Unilateral T cell maturation arrest in the thymus of CBA/H mice as a long-term effect after neutron irradiation

Thymuses of CBA/H mice were investigated up to 570 days after whole-body irradiation with 2.5 Gy fast fission neutrons or 6.0 Gy X rays. A number of these thymuses, observed 220-270 days after neutron irradiation, have two equal sized lobes, one of which has an abnormal T cell distribution. The present paper reports on the distribution of lymphoid and stromal cell types in these thymuses. For this purpose, we employed immunohistology using the indirect immunoperoxidase method. We incubated frozen sections of these aberrant thymuses with monoclonal antibodies directed to cell surface differentiation antigens on lymphoid cells, such as Thy-1, T-200, MT-4, Lyt-1, Lyt-2, and MEL-14; monoclonal antibodies directed to major histocompatibility complex (MHC) antigens, such as I-A and H-2K; and monoclonal antibodies directed to determinants in various thymic stromal cell types. The results of this study show a T cell differentiation arrest in only one of the two thymic lobes. T cells in the aberrant lobe express Thy-1, T-200, and MEL-14 antigens but are MT-4- and Lyt-1-. In some lobes, a weak Lyt-2 expression was observed. The observed T cell maturation arrest is mainly restricted to the cortex since in the medulla, in addition to cells with an aberrant cortical phenotype, normal T cell phenotypes are observed. This indicates that cortex and medulla have independent generation kinetics in T cell maturation. The stromal cell composition in these abnormal lobes is not different from that in the normal lobe, but the size of the medulla tends to be smaller. Furthermore, the I-A expression on the cortical epithelial cells does not reveal the characteristic reticular staining pattern that is observed in the normal lobe, since the I-A determinants are not strictly confined to the epithelial cells. In addition, cortical lymphoid and stromal cells in these lobes are slightly H-2K+. These alterations in MHC expression in the cortex are discussed in relation to the observed T cell maturation arrest.     

Collateral presentation of antigens as physiological prototype for lymph node metastases

The formation of lymphogenic metastases remains enigmatic. In particular, the much more pronounced predilection of carcinomas than of sarcomas to metastasizing into regional lymph nodes is an unsolved problem. We suggest that this difference could be due to the ability of epitheliocytes for a hypothetical process termed by us “collateral presentation of antigens”. Under conditions of infection of epithelium with intracellular pathogens or during inflammation, epithelial cells acquire a special receptor phenotype, undergo the epithelial-mesenchymal transition, and migrate along lymphatic vessels into lymph nodes where they present antigen to immunocytes. The collateral presentation of antigens can be of significant biological importance in the case of insufficient classical pathway of antigen presentation (by dendritic cells) or on disturbance in the death mechanisms of the infected cells. Depending on conditions of induction of the epithelial-mesenchymal transition and on possible ability of epitheliocytes to express MHC II with co-stimulating molecules, two pathways, “container-mediated” and “MHC II-dependent”, of antigen presentation in lymph nodes resulting in development of immunogenesis or anergy of immunocytes are supposed to exist. All pathways of delivery of the epithelial cells into lymph nodes and of antigen presentation by epitheliocytes terminate by death of these cells. The lymphogenic metastasizing realizes the same mechanism under conditions of tumor disease; however, this is not associated with cell death, but they actively colonize the lymph node. The proposed hypothesis allows us to explain the metastasizing of sarcomas into lymph nodes. The main prerequisite for lymphogenic metastasizing seems to be related with the mesenchymal-epithelial transition of sarcoma cells promoting their involvement in the presentation of antigens.     

CD1 and major histocompatibility complex II molecules follow a different course during dendritic cell maturation

The maturation of dendritic cells is accompanied by the redistribution of major histocompatibility complex (MHC) class II molecules from the lysosomal MHC class II compartment to the plasma membrane to mediate presentation of peptide antigens. Besides MHC molecules, dendritic cells also express CD1 molecules that mediate presentation of lipid antigens. Herein, we show that in human monocyte-derived dendritic cells, unlike MHC class II, the steady-state distribution of lysosomal CD1b and CD1c isoforms was unperturbed in response to lipopolysaccharide-induced maturation. However, the lysosomes in these cells underwent a dramatic reorganization into electron dense tubules with altered lysosomal protein composition. These structures matured into novel and morphologically unique compartments, here termed mature dendritic cell lysosomes (MDL). Furthermore, we show that upon activation mature dendritic cells do not lose their ability of efficient clathrin-mediated endocytosis as demonstrated for CD1b and transferrin receptor molecules. Thus, the constitutive endocytosis of CD1b molecules and the differential sorting of MHC class II from lysosomes separate peptide- and lipid antigen-presenting molecules during dendritic cell maturation.     

Cortical thymocyte differentiation in thymomas: an immunohistologic analysis of the pathologic microenvironment

Four monoclonal antibodies (BH11, T2/30, AG3, and BC3) were produced against different epithelial components of the normal thymus. An immunohistologic study was performed on 13 thymomas by the use of these and other stromal and lymphocyte-specific reagents. The aim of this study was to find possible relationships between the proliferating thymoma epithelial cell type and the T cell composition of thymomas. Our results indicate that cortical T cell differentiation is present in thymomas, and that this differentiation is induced in the absence of detectable levels of MHC class II antigens on the epithelial component in most cases. The role of the MHC class II antigens cannot be excluded, however, because these antigens were always present on macrophages. Analysis of the selected group of thymomas, each of which contained epithelial cells homogeneously stained by at least one of the described monoclonal antibodies, showed that the cortical type T cell inducer capacity of thymomas is independent of the epithelial type predominant in the tumor.     

Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells

In order for cytotoxic T cells to initiate immune responses, peptides derived from internalized antigens must be presented to the cytotoxic T cells on major histocompatibility complex (MHC) class I molecules. Here we show that dendritic cells, the only antigen-presenting cells that initiate immune responses efficiently, have developed a unique membrane transport pathway linking the lumen of endocytic compartments and the cytosol. Endosome-to-cytosol transport is restricted to dendritic cells, specific to internalized antigens and selective for the size of the transported molecules. Thus, in dendritic cells, internalized antigens gain access to the cytosolic antigen-processing machinery and to the conventional MHC class I antigen-presentation pathway.     

MHC class II compartments in human dendritic cells undergo profound structural changes upon activation

Immature dendritic cells efficiently capture exogenous antigens in peripheral tissues. In an inflammatory environment, dendritic cells are activated and become highly competent antigen-presenting cells. Upon activation, they lose their ability for efficient endocytosis and gain capability to migrate to secondary lymphoid organs. In addition, peptide loading of MHC class II molecules is enhanced and MHC class II/peptide complexes are redistributed from an intracellular location to the plasma membrane. Using immuno-electron microscopy, we show that activation of human monocyte-derived dendritic cells induced striking modifications of the lysosomal multilaminar MHC class II compartments (MIICs), whereby electron-dense tubules and vesicles emerged from these compartments. Importantly, we observed that MHC class II expression in these tubules/vesicles transiently increased, while multilaminar MIICs showed a strongly reduced labeling of MHC class II molecules. This suggests that formation of the tubules/vesicles from multilaminar MIICs could be linked to transport of MHC class II from these compartments to the cell surface. Further characterization of endocytic organelles with lysosomal marker proteins, such as the novel dendritic cell-specific lysosomal protein DC-LAMP, HLA-DM and CD68, revealed differential sorting of these markers to the tubules and vesicles .     

Repopulation of the mouse thymus after sublethal fission neutron irradiation. II. Sequential changes in the thymic microenvironment

The stromal cells of the thymus of sham-irradiated and sublethal fission neutron-irradiated CBA/H mice were analyzed with immunohistology, using monoclonal antibodies directed to I-A and H-2K antigens as well as specific determinants for cortical and medullary stromal elements. In the control thymuses, I-A expression in the thymus shows a reticular staining pattern in the cortex and a confluent staining pattern in the medulla. In contrast, H-2K expression is mainly confluently located in the medulla. Whole body irradiation with 2.5 Gy fission neutrons reduces within 24 hr the cortex to a rim of vacuolized "nurse cell-like" epithelial cells, largely depleted of lymphoid cells. The localization of I-A antigens changes in the cortex and I-A determinants are no longer associated with or localized on epithelial reticular cells. Medullary stromal cells, however, are more or less unaffected. A high rate of phagocytosis is observed during the first 3 days after irradiation. About 5 days after irradiation, the thymus becomes highly vascularized and lymphoid cells repopulate the cortex. The repopulation of the thymic cortex coincides with the appearance of a bright H-2K expression in the cortex which is associated with both stromal cells as well as lymphoid blasts. During the regeneration of the thymus, the thymic stromal architecture is restored before the expression of cell surface-associated reticular MHC staining patterns. The observed sequential changes in the thymic microenvironment are related to the lymphoid repopulation of the thymus.     

A modular and combinatorial view of the antigen cross-presentation pathway in dendritic cells

Major histocompatibility complex class I (MHC I) presentation of exogenous antigens (cross-presentation) by dendritic cells (DC) is essential for CD8 T-cell immunity. Most cells use MHC I molecules to present peptides derived from endogenous proteins processed in the cytosol by the proteasome. The resulting peptides are translocated into the endoplasmic reticulum for loading onto MHC I molecules, and these complexes are then transported to the cell surface. In cross-presenting DC, these steps have been proposed to occur along two major tracks. In the 'endocytic' track, exogenous antigen processing and loading occur within endosomal compartments, using MHC I molecules recycled from the plasma membrane and transported back to the surface. In the 'cytosolic' track, antigens are translocated from endosomes to the cytosol, accessing the endogenous MHC I presentation pathway. This dichotomy now appears too simplistic. Some steps may occur in locations belonging to the endosomal track and others in the cytosolic track, or in hybrid compartments combining features of both. We propose a 'modular' view of cross-presentation, whereby processing, loading and MHC I transport represent modules that can occur in one or more locations. Cross-presentation of each MHC I-peptide complex may result from combining one or more options for each of these modules.     

Cathepsin S controls MHC class II-mediated antigen presentation by epithelial cells in vivo

Epithelial cells at environmental interfaces provide protection from potentially harmful agents, including pathogens. In addition to serving as a physical barrier and producing soluble mediators of immunity, such as cytokines or antimicrobial peptides, these cells are thought to function as nonprofessional APCs. In this regard, intestinal epithelial cells are particularly prominent because they express MHC class II molecules at the site of massive antigenic exposure. However, unlike bone marrow-derived professional APC, such as dendritic cells or B cells, little is known about the mechanisms of MHC class II presentation by the nonprofessional APC in vivo. The former use the lysosomal cysteine protease cathepsin S (Cat S), whereas thymic cortical epithelial cells use cathepsin L (Cat L) for invariant chain degradation and MHC class II maturation. Unexpectedly, we found that murine Cat S plays a critical role in invariant chain degradation in intestinal epithelial cells. Furthermore, we report that nonprofessional APC present a class II-bound endogenous peptide to naive CD4 T cells in vivo in a Cat S-dependent fashion. These results suggest that in vivo, both professional and nonprofessional MHC class II-expressing APC use Cat S, but not Cat L, for MHC class II-mediated Ag presentation.     

Human neonatal dendritic cells are competent in MHC class I antigen processing and presentation

Neonates are clearly more susceptible to severe disease following infection with a variety of pathogens than are adults. However, the causes for this are unclear and are often attributed to immunological immaturity. While several aspects of immunity differ between adults and neonates, the capacity of dendritic cells in neonates to process and present antigen to CD8+ T cells remains to be addressed. We used human CD8+ T cell clones to compare the ability of neonatal and adult monocyte-derived dendritic cells to present or process and present antigen using the MHC class I pathway. Specifically, we assessed the ability of dendritic cells to present antigenic peptide, present an HLA-E-restricted antigen, process and present an MHC class I-restricted antigen through the classical MHC class I pathway, and cross present cell-associated antigen via MHC class I. We found no defect in neonatal dendritic cells to perform any of these processing and presentation functions and conclude that the MHC class I antigen processing and presentation pathway is functional in neonatal dendritic cells and hence may not account for the diminished control of pathogens.     

Luminal antigens access late endosomes of intestinal epithelial cells enriched in MHC I and MHC II molecules: in vivo study in Crohn's ileitis

In contrast to healthy conditions, intestinal epithelial cells (IECs) stimulate proinflammatory CD4+ and CD8+ T cells during Crohn's disease (CD). The underlying regulatory mechanisms remain unknown. Here we investigated the epithelial expression of major histocompatibility complex (MHC) I and MHC II and its interference with endocytic pathways, in vivo. During ileoscopy, ovalbumin (OVA) was sprayed onto ileal mucosa of CD patients (ileitis and remission) and controls. The epithelial traffic of OVA and MHC I/II pathways were studied in biopsies using fluorescence and electron microscopy. We found MHC I and MHC II to accumulate within multivesicular late endosomes (MVLE) of IECs. Faint labeling for these molecules was seen in early endosomes and lysosomes. MVLE were entered by OVA 10 min after exposure. Exosomes carrying MHC I, MHC II, and OVA were detected in intercellular spaces of the epithelium. OVA trafficking and labeling patterns for MHC I and MHC II in IECs showed no differences between CD patients and controls. Independent of inflammatory stimuli, MHC I and MHC II pathways intersect MVLE in IECs, which were efficiently targeted by luminal antigens. Similar to MHC II-enriched compartments in professional antigen presenting cells, these MVLE might be critically involved in MHC I- and MHC II-related antigen processing in IECs and the source of epithelial-released exosomes. The access of luminal antigens to MHC I in MVLE might indicate that the presentation of exogenous antigens by IECs must not be restricted to MHC II but might also occur as "cross-presentation" via MHC I to CD8+ T cells.     

Novel regulation of MHC class II function in B cells

The presence of post-translational regulation of MHC class II (MHC II) under physiological conditions has been demonstrated recently in dendritic cells (DCs) that potently function as antigen-presenting cells (APCs). Here, we report that MARCH-I, an E3 ubiquitin ligase, plays a pivotal role in the post-translational regulation of MHC II in B cells. MARCH-I expression was particularly high in B cells, and the forced expression of MARCH-I induced the ubiquitination of MHC II. In B cells from MARCH-I-deficient mice (MARCH-I KO), the half-life of surface MHC II was prolonged and the ubiquitinated form of MHC II completely disappeared. In addition, MARCH-I-deficient B cells highly expressed exogenous antigen-loaded MHC II on their surface and showed high ability to present exogenous antigens. These results suggest that the function of MHC II in B cells is regulated through ubiquitination by MARCH-I.     

Rapid changes in shape and number of MHC class II expressing cells in rat airways after Mycoplasma pulmonis infection

Mycoplasma pulmonis infection in rodents causes a chronic inflammatory airway disease with a strong immunological component, leading to mucosal remodeling and angiogenesis. We sought to determine the effect of this infection on the shape and number of dendritic cells and other major histocompatibility complex (MHC) class II expressing cells in the airway mucosa of Wistar rats. Changes in the shape of subepithelial OX6 (anti-MHC class II)-immunoreactive cells were evident in the tracheal mucosa 2 days after intranasal inoculation with M. pulmonis. By 1 week, the shape of the cells had changed from stellate to rounded (mean shape index increased from 0.42 to 0.77). The number of OX6-positive cells was increased 6-fold at 1 week and 16-fold at 4 weeks. Coincident with these changes, many columnar epithelial cells developed OX6 immunoreactivity, which was still present at 4 weeks. We conclude that M. pulmonis infection creates a potent immunologic stimulus that augments and transforms the OX6-immunoreactive cell population in the airways by changing the functional state of airway dendritic cells, initiating an influx of MHC class II expressing cells, and activating expression of MHC class II molecules by airway epithelial cells.     

Effects of lipofectin-antigen complexes on major histocompatibility complex class I-restricted antigen presentation pathway in murine dendritic cells and on dendritic cell maturation

We previously reported that exogenous antigens complexed with the cationic liposome lipofectin (LF) were efficiently presented via major histocompatibility complex (MHC) class I molecules on pulsed dendritic cells (DCs) in vitro. In the present study, we demonstrated that MHC class I-restricted antigen presentation on DC2.4 cells, a murine immature DC line, treated with LF-antigen complexes was remarkably suppressed through the inhibition of endocytosis, proteasome catalysis, and Golgi transport. We also found that LF did not influence expression of interleukin-12 p40 mRNA, MHC molecules, or co-stimulatory molecules in DC2.4 cells. These findings suggest that an antigen-loading procedure using LF could enhance delivery of exogenous antigens to the classical MHC class I pathway in DCs, but it does not initiate DC maturation.     

Forming a Complex with MHC Class I Molecules Interferes with Mouse CD1d Functional Expression

CD1d molecules are structurally similar to MHC class I, but present lipid antigens as opposed to peptides. Here, we show that MHC class I molecules physically associate with (and regulate the functional expression of) mouse CD1d on the surface of cells. Low pH (3.0) acid stripping of MHC class I molecules resulted in increased surface expression of murine CD1d on antigen presenting cells as well as augmented CD1d-mediated antigen presentation to NKT cells. Consistent with the above results, TAP1-/- mice were found to have a higher percentage of type I NKT cells as compared to wild type mice. Moreover, bone marrow-derived dendritic cells from TAP1-/- mice showed increased antigen presentation by CD1d compared to wild type mice. Together, these results suggest that MHC class I molecules can regulate NKT cell function, in part, by masking CD1d.     

TCR signaling for initiation and completion of thymocyte positive selection has distinct requirements for ligand quality and presenting cell type

Thymocyte selection involves signaling by TCR engaging diverse self-peptide:MHC molecule ligands on various cell types in the cortex and medulla. Here we separately analyze early and late stages of selection to better understand how presenting cell type, ligand quality, and the timing of TCR signaling contribute to intrathymic differentiation. TCR transgenic CD4+CD8+ thymocytes (double positive (DP)) from MHC-deficient mice were stimulated using various presenting cells and ligands. The resulting CD69high cells were isolated and evaluated for maturation in reaggregate cultures with wild-type or MHC molecule-deficient thymic stroma with or without added hemopoietic dendritic cells (DC). Production of CD4+ T cells required TCR signaling in the reaggregates, indicating that transient recognition of self-ligands by DP is inadequate for full differentiation. DC bearing a potent agonist ligand could initiate positive selection, producing activated thymocytes that matured into agonist-responsive T cells in reaggregates lacking the same ligand. DC could also support the TCR signaling necessary for late maturation. These results argue that despite the negative role assigned to DC in past studies, neither the peptide:MHC molecule complexes present on DC nor any other signals provided by these cells stimulate only thymocyte death. These findings also indicate that unique epithelial ligands are not necessary for positive selection. They provide additional insight into the role of ligand quality in selection events and support the concept that following initiation of maturation from the DP state, persistent TCR signaling is characteristic of and perhaps required by T cells.     

Cross-presentation in viral immunity and self-tolerance

T lymphocytes recognize peptide antigens presented by class I and class II molecules encoded by the major histocompatibility complex (MHC). Classical antigen-presentation studies showed that MHC class I molecules present peptides derived from proteins synthesized within the cell, whereas MHC class II molecules present exogenous proteins captured from the environment. Emerging evidence indicates, however, that dendritic cells have a specialized capacity to process exogenous antigens into the MHC class I pathway. This function, known as cross-presentation, provides the immune system with an important mechanism for generating immunity to viruses and tolerance to self.     

Apoptosis paves the detour path for CD8 T cell activation against intracellular bacteria

Intracellular bacteria such as Mycobacterium tuberculosis primarily infect macrophages. Within these host cells, the pathogens are confined to phagosomes and their antigens are secluded from the classical MHC I presentation pathway. Moreover, macrophages fail to express certain antigen presenting molecules like CD1 proteins. As a result of this intracellular lifestyle, the pathways for the induction of MHC I- and CD1-restricted CD8 T cells by such microorganisms remain elusive. Based on recent findings in tuberculosis and salmonellosis, we propose a new detour pathway for CD8 T cell activation against intracellular bacteria through apoptotic blebs from infected macrophages. Pathogen-derived antigens including proteins and lipids are delivered from infected cells to non-infected dendritic cells. Subsequently, these professional antigen presenting cells display microbial antigens through MHC I and CD1 to T cells. Thus, cross-priming mediated by apoptotic vesicles is not just a matter of antigen distribution, but an intrinsic immunological function due to the nature of phagosomally located intracellular bacteria. We consider infection-induced apoptosis the conditio sine qua non for antigen-specific CD8 T cell activation by phagosome-enclosed pathogens. This important new function of cell death in antibacterial immunity requires consideration for rational vaccine design.     

Ligand exchange of major histocompatibility complex class II proteins is triggered by H-bond donor groups of small molecules

Hydrogen bonds (H-bonds) are crucial for the stability of the peptide-major histocompatibility complex (MHC) complex. In particular, the H-bonds formed between the peptide ligand and the MHC class II binding site appear to have a great influence on the half-life of the complex. Here we show that functional groups with the capacity to disrupt hydrogen bonds (e.g. -OH) can efficiently catalyze ligand exchange reactions on HLA-DR molecules. In conjunction with simple carrier molecules (such as propyl or benzyl residues), they trigger the release of low affinity ligands, which permits the rapid binding of peptides with higher affinity. Similar to HLA-DM, these compounds are able to influence the MHC class II ligand repertoire. In contrast to HLA-DM, however, these simple small molecules are still active at neutral pH. Under physiological conditions, they increase the number of "peptide-receptive" MHC class II molecules and facilitate exogenous peptide loading of dendritic cells. The drastic acceleration of the ligand exchange on these antigen presenting cells suggests that, in general, availability of H-bond donors in the extracellular milieu controls the rate of MHC class II ligand exchange reactions on the cell surface. These molecules may therefore be extremely useful for the loading of antigens onto dendritic cells for therapeutic purposes.     

Phenotypic and functional characterization of human thymic stromal cell lines.

To establish new tools for studying human thymic stromal cells, we transfected adherent cells from a human postnatal thymus using a plasmid encoding SV40 large T antigen. Among the cell lines obtained, we characterized four epithelial cell lines (LT-TEC1 to LT-TEC4) and one thymic myoid cell line (MITC). Several morphological, functional and phenotypic differences were observed between these 2 cell types. Epithelial cells were heterogeneous and larger than myoid cells. Untreated LT-TEC lines expressed MHC class I, ICAM-1 and LFA-3 antigens and not MHC class II antigens, similarly to primary thymic epithelial cells (PTEC), while MITC line expressed only class I and LFA-3 antigens. After IFN-gamma treatment, MHC class II and ICAM-1 antigens were markedly upregulated in LT-TEC lines but not in MITC, indicating the absence or a dysfunction of regulatory factors in MITC line. Myoid cells expressed mRNA for all the subunits of the acetylcholine receptor (AChR) while epithelial cells expressed only the alpha, beta and epsilon subunits. Strikingly, LT-TEC produced much more C-C chemokines and IL-6 than MITC cells, while these latter produced higher levels of IL-8 and TNF-alpha. Altogether, these results reveal phenotypic and functional differences between these two stromal cell types, suggesting a potential involvement of myoid cells in the thymic function.