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The mechanisms underlying age-associated thymic involution are unknown. In mice, thymic involution shows mouse strain--dependent genetic variation. Identification of the underlying genes would provide mechanistic insight into this elusive process. We previously showed that responsiveness of hematopoietic stem and progenitor cells (HSPCs) to transforming growth factor-beta 2, a positive regulator of HSPC proliferation, is regulated by a quantitative trait locus (QTL) on chr. 4, Tb2r1. Interestingly, [Tgfb2.sup.+/-] mice have delayed thymic involution. Therefore, we tested the hypothesis that a QTL on chr. 4 might regulate thymic involution. Aged, but not young, B6.D2-chr.4 congenic mice, where the telomeric region of chr. 4 was introgressed from DBA/2 to C57BL/6 mice, had larger thymi, and better maintenance of early thymic precursors than C57BL/6 control mice. These observations unequivocally demonstrate that the telomeric region of chr. 4 contains a QTL, Til (thymic involution 1) that regulates thymic involution, and suggest the possibility that Ti1 may be identical to Tb2r1. doi: 10.1093/gerona/glq041

T cell egress from the thymus is essential for adaptive immunity, yet the requirements for and sites of egress are incompletely understood. We have shown that transgenic expression of sphingosine-1-phosphate receptor-1 (S1P1) in immature thymocytes leads to their perivascular accurnulation and premature release into circulation. Using an intravascular procedure to label emigrating cells, we found that mature thymocytes exit via blood vessels at the corticomedullary junction. By deleting sphingosine kinases in neural crest-derived pericytes, we provide evidence that these specialized vessel-ensheathing cells contribute to the S1P that promotes thymic egress. Lymphatic endothelial cell-derived S1P was not required. These studies identify the major thyrnic egress route and suggest a role for pericytes in promoting reverse transmigration of celts across blood vessel endothelium. doi: 10.1126/science.1188222

Although the polysialyltransferase ST8Sia IV is expressed in both primary and secondary human lymphoid organs, its product, polysialic acid (polySia), has been largely overlooked by immunologists. In contrast, polySia expression and function in the nervous system has been well characterized. In this context, polySia modulates cellular adhesion, migration, cytokine response, and contactdependent differentiation. Provocatively, these same processes are vital components of immune development and function. We previously established that mouse multipotent hematopoietic progenitors use ST8Sia IV to express polySia on their cell surfaces. Here, we demonstrate that, relative to wild-type controls, ST8Sia IV-/- mice have a 30% reduction in total thymocytes and a concomitant deficiency in the earliest thymocyte precursors. T-cell progenitors originate in the bone marrow and are mobilized to the blood at regular intervals by unknown signals. We performed in vivo reconstitution experiments in which ST8Sia IV-/- progenitors competed with wild-type cells to repopulate depleted or deficient immune subsets. Progenitors lacking polySi exhibited a specific defect in T-cell development because of an inability to access the thymus. This phenotype probably reflects a decreased capacity of the ST8Sia IV-/- progenitors to escape from the bone marrow niche. Collectively, these results provide evidence that polySia is involved in hematopoietic development. bone marrow niche | hematopoiesis | mobilization | development

The location of leukocytes in different microenvironments is intimately connected to their function and, in the case of leukocyte precursors, to the executed differentiation and maturation program. Leukocyte migration within lymphoid organs has been shown to be mediated by constitutively expressed chemokines, but how the bioavailability of these homeostatic chemokines is regulated remains unknown. Here, we report in vivo evidence for the role of a nonsignaling chemokine receptor in the migration of leukocytes under physiological, i.e., noninflammatory, conditions. We have studied the in vivo role of the silent chemokine receptor CCX-CKR1 by both loss- and gain-of-function approaches. CCXCKR1 binds the constitutively expressed chemokines CC chemokine ligand (CCL)19, CCL21, and CCL25. We find that CCX-CKR1 is involved in the steady-state homing of CD[11c.sup.+][MHCII.sup.high] dendritic cells to skin-draining lymph nodes, and it affects the homing of embryonic thymic precursors to the thymic anlage. These observations indicate that the silent chemokine receptor CCX-CKR1, which is exclusively expressed by stroma cells, but not hematopoietic cells themselves, regulates homeostatic leukocyte migration by controlling the availability of chemokines in the extracellular space. This finding acids another level of complexity to our understanding of leukocyte homeostatic migration. cell trafficking | homeostasis | lymphopoiesis | thymus

The spliceosome cycle consists of assembly, catalysis, and recycling phases. Recycling of postspliceosomal U4 and U6 small nuclear ribonucleoproteins (snRNPs) requires p110/SART3, a general splicing factor. In this article, we report that the zebrafish earl grey (egy) mutation maps in the p110 gene and results in a phenotype characterized by thymus hypoplasia, other organ-specific defects, and death by 7 to 8 days postfertilization. U4/U6 snRNPs were disrupted in egy mutant embryos, demonstrating the importance of p110 for U4/U6 snRNP recycling in vivo. Surprisingly, expression profiling of the egy mutant revealed an extensive network of coordinately up-regulated components of the spliceosome cycle, providing a mechanism compensating for the recycling defect. Together, our data demonstrate that a mutation in a general splicing factor can lead to distinct defects in organ development and cause disease. small nuclear RNA | small nuclear ribonucleoprotein | splicing | genetic screen | thymus

The role that Six genes and Eya1 play in the development of the third pharyngeal pouch into the thymus/parathyroid is studied.

Wnt signaling is essential for T cell development in the thymus, but the stages in which it occurs and the molecular mechanisms underlying Wnt responsiveness have remained elusive. Here we examined Wnt signaling activity in both human and murine thymocyte populations by determining [beta]-catenin levels, Tcf-reporter activation and expression of Wnt-target genes. We demonstrate that Wnt signaling occurs in all thymocyte subsets, including the more mature populations, but most prominently in the double negative (DN) subsets. This differential sensitivity to Wnt signaling was not caused by differences in the presence of Wnts or Wnt receptors, as these appeared to be expressed at comparable levels in all thymocyte subsets. Rather, it can be explained by high expression of activating signaling molecules in DN cells, e.g., [beta]-catenin, plakoglobin, and long forms of Tel-l, and by low levels of inhibitory molecules. By blocking Wnt signaling from the earliest stage onwards using overexpression of Dickkopf, we show that inhibition of the canonical Wnt pathway blocks development at the most immature DN1 stage. Thus, responsiveness to developmental signals can be regulated by differential expression of intracellular mediators rather than by abundance of receptors or ligands. double negative cells | T cell development | [beta]-catenin | Dickkopf

The thymus harbors an organ-typical dense network of branching and anastomosing blood vessels. To address the molecular basis for morphogenesis of this thymus-specific vascular pattern, we have inactivated a key vascular growth factor, VEGF-A, in thymus epithelial cells (TECs). Both Vegf-A alleles were deleted in TECs by a complementation strategy termed nude mouse [mutated in the transcription factor Foxnl (forkhead box N1)] blastocyst complementation. Injection of Foxn[1.sup.+/+] ES cells into Foxn[1.sup.nu/nu] blastocysts reconstituted a functional thymus. By dissecting thymus stromal cell subsets, we have defined, in addition to medullary TECs (mTECs) and cortical TECs (cTECs), another prominent stromal cell subset designated cortical mesenchymal cells (cMes). In chimeric thymi, mTECs and cTECs but not cMes were exclusively ES cell-derived. According to this distinct origin, the Vegf-A gene was deleted in mTECs and cTECs, whereas cMes still expressed Vegf-A. This genetic mosaic was associated with hypovascularization and disruption of the organ-typical network of vascular arcades. Thus, vascular growth factor production by TECs is required for normal thymus vascular architecture. These experiments provide insights into Foxn1-dependent and Foxn1-independent stromal cell development and demonstrate the value of this chimeric approach to analyzing gene function in thymus epithelium. mesenchyme | nude mice | thymus development | vascular endothelial growth factor | nude mouse blastocyst complementation

Cloned Pax5-deficient pro-B cells placed into RAG2-deficient mice were found to give long-term reconstitution of the thymus and form mature T cells expressing alpha/Beta-T-cell receptors. Pax5 is thus implicated in the control of B-lineage commitment, and not immunoglobulin DJ rearrangement or expression of E2A, EBF, lambda5, VpreB, Igalpha and IgBeta.

After initial seeding by thymic emigrants, homeostatic regulation of the T cell pool has been thought to occur entirely within the periphery. Here we report that the degree of thymic emigration directly affects the number and the CD4/CD8 ratio of peripheral T cells. We demonstrate that the increase in T cell pool size caused by the engraftment of 2, 6, or 9 thymic lobes correlates almost exactly with the number of emigrants exported from those grafts in the previous 3 weeks, regardless of how long the graft has been in place. The extent of the increase supports the concept of a 3-week period after thymic export in which emigrant T cells are exempt from peripheral T cell homeostasis. This apparent exclusion of recent thymic emigrants from the niche-based regulation of peripheral T cell numbers ensures repertoire turnover throughout adult life and provides the basis of a direct and previously unrecognized role for the thymus in the regulation of peripheral T cell homeostasis.

The Ah receptor (AhR) mediates dioxin toxicity in skin, immune system, testis and liver, and inhibition/induction of proliferation by intracellular AhR has been studied in developing hepatoma and thymus cells. AhR is a basic helix-loop-helix (bHLH)/Paer/AhR/Arnt/Sim (PAS) transcription factor and induces the p27(super.Kip1) cyclin/cdk inhibitor by altering Kip1 transcription in a direct way with no need for ongoing cell proliferation or protein synthesis.

Experiments were conducted to study the effects of ethanol on DNA phosphate charge neutralization and condensation of DNA molecules. Findings have indicated that the hydrodynamic radius R (sub h) and the radius of gyration R (sub g) are clear indicators of changing condensation behavior. At low alcohol concentrations, the Gaussian coil exhibited an abrupt shape transition at 40% ethanol concentration. The Gaussian chain behavior manifested anew when the alcohol concentration was raised.

The ability of the thymus to reconstitute T cells expressing CD4 cell-surfaces antigens, lost during HIV infection, may be affected by age-related involution. Thymic output has been quantified through measurements of the excisional DNA products of TCR-gene rearrangement. Thymic function was found to decline with age, but considerable output is maintained into late adulthood.

We performed a comprehensive analysis oft cell receptor (TCR) [Gamma] rearrangements in T cell precursors of the mouse adult thymus. Using a sensitive quantitative PCR method, we show that [TCR.sub.[Gamma]] rearrangements are present in [CD44.sup. +][CD25.sup. +] Pro-T thymocytes much earlier than expected. [TCR.sub.[Gamma]] rearrangements increase significantly from the Pro-T to the [CD44.sup.-][CD25.sup. +] Pre-T cell transition, and follow different patterns depending on each [V.sub.[Gamma]] gene segment, suggesting that ordered waves of [TCR.sub.[Gamma]] rearrangement exist in the adult mouse thymus as has been described in the fetal mouse thymus. Recombinations of [TCR.sub.[Gamma]] genes occur concurrently with [TCR.sub.[Delta]] and D-J[Beta] rearrangements, but before V[Beta] gene assembly. Productive [TCR.sub.[Gamma]] rearrangements do not increase significantly before the Pre-T cell stage and are depleted in [CD4.sup. +][CD8.sup. +] double-positive cells from normal mice. In contrast, double-positive thymocytes from TCR[Delta]-/- mice display random proportions of [TCR.sub.[Gamma]] rearranged alleles, supporting a role for functional [TCR.sub.[Gamma]]/[Delta] rearrangements in the [Gamma][Delta] divergence process.

Thymocyte and thymic epithelial cell (TEC) development are interdependent processes. Although lineage relationships among progressively maturing thymocyte subsets have been characterized, the developmental relationships among TEC subsets are obscure. Because epithelial cells express distinct keratin (K) species as a function of differentiation stage and proliferative status, we used K expression patterns to identify mouse TEC subsets and determine their lineage relationships. As expected, cortical and medullary TEC subsets express distinct K expression patterns in the normal thymus. However, we detected two distinct cortical TEC subsets, a major K[8.sup.+]K[5.sup.-] subset and a minor K[8.sup.+]K[5.sup.+] subset, which is highly represented at the cortico-medullary junction. Both cortical TEC subsets are also present in recombination activating gene 1 (RAG[-1.sup.-/-]) and TCR[Beta]x[[Delta].sup.-/-] thymi in which T-cell development is blocked at the CD[4.sup.-]CD[8.sup.-]CD[25.sup.+]CD[44.sup.-] pre-T cell stage. In contrast, K[8.sup.+]K[5.sup.+] TECs predominate in the thymi of human CD3[Epsilon] transgenic mice in which thymocyte development is blocked at an earlier CD[4.sup.-]CD[8.sup.-]CD[25.sup.-]CD[44.sup.+] stage. Transplantation of newborn human CD3[Epsilon] transgenic thymi under the kidney capsule of RAG[-1.sup.-/-] mice results in the emergence of K[8.sup.+]K[5.sup.-] TECs concomitant with the appearance of CD[25.sup.+] thymocytes. Together, the data suggest that cortical TEC development proceeds from a K[8.sup.+]K[5.sup.+] precursor subset to a K[8.sup.+]K[5.sup.-] stage in a differentiation process concomitant with T-cell lineage commitment.

A rearranged T cell receptor (TCR) V[Alpha] and J[Alpha] gene from a cytochrome c-specific T cell hybridoma was introduced into the genomic J[Alpha] region. The introduced TCR [Alpha] chain gene is expressed in a majority of CD3 positive and CD4 CD8 double-negative immature thymocytes. However, only a few percent of the double-positive and single-positive thymocytes express this TCR [Alpha] chain. This decrease is caused by a rearrangement of TCR [Alpha] chain locus, which deletes the introduced TCR gene. Analysis of the mice carrying the introduced TCR [Alpha] chain and the transgenic TCR [Beta] chain from the original cytochrome c-specific T cell hybridoma revealed that positive selection efficiently rescues double-positive thymocytes from the loss of the introduced TCR [Alpha] chain gene. In the mice with negatively selecting conditions, T cells expressing the introduced TCR [Alpha][Beta] chains were deleted at the double-positive stage. However, a large number of thymocytes escape negative selection by using an endogenous TCR [Alpha] chain created by secondary rearrangement maintaining normal thymocyte development. These results suggest that secondary rearrangements of the TCR [Alpha] chain gene play an important role in the formation of the T cell repertoire.

Interaction of the T cell receptor (TCR) with peptide/major histocompatibility complexes (MHC) in the thymus is of critical importance for developing thymocytes. In a previous study, we described an antagonist peptide that inhibited negative selection of transgenic thymocytes induced by an agonist peptide. In this study we show that this antagonist peptide can induce positive selection of [CD8.sup.+] thymocytes more efficiently than the agonist or the weak agonist peptides, whereas the opposite is true for their ability to cause negative selection. The intracellular signals induced in thymocytes by such peptides after TCR ligation was examined in [CD4.sup.+][8.sup.+] double-positive thymocytes from F5/[[Beta].sub.2][m.sup.[convolution]]/Rag-[1.sup.[convolution]] transgenie mice. TCR ligation with either the agonist, weak agonist, or antagonist peptide variants resulted in hyperphosphorylation of CD3[Zeta], CD3[Epsilon], ZAP-70, Syk, Vav, SLP-76, and pp36-38. The extent of phosphorylation of these intracellular proteins correlated with the efficiency with which the peptide analogs induced apoptosis of immature thymocytes. Unexpectedly, there was no correlation between the upstream TCR signaling pathways analyzed and the capacity of the different peptides to induce positive selection.

Generation and negative selection of [NK1.1.sup.+][Alpha]/[Beta]T cell receptor [(TCR).sup.+] thymocytes were analyzed using TCR-transgenic (B10.D2 x DO10)[F.sub.1] and (C57BL/6 x DO10)[F.sub.1] mice and [Rag-1.sup.-/-]/DO10 mice, which had been established by breeding and backcrossing between [Rag.sup.-1-/-] and DO10 mice. Almost all T cells from these mice were shown to bear V[Alpha]13/V[Beta]8.2 that is specific for chicken ovalbumin (cOVA) and restricted to I-[A.sup.d]. A normal proportion of the [NK1.1.sup.+] V[Alpha]13/V[[Beta]8.2.sup.+] thymocytes was generated in these mice. However, the actual cell number of both [NK1.1.sup.+] and [NK1.1.sup.-] thymocytes in I-[A.sup.d/d] mice (positive selecting background) was larger than that in I-[A.sup.b/d] mice (negative selecting background). Markedly low but significant proportions of [NK1.1.sup.+] V[Alpha]13/V[[Beta]8.2.sup.+] cells were detected in the spleens from I-[A.sup.d/d] and I-[A.sup.b/d] mice. It was shown that the splenic [NK1.1.sup.+] T cells of the I-[A.sup.b/d] mice were anergized against stimulation through TCR. When (B10.D2 x DO10)[F.sub.1] and (C57BL/6 x DO10)[F.sub.1] mice were given cOVA, extensive or intermediate elimination of [NK1.1.sup.+][Alpha]/[[Beta]TCR.sup.+] thymocytes was induced in I-[A.sup.d/d] or I-[A.sup.b/d] mice, respectively. However, the clonal elimination was not as complete as that seen in the major [NK1.1.sup.-] thymocyte population. The present findings indicate that normal generation of [NK1.1.sup.+][Alpha]/[[Beta]TCR.sup.+] thymocytes occurs in the absence of V[Alpha]14-J[Alpha]281 and that substantial negative selection operates on the [NK1.1.sup.+][Alpha]/[[Beta]TCR.sup.+] cells.

We have identified a spontaneous mutation in mice, which we term HD for 'helper T cell deficient.' This mouse is distinguished by the virtual absence of peripheral T cells of the [CD4.sup.+][8.sup.-] major histocompatibility complex (MHC) class II-restricted T helper subset due to a specific block in thymic development. The developmental defect is selective for [CD4.sup.+][8.sup.-] cells; the maturation of [CD4.sup.-][8.sup.+] and [Gamma][Delta] T cells is normal. The autosomal recessive mutation underlying the HD phenotype is unrelated to MHC class II, since it segregates independently of the MHC class II locus. Moreover, the HD phenotype is not caused by a defect of the CD4 gene. Bone marrow transfer experiments demonstrate that the defect is intrinsic to cells of the hematopoietic lineage, i.e., most likely to developing thymocytes themselves. The frequency of [CD4.sup.+][8.sup.low] intermediate cells is markedly increased in HD mice, suggesting that class II-restricted thymocytes are arrested at this stage. This is the first genetic defect of its kind to be described in the mouse and may prove highly informative in understanding the molecular pathways underlying lineage commitment.

Research was conducted to analyze the function of epithelial V-like antigen (EVA), a gene encoding new member of the immunoglobulin superfamily. Results showed that EVA is expressed in the thymus and in many epithelial structures early in embryogenesis, has a strong homology to the myelin protein zero and is poorly soluble in nonionic detergents. These results suggest the potential role of EVA as a homotypic adhesion molecule in thymus development, particularly in the early stages of organogenesis.

Thymocytes are positively or negatively selected depending on interactions between their T cell receptors (TCR) and peptides presented by major histocompatibility complex molecules. We have previously shown that apoptosis of thymocytes from an [Alpha][Beta] TCR-transgenic mouse (F5), induced by antigenic peptide, can be inhibited specifically by an antagonist peptide variant in an in vitro culture model. We have now extended these experiments by demonstrating that the antagonist peptide can inhibit natural negative selection of maturing thymocytes, induced by endogenously expressed antigen, in fetal thymic organ cultures (FTOC). This inhibition resulted in the rescue and maturation of thymocytes that would otherwise have been deleted. Mature T cells generated in these cultures were able to respond to antigen by producing limited quantities of interferon-[Gamma], but unlike T cells from control FTOC, they required exogenous interleukin-2 to generate cytolytic effector cells. Interestingly, the antagonist peptide also accelerated the development of F5 thymocytes in the absence of the negatively selecting ligand. These data suggest that the developmental fate of a thymocyte may be determined by the recognition of multiple distinct peptide ligands during thymic selection. Alterations in the profiles of selecting peptides presented in the thymus would thus have profound effects on the size and autoreactive potential of the T cell repertoire generated.

Major histocompatibility complex (MHC) class II molecules are assembled with the assistance of Ii in the endoplasmic reticulum (ER) and transported to an endocytic compartment where Ii undergoes rapid degradation [...], Degradation of invariant chain (Ii) is a critical step in major histocompatibility complex class II-restricted antigen presentation. Cathepsin L was found to be necessary for Ii degradation in cortical thymic epithelial cells (cTECs), but not in bone marrow (BM) -- derived antigen-presenting cells (APCs). Consequently, positive selection of [CD4.sup.+] T cells was reduced. Because different cysteine proteinases are responsible for specific Ii degradation steps in cTECs and BM-derived APCs, the proteolytic environment in cells mediating positive and negative selection may be distinct. The identification of a protease involved in class II presentation in a tissue-specific manner suggests a potential means of manipulating [CD4.sup.+] T cell responsiveness in vivo.

The thymus, thyroid, and parathyroid glands in vertebrates develop from the pharyngeal region, with contributions both from pharyngeal endoderm and from neural crest cells in the pharyngeal arches. Hoxa3 mutant homozygotes have defects in the development of all three organs. Roles for the Hoxa3 paralogs, Hoxb3 and Hoxd3, were investigated by examining various mutant combinations. The thyroid defects seen in Hoxa3 single mutants are exacerbated in double mutants with either of its paralogs, although none of the double-mutant combinations resulted in thyroid agenesis. The results indicate that the primary role of these genes in thyroid development is their effect on the development and migration of the ultimobranchial bodies, which contribute the parafollicular or C-cells to the thyroid. Hoxb3,Hoxd3 double mutants show no obvious defects in the thymus or parathyroids. However, the removal of one functional copy of Hoxa3 from the Hoxb3,Hoxd3 double mutants ([Hoxa3.sup.+/-],[Hoxb3.sup.-/-],[Hoxd3.sup.-/-]) results in the failure of the thymus and parathyroid glands to migrate to their normal positions in the throat. Very little is known about the molecular mechanisms used to mediate the movement of tissues during development. These results indicate that Hoxa3, Hoxb3, and Hoxd3 have highly overlapping functions in mediating the migration of pharyngeal organ primordia. In addition, Hoxa3 has a unique function with respect to its paralogs in thymus, parathyroid, and thyroid development. This unique function may be conferred by the expression of Hoxa3, but not Hoxb3 nor Hoxd3, in the pharyngeal pouch endoderm.

Mature T lymphocytes display a biased repertoire of T cell receptors (TCRs), enriched for those that can recognize foreign antigens presented by the animal's own major histocompatibility complex (MHC) molecules [...], The relation between an antigenic peptide that can stimulate a mature T cell and the natural peptide that promoted selection of this cell in the thymus is still unknown. An experimental system was devised to address this issue in vivo -- mice expressing neopeptides in thymic stromal cells after adenovirus-mediated delivery of invariant chain-peptide fusion proteins. In this system, selection of T cells capable of responding to a given antigenic peptide could be promoted by the peptide itself, by closely related analogs lacking agonist and antagonist activity, or by ostensibly unrelated peptides. However, the precise repertoire of T cells selected was dictated by the particular neopeptide expressed.

Allelic exclusion at the T-cell receptor [Alpha] chain locus is incomplete resulting in the generation of T cells that express two T-cell receptors. The potential involvement of such T cells in autoimmunity has been suggested [Padovan, E., Casorati, G., Dellabona, P., Meyer, S., Brockhaus, M. & Lanzavecchia, A. (1993) Science 262, 422-424; Heath, W. R. & Miller, J. F. A. P. (1993) J. Exp. Med. 178, 1807-1811]. Here we show that expression of a second T-cell receptor can rescue T cells with autospecific receptors from thymic deletion and allow their exit into the periphery. Dual receptor T cells, created by constitutive expression of two transgenic T-cell receptors on a Rag[1.sup.-/-] background, are tolerant to self by maintaining low levels of autospecific receptor, but self-reactive effector function (killing) can be induced through activation via the second receptor. This opens the possibility that T cells carrying two receptors in the periphery of normal individuals contain putatively autoreactive cells that could engage in autoimmune effector functions after recognition of an unrelated environmental antigen.

The thymus of mice lacking antigen CD30 receptors have increased amounts of thymocytes. Killing of thymocytes by CD3 cross-linking was ineffective both in vivo and in vitro. Breeding the CD30 mutation separately into alpha-beta-TCR- or alpha-delta-TCR-transgenic mice revealed a substantial impairment of negative but not in positive selection of T cells. Findings indicated that the CD30 receptor is implicated in cell death signaling.

Programmed cell death is vital for eliminating unwanted cells in multicellular organisms, and its dysregulation is now widely recognized in autoimmune and neoplastic disease pathogenesis (1). In self-reactive T cells, [...], T cell hybridomas require the immediate-early gene NGFI-B (nur77) for T cell receptor (TCR)--mediated apoptosis, a model for negative selection of self-reactive T cells. TCR-mediated death was examined in mice bearing an NGFI-B loss-of-function mutation, either by administration of antibodies to CD3 (anti-CD3) or in two well-characterized transgenic models expressing self-reactive TCRs. Both the extent and the rate of thymocyte death were unimpaired. Anti-CD3--induced death was normal in [CD4.sup.+] peripheral T cells, in which death is mediated predominantly by the Fas signaling pathway. Thus, no unique requirement for NGFI-B is observed for thymic or peripheral T cell death.

Analysis of the mechanism of action of calcitonin gene-related peptide (CGRP) and dehydroepiandrosterone (DHEA) on the mitogen-activated growth of thymocytes in mouse thymus reveals that individually or together DHEA and CGRP do not activate thymocyte growth when concanavalin A (ConA) is not present. DHEA and CGRP suppress thymocyte growth in ConA-induced thymocyte proliferation assays. The growth response of the thymocytes is more to a CGRP antagonist than to ConA alone. CGRP also influences CD4+ T cell growth and this pathway may mediate the effect of DHEA on the CD4+ T cell growth.

The efficacy of positive selection is assessed using in vitro chimeras established between normal thyme and those lacking major histocompatibility complex (MHC) molecules. For a range of chimerism, the formation of mature single-positive thymocytes varied directly with wild-type stromal fractions. T-cell receptor-transgenic thymoyctes are developed similarly in graded chimeras that express selecting and nonselecting MHC alleles. A rate-limiting step occurs in positive selection, wherein interaction between each thymocyte and one stromal cell niche takes place.

Apoptosis occurs in the normal thymus due to absence of positive selection and not due to the presence of negative selection. Apoptosis or T-cell death occurs in immature thymus due to the negative selection of the molecules of the major histocompatibility complex (MHC). The process of apoptosis in normal thymus is investigated by the method of detecting cells with DNA strand breaks. Apoptic cells are distributed all over the cortex and are not independent of F4/80+ macrophages.

Scid/scid mince and normal mice were studied to detrermine the colonization of murine fetal thymus lobes by donor cells from human umbilical cord blood or human bone marrow. The cells were cultured to yield double positive CD8+ CD8+ cells. CD8+ or CD4+ single-positive T cells undergo accumulation. The existence of CD7+ progenitor T cells determined colonization, and the development of the progenitor T cells is enhanced by thymic organ culture.

An analysis of flow linear dichroism and circular dichroism spectroscopy reveals the analogy between AT-specific minor-groove binder 4',6- diaminino -2-phenylindole (DAPI) and the complex formed when 2,5-bis (4-amidinophenyl) furan (APF) binds to calf thymus DNA, poly (dA-dT)2 and poly(dG-dC)2. Spectroscopic data obtained illustrate the binding of APF to calf thymus DNA and poly (dG-dT)2 at an angle of 46 degrees between its symmetrical axis and DNA helix axis.

The sever-combined immunodeficiency (SCID)-hu mouse model was used to determine the effects of virus infection on human tissue, especially the thymus. The SCID-hu mouse model was created by implantation of the mouse kidney capsule with human fetal liver and thymus. These implants were then inoculated with HIV-1. Results indicated significant depletion of the CD4 cells, a few weeks after infection. This results point to the growing importance of SCID-hu mouse in in vivo studies of HIV-1-induced pathology.

Both mesenchymal cells and major histocompatibility complex class II+ epithelial cells are needed to effect the growth of T-cells in the thymic stroma. In vitro testing of the factors necessary for T-cell development in the thymus showed that both types of cells and fibroblasts are essential. The mesenchyme's influence is probably confined to one stage of T-cell development and is incapable of acting without other cellular interactions.

Severe combined immunodeficiency (SCID) mice implantedwith human fetal tissue can support the replication of human cytomegaloviruses (CMV). An investigation was conducted to determine whether the SCID-hu system could provide a model for human CMV infections and to identify the cellular targets of CMV. The results showed that SCID-hu mice could provide a good modelsystem for human CMV infections. The virus targeted the epithelial cells of thethymic medulla. Experiments also showed that ganciclovir is an effective suppressant of CMV replication in the tissue implant.

The existence of the thymus was recognised by ancient scientists and doctors, although its vital role in the development and maintenance of the competent T cell repertoire has been fully appreciated only comparatively recently. Current research is now moving on to explore the role of the thymus in interactions between the brain, the nervous system and the immune response. This is an exciting new field which should lead to advances in the management, treatment and prognosis of disease in man and animals.

Thymic major histocompatibility complex-B (MHC-B) may affect B-reactive T cells. This was investigated, along with the ability of thymic epithelial cell (TEC) lines to generate cytotoxic T-lymphocytes (CTLs) when injected into bone marrow chimeras of the thymus. One TEC line succeeded in selecting CTLs specific to vesicular stomatitis virus and influenza antigens.

A transgenic mouse strain, designated DO10, was designed to recognize chicken ovalbumin within the context of a class II major histocompatibility molecule, I-Ad. This system was used to isolate transgenic thymocytes for coculture with I-Ad-expressing fibroblasts, B cells and thymic nurse cells comprising an in vitro system to study the process of clonal deletion. The results of in vitro analysis showed that most cells can induce clonal deletion once the peptide antigen has been presented to the cell surface. Therefore, clonal deletion is dependent on the stage at which the thymocytes are vulnerable to the onset of apoptosis, rather than on the peptide antigen-presenting cells.

The role of medullary thymic epithelium in toleragenesiswas investigated by transplanting third and fourth branchial clefts of mice embryos to athymic nude mice. The grafts expressed the major histocompatibilitycomplex (MHC) class I antigen H-2Kb only on medullary epithelial cells. Resultsshow that T-cells arising from the branchial clefts have in vivo tolerance to Kb antigen-bearing skin grafts. However, cells maintained the capability to mount an immunologic response in vitro to cells expressing the same antigen. These results indicate that the thymic medullary epithelium is responsible for tolerance induction in T-lymphocytes.

We purified native WASp (Wiskott-Aldrich Syndrome protein) from bovine thymus and studied its ability to stimulate actin nucleation by Arp2/3 complex. WASp alone is inactive in the presence or absence of 0.5 [micro]M GTP-Cdc42. Phosphatidylinositol 4,5 bisphosphate [(PIP.sub.2)] micelles allowed WASp to activate actin nucleation by Arp2/3 complex, and this was further enhanced twofold by GTP-Cdc42. Filaments nucleated by Arp2/3 complex and WASp in the presence of [PIP.sub.2] and Cdc42 concentrated around lipid micelles and vesicles, providing that Cdc42, was GTP-bound and prenylated. Thus, the high concentration of WASp in neutrophils (9 [micro]M) is dependent on interactions with both acidic lipids and GTP-Cdc42 to activate actin nucleation by Arp2/3 complex. The results also suggest that membrane binding increases the local concentrations of Cdc42 and WASp, favoring their interaction. Key words: prenylation * membrane * GBD * neutrophil * thymus

During selection of a T cell repertoire within the thymus, [CD4.sup.+] [CD8.sup.+] (double positive, DP) thymocytes with T cell antigen receptors (TCRs) that recognize complexes of host major histocompatibility complex [...], Most developing thymocytes undergo apoptosis because they cannot interact productively with molecules encoded by the major histocompatibility complex. Here, we show that mice lacking the orphan nuclear hormone receptor ROR[Gamma] lose thymic expression of the anti-apoptotic factor Bcl-xL. ROR[Gamma] thus regulates the survival of [CD4.sup.+] [8.sup.+] thymocytes and may control the temporal window during which thymocytes can undergo positive selection. ROR[Gamma] was also required for development of lymph nodes and Peyer's patches, but not splenic follicles. In its absence, there was loss of a population of [CD3.sup.-][CD4.sup.+][CD45.sup.+] cells that normally express ROR[Gamma] and that are likely early progenitors of lymphoid organs. Hence, ROR[Gamma] has critical functions in T cell repertoire selection and lymphoid organogenesis.

The identity of cells that mediate positive selection of [CD8.sup.+] T cells was investigated in two T cell receptor (TCR) transgenic systems. Irradiated [[Beta].sub.2]-micro-globulin mutant mice or mice with mutations in both the [K.sup.b] and [D.sup.b] genes were repopulated with fetal liver cells from class [I.sup.+] TCR transgenic mice. In the case of the 2C TCR, mature transgene-expressing [CD8.sub.+] T cells appeared in the thymuses of the chimeras and in larger numbers in the peripheral lymphoid organs. These [CD8.sub.+] T cells were functional, exhibited a naive, resting phenotype, and were mostly thymusdependent. Their development depended on donor cell class I expression. These results establish that thymic hematopoietic cells can direct positive selection of [CD8.sup.+] T cells expressing a conventional TCR. In contrast, no significant development of HY (male antigen)-[TCR.sup.+] [CD8.sup.+] T cells was observed in class [I.sup.+] into class I-deficient chimeras. These data suggest that successful positive selection directed by hematopoietic cells depends on specific properties of the TCR or its thymic ligands. The possibility that hematopoietic cell-induced, positive selection occurs only with TCRs that exhibit relatively high avidity interactions with selecting ligands in the thymus is discussed.