Mary A. Ritter

The thymic microenvironment

Abstract The thymus has been recognized as a site of primary immune function for more than three decades and yet, despite intense research, many of the central questions regarding its function in T-lymphocyte maturation have remained unanswered. Current cellular and molecular analytical techniques are finally proving to be sophisticated enough to define the cells which comprise the thymic framework and dissect their complex interactions with developing thymocytes. In a series of articles over the coming months Immunology Today will be investigating the complex interrelationship between the thymic microenvironment and the developing T cell. As a foundation for these, Richard Boyd and colleagues provide a detailed overview of the basic thymic components, backed up by an extensive bibliography and full colour poster. The latest research trends are then summarized by Donald Palmer, Adrian Hayday and Michael Owen, followed by an examination of the mutual interdependence of thymic stroma and thymocyte by Mary Ritter and Richard Boyd.

Thymic involution with ageing: obsolescence or good housekeeping?

Abstract The thymus undergoes premature ageing in comparison with other organs of the immune system, with involution starting soon after birth and continuing throughout life. Does this reflect a failure of repair or is the process of adaptive value? Here, Andrew George and Mary Ritter apply a cost-benefit analysis, and conclude that the scaling down of this organ has been evolutionarily advantageous to our ancestors.

Development in the thymus: it takes two to tango

Intrathymic T-cell development is dependent upon signals provided by the thymic stromal cell microenvironment. However, loss of thymic T cells in natural and experimentally induced situations is associated with a reduction in the surrounding epithelium, suggesting an interdependence between thymocytes and their microenvironment. Here, the authors review the evidence in favour of this intrathymic symbiosis, and hypothesize that T cells may provide maturation and survival signals that are necessary for the development and maintenance of their microenvironment.

CD205 (DEC-205): A recognition receptor for apoptotic and necrotic self

CD205 is an endocytic receptor that is expressed at high levels by cortical thymic epithelial cells and by dendritic cell (DC) subsets, including the splenic CD8+ DC population that is responsible for cross-presentation of apoptotic cell-derived antigens. Antigen endocytosed via CD205 enters the MHC class I and MHC class II antigen presentation pathways and is subsequently presented to both CD4+ and CD8+ T cells. Despite the known role of CD205 in antigen uptake, the nature of the ligands bound by CD205 has not been determined, and most studies have relied on the use of monoclonal antibodies as surrogate ligands. To go beyond this approach, we created a panel of CD205–IgG fusion proteins spanning the extracellular portion of CD205 and used these to identify the physiological distribution of CD205 ligands. Our data demonstrate that two areas of the CD205 molecule, within C-type lectin-like domains (CTLDs) 3 + 4 and 9 + 10, recognise ligands expressed during apoptosis and necrosis of multiple cell types, and are additionally expressed by live cells of the dendritic cell line DC2.4. Thus, CD205 acts as a recognition receptor for dying cells, potentially providing an important pathway for the uptake of self-antigen in intrathymic and peripheral tolerance.

Importance of the linker in expression of single-chain Fv antibody fragments: optimisation of peptide sequence using phage display technology

We have investigated the potential for enhancing the production of functional single-chain Fv antibody fragments (sFv), by altering the sequence of the linker that joins the variable domains of the molecule. To identify new functionally improved linkers we have used a phage display library containing a random sequence of six amino acids in the linker. Multiple rounds of panning on the antigen led to the selection of six different linker sequences that enhanced the binding of phage to the antigen. Five of the linkers also improved the secretion of soluble sFv by approximately five-fold. Analysis of the predominant linker sequence isolated showed that this improvement is not due to an increased affinity for the antigen, nor to alterations in the toxicity to bacteria. However the linker did affect the denaturation of the sFv in urea. It is therefore possible that the novel sequence helps in the refolding or secretion of the molecule.

One for all and all for one: thymic epithelial stem cells and regeneration

It has long been believed that the thymic epithelial microenvironment originates from both the endodermal and ectodermal germ cell layers. However, a growing body of evidence indicates that such a dual origin is not the case, and that the diverse thymic epithelial populations all develop from a common epithelial stem cell. This article explores these data, investigates the identity of such cells and the signals that might control their expansion and differentiation, and considers the possibility of stem cell transplantation for thymic regeneration.

Altered expression and endocytic function of CD205 in human dendritic cells, and detection of a CD205–DCL-1 fusion protein upon dendritic cell maturation

CD205 (DEC‐205) is a member of the macrophage mannose receptor family of C‐type lectins. These molecules are known to mediate a wide variety of biological functions including the capture and internalization of ligands for subsequent processing and presentation by dendritic cells. Although its ligands await identification, the endocytic properties of CD205 make it an ideal target for those wishing to design vaccines and targeted immunotherapies. We present a detailed analysis of CD205 expression, distribution and endocytosis in human monocyte‐derived dendritic cells undergoing lipopolysaccharide‐induced maturation. Unlike other members of the macrophage mannose receptor family, CD205 was up‐regulated upon dendritic cell maturation. This increase was a result of de novo synthesis as well as a redistribution of molecules from endocytic compartments to the cell surface. Furthermore, the endocytic capacity of CD205 was abrogated and small amounts of the recently identified CD205–DCL‐1 fusion protein were detected in mature DC. Our results suggest that CD205 has two distinct functions – one as an endocytic receptor on immature dendritic cells and a second as a non‐endocytic molecule on mature dendritic cells – and further highlight its potential as an immuno‐modulatory target for vaccine and immunotherapy development.

Expression of The αβ 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 αβ T-cell receptor (TCRαβ) has been examined by immunohistology. In these mice, a defined mutation was introduced into the TCRc 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 αβ TCR but not the γδ TCR is obligatory for establishing the thymic medulla and suggests that the growth of medullary epithelial cells may require contact with TCRαβ-expressing cells.

In vivo selection of sFv from phage display libraries

The development of phage display technology has facilitated the development of many new and sometimes novel antibody based reagents for scientific research. However, present methods for selection from phage-sFv display libraries are limited to selection against purified antigens or ex vivo cells of known origin and phenotype. Existing methods therefore preclude the isolation of sFv against unknown molecules in their natural environment, where expression is complex and subject to diverse control mechanisms. Since such a complex environment is difficult to mimic in vitro, the development of an in vivo selection procedure would greatly enhance the selection from phage display antibody libraries and lead to the development of reagents against cell surface molecules in their natural environment. This would be particularly advantageous for isolation of sFv against vascular endothelium which can readily change phenotype when cultured and is believed to express molecules in a tissue specific manner and in response to different stimuli. We describe here the development of an in vivo selection procedure in the mouse and demonstrate its potential for the selection of sFv from a phage-sFv library. The target antigen for one sFv is expressed solely on the thymic endothelium, while the second, a 165-170 kDa molecule in present on both thymic endothelium and the perivascular epithelium.

Myasthenia gravis thymus: Clinical, histological and culture correlations

This paper attempts to quantitate immunohistological changes in the myasthenia gravis (MG) thymus and to correlate them with clinical and culture parameters in 40 untreated young onset patients covering a wide range of durations and serum anti-acetylcholine receptor (AChR) antibody titers. Total cellularities of both the thymic cortex and the medulla declined significantly with age. There was some hyperplasia of subcapsular and of medullary epithelial cells, often at the expense of cortex. A combined index of all hyperplastic changes correlated significantly with serum anti-AChR titre. Otherwise histological indices, e.g. of germinal centres (GC) were largely unrelated to any clinical parameters, especially duration of symptoms. Specific anti-AChR synthesis in culture (very closely related to serum titer) correlated better with the medullary lymph node-type T-cell areas; these were more widely prevalent and MG-specific. In contrast, basal and mitogen-stimulated total IgG productivity followed the GC indices more closely. We propose that the variability of GC is due to their dependence on extraneous immune complexes, and we discuss whether they or the T-cell areas are primary or secondary abnormalities. Finally, we conclude that autosensitization in MG with thymic hyperplasia and neoplasia probably arises through separate mechanisms.