Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self.

Expression of peripheral antigens in the thymus has been implicated in T cell tolerance and autoimmunity. Here we identified medullary thymic epithelial cells as being a unique cell type that expresses a diverse range of tissue-specific antigens. We found that this promiscuous gene expression was a cell-autonomous property of medullary epithelial cells and was maintained during the entire period of thymic T cell output. It may facilitate tolerance induction to self-antigens that would otherwise be temporally or spatially secluded from the immune system. However, the array of promiscuously expressed self-antigens appeared random rather than selected and was not confined to secluded self-antigens.

Sampling of complementing self-antigen pools by thymic stromal cells maximizes the scope of central T cell tolerance.

Expression of peripheral antigens in the thymus has been implicated in T cell tolerance and autoimmunity, yet the identity of cells involved remains elusive. Here we show that antigen expression in a minor fraction of medullary thymic epithelial cells leads to deletion of specific CD4 T cells. Strikingly, this deletion is not dependent on cross-presentation by hemopoietic antigen-presenting cells, which have been ascribed a predominant role in negative selection. By contrast, when the same antigen enters the thymus via the blood stream, negative selection is strictly dependent on antigen presentation by hemopoietic cells. These findings imply that the (re)-presentation of "self" by thymic stromal cells is non-redundant, and that different thymic antigen-presenting cells instead cover complementing sets of self-antigens, thus maximizing the scope of central tolerance

Self-antigen presentation by thymic stromal cells: a subtle division of labor.

Self-antigen-MHC complexes expressed by thymic stromal cells serve as ligands for TCR-mediated positive and negative selection, resulting in a self-MHC-restricted, self-tolerant T cell repertoire. It has recently become apparent that thymic stromal cells differ in their accessibility to antigen as well as their ability to process and present antigen. These differences result in the sampling by thymic stromal cells of largely nonoverlapping self-antigen pools and the display of self-peptide profiles specific for each cell type. In conjunction with single or serial cell-cell interactions between thymocytes and stromal cells, such differences in self-antigen display allow for maximal (re)presentation of 'self' in the thymus and optimize the efficacy of positive and negative selection.

"Promiscuous" expression of tissue antigens in the thymus: a key to T-cell tolerance and autoimmunity?

Induction and maintenance of self-tolerance in the developing and mature T cell repertoire is mediated by multiple mechanisms operating both in the thymus ("central tolerance") and in peripheral lymphoid and nonlymphoid organs ("peripheral tolerance"). The thymus is viewed as the prime site of T cell tolerance induction to ubiquitous proteins and abundant blood-borne antigens entering the thymus via the circulation. By contrast, tolerance to self-antigens that are confined to specific tissues has been ascribed to a variety of mechanisms acting on peripheral T cells. Based on the recent finding that intrathymic expression of "tissue-specific" antigens is a common occurrence the prevailing notion that tolerance induction in the thymus applies only to a limited set of "abundant" proteins may have to be revised. Interestingly, this "promiscuous" expression of tissue antigens in the thymus appears to be a unique property of thymic epithelial cells rather than bone marrow derived antigen-presenting cells, implying cell type specific regulation rather than basal leakiness as a mechanism of "promiscuous" gene transcription. We summarize recent experimental evidence supporting this novel concept and discuss implications for autoimmunity.

Shaping of the autoreactive T-cell repertoire by a splice variant of self protein expressed in thymic epithelial cells.

Intrathymic expression of tissue-specific self antigens may be involved in immunological tolerance and protection from autoimmune disease. We have analyzed the role of T-cell tolerance to proteolipid protein (PLP), the main protein of the myelin sheath, in susceptibility to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Intrathymic expression of PLP was largely restricted to the shorter splice variant, DM20. Expression of DM20 by thymic epithelium was sufficient to confer T-cell tolerance to all epitopes of PLP in EAE-resistant C57BL/6 mice. In contrast, the major T-cell epitope in SJL/J mice was only encoded by the central nervous system-specific exon of PLP, but not by thymic DM20. Thus, lack of tolerance to this epitope offers an explanation for the exquisite susceptibility of SJL/J mice to EAE. As PLP expression in the human thymus is also restricted to the DM20 isoform, these findings have implications for selection of the autoimmune T-cell repertoire in multiple sclerosis.

Tolerance and determinant hierarchy.

The overall T cell response to a multideterminant antigen consists of the sum of responses to a limited number of different determinants on the protein. Antigen-presenting cells (APCs) are crucial in delimiting the determinants on the protein to which a response will be mounted. This influence is apparent at two levels. First, the determinants that are generated and displayed by APCs in the thymus are pivotal in shaping the T cell repertoire that will be available for responding to antigen determinants in the periphery. Second, antigen processing affects the selection of determinants that become displayed by the various peripheral APC populations that are involved in inducing and promoting a T cell response. We have studied the effect of the display hierarchy on tolerance induction to individual determinants in transgenic mice expressing different serum levels of hen egg lysozyme. We have also analysed aspects of the processing machinery that contribute to shaping the hierarchy of determinant display on MHC class II molecules: proteolysis and reduction of disulfide bonds.

CD4 T cell tolerance to human C-reactive protein, an inducible serum protein, is mediated by medullary thymic epithelium.

Inducible serum proteins whose concentrations oscillate between nontolerogenic and tolerogenic levels pose a particular challenge to the maintenance of self-tolerance. Temporal restrictions of intrathymic antigen supply should prevent continuous central tolerization of T cells, in analogy to the spatial limitation imposed by tissue-restricted antigen expression. Major acute-phase proteins such as human C-reactive protein (hCRP) are typical examples for such inducible self-antigens. The circulating concentration of hCRP, which is secreted by hepatocytes, is induced up to 1,000-fold during an acute-phase reaction. We have analyzed tolerance to hCRP expressed in transgenic mice under its autologous regulatory regions. Physiological regulation of basal levels (<10(-9) M) and inducibility (>500-fold) are preserved in female transgenics, whereas male transgenics constitutively display induced levels. Surprisingly, crossing of hCRP transgenic mice to two lines of T cell receptor transgenic mice (specific for either a dominant or a subdominant epitope) showed that tolerance is mediated by intrathymic deletion of immature thymocytes, irrespective of widely differing serum levels. In the absence of induction, hCRP expressed by thymic medullary epithelial cells rather than liver-derived hCRP is necessary and sufficient to induce tolerance. Importantly, medullary epithelial cells also express two homologous mouse acute-phase proteins. These results support a physiological role of "ectopic" thymic expression in tolerance induction to acute-phase proteins and possibly other inducible self-antigens and have implications for delineating the relative contributions of central versus peripheral tolerance.

Two separable T cell receptor signals reconstitute positive selection of CD4 lineage T cells in vivo.

Positive selection is an obligatory step during intrathymic T cell differentiation. It is associated with rescue of short-lived, self major histocompatibility complex (MHC)-restricted thymocytes from programmed cell death, CD4/CD8 T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TCR) signaling during positive selection can be closely mimicked by targeting TCR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. We show that selection of CD4 T cell lineage cells in mice deficient for MHC class I and MHC class II expression can be reconstituted in vivo by two separable T cell receptor signaling steps, whereas a single TCR signal leads only to induction of short-lived CD4+CD8lo intermediates. These intermediates remain susceptible to a second TCR signal for 12-48 h providing an estimate for the duration of positive selection in situ. While both TCR signals induce differentiation steps, only the second one confers long-term survival on immature thymocytes. In further support of the two-step model of positive selection we provide evidence that CD4 T cell lineage cells rescued by a single hybrid antibody pulse in MHC class II-deficient mice are pre-selected by MHC class I.

A filarial cysteine protease inhibitor down-regulates T cell proliferation and enhances interleukin-10 production.

Filarial nematodes are a cause of chronic debilitating diseases in the tropics. A hallmark of filariasis is the marked down-regulation and polarization of host immune responses, yet molecular constituents of parasites causing this state have remained undefined. We describe a 17-kDa antigen (Av17) of the rodent filarial parasite Acanthocheilonema viteae, which shows amino acid homologies to cystatin C, a major cysteine protease inhibitor belonging to family 2 of the cystatin superfamily. Av17 is released by filariae in vitro. Exported molecules of A. viteae worms are shown to markedly suppress mitogen-induced T cell proliferation of mice and jirds. Av17 accounts for 45.5% of this suppressive activity in the murine system. Recombinant Av17 (rAv17), expressed in Escherichia coli, exhibits biological activity as a cysteine protease inhibitor and was used to examine the immunomodulatory effects, rAv17 induces down-regulation of murine T cell responses to mitogens, to T cell receptor cross-linking by anti-CD3 antibodies and to specific antigens, and at the same time up-regulation of interleukin-10. Hence, this filarial cystatin is a likely effector molecule of immunomodulation and a potential target for antifilarial intervention.

Selection of a broad repertoire of CD4+ T cells in H-2Ma0/0 mice.

According to past reports, H-2Ma0/0 mice express a single major histocompatiblity complex class II molecule, A(b), heavily loaded with a single peptide derived from the invariant chain, CLIP. Despite the highly restricted diversity of the class II:peptide complexes expressed on thymic stromal cells in the mutant animals, a large and diverse population of CD4+ T cells is positively selected. However, two important issues remained unresolved and are addressed here: Just how preponderant is CLIP occupancy of the class II molecules from H-2M0/0 mice? How extensive and functionally competent is the CD4+ population selected in the mutant animals? Our results argue that a single class II:peptide complex can select a very broad, though not complete, repertoire of CD4+ T cells.

The MHC class II-restricted T cell response of C57BL/6 mice to human C-reactive protein: homology to self and the selection of T cell epitopes and T cell receptors.

The T cell response of C57BL/6 mice to human C-reactive protein (hCRP), an inducible acute phase protein, was analysed. Two I-A(b)-restricted epitopes at positions 79 95 (epitope A) and 87-102 (epitope B) were identified using a panel of CD4+ T cell clones. Human C-reactive protein shares considerable homology with mouse C-reactive protein and mouse serum amyloid P component. Interestingly, the two epitopes map to the region of lowest homology between human CRP and its mouse homologues. Human CRP-specific T cell clones express a restricted T cell receptor (TCR) repertoire, both with regard to usage of TCR germline gene segments (V alpha, J alpha, V beta, J beta) and certain TCR alpha beta combinations. Therefore, epitope-A specific clones preferentially use TCR V beta8.3 and V alpha3 J alpha15 V beta8.3-J beta2.3 and epitope-B specific clones use V beta2 and V alpha1-J alpha24/30-V beta2. This bias is even more pronounced when TCR usage is correlated with epitope fine specificity. A role for homology of hCRP to self components in selecting these particular T cell epitopes and TCR is discussed.

Two genetically separable steps in the differentiation of thymic epithelium.

The development of the thymus depends initially on epithelial-mesenchymal and subsequently on reciprocal lympho-stromal interactions. The genetic steps governing development and differentiation of the thymic microenvironment are unknown. With the use of a targeted disruption of the whn gene, which recapitulates the phenotype of the athymic nude mouse, the WHN transcription factor was shown to be the product of the nude locus. Formation of the thymic epithelial primordium before the entry of lymphocyte progenitors did not require the activity of WHN. However, subsequent differentiation of primitive precursor cells into subcapsular, cortical, and medullary epithelial cells of the postnatal thymus did depend on activity of the whn gene. These results define the first genetically separable steps during thymic epithelial differentiation.

Insertional mutagenesis affecting programmed cell death leads to thymic hyperplasia and altered thymopoiesis.

The mouse mutant Ft displays thymic hyperplasia and fused toes (Ft) of the forelimbs. Both phenotypic abnormalities are caused by transgene insertion in the D region of chromosome 8. While the forelimb defect is probably caused by developmentally dysregulated programmed cell death, the mechanism underlying thymic hyperplasia has not been characterized. In this work, we show that expansion of the thymocyte compartment progresses with time, is polyclonal, and affects all major thymocyte subsets, including the earliest CD4-8- subset, i.e., CD44+ CD25- cells; hyperplasia is not an autonomous property of mutant T cells, but is caused indirectly by a primary defect in thymic stromal. The rate of cell division and the cell turnover of immature CD4-8- and CD4+8+ thymocytes under steady state conditions are not altered in hyperplastic Ft thymi. Immature CD4+8+ thymocytes of mutant mice, however, are less susceptible to induction in vitro of programmed cell death by different modes (TCR cross-linking, cortisone, or radiation). Increased production of thymocytes results in increased export of T cells, yet the size and composition of the peripheral T cell pool are normal. Overproduction of immature CD4+8+ thymocytes is offset partly by a reduced conversion rate of CD4+8+ double positive to single positive thymocyte growth control by epithelial cells, and may serve as a model to study the regulation of early thymopoiesis.

Tolerance and immunity to the inducible self antigen C-reactive protein in transgenic mice.

The understanding of immunological tolerance has been greatly aided by the development of transgenic animal models in which expression of a specific T cell receptor (or B cell receptor) and its cognate self antigen is experimentally controlled. In most cases, expression of the self antigen was constitutive and did not allow for variation of its time- and dose-dependent expression pattern, parameters which are known to influence the balance of tolerance versus immunity. We describe a transgenic model in which expression of human C-reactive protein (hCRP), an acute-phase protein, is tightly controlled at basal levels (female mice express around 10(-9) M and male mice 5 x 10(-7) M circulating hCRP) and is highly inducible (induction factor of 25-500). T cells from C57BL/6 mice recognize two epitopes of hCRP termed A (residues 79-95) and B (residues 87-102). Different efficacies of presentation in vitro and in vivo identify epitope A as sub-dominant and epitope B as dominant. T cells of non-induced hCRP transgenic mice are tolerant to the dominant epitope, but reactive to the subdominant epitope. A hCRP-specific IgG antibody response is detectable in transgenic mice, but is weaker than in littermates. Upon induction of hCRP, both T cell epitopes are presented by thymic and splenic antigen-presenting cells (APC) in vivo. Kinetics of presentation by splenic APC closely match serum kinetics of hCRP, whereas presentation in the thymus is considerably prolonged. This model enables epitope-specific T cell tolerance to be studied as a function of time- and dose-dependent expression of the self antigen.

Clonal deletion of major histocompatibility complex class I-restricted CD4+CD8+ thymocytes in vitro is independent of the CD95 (APO-1/Fas) ligand.

The CD95 (APO-1/Fas) ligand (CD95L) mediates apoptosis in sensitive target cells, Ca(2+)-independent cytotoxicity of cells from perforin knock-out mice, and peripheral deletion of activated T cells through engagement of its cognate receptor CD95. Double-positive thymocytes show a high constitutive expression of CD95. Therefore, we used a model system and investigated whether negative selection through apoptosis might involve CD95/CD95L. We analyzed whether CD95L may induce antigen-specific deletion of double-positive thymocytes from mice transgenic for a lymphocytic choriomeningitis virus (LCMV)/H2b-specific T cell receptor (TCR). These cells are deleted in vitro upon addition of the LCMV-peptide 33-41 in a major histocompatibility complex-class I-restricted fashion. Deletion was not blocked by soluble mouse and human CD95-Fc receptor decoys. CD95-Fc receptor decoys, however, were effective in blocking apoptosis induced by mouse CD95L-transfected L929 cells in sensitive CD95+ target cells and in thymocytes. These results suggest that TCR-induced deletion of immature thymocytes in vitro is independent of CD95L. Thus, our data argue against a role of CD95L in negative selection of MHC-class I-restricted autoreactive thymocytes.

Intrathymic T cell receptor (TcR) targeting in mice lacking CD4 or major histocompatibility complex (MHC) class II: rescue of CD4 T cell lineage without co-engagement of TcR/CD4 by MHC class II.

A critical step during intrathymic T cell development, termed positive selection, is associated with rescue of short-lived, immature thymocytes from programmed cell death, T cell lineage commitment, and induction of lineage-specific differentiation programs. T cell receptor (TcR)-major histocompatibility complex (MHC) interactions during positive selection can be closely mimicked by targeting TcR on immature thymocytes to cortical epithelial cells in situ via hybrid antibodies. Here, we show that antibody-mediated TcR signaling in mice deficient for CD4 or MHC class II expression induces polyclonal differentiation of the CD4 T cell lineage. Following a single TcR signal pulse in situ, a temporal sequence of phenotype changes can be discerned: CD69 up-regulation (< 1 day), CD8 down-regulation, TcR up-regulation (1-1.5 days) and down-regulation of the heat-stable antigen (1.5-2 days). Differentiation of phenotypically and functionally mature CD4 T cells in situ is attained within 3 days. Rescue of CD4 lineage T cells in the absence of TcR/CD4 co-engagement by MHC class II in this experimental system supports the stochastic/selective model of T cell lineage commitment.

Presentation and intercellular transfer of self antigen within the thymic microenvironment: expression of the E alpha peptide-I-Ab complex by isolated thymic stromal cells.

Expression of a self peptide derived from the alpha chain of MHC class II (I-Ed) in association with I-Ab was studied in the murine thymic microenvironment. Previous work using the mAb Y-Ae which specifically recognizes the E alpha-I-Ab complex had reported differential expression between the thymic medulla and the cortex of this peptide-MHC complex: MHC class II-positive stromal cells in the medulla were strongly positive, whereas this complex was barely detectable on cortical epithelial cells (cEpC) in situ. This difference in presentation of an abundant self peptide is intriguing, since the self protein from which this peptide is derived and the presenting MHC molecule are strongly expressed in both compartments. In this report we show by cell surface phenotype and functional assays that isolated cEpC express the E alpha-I-Ab complex at significant although lower levels than medullary dendritic cells (DC), when examined ex vivo. These results support the notion that cEpC and bone marrow-derived stromal cells present a similar set of self peptide-MHC complexes in situ. In addition, we detect intercellular transfer in situ of the E alpha determinant from radioresistant stromal cells to thymic DC, a mechanism which may enhance the efficacy of tolerance induction by spreading self antigens with the thymic microenvironment.

Programmed cell death is affected in the novel mouse mutant Fused toes (Ft).

We have identified a novel dominant mouse mutant that is characterised by fused toes on the fore limbs and a thymic hyperplasia, in heterozygous animals. Homozygosity of the mutation leads to malformation of the developing brain, lost of the genetic control of left-right asymmetry and to death around day 10 of development. Analysis of both limb development and induction of apoptosis in immature thymocytes in vitro suggest that programmed cell death is affected by the mutation. Since the mutation was caused via a transgene insertion we were able to map it to the D region on mouse chromosome 8. So far, no mutation that affects programmed cell death has been mapped to this chromosome. Thus, this mutation will allow the identification of a novel gene involved in programmed cell death during mammalian development.