Role In Negative Selection Research Articles

Cutting edge: Extracellular signal-related kinase is not required for negative selection of developing T cells.

Signals initiated through the TCR during development can result in either survival and differentiation or cell death. High affinity signals that induce death elicit a robust yet transient activation of signaling pathways, including Erk, whereas low affinity ligands, which promote survival, generate a gradual and weaker activation of the same pathways. It was recently demonstrated that Erk localizes to distinct cellular locations in response to high and low affinity ligands. Although a requirement for Erk in positive selection is well established, its role in negative selection is controversial and, thus, the importance of Erk relocalization during development is not understood. In this study, we examined the role of Erk in negative selection using mice that are genetically deficient in both Erk1 and Erk2 in T cells. Results from three different models reveal that thymocyte deletion remains intact in the absence of Erk.

Importance of MHCII expression on thymic epithelium versus dendritic cells for the positive and negative selection of CD4 T Cells (138.28)

Abstract Development of T cells from T cell precursors occurs in the thymus through interactions of T lineage cells with thymic stromal populations. These stromal populations are distributed in an ordered fashion that defines thymic architecture and provides the environment for thymic differentiation. The successful generation of a mature and self-tolerant CD4 T cell repertoire requires that developing T cells pass at least two checkpoints; positive and negative selection. Positive selection has been reported to be mediated by cortical epithelial cells, allowing T lineage cells that have rearranged a T cell receptor capable of recognizing self peptide-MHCII complexes at appropriately affinity, to survive and commit to the CD4 lineage. Positively selected cells can then move into the medulla where they must pass negative selection, which acts to remove highly self reactive cells. MHCII is expressed in the medulla on both medullary epithelial cells and dendritic cells and plays a critical role in negative selection. To further examine the cell populations in which MHCII expression is important in CD4 T cell development in unmanipulated mice, we are characterizing the effect of selective inactivation of MHCII, using a panel of mice expressing cre recombinase specifically in epithelial cells or in dendritic cells in combination with an MHCII floxed allele.

The adaptive phenotype of cortical thymic epithelial cells

Epithelial cells in the thymus are required for positive and negative selection of developing thymocytes. Although medullary epithelial cells play a major role in negative selection owing to their facility of expressing peripheral self-antigens, the adaptive features of cortical epithelial cells are largely unknown. A paper in this issue of the European Journal of Immunology shows that the putative serine protease Prss16 affects positive selection of a subset of CD4(+) T cells. A survey of chordate genomes indicates that the Prss16 gene emerged in vertebrates as a paralogue of evolutionarily older members of the serine carboxypeptidase 28 family; thus, Prss16 is not associated with the appearance of the adaptive immune system and the thymus in jawed vertebrates. Nevertheless, it appears that Prss16 has later evolved as an essential contributor to the MHC class II peptide/ligand repertoire.

RANKL signaling regulates the development of the immune system and immune tolerance

The receptor activator of the nuclear factor κB (NF-κB) ligand (RANKL), which is a member of the tumor necrosis factor (TNF) family, is crucial for the development and regulation of the immune system and also for bone formation. Previous analyses conducted on RANKL- or RANK-deficient mice have revealed that these molecules play an essential role in the development of lymph nodes but not Peyer's patches. Although the details of the mechanism by which RANKL signaling controls lymph node development have not been elucidated, it is known that this mechanism differs from that by which signals are transduced from lymphotoxin, which is another TNF family member that performs the same function. We and other researchers have recently found that RANKL signaling is crucial for the establishment of self-tolerance and the suppression of excessive immune responses. RANKL expression in keratinocytes was found to increase with ultraviolet (UV) irradiation or inflammation. Upregulated RANKL expression activates the epidermal dendritic cells (DCs) and consequently induces the proliferation of regulatory T cells, which in turn suppress autoimmune reactions. In addition, we recently found that RANKL signaling controls the development of medullary thymic epithelial cells, which play a critical role in negative selection in the thymus. The signaling pathways of the TNF receptors RANK and CD40 seem to partially overlap. These findings suggest that functional redundancies may also exist among the signaling pathways of TNFs involved in other physiological phenomena.

Dendritic cells in the thymus contribute to T-regulatory cell induction

Central tolerance is established through negative selection of self-reactive thymocytes and the induction of T-regulatory cells (T(R)s). The role of thymic dendritic cells (TDCs) in these processes has not been clearly determined. In this study, we demonstrate that in vivo, TDCs not only play a role in negative selection but in the induction of T(R)s. TDCs include two conventional dendritic cell (DC) subtypes, CD8(lo)Sirpalpha(hi/+) (CD8(lo)Sirpalpha(+)) and CD8(hi)Sirpalpha(lo/-) (CD8(hi)Sirpalpha(-)) [corrected] which have different origins. We found that the CD8(hi)Sirpalpha(+) DCs represent a conventional DC subset that originates from the blood and migrates into the thymus. Moreover, we show that the CD8(lo)Sirpalpha(+) DCs demonstrate a superior capacity to induce T(R)s in vitro. Finally, using a thymic transplantation system, we demonstrate that the DCs in the periphery can migrate into the thymus, where they efficiently induce T(R) generation and negative selection.

Evolutionary History of a Specialized P450 Propane Monooxygenase

The evolutionary pressures that shaped the specificity and catalytic efficiency of enzymes can only be speculated. While directed evolution experiments show that new functions can be acquired under positive selection with few mutations, the role of negative selection in eliminating undesired activities and achieving high specificity remains unclear. Here we examine intermediates along the ‘lineage’ from a naturally occurring C 12–C 20 fatty acid hydroxylase (P450 BM3) to a laboratory-evolved P450 propane monooxygenase (P450 PMO) having 20 heme domain substitutions compared to P450 BM3. Biochemical, crystallographic, and computational analyses show that a minimal perturbation of the P450 BM3 fold and substrate-binding pocket accompanies a significant broadening of enzyme substrate range and the emergence of propane activity. In contrast, refinement of the enzyme catalytic efficiency for propane oxidation (∼ 9000-fold increase in k cat/ K m) involves profound reshaping and partitioning of the substrate access pathway. Remodeling of the substrate-recognition mechanisms ultimately results in remarkable narrowing of the substrate profile around propane and enables the acquisition of a basal iodomethane dehalogenase activity as yet unknown in natural alkane monooxygenases. A highly destabilizing L188P substitution in a region of the enzyme that undergoes a large conformational change during catalysis plays an important role in adaptation to the gaseous alkane. This work demonstrates that positive selection alone is sufficient to completely respecialize the cytochrome P450 for function on a nonnative substrate.

Evidence for De Novo Expression of Thymic Insulin by Peripheral Bone Marrow‐derived Cells

Thymic expression of insulin has been suggested to play a major role in negative selection of autoreactive T cells and tolerance induction against pancreatic beta cells. Furthermore, the expression of insulin in peripheral antigen-presenting cells (APC) has been clearly demonstrated but whether thymic negative selection and tolerance induction also depends on peripheral influx of self-antigens (Ag) remains to be conclusively demonstrated. In this study, we wanted to test whether peripheral influx of insulin expressing cells might contribute to negative selection. In order to address this question, we used mice deficient in the Ins1 and Ins2 genes. Embryonic thymi either deficient in both insulin genes or expressing Ins2 were dissected and transplanted under the kidney capsule of athymic nude mice recipients. After indicated time points, grafted thymi were removed and analysed for insulin re-expression and for the emergence of autoreactive T cells. The analysis revealed a re-expression of Ins2 in grafted insulin deficient thymi suggesting that self-Ag expression in the thymus is not only intrinsically regulated but peripheral influx of APC capable of expressing insulin might contribute to thymic selection and tolerance induction.

The Autoimmune Regulator Directly Controls the Expression of Genes Critical for Thymic Epithelial Function

The autoimmune regulator (Aire) gene plays an essential role in negative selection of T cells and deletion of autoreactive T cells in the thymus. The defect in thymic selection in Aire(-/-) mice was attributed to the repressed expression of tissue-specific Ags in the thymic epithelial cells and defective Ag presentation; however, the molecular mechanism underlying these functions has been elusive. Using the chromatin immunoprecipitation technique, we demonstrate here that Aire binds in vivo to specific DNA sequence motifs and directly regulates thymic expression of genes important for thymic functions including expression of autoantigens, cytokines, transcription factors, and posttranslational modifiers. These results unambiguously established Aire as a key transcriptional regulator of the immune system.

Developmental kinetics, turnover, and stimulatory capacity of thymic epithelial cells

AbstractDespite the importance of thymic stromal cells to T-cell development, relatively little is known about their biology. Here, we use single-cell analysis of stromal cells to analyze extensive changes in the number and composition of thymic stroma throughout life, revealing a surprisingly dynamic population. Phenotypic progression of thymic epithelial subsets was assessed at high resolution in young mice to provide a developmental framework. The cellular and molecular requirements of adult epithelium were studied, using various mutant mice to demonstrate new cross talk checkpoints dependent on RelB in the cortex and CD40 in the medulla. With the use of Ki67 and BrdU labeling, the turnover of thymic epithelium was found to be rapid, but then diminished on thymic involution. The various defects in stromal turnover and composition that accompanied involution were rapidly reversed following sex steroid ablation. Unexpectedly, mature cortical and medullary epithelium showed a potent capacity to stimulate naive T cells, comparable to that of thymic dendritic cells. Overall, these studies show that the thymic stroma is a surprisingly dynamic population and may have a more direct role in negative selection than previously thought.

Chromosomal clustering of genes controlled by the aire transcription factor

Autoimmune regulator (aire) is a transcription factor that controls the self-reactivity of the T cell repertoire. Although previous results indicate that it exerts this function in part by promoting ectopic expression of a battery of peripheral-tissue antigens in epithelial cells of the thymic medulla, recent data argue for additional roles in negative selection of thymocytes by medullary cells. As one approach to exploring such roles, we performed computational analyses of microarray data on medullary RNA transcripts from aire-deficient versus wild-type mice, focusing on the genomic localization of aire-controlled genes. Our results highlight this molecule's transcriptional activating and silencing roles and reveal a significant degree of clustering of its target genes. On a local scale, aire-regulated clusters appeared punctate, with aire-controlled and aire-independent genes often being interspersed. This pattern suggests that aire's action may not be a simple reflection of the wide action of a chromatin remodeling enzyme. Analysis of the identity of certain of the clustered genes was evocative of aire's potential roles in antigen presentation and the coordination of intrathymic cell migration: for example, major histocompatibility complex class I and class II gene products and certain chemokine genes are targets of aire-regulated transcription.

Distribution Properties of Polymononucleotide Repeats in Molluscan Genomes

A total of 635 DNA sequences from 35 species of mollusks were used as taxonomic support to investigate several distribution features of polymononucleotides in genomic regions of different functionality. We show that all polymononucleotide types in mollusks fit to expectations in exons but not in nonexonic regions, in agreement with a leading role of negative selection on expansions/contractions of transcription-linked poly-(A/T) repeats. The fit of all repeat length types to an exponential decay precludes the existence of a threshold size for replication slippage, a popular but unsatisfactorily explained concept in mutation models for single repeats. The genomic density of poly-(A/T) repeats is not correlated with the DNA content of species, suggesting that the differential density of repeats between species could be better explained by the species-specific performance of its repair mechanisms. This research allows a better understanding of the distribution patterns of single repeats in eukaryotes.

Dynamics of T cell activation threshold tuning

T lymphocytes are believed to alter their sensitivity to TCR stimulation by means of a tunable cellular activation threshold. We present two modelling examples which show that the concept of a tunable threshold can be made mechanistically plausible. The tunable threshold is treated as an emergent property of the dynamics of the T cell's signalling machinery. In addition, we discuss how the dynamic properties of activation threshold tuning can be determined experimentally with the aid of these two models. We propose a novel ‘avidity selection’ mechanism for the initial stages of the immune response, based on the properties of the T cell activation threshold tuning mechanism we propose for the commitment to differentiation. Our main finding is that activation threshold tuning allows T cells to respond to relevant ligands with a detection threshold that is (i) uniform across both the T cell repertoire and the secondary lymphoid tissues, while (ii) retaining tolerance to autostimulation. Our analysis indicates that central tolerance enhances the efficiency of peripheral tolerance, casting new light on the role of negative selection in the thymus.

LIGHT (a cellular ligand for herpes virus entry mediator and lymphotoxin receptor)-mediated thymocyte deletion is dependent on the interaction between TCR and MHC/self-peptide.

Negative selection serves as a major mechanism to maintain self-tolerance. We previously reported that LIGHT (a cellular ligand for herpes virus entry mediator and lymphotoxin receptor), a TNF family member, plays an important role in thymocyte development via promoting apoptosis of double-positive thymocytes. Here, we demonstrated that LIGHT-mediated deletion of thymocyte requires the strong interaction of TCR with MHC/self-peptide. Transgenic mice overexpressing LIGHT in thymocytes were bred with a transgenic mouse line expressing a TCR recognizing the H-Y male Ag in the context of H-2b class I MHC molecules. In male H-Y/LIGHT double-transgenic mice, more efficient negative selection of H-Y T cells occurred, and total thymocyte number was further reduced compared with H-Y/negative littermates. In contrast, the presence of LIGHT transgene had no evident impact on the thymocyte development of female H-Y/LIGHT double-transgenic mice. Taken together, LIGHT plays a role in negative selection of thymocytes via inducing the apoptosis of thymocytes bearing high affinity TCR during negative selection.

Thymocyte development in early growth response gene 1-deficient mice.

Early growth response gene 1 (Egr1) codes for a transcriptional regulator that contains a zinc-finger DNA binding domain. Egr1 expression is induced by a variety of extracellular stimuli including TCR-ligand interactions. Its pattern of expression in the thymus and dependence on ERK activation have led to speculation that it has a role in T cell development, but the exact nature of this role has been undefined. To more clearly define the role of Egr1 in thymocyte development, we have analyzed thymocytes from Egr1-deficient mice. We find that thymuses from Egr1-deficient mice contain twice as many cells as age-matched controls, and the increase in thymocyte number is apparent at the early CD4/CD8 double negative stage of development. Subsequent maturation to the CD4/CD8 double positive stage and survival of the double positive cells both appear normal in Egr1-deficient animals. We also find that Egr1 promotes positive selection of both CD4 and CD8 single positive cells without playing a major role in negative selection. Egr1 influences positive selection by enhancing expression of the helix-loop-helix inhibitor Id3 and the anti-apoptosis molecule bcl-2. Thus, Egr1 translates developmental signals into appropriate changes in gene expression at multiple stages of thymocyte development.

Different role of Apaf-1 in positive selection, negative selection and death by neglect in foetal thymic organ culture.

Apoptotic protease-activating factor 1 (Apaf-1) is a component of the apoptosome which is required for the activation of procaspase-9. As Apaf-1 knockout (KO) (Apaf-1-/-) mice die before birth, the role of Apaf-1 during thymic selection was investigated using 5 day foetal thymic organ culture (FTOC) of thymi obtained at gestational day 15. There was a lower ratio of CD4 single-positive (SP) to CD8 SP cells and decreased apoptosis of CD4+CD8+ (DP) thymocytes from Apaf-1-/- mice compared with wild-type. To determine if these defects resulted in increased production of neglected thymocytes, the Apaf-1-/- mice were crossed with the T-cell receptor (TCR)-alpha-chain KO mice. There was no difference in thymocyte development in the thymi of TCR-alpha-/-Apaf-1-/- and TCR-alpha-/-Apaf-1+/+ mice 5 days after FTOC. To determine if Apaf-1 is involved in apoptosis during death by negative or positive selection, FTOC of the thymus of Apaf-1-/- Db/HY TCR-alphabeta transgenic (Tg) mice was carried out. There was decreased apoptosis of the HY clonal-specific M33+ thymocytes and an increased percentage of the autoreactive CD8+M33+ thymocytes in male, but not female Apaf-1-/- Db/HY TCR Tg mice. Our data suggest that Apaf-1 is not involved in positive selection or death by neglect, but may have a partial role in negative selection during early thymic T-cell development.

The critical role of LIGHT, a TNF family member, in T cell development.

Negative selection refers to the selective deletion of autoreactive thymocytes but its molecular events have not been well defined. In this study, we demonstrate that a cellular ligand for herpes virus entry mediator and lymphotoxin receptor (LIGHT), a newly identified member of the TNF superfamily, may play a critical role in negative selection. Using TCR transgenic mice, we find that the blockade of LIGHT signaling in vitro and in vivo prevents negative selection induced by peptide and intrathymically expressed Ags, resulting in the rescue of thymocytes from apoptosis. Furthermore, the thymi of LIGHT transgenic mice show severe atrophy with remarkably reduced CD4(+)CD8(+) double-positive cells caused by increased apoptosis, suggesting that LIGHT can delete immature T cells in vivo. Taken together, these results demonstrate a critical role of LIGHT in thymic negative selection of the T cell repertoire.

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

Negative selection in the thymus includes semimature T cells.

The thymic medulla plays a key role in negative selection (self-tolerance induction) and contains differentiated T cells en route to the extrathymic environment. However, being relatively mature, medullary T cells are thought to be beyond the stage of tolerance induction. This paradox is resolved by the finding that medullary T cells (CD4+8- thymocytes) comprise two distinct subsets. Medullary thymocytes expressing a fully mature (HSAlo) phenotype are strongly resistant to tolerance induction, whereas cells with a semimature (HSAhi) phenotype are tolerance susceptible. These findings suggest that the differentiated T cells reaching the medulla from the cortex remain sensitive to tolerance induction for a brief period before acquiring a fully mature tolerance-resistant phenotype. The semimature subset of medullarsy T cells displays unique requirements for tolerance induction; depending upon the conditions used, tolerizing these cells can involve either a Fas (CD95)-dependent or a Fas-independent pathway.

Engraftment of rat bone marrow and its role in negative selection of murine T cells in mice conditioned with a modified nonmyeloablative regimen

Abstract: We have recently demonstrated that xenogeneic (rat) bone marrow engraftment and donor‐specific tolerance can be induced in mice conditioned with a nonmyeloablative regimen consisting of administration of anti‐CD4, ‐CD8, ‐Thy‐1.2, and ‐NK1.1 mAbs on days ‐6 and ‐1, 3 Gy whole body irradiation (WBI) on day 0, and 7 Gy thymic irradiation on day 0, followed by injection of T cell‐depleted (TCD) F344 BMC. We have now investigated the mechanism of tolerance in these mixed xenogeneic chimeras by evaluating the capacity of rat bone marrow‐derived cells to induce clonal deletion of mouse T cell subsets. Both Vβ5+ and Vβ11+ murine T cell subsets were markedly depleted in peripheral blood of chimeras but not of normal B1O mice. These results demonstrate that rat molecules contribute to the ligand for negative selection of these Vβ families of mouse T cells, and that rat bone marrow‐derived cells participate in negative selection of the murine T cell repertoire, suggesting that clonal deletion is probably a major mechanism of tolerance induction in this model. In addition, based on results in a similar allogeneic BMT model, we hypothesized that, if sufficient quantities of mAbs were administered, thymic irradiation could be eliminated from this regimen without compromising the ability to induce mixed xenogeneic chimerism and tolerance. We now demonstrate induction of comparable multilineage mixed xenogeneic chimerism in animals receiving this conditioning regimen with or without 7 Gy thymic irradiation. Thus, if a similar approach were applied clinically, the potential toxicity of this regimen might be reduced further by the elimination of thymic irradiation.

Prevention by liver transplantation of the graft-versus-host reaction and allograft rejection in a rat model of small bowel transplantation.

In the rat combination of DA (MHC haplotype RT1av1) donor into PVG(RT1c) recipient, liver grafts are not rejected and allow the acceptance of other organ grafts from the same donor strain. Here we show that an existing DA liver graft allows the acceptance of a DA small bowel graft in the PVG; the liver graft also prevented the graft-versus-host reaction normally associated with small bowel grafting. In addition, a liver graft could suppress the GVHR in F1 hybrid recipients of parental lymphoid cells, a classic GVHR model. GVHR suppression was immunologically specific and required that donor lymphocytes and liver be of the same strain. Besides their clinical implications, these results demonstrate the capacity of the liver for tolerance induction and suggest that it may play a physiological role in negative selection of T cells.