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Results for “thymic selection”

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PubMed1994

Isolation of P1 bacteriophage clones containing large contiguous segments of the human and mouse loci for the T-cell coreceptor molecule CD8.

The T-lymphocyte coreceptor molecules CD8 (composed of CD8 alpha and CD8 beta chains) and CD4 undergo a complex pattern of regulated expression during T-cell maturation. In the thymus, the most immature cells progress from expressing neither molecule (the double-negative [DN] stage) to an intermediate stage at which both are coexpressed (the double-positive [DP] stage). As a result of thymic selection and further differentiation, DP cells give rise to the most mature thymic cells and peripheral T cells that express either CD8 or CD4 (the single-positive [SP] stage). Our previous studies of the transcriptional regulatory mechanisms controlling CD8 alpha expression during the DN-->DP and DP-->SP transitions suggest the existence of important cis-acting elements located a considerable distance from the CD8 alpha gene and that these elements might serve to regulate both CD8 alpha and CD8 beta. While both genes and intergenic DNA span approximately 60 kb in the mouse, the relevant cis elements could lie either within or beyond this region. As a result, we sought to isolate large contiguous segments of DNA in P1 bacteriophage that covered at least this region from the mouse and human CD8 locus. Our initial physical characterization of these clones demonstrates the value of the P1 system as all isolated clones were found to contain single contiguous 85- to 95-kb segments of DNA that are faithful replicas of the chromosomal locus. The presence of abundant native flanking DNA both upstream and downstream of the intact coding regions will make these clones extremely useful for identifying physiological CD8 cis-active regulatory elements by virtue of their ability to direct appropriate lineage- and stage-specific expression in transfected and transgenic T cells.

Animals↗
PubMed2000

Transgenic expression of Ly-49A in thymocytes alters repertoire selection.

A T cell-specific Ly-49A transgene inhibits TCR-mediated activation in the presence of H-2Dd. Expression of this transgene by developing thymocytes impairs negative selection evidenced by a failure to delete potentially autoreactive T cells and development of a graft-vs-host-disease-like syndrome. In mice carrying both the Ly-49A and a class II-restricted TCR transgene, positive selection was lost, but only when H-2Dd was present on thymic epithelium. These results are consistent with models suggesting that thymic selection is dependent on the perceived intensity of TCR signaling. More interestingly, these results show that Ly-49A does not simply provide a strict on/off switch for T cell responses. Since Ly-49A may shift the signaling threshold of TCR-induced triggering, inducible expression of Ly-49A may regulate peripheral memory/activated T cells by raising the threshold for T cell reactivation.

Animals↗
PubMed2001

Major histocompatibility complex class II-positive cortical epithelium mediates the selection of CD4(+)25(+) immunoregulatory T cells.

CD4(+)25(+) T cells are a unique population of immunoregulatory T cells which are critical for the prevention of autoimmunity. To address the thymic selection of these cells we have used two models of attenuated thymic deletion. In K14-A(beta)(b) mice, major histocompatibility complex (MHC) class II I-A(b) expression is limited to thymic cortical epithelium and deletion by hematopoietic antigen-presenting cells does not occur. In H2-DMalpha-deficient mice, MHC class II molecules contain a limited array of self-peptides resulting in inefficient clonal deletion. We find that CD4(+)25(+) T cells are present in the thymus and periphery of K14-A(beta)(b) and H2-DMalpha-deficient mice and, like their wild-type counterparts, suppress the proliferation of cocultured CD4(+)25(-) effector T cells. In contrast, CD4(+)25(+) T cells from MHC class II-deficient mice do not suppress responder CD4(+) T cells in vitro or in vivo. Thus, development of regulatory CD4(+)25(+) T cells is dependent on MHC class II-positive thymic cortical epithelium. Furthermore, analysis of the specificities of CD4(+)25(+) T cells in K14-A(beta)(b) and H2-DMalpha-deficient mice suggests that a subset of CD4(+)25(+) T cells is subject to negative selection on hematopoietic antigen-presenting cells.

Animals↗
PubMed1992

Interleukins modulate glucocorticoid-induced thymocyte apoptosis.

Glucocorticoid hormones, calcium ionophores and anti-CD3 monoclonal antibodies induce apoptosis in mouse thymocytes. This type of cell death, which is characterized by an extensive DNA fragmentation into oligonucleosomal subunits, occurs in the intrathymic process of negative selection, and is involved in the deletion of autoreactive T-cells during thymic maturation. A number of cytokines are able to modulate apoptosis, and interleukins, including interleukin-1, interleukin-2, and interleukin-4, play a crucial role in thymic maturation and T-cell development. We tested the effects of several cytokines on the glucocorticoid hormone-induced apoptosis of mouse thymocytes in vitro, and demonstrated that interleukin-1 alpha, interleukin-2, and interleukin-4 inhibit the apoptosis induced by dexamethasone, but that interleukin-3 and interleukin-6 exert no noteworthy effect. Dose-response experiments indicated that interleukin-4 is more potent than interleukin-1 alpha and interleukin-2 in inhibiting dexamethasone-induced apoptosis. Furthermore, interleukin-4 fully inhibited the DNA fragmentation induced by the protein kinase-C activator 12-O-tetradecanoylphorbol-13-acetate, but was ineffective against apoptosis induced by the calcium ionophore A23187. These results suggest that interleukins regulate the thymic selection process by acting as modulators of the negative selection process.

Animals↗
PubMed1991

Transgenic mice as immune system models.

In the past year, transgenic mice have continued to be powerful models for the investigation of various features of the immune system, particularly for studies of lymphocyte differentiation and tolerance. Major achievements have included the definition of intra-thymic selection events in T-cell differentiation, and the demonstration of extra-thymic tolerance resulting from clonal energy.

Animals↗
PubMed1994

Small cortical thymocytes are subject to positive selection.

To determine the developmental stages at which positive selection can act to produce mature T cells, CD4+8+3lo thymocytes of large dividing type and of small nondividing type were sorted and transferred into the thymus of nonirradiated Thy-1 congenic recipient mice. In contrast to earlier studies, the small as well as the large thymocytes produced mature CD4+8-3hi and CD4-8+3hi progeny, although production was less efficient from the small cells. The relative efficiency of small cells was increased and was close to that of large cells when bcl-2/anti-HY T cell receptor (TCR) alpha beta transgenic donors were used to improve cell survival, overcome stress effects of the transfer process, and increase the frequency of selectable cells. The results from transferring small CD4+8+3lo thymocytes expressing a TCR transgene from a nonselecting to a selecting thymic MHC environment also confirmed that the small cells were capable of being selected and maturing. Thus the developmental window available for positive selection includes the small CD4+8+3lo thymocytes. The results also showed a striking difference in the kinetics of production of mature progeny from the transferred CD4+8+3lo precursors. CD4+8-3hi cells appeared several days before CD4-8+3hi cells, apparently because the CD4-8+ lineage cells spent several days in transit as CD4+8+3hi intermediates before losing CD4. Most CD4+8- lineage cells on the other hand, either passed very rapidly through this intermediate stage, or lost CD8 before increasing the expression of CD3.

Animals↗
PubMed1997

Positive and negative CD4+ thymocyte selection by a single MHC class II/peptide ligand affected by its expression level in the thymus.

The central event in thymic selection of T cells bearing alpha beta TCRs is their interaction with self-peptides bound to self-MHC molecules. With the use of transgenic mouse lines expressing a single peptide/MHC class II complex, we show that CD4+ T cells with the preferential usage of particular TCR V(alpha)s and V(beta)s were selected to mature on this complex in lines with the lower expression, whereas such CD4+ T cells were eliminated in the thymus in a line with the relatively high expression. When a low expressing line was crossed with a high expressing line, the frequency of CD4+ T cells selected by this complex markedly decreased. Thus, these results suggest that a single peptide/MHC class II complex, being affected by its cell surface density in the thymus, can serve as both positively and negatively selecting ligand in vivo.

Amino Acid Sequence↗
PubMed2005

1Alpha,25-dihydroxyvitamin D3 restores thymocyte apoptosis sensitivity in non-obese diabetic (NOD) mice through dendritic cells.

AIMS/HYPOTHESIS: Resistance of NOD thymocytes to apoptosis-inducing signals is restored by 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3), a therapy preventing diabetes in NOD mice. We studied whether modulation of thymocyte apoptosis is due to direct effects on thymic T lymphocytes or indirect effects via thymic dendritic cells, since both cell types constitute known targets for 1alpha,25OH2D3. METHODS AND RESULTS: Female NOD mice were treated with 1alpha,25OH2D3 (5microg/kg/2d) from 21 to 70 days. Vehicle-treated NOD and NOR mice served as controls. Analysis of thymic T lymphocytes from 1alpha,25OH2D3)-treated mice revealed a decrease in number of apoptosis-resistant CD4+CD8+ and CD4+CD8-HSA(high) T lymphocyte subsets, higher pro-apoptotic IL-2 and FasL, and lower anti-apoptotic Bclx-L mRNA expression levels. Thymic dendritic cells from 1alpha,25OH2D3-treated NOD mice had increased CD8alpha+FasL+ and CD80+/86+ expression compared to control NOD mice. In a syngeneic co-culture system of thymocytes and thymic dendritic cells, apoptosis levels were 20% higher only in co-cultures where both T cell- and dendritic cell-compartments originated from 1alpha,25OH2D3-treated mice. Activation-induced cell death-sensitivity in peripheral T lymphocytes was comparable to levels present in NOR mice, confirming better thymic selection in 1alpha,25OH2D3-treated mice. CONCLUSION/INTERPRETATION: We conclude that 1alpha,25OH2D3 needs both thymic T cell- and dendritic cell-compartments to exert its apoptosis-restorative effects in NOD thymocytes.

Animals↗
PubMed1999

Evolution of antigen-specific T cell receptors in vivo: preimmune and antigen-driven selection of preferred complementarity-determining region 3 (CDR3) motifs.

Antigen (Ag)-driven selection of helper T cells (Th) in normal animals has been difficult to study and remains poorly understood. Using the major histocompatibility complex class II- restricted murine response to pigeon cytochrome c (PCC), we provide evidence for both preimmune and Ag-driven selection in the evolution of Ag-specific immunity in vivo. Before antigenic challenge, most Valpha11(+)Vbeta3(+) Th (70%) express a critical complementarity-determining region 3 (CDR3) residue (glutamic acid at TCR-alpha93) associated with PCC peptide contact. Over the first 5 d of the primary response, PCC-responsive Valpha11(+)Vbeta3(+) Th expressing eight preferred CDR3 features are rapidly selected in vivo. Clonal dominance is further propagated through selective expansion of the PCC-specific cells with T cell receptor (TCR) of the "best fit." Ag-driven selection is complete before significant emergence of the germinal center reaction. These data argue that thymic selection shapes TCR-alpha V region bias in the preimmune repertoire; however, Ag itself and the nongerminal center microenvironment drive the selective expansion of clones with preferred TCR that dominate the response to Ag in vivo.

Animals↗
PubMed2002

Morphologic transformation of the thymus in developing zebrafish.

The morphologic transformation of the developing zebrafish thymus from 1 week postfertilization (wpf) to 15 wpf is described. The thymus overall morphology changed from a small pouch-like shape at 1 wpf to a conical shape between 2 and 3 wpf before acquiring a more complex shape from 4 wpf onward. Rapid growth rate along the lateral axis at the region near the pharyngeal epithelium occurred between 1 and 2 wpf, whereas rapid growth rate along the dorsal-ventral axis occurred between 3 and 6 wpf. Expansion of thymocyte population beginning from 1 wpf became more evident by 2 to 3 wpf, as indicated by the apparent increase of different sizes of lymphocytes, recombination activating gene-1 (rag-1), and T-cell antigen receptor alpha chain constant region (TCRAC) -positive cells. Tissue section in situ hybridization (ISH) analysis with rag-1 probe reveals that cortex-medullary regionalization has begun between 1 and 2 wpf as rag-1 expression clearly demarcated the cortex, whereas the medulla was rag-1 negative. The presence of TCRAC-positive cells in the medulla by 2-3 wpf, suggests that the thymic selection processes had begun. The zebrafish thymus is morphologically mature by 3 wpf. Early signs of thymic involution were observed in zebrafish aged 15 wpf.

Animals↗
PubMed1995

Characterization of mouse CD53: epitope mapping, cellular distribution and induction by T cell receptor engagement during repertoire selection.

The pan-leukocyte antigen CD53 is a member of the poorly understood transmembrane 4 superfamily (TM4SF) of cell membrane glycoproteins. CD53 is proposed to play a role in thymopoiesis, since rat CD53 is expressed on immature CD4-8-thymocytes and the functionally mature single-positive subset, but is largely absent from the intermediate CD4+8+ cells. We have characterized CD53 in the mouse through the production of two new monoclonal antibodies, MRC OX-79 and OX-80, which were raised against the RAW 264 cell line and screened on recombinant CD53 fusion proteins. The epitopes recognized by both antibodies are dependent on disulfide bonding and map to the major extracellular region of CD53, requiring the presence of a single threonine residue at position 154. Mouse CD53 has a molecular mass of 35-45 kDa and is expressed on virtually all peripheral leukocytes, but not on cells outside the lymphoid or myeloid lineages. CD53 expression distinguishes subpopulations of thymocytes in the mouse and resembles the expression pattern of rat CD53. Amongst the immature CD4-8-thymocytes, mouse CD53 is clearly detectable on the earliest CD44high25- subset, but down-regulated on the later CD44high25+, CD44low25+ and CD44low25- stages. Also, the subsequent transient TcR-/low CD4-8+ cells and most CD4+8+ thymocytes express little or no CD53. This is consistent with the idea that cells which are committed to enter the selectable CD4+8+ compartment switch off CD53. The effect of T cell receptor (TcR) engagement on the re-expression of CD53 on CD4+8+ thymocytes was studied both ex vivo and in vitro using F5 mice, transgenic for the H-2b/influenza nucleoprotein-peptide-specific TcR, back-crossed onto an H-2q or H-2b background of RAG-2-deficient mice. CD4+8+ thymocytes from non-selecting H-2q F5 mice are CD53 negative, but in vitro stimulation through the TcR dramatically induces CD53 expression. In contrast, a fraction of CD4+8+ thymocytes from positively selecting H-2b F5 transgenic mice express CD53. Therefore TcR engagement by selecting major histocompatibility complex peptide complexes, or surrogate ligands, induces CD53 expression on otherwise CD53-negative, non-selected CD4+8+ thymocytes. Whether CD53 itself participates as a signaling molecule in further stages of thymic selection is still a matter of speculation.

Amino Acid Sequence↗
PubMed2003

CD45 isoform expression in autoimmune myasthenia gravis.

In myasthenia gravis (MG), humoral and cellular immune mechanisms are involved in the autoimmune pathogenesis. In this study, we investigated the role of the CD45 molecule in MG, having recently reported an association in multiple sclerosis. CD45, a protein-tyrosine phophatase receptor type C (PTPRC), is essential for both thymic selection and peripheral activation of T and B cells. Our aims were to determine (a) the prevalence of a functional mutation in the CD45 gene (exon 4 77C --> G; prevalence analysis), and (b) the distribution of memory (CD45RO+) and naive (CD45RA+) T cells in the peripheral blood (subset analysis). T cells from 78 patients with generalised MG were stained with monoclonal antibodies against CD45RO, CD45RA, CD4 and CD8 and quantified by four-colour flow cytometry. The control panel for the prevalence analysis (a) consisted of 303 healthy individuals. (b) From those, 67 age- and sex-matched probands were randomly selected as controls for the subset analysis. Patients were stratified according to their MG onset age, thymic pathology and immunosuppressive treatment. Statistical analysis was performed by Fisher's exact test, asymptotic chi2 test, the two-sided Mann-Whitney test and Spearman's correlation coefficient. As a result, the 77C --> G mutation in exon 4 of the CD45 gene was found in 1 of 78 patients versus none of the 303 controls. Thus, no association was detected with this single nucleotide polymorphism in MG patients overall. Surprisingly, however, ratios of CD45RO+ to CD45RA+ T cells were lower among CD8+ T cells from patients with late-onset MG (P = 0.023). Thymoma patients also showed a similar trend among CD4+ and CD8+ T-cells, as expected. These differences were not related to immunosuppressive drug treatment or thymectomy (in the 67 informative patients). Since there is no other evidence for increased thymopoiesis in late-onset MG, we propose an altered subset balance in the circulation.

CD4-Positive T-Lymphocytes↗
PubMed1997

Down-modulation of CD8 beta-chain in response to an altered peptide ligand enables developing thymocytes to escape negative selection.

Mice expressing a Kb-restricted transgenic T cell receptor (TCR) and a naturally occurring MHC class I variant molecule, Kbm8, were used to study thymic selection. The transgenic TCR was specific for the major peptide determinant from ovalbumin (OVA(257-264)), while Kbm8 has a mutation that alters the position 2 binding pocket of the Kb molecule, abolishing antigenic peptide presentation and positive selection of transgenic T cells. Peptide presentation was restored by identifying a position 2 analog peptide with Kbm8-binding capacity. In combination with Kbm8, the E2 peptide variant was capable of deleting immature double-positive thymocytes in suspension culture. Similarly, addition of exogenous E2 peptide to fetal thymic organ culture resulted in efficient deletion of double-positive thymocytes. However, there remained a population of CD8 single-positive T cells that exhibited impaired responsiveness to the antigenic peptide and lacked expression of the CD8 beta-chain. These results suggest a mechanism whereby developing thymocytes bearing an alphabetaTCR can modify their expression of the CD8 coreceptor to escape thymic deletion and achieve self-tolerance.

Animals↗
PubMed1996

A model for developmentally acquired thymus-dependent tolerance to central and peripheral antigens.

Current models of tolerance to peripheral, tissue-specific antigens contain some major caveats. First, they consider peripheral tolerance independently from intrathymic T cell selection, a dichotomy that is challenged by observations on TE-induced tolerance. Second, they do not account for the fact that vertebrates are more readily tolerised in development than in adult life. Third, they do not explain the fact that embryonic/neonatal tolerance to foreign tissues can only be induced by HC or TE. A model of thymic selection and peripheral tolerance is developed here that resolves those problems, by assuming two classes of T cell effector functions, one being regulatory and the other aggressive. Three postulates are required: (1) both epithelial and hemopoietic cellular compartments of the thymic stroma can support both positive and negative selection of T cells, but with vastly different avidity requirements and efficiency; (2) positively selected T cells with the highest avidity that escape deletion are activated intrathymically and irreversibly committed for regulatory effector functions; (3) the functional phenotype of all other thymic emigrants is determined in the periphery upon encounter with antigen. Functional commitment in the periphery depends on the maturity stage (RTE or PMR) of the immunocompetent cell, on the nature of the antigen-presenting cells, and on the effector classes of other T lymphocytes interacting on the same presenting cell. This model explains a number of observations on experimental autoimmune disease and transplantation tolerance, and it contains several readily testable predictions.

Animals↗
PubMed2007

Bcl10/Malt1 signaling is essential for TCR-induced NF-kappaB activation in thymocytes but dispensable for positive or negative selection.

During T cell development in the thymus, high-affinity/avidity TCR engagement induces negative selection by apoptosis, while lower affinity/avidity TCR interactions lead to positive selection and survival of thymocytes. Yet, the mechanisms that discriminate between positive and negative selection are not fully understood. One major regulator of survival and apoptosis in lymphoid cells is the transcription factor NF-kappaB. Several reports have indicated key roles for NF-kappaB in positive and negative selection. In peripheral T cells, TCR ligation activates NF-kappaB through a selective pathway that involves protein kinase Ctheta, Bcl10, and Malt1. While protein kinase Ctheta is dispensable for thymic TCR signaling, the molecular roles of Bcl10 and Malt1 in thymocytes have not been investigated. In the present study, we show that both Bcl10 and Malt1 are essential for TCR signaling in thymocytes as a genetic disruption of either molecule blocks TCR-induced NF-kappaB activation in these cells. To investigate the function of this pathway in thymic selection, we introduced the Bcl10 or Malt1 mutations into three well-established TCR transgenic mouse models. Surprisingly, using several in vivo or in vitro assays, we were unable to demonstrate a role for TCR-induced NF-kappaB activation in either positive or negative selection. Thus, while TCR signaling to NF-kappaB controls the activation of mature T cells, we suggest that this pathway is not involved in the positive or negative selection of thymocytes.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Allelic exclusion of mouse T cell receptor alpha chains occurs at the time of thymocyte TCR up-regulation.

We report a detailed analysis of TCR V alpha and V beta chain expression on immature versus mature thymocytes of normal, TCR beta-transgenic, and TCR alpha-hemizygous mice. Chain pairing between TCR V alpha and V beta chains is random on immature thymocytes, but individual chain pairs are selected in mature thymocytes. This indicates that V alpha-V beta chain pairing preferences are determined during thymic selection, and not as a result of structural constraints. Dual V alpha chain expression is found frequently on immature, but not mature thymocytes. It is not found in TCR alpha-hemizygous mice, showing that cell surface expression of dual alpha chains is caused by lack of allelic exclusion in immature thymocytes. Down-regulation of one of the alpha chains occurs concurrently with differentiation from TCRlo, CD69- to TCRhi, CD69+ phenotype, suggesting that it is associated with positive selection of the functional TCR.

Alleles↗
PubMed2001

A kinetic window constricts the T cell receptor repertoire in the thymus.

To characterize the ligand binding properties of a naive T cell repertoire capable of responding to a foreign antigen, we analyzed T cell populations from T cell receptor (TCR) beta transgenic mice using a novel, single cell peptide/major histocompatibility complex (MHC) tetramer dissociation assay. The largely CD4+CD8(-/low) antigen-specific thymocyte repertoire exhibited a broad, bimodal distribution of tetramer binding half-lives (t(1/2)s), with a significant underrepresentation in the intermediate half-life range in which the majority of the peripheral repertoire lies. Thus, cells with the potential to bind foreign antigen with the lowest and highest stability are likely to be selectively removed from the repertoire prior to their establishment in the periphery. These studies provide direct evidence that thymic selection biases the naive peripheral T cell repertoire toward TCR-ligand interactions that fall within a moderate half-life "window."

Amino Acid Sequence↗
PubMed2005

AIRE recruits multiple transcriptional components to specific genomic regions through tethering to nuclear matrix.

Thymic selection requires that diverse self antigens be presented to developing thymocytes by stromal cells. Consistent with this function, medullary thymic epithelial cells have been shown to express a large number of genes, many of which are tissue restricted. Autoimmune regulator (AIRE) is a nuclear protein, which has recently been identified as a regulator of this process, however, the mechanism by which AIRE functions is not well understood. Here we use a transrepression assay to demonstrate that AIRE interacts with multiple components of the transcription complex including a novel interaction with the UBA domain protein, GBDR1. When AIRE is expressed in cultured human thymic epithelial cells, it tightly associates with nuclear matrix, suggesting that AIRE responsive genes may be localized to specific regions. Using a mathematical approach we have re-analyzed an Affymetrix dataset identifying AIRE responsive genes and show that they tend to localize to specific regions of the genome. Together, these data suggest that AIRE regulates gene expression by recruiting components of the transcription complex to specific regions of the genome via interactions with nuclear matrix.

Animals↗