamoyl transferase (OCT) showed a constant rise in all groups of mice after LPS injection. However, at 24 hr after LPS injection, the AST level showed the greatest rise in mice with 4-week tumour transplants. By contrast, OCT, which is liberated only from hepatocytes, showed the greatest rise in non-tumour-bearing mice.