Ultrastructural analysis of thymic nurse cell epithelium

Thymic nurse cells (TNC), a paradigmatic cell type of cortical epithelium, are large lymphoid-epithelial cell complexes of thymocytes enclosed within vacuoles lined by the epithelial cell membrane. TNC express major histocompatibility complex (MHC) class I and class II molecules on their surface and vacuole-lining membranes at high density and it was suggested that TNC provide an optimal microenvironment for positive selection of T cells. In this report we present electron microscopical data demonstrating that chicken TNC display morphological structures of exocytosis previously shown for hormone-secreting cells. In TNC, however, exocytosis is restricted to the capillary cleft between the epithelial cell and engulfed thymocytes. Thus, besides physical contact between the epithelial cell and enclosed thymocytes, TNC may additionally influence the development of thymocytes through release of soluble factors in a restricted microenvironment. By employing the 3-(2,4-dinitroanilino)-3-amino-N-methyl-propylamine technique which at the ultrastructural level detects acidic organelles involved in processing of antigens presented by MHC class II molecules, we also show that TNC contain acidic compartments similar to classical antigen-presenting cells, i.e. early and late endosomes and lysosomes, albeit in a lower amount than in thymic dendritic cells. This fact provides evidence that TNC not only are capable of antigen presentation but also possess the intracellular machinery for antigen processing.     

Thymic epithelial cells derived from the cultures of thymic nurse cells

Thymic epithelial cells are derived from the cultures of thymic nurse cells. These cultures are free from fibroblasts and macrophages. The epithelial nature of these cells is confirmed by demonstrating the presence of keratin filaments in them. These epithelial cells show heterogeneity in shape, size and distribution of keratin filaments. They contain nonspecific esterase(s) molecules and express both I-A and H-2K antigens.     

The limited immunocompetence of thymocytes within murine thymic nurse cells

Thymic nurse cells, cortical epithelial cells enclosing 20-200 lymphocytes, were prepared from mouse thymus by enzyme digestion and repetitive sedimentation. Individual nurse cells were then isolated free of any exogenous thymocytes by micromanipulation, and the endogeneous thymocytes released from inside the nurse cells by a brief period of culture. The thymocytes from within individual nurse cells were tested, at the one cell/well level, for their capacity to proliferate in high cloning efficiency mitogen-stimulated limiting dilution cultures. The resultant clones were tested for their cytolytic capacity in a lectin-mediated isotype-release assay. Most intra-nurse cell thymocytes were unresponsive, like typical cortical thymocytes, but an average of 1/30, or around 2-6 lymphocytes/nurse cell, were able to proliferate in response to concanavalin A. The clones produced were of a relatively small size, similar to those characteristic of helper-lineage T cells. No cytolytic clones at all were obtained, despite stringent positive controls showing an efficient cytolytic response from known sources of cytolytic precursor cells. This finding disagrees with earlier studies on nurse cell lymphocytes, where there may have been a possibility of contamination with exogenous thymocytes. These results suggest either that the nurse cell represents a selective environment for helper-lineage T cell differentiation, or that further steps after the nurse cell stage are needed to produce mature cytolytic-lineage T cells.     

Analysis of the thymic microenvironment by monoclonal antibodies with special reference to thymic nurse cells

Two hybridoma cell lines secreting monoclonal antibodies against stromal tissues of mouse thymus were produced using the spleen cells of BALB/C mice immunized with newborn thymic homogenate of C57BL/6 mice emulsified in Freund's complete adjuvant. The monoclonal antibody Th-3 reacted with stromal cells in the thymic cortex and the monoclonal antibody Th-4 reacted with stromal cells in the thymic medulla. The stromal cells revealed by Th-3 showed a meshwork structure in the cortex, and formed a monolayered border at the cortical surface and around the vasculature. Each mesh of this structure was connected to each other, forming a complex labyrinth and being open toward the medullary area. Neither lymphoid cells nor any cells in any other organs were reacted with this Th-3 antibody. However, the reactivity of Th-3 with the thymic cortical stromal cells was observed not only in C57BL/6 mice which had been used as source of antigen, but also in other strains of mice such as C3H and BALB/C. Immunoelectron microscopy revealed that Th-3 monoclonal antibody was reactive with some component, diffusely present in the cytoplasm of cortical epithelial cells. The pattern of Th-3 positive meshwork in the thymic cortex was quite similar to that stained by either anti-IA or anti-IE antibody, but the Th-3 positive reaction was not inhibited by these anti-IA and anti-IE antibodies. Thymic nurse cells prepared by the method of Wekerle were positive for Th-3 antibody. On the contrary, Th-4 reacted only with epithelial cells in the thymic medulla. It was suggested that Th-3 monoclonal antibody detected some antigen specific to so called thymic nurse cells.     

The murine thymic nurse cell: an isolated thymic microenvironment

The thymic nurse cell (TNC) consists of an epithelial cell enclosing lymphoid elements and is found in enzymic digests of the thymus. Although these structures have been implicated in the normal intrathymic development of T lymphocytes, little is known about the in situ structure of this unusual cell complex. In this study, various dyes were introduced into the intact thymus and their differential permeability was used to demonstrate that the TNC exists as a sealed structure in situ. The lymphocytes within the TNC were shown to be isolated from the general thymic environment. Preliminary studies on these lymphocytes and the physiology of their active release from individual, micromanipulated TNC in microcultures are reported.     

Thymic nurse cells and thymic repopulation after whole body sublethal irradiation in mice

Thymic Nurse Cells (TNCs) are lymphoepithelial complexes which are thought to play a role in the early stages of the intrathymic differentiation pathway. Therefore, their repopulation kinetics were analyzed in mice after sublethal whole-body irradiation. Changes of the number of TNCs per thymus were parallel with the evolution of the whole thymocyte population. Particularly, a first wave of TNCs restoration was followed by a secondary depletion and a final recovery. This suggests that TNCs restoration is related to the proliferating progeny of intrathymic radioresistant thymocytes. When normal bone marrow cells were grafted intravenously after irradiation, no secondary depletion was found. This pattern of restoration was obviously related to thymic repopulation by cells which were derived from the inoculated bone marrow. Homing studies with FITC labelled bone marrow cells showed that inoculated bone marrow cells did not penetrate TNCs early after irradiation. Later on, when immigrant cells started to proliferate, they were found preferentially within TNCs before spreading in the whole thymus. The results indicate that interactions between immature thymocytes and epithelial cells within TNCs are critical for the first steps of intrathymic lymphopoiesis.     

Helper T cell activity demonstrated by thymic nurse T cells (TNC-T).

Culture supernatants of mixed lymphocyte reactions (MLR) between AKR thymic nurse T cells (TNC-T) as responders and irradiated DBA/2 spleen cells as stimulators, induce significant proliferation of concanavalin A (Con A)-stimulated DBA/2 thymocytes. The culture supernatants of MLRs using extra-TNC thymocytes (ET) as responders do not show this activity, and the addition of thymus epithelium did not enable a response to take place. Again, unlike ET, TNC-T cells could also give a positive allogeneic response. Irradiated AKR TNC-T cells stimulated allogeneic BALB/c spleen B cells to produce immunoglobulin. These observations indicate that cells in the TNC-T pool are more mature than those in the ET pool.     

Effect of interferon-gamma and tumor necrosis factor-alpha on lymphoepithelial interactions within thymic nurse cells

Isolated thymic nurse cells (TNC) represent a specialized microenvironment in vivo where thymocytes interact specifically with subcapsular epithelial cells. They are thought to play a critical role in the process of T cell differentiation. We demonstrate that recombinant murine interferon-gamma and recombinant human tumor necrosis factor-alpha can act on these interactions: they stimulate TNC-derived epithelial cells to establish interactions with thymocytes in vitro and to form new lymphoepithelial complexes. This phenomenon is partially inhibited by anti-Ia monoclonal antibodies. Implications of these findings for normal intrathymic differentiation are discussed.     

Establishment of mouse thymic nurse cell clones from a spontaneous BALB/c thymic tumor

This is the first report on the establishment of the readily identifiable and functioning thymic nurse cell (TNC) clones from the mouse thymus. In the course of the culture of an epithelial cell line using a medium with a low concentration of Ca2+ from the spontaneous BALB/c thymic tumor, lymphocytes as well as thymic stromal cells which were not apparently typical epithelial cells by light microscopy seemed to grow relatively well in one flask. The culture medium was exchanged with regular Dulbecco's modified Eagle's medium in the third week, and from that flask TNC clones were established together with lymphoblast cell clones. The established cells (IT-79MTNC3) were easily identified as TNC. They formed complexes with simultaneously established lymphoblast cells and were remarkably similar to those of fresh TNC and thymocytes. Cloned TNC could express Ia antigens. In co-culture experiments, cloned TNC together with recombinant interleukin 2 (rIL 2) appeared to support the growth of fetal thymocytes which with rIL 2 alone failed to proliferate. The supernatant of IT-79MTNC3 was previously found to contain the growth factor for some T cell clones. It remains to be solved whether the TNC affects fetal thymocytes through direct contact or secretes an active growth-promoting factor. Experiments along this line are now in progress.     

Human scavenger receptor B1 is involved in recognition of apoptotic thymocytes by thymic nurse cells

Recognition and uptake of apoptotic cells by neighboring phagocytes is essential for the clearance of dying cells without accompanying inflammation or tissue damage. In the thymus, many apoptotic cells are generated in the process of negative selection, and both thymic macrophages (professional phagocytes) and nursing thymic epithelial cells (nursing TEC; nonprofessional phagocytes) recognize and ingest them. However the receptors responsible for this recognition and uptake have not been identified. In the present study, we have established a human nursing TEC line and examined the expression of several genes of the scavenger receptor family considered to be potential receptors for apoptotic cells. Human scavenger receptor-B1 (hSR-B1)/CLA-1, previously shown to recognize apoptotic cells, was strongly expressed in nursing TEC, whereas there was little or no expression of the other scavenger receptors tested: scavenger receptor class A, CD36, or CD68. Suppression of hSR-B1/CLA-1 expression using antisense oligonucleotides decreased the binding of apoptotic thymocytes to nursing TEC by more than 40%. These results indicate that hSR-B1/CLA-1 may play a major role in the clearance of apoptotic cells in the thymus, mediating the recognition and ingestion of apoptotic thymocytes by nursing TEC.     

Cell surface marker analysis of mouse thymic dendritic cells.

Cell surface markers of mouse thymic dendritic cells have been studied by flow cytometry after isolation by collagenase digestion, separation of the low-density cell fraction and differential adherence. The dendritic cell preparation had a purity of greater than 90%, the contaminating population being essentially composed of thymocytes, macrophages constituting less than 1%. Dendritic cells displayed high forward and low-intermediate side angle scatter, and expressed high levels of major histocompatibility complex (MHC) class I and class II molecules, the heat-stable antigen (HSA), the adhesion molecules Pgp-1 (CD44), LFA-1, ICAM-1 and low levels of Mac-1 and the leukocyte common antigen CD45. Thymic dendritic cells are negative for the stem cell antigen-2 (Sca-2), the B cell-specific form of CD45 (B220), the mouse macrophage markers Fc receptor and F4/80, and the granulocyte marker Gr-1. However, although they do not express the T cell markers Thy-1, CD2, CD3, CD4 and CD5, 20%-30% of dendritic cells are positive for the interleukin 2 receptor alpha chain (CD25), and about 30% express intermediate levels of CD8. These results are discussed with regard to the functional significance of the expression of CD8 by thymic dendritic cells, and the existence of different dendritic cell subpopulations in the murine thymus.     

Unbiased analysis, enrichment and purification of thymic stromal cells

The microenvironment of the thymus consists of functionally discrete niches composed of distinct stromal cell subsets. Clinically relevant changes affecting T-cell differentiation occur within these niches with age and injury caused by irradiation and chemotherapy treatments. The study of thymic stromal cells has been hampered by the technical difficulty in isolating significant numbers of this important population. Here we present an improved protocol for enzymatic isolation of stromal cells that enables comparative flow cytometric analyses and their purification for downstream cellular or molecular analysis. Fractions analyzed throughout enzymatic digestion of the thymus revealed that various stromal subsets are isolated at characteristic intervals. This highlights the importance of pooling all cells isolated from the thymus for numerical and phenotypic analysis to avoid biased representation of subpopulations. We also describe refined magnetic bead separation techniques that yield almost pure preparations of CD45(-) stroma. Sorting of these suspensions using defined markers enabled purification of the major epithelial subsets, confirmed by keratin staining and PCR analysis. This three-step procedure represents a rapid, reproducible method for the unbiased purification of the stromal cells that direct thymic T-cell differentiation.     

The human thymus microenvironment: in vivo identification of thymic nurse cells and other antigenically-distinct subpopulations of epithelial cells.

We have studied the human thymus microenvironment in order to identify subsets of cells that may be responsible for the induction of different aspects of T-lymphocyte differentiation, education and MHC restriction. Using immunofluorescence on tissue sections and cell suspensions we have found MHC products (HLA-A, B, C and DR) to be present throughout the thymus epithelium whilst human T-cell antigens are absent from all non-lymphoid cells. In contrast, Thy-1 antigen (expressed on approximately 1% paediatric human thymocytes) has a differential expression amongst thymic epithelial cells, being confined to those in the subcapsular cortex and to 'thymic nurse cells' (TNC). The former represent the site to which thymocyte precursors first migrate upon entering the thymus. The latter are large epithelial cells, located within the cortex, whose plasma membrane totally enclose a number of thymus lymphocytes; these cells are therefore good candidates for the mediators of direct contact (stromal) induced thymocyte maturation.     

Demonstration of keratin filaments in thymic nurse cells (TNC) and alloreactivity of TNC-T cell population

In vitro cultured epithelial reticular cells from foetal thymic explants and thymic nurse cells (TNC) demonstrate the presence of keratin filaments in the cytoplasm. Keratin-positive cells of cultured thymic epithelium are heterogeneous in size and shape as are TNC. T cells inside the nurse cells (TNC-T) ooze out after 18 h of incubation at 37 degrees C. The alloreactivity of these T cells was compared with that of thymocytes and lymph node (LN) cells in a miniaturised mixed lymphocyte reaction using hanging drops set up in Terasaki plates. AKR TNC-T cells and LN cells showed significant proliferative response when stimulated by BALB/c LN cells. No significant proliferative response was shown by non-TNC thymocytes. The data suggest that TNC harbour a mature sample of the T-cell population as judged by alloreactivity.     

Heterogeneity of thymic dendritic cells

Thymus is the site of generation and selection of T-lymphocytes. It also contains phenotypically and functionally distinct dendritic cell (DC) populations, including conventional DC (cDC) and plasmacytoid DC (pDC). Thymic cDC are heterogeneous and contain two subsets: a major subset derived from the precursors within thymus, and a minor subset presumably of extrathymic origin. Increasing evidence suggest that thymic cDC can cross-present self-antigens to developing thymocytes and play an important role in thymocyte negative selection and central tolerance induction. Thymic pDC can produce type-I interferon upon appropriate activation. However, their role in a steady state thymus is currently unclear.     

Establishment of a murine thymic epithelial cell line capable of inducing both thymic nurse cell formation and thymocyte apoptosis

A thymic epithelial cell (TEC) line (B/c. TEC-L1) was established from a normal thymus of a 4-week BALB/c mouse. The B/c. TEC-L1 had an epithelial morphology showing a contact-inhibited cobblestone-like arrangement with occasional desmosome-like structures at the adjacent cellular membranes. B/c.TEC-L1 cells showed positive staining for desmosomal glycoprotein, cytokeratin, thymosin alpha 1 beta 3, and I-Ad, and MHC class I antigens. The doubling time was 24 hours, and the chromosome number ranged from 52 to 78 with the mode of 70. Coculture of B/c.TEC-L1 cells with syngeneic, peanut agglutinin-agglutinated (PNA+) thymocytes in suspension at 37 degree C was followed by the formation of TEC thymocyte rosettes, after which the reconstitution of thymic nurse cells ensued. At 4 degrees C, PNA+ thymocytes bound to the B/c.TEC-L1 cell but did not form thymic nurse cells. PNA- thymocytes, although to a lesser degree than PNA+ cells, bound to the TECs at 37 degrees C, but at 4 degrees C few cells bound to the TECs. Allogeneic thymocytes also bound to the TECs at 37 degrees C. When the PNA+ thymocytes were cultured on the B/c.TEC-L1 monolayer, the small ones chiefly adhered on the surface of the TECs, while underneath the TECs the relatively large thymocytes (including cells in mitosis) predominated. Although the PNA- thymocytes bound to the surface of the monolayer within a few hours after coculture, by 24 hours nearly all cells disappeared. It is presumed that the thymocytes creeping underneath the B/c.TEC-L1 monolayer and those enveloped within the thymic nurse cell reconstituted in the suspension culture; both may be placed in circumstances analogous to the thymic microenvironment, wherein immature thymocytes appear to contact TECs directly and to be exposed to higher concentrations of thymic hormones and other soluble factors. Additionally, cell death in the PNA+ thymocytes was also observed in the coculture with B/c.TEC-L1 cells. The PNA+ cells revealed the morphological changes termed "apoptosis" characterized by chromatin condensation and nuclear fragmentation.     

Murine thymic plasmacytoid dendritic cells

We report herein heterogeneous murine thymic cell subsets expressing CD11c and B220 (CD45R). The CD11c(+)B220(+) subset expresses Ly6C(high) and MHC class II(low) in contrast with previously described thymic DC (CD11c(+)B220(-) cells). Freshly isolated thymic CD11c(+)B220(+) cells show typical plasmacytoid morphology which differentiates to mature DC, in vitro with CpG oligodeoxynucleotides (ODN) 2216; we term this subset thymic plasmacytoid DC (pDC). These thymic pDC are highly sensitive to spontaneous apoptosis in vitro and induce low T cell allo-proliferation activity. Thymic pDC express low TLR2, TLR3 and TLR4 mRNA, normally found on human immature DC, and high TLR7 and TLR9 mRNA, normally found on human pDC. Thymic pDC also produce high amounts of IFN-alpha following culture with CpG ODN 2216 (TLR9 ligands) as compared with the previously defined thymic DC lineage which expresses low TLR9 mRNA and produce high IL-12 (p40) with CpG ODN 2216. These results indicate that thymic pDC are similar to IFN-producing cells as well as human pDC. The TLR and cytokine production profiles are consistent with a nomenclature of pDC. The repertoire of this cell lineage to TLR9 ligands demonstrate that such responses are determined not only by the quantity of expression, but also cell lineage.     

Medullary thymic epithelial stem cells: role in thymic epithelial cell maintenance and thymic involution

The thymus consists of two distinct anatomical regions, the cortex and the medulla; medullary thymic epithelial cells (mTECs) play a crucial role in establishing central T-cell tolerance for self-antigens. Although the understanding of mTEC development in thymic organogenesis as well as the regulation of their differentiation and maturation has improved, the mechanisms of postnatal maintenance remain poorly understood. This issue has a central importance in immune homeostasis and physiological thymic involution as well as autoimmune disorders in various clinicopathological settings. Recently, several reports have demonstrated the existence of TEC stem or progenitor cells in the postnatal thymus, which are either bipotent or unipotent. We identified stem cells specified for mTEC-lineage that are generated in the thymic ontogeny and may sustain mTEC regeneration and lifelong central T-cell self-tolerance. This finding suggested that the thymic medulla is maintained autonomously by its own stem cells. Although several issues, including the relationship with other putative TEC stem/progenitors, remain unclear, further examination of mTEC stem cells (mTECSCs) and their regulatory mechanisms may contribute to the understanding of postnatal immune homeostasis. Possible relationships between decline of mTECSC activity and early thymic involution as well as various autoimmune disorders are discussed.