Differentiation patterns of CD4/CD8 thymocyte subsets in cocultures of fetal thymus and lymphohemopoietic cells from c-fos transgenic and normal mice.

This study examined the involvement of c-fos protooncogene in thymocyte development from lymphohemopoietic T cell progenitors, within the thymic microenvironment. We first analyzed the thymocytes developing in vitro in the fetal thymus from the c-fos transgenic mice and found a high proportion of CD4+ single positive (SP) cells. We then seeded either fetal liver or bone marrow (BM) cells from normal donors onto lymphocyte-depleted fetal thymus explants of c-fos transgenic mice. The results showed an increased proportion of mature CD4+ SP and decreased CD4+CD8+ double positive (DP) cells. A similar pattern of CD4/CD8 thymocyte subsets was observed when either thymus or BM cells from c-fos transgenic mice developed within a normal thymic stroma. The kinetics of thymocyte development in organ culture (from Days 3 to 11) suggested that the SP cells obtained under these conditions may have bypassed the CD4+CD8+ DP phase. It appears that the altered pattern of thymocyte development manifested in adult c-fos transgenic mice can be induced by the early embryonic thymic stroma, and may also involve cells in the lymphohemopoietic tissues.     

Presymptomatic biochemical changes in hindlimb muscle of G93A human Cu/Zn superoxide dismutase 1 transgenic mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is primarily a motor neuron disorder. Intriguingly, early muscle denervation preceding motor neuron loss is observed in mouse models of ALS. Enhanced muscle vulnerability to denervation process has been suggested by accelerated muscle deterioration following peripheral nerve injury in an ALS mouse model. Here we provide evidence of biochemical changes in the hindlimb muscle of young, presymptomatic G93A hSOD1 transgenic mice. In this report, we demonstrate that cdk5 activity is reduced in hindlimb muscle of 27-day-old G93A hSOD1 transgenic mice. In vitro analysis revealed mutant hSOD1-mediated suppression of cdk5 activity. Furthermore, the decrease in muscle cdk5 activity was accompanied by a significant reduction in MyoD and cyclin D1 levels. These early muscle changes raise the possibility that the progressive deterioration of muscle function is potentiated by altered muscle biochemistry in these mice at a very young, presymptomatic age.     

IL-10 transgenic mice present a defect in T cell development reminiscent of SCID patients.

To analyze the effect of IL-10 overexpressed by APCs as observed in some SCID patients, we have expressed the human IL-10 cDNA under the control of the murine MHC class II promoter in transgenic mice. Similar to SCID patients, these mice presented a defect in T cell maturation characterized by a rapid thymic aplasia that started after birth. The blockage in T cell maturation was strictly restricted to TCR-alpha beta T cells as the absolute number of thymic dendritic, TCR-gamma delta and NK1.1 T cells were equivalent to control littermates. Crossing IL-10 transgenic mice with TCR transgenic mice or treatment with staphylococcal enterotoxin B showed that the defect was not related to the impairment of positive or negative selection. However, repopulating of IL-10 transgenic mouse-fetal thymic organ culture with different stages of triple negative T cells isolated from control mice showed that the blockage occurred specifically at the pre-T cell stage and was reverted by treatment with blocking anti-IL-10 mAbs. These results demonstrate that IL-10 regulates T cell maturation and that dysregulation of IL-10 expression can lead to severe T cell immunodeficiency.     

Regulation of thymic NKT cell development by the B7-CD28 costimulatory pathway

Invariant NKT (iNKT) cells are a population of TCRalphabeta-expressing cells that are unique in several respects. In contrast to conventional T cells, iNKT cells are selected in the thymus for recognition of CD1, rather than conventional MHC class I or II, and are selected by CD1-expressing double-positive thymocytes, rather than by the thymic stromal cells responsible for positive selection of conventional T cells. We have probed further the requirements for thymic iNKT cell development and find that these cells are highly sensitive to B7-CD28 costimulatory interactions, as evidenced by the substantially decreased numbers of thymic iNKT cells in CD28 and in B7 knockout mice. In contrast to the requirement for CD1, B7-CD28 signaling does not affect early iNKT cell lineage commitment, but exerts its influence on the subsequent intrathymic expansion and differentiation of iNKT cells. CD28 wild-type/CD28-deficient mixed bone marrow chimeras provided evidence of both cell-autonomous and non-cell-autonomous roles for CD28 during iNKT cell development. Paradoxically, transgenic mice in which thymic expression of B7 is elevated have essentially no measurable thymic iNKT cells. Taken together, these results demonstrate that the unique pathway involved in iNKT cell development is marked by a critical role of B7-CD28 interactions and that disruption or augmentation of this costimulatory interaction has substantial effects on iNKT cell development in the thymus.     

Generation and characterization of Tbx1-AmCyan1 transgenic reporter mouse line that selectively labels developing thymus primordium

Thymus development is a complicated process that includes highly dynamic morphological changes and reciprocal tissue interactions between endoderm-derived epithelial cells of the anterior foregut and neural crest-derived mesenchymal cells. We generated and characterized a Tbx1-AmCyan1 reporter transgenic mouse to visualize thymus precursor cells during early embryonic development. In transgenic embryos, AmCyan1 fluorescence was specifically detected in the endoderm of the developing 3rd and 4th pharyngeal pouches and later in thymus epithelium until E14.5. Cells expressing AmCyan1 that were isolated based on AmCyan1 fluorescence expressed endodermal, thymic, and parathyroid markers, but they did not express neural crest or endothelial markers; these findings indicated that this transgenic mouse strain could be used to collect thymic or parathyroid precursor cells or both. We also showed that in nude mice, which exhibit defects in thymus development, the thymus precursors were clearly labeled with AmCyan1. In summary, these AmCyan1-fluorescent transgenic mice are useful for investigating early thymus development.     

Culture and characterization of thymic epithelium from autoimmune NZB and NZB/W mice

Autoimmune NZB and NZB/W mice display early abnormalities in thymus histology, T cell development, and mature T cell function. Abnormalities in the subcapsular/medullary thymic epithelium (TE) can also be inferred from the early disappearance of thymulin from NZB. It has also been reported that NZB thymic epithelial cells do not grow in culture conditions that support the growth of these cells from other strains of mice. In order to study the contribution of TE to the abnormal T cell development and function in NZB and NZB/W mice, we have devised a culture system which supports the growth of TE cells from these mice. The method involves the use of culture vessels coated with extracellular matrix produced by a rat thymic epithelial cell line. TEA3A1, and selective low-calcium, low-serum medium. In addition TEA3A1 cells have been used as an antigen to generate monoclonal antibodies specific for subcapsular/medullary TE. These antibodies, as well as others already available, have been used to show that the culture conditions described here select for cells displaying subcapsular/medullary TE markers, whereas markers for cortical TE and macrophages are absent.     

The GTPase Rho has a critical regulatory role in thymus development.

The present study employs a genetic approach to explore the role of Rho GTPases in murine thymic development. Inactivation of Rho function in the thymus was achieved by thymic targeting of a transgene encoding C3 transferase from Clostridium botulinum which selectively ADP-ribosylates Rho within its effector domain and thereby abolishes its biological function. Thymi lacking functional Rho isolated from C3 transgenic mice were strikingly smaller and showed a marked (90%) decrease in cellularity compared with their normal litter mates. We also observed a similar decrease in levels of peripheral T cells in C3 transgenic mice. Analysis of the maturation status of thymocytes indicated that differentiation of progenitor cells to mature T cells can occur in the absence of Rho function, and both positive and negative selection of T cells appear to be intact. However, transgenic mice that lack Rho function in the thymus show maturational, proliferative and cell survival defects during T-cell development that severely impair the generation of normal numbers of thymocytes and mature peripheral T cells. The present study thus identifies a role for Rho-dependent signalling pathways in thymocyte development. The data show that the function of Rho GTPases is critical for the proliferative expansion of thymocytes. This defines a selective role for the GTPase Rho in early thymic development as a critical integrator of proliferation and cell survival signals.     

Restricted STAT5 activation dictates appropriate thymic B versus T cell lineage commitment

The molecular mechanisms regulating lymphocyte lineage commitment remain poorly characterized. To explore the role of the IL7R in this process, we generated transgenic mice that express a constitutively active form of STAT5 (STAT5b-CA), a key downstream IL7R effector, throughout lymphocyte development. STAT5b-CA mice exhibit a 40-fold increase in pro-B cells in the thymus. As documented by BrdU labeling studies, this increase is not due to enhanced B cell proliferation. Thymic pro-B cells in STAT5b-CA mice show a modest increase in cell survival ( approximately 4-fold), which correlates with bcl-x(L) expression. However, bcl-x(L) transgenic mice do not show increases in thymic B cell numbers. Thus, STAT5-dependent bcl-x(L) up-regulation and enhanced B cell survival are not sufficient to drive the thymic B cell development observed in STAT5b-CA mice. Importantly, thymic pro-B cells in STAT5b-CA mice are derived from early T cell progenitors (ETPs), suggesting that STAT5 acts by altering ETP lineage commitment. Supporting this hypothesis, STAT5 binds to the pax5 promoter in ETPs from STAT5b-CA mice and induces pax5, a master regulator of B cell development. Conversely, STAT5b-CA mice exhibit a decrease in the DN1b subset of ETPs, demonstrating that STAT5 activation inhibits early T cell differentiation or lineage commitment. On the basis of these findings, we propose that the observed expression of the IL-7R on common lymphoid progenitors, but not ETPs, results in differential STAT5 signaling within these distinct progenitor populations and thus helps ensure appropriate development of B cells and T cells in the bone marrow and thymic environments, respectively.     

Thymic and postthymic regulation of diabetogenic CD8 T cell development in TCR transgenic nonobese diabetic (NOD) mice

Natural development of diabetes in nonobese diabetic (NOD) mice requires both CD4 and CD8 T cells. Transgenic NOD mice carrying alphabeta TCR genes from a class I MHC (Kd)-restricted, pancreatic beta cell Ag-specific T cell clone develop diabetes significantly faster than nontransgenic NOD mice. In these TCR transgenic mice, a large fraction of T cells express both transgene derived and endogenous TCR beta chains. Only T cells expressing two TCR showed reactivity to the islet Ag. Development of diabetogenic T cells is inhibited in mice with no endogenous TCR expression due to the SCID mutation. These results demonstrate that the expression of two TCRs is necessary for the autoreactive diabetogenic T cells to escape thymic negative selection in the NOD mouse. Further analysis with MHC congenic NOD mice revealed that diabetes development in the class I MHC-restricted islet Ag-specific TCR transgenic mice is still dependent on the presence of the homozygosity of the NOD MHC class II I-Ag7.     

Tetanus toxin fragment C fusion facilitates protein delivery to CNS neurons from cerebrospinal fluid in mice

To improve protein delivery to the CNS following intracerebroventricular administration, we compared the distribution of a human Cu/Zn superoxide dismutase:tetanus toxin fragment C fusion protein (SOD1:TTC) in mouse brain and spinal cord with that of tetanus toxin fragment C (TTC) or human SOD1 (hSOD1) alone, following continuous infusion into the lateral ventricle. Mice infused with TTC or SOD1:TTC showed intense anti-TTC or anti-hSOD1 labeling, respectively, throughout the CNS. In contrast, animals treated with hSOD1 revealed moderate staining in periventricular tissues. In spinal cord sections from animals infused with SOD1:TTC, the fusion protein was found in neuron nuclear antigen-positive (NeuN+) neurons and not glial fibrillary acidic protein-positive (GFAP+) astrocytes. The percentage of NeuN+ ventral horn cells that were co-labeled with hSOD1 antibody was greater in mice treated with SOD1:TTC (cervical cord = 73 +/- 8.5%; lumbar cord = 62 +/- 7.7%) than in mice treated with hSOD1 alone (cervical cord = 15 +/- 3.9%; lumbar cord = 27 +/-4.7%). Enzyme-linked immunosorbent assay for hSOD1 further demonstrated that SOD1:TTC-infused mice had higher levels of immunoreactive hSOD1 in CNS tissue extracts than hSOD1-infused mice. Following 24 h of drug washout, tissue extracts from SOD1:TTC-treated mice still contained substantial amounts of hSOD1, while extracts from hSOD1-treated mice lacked detectable hSOD1. Immunoprecipitation of SOD1:TTC from these extracts using anti-TTC antibody revealed that the recovered fusion protein was structurally intact and enzymatically active. These results indicate that TTC may serve as a useful prototype for development as a non-viral vehicle for improving delivery of therapeutic proteins to the CNS.     

Effects of a constitutively active form of calcineurin on T cell activation and thymic selection

Calcineurin is a calcium/calmodulin-dependent phosphatase whose activity is required for the induction of T cell lymphokine production and proliferation. Although its specific role in T cell development is less well defined, studies with the immunosuppressive drugs cyclosporin A and FK-506 suggest that it is involved in both positive and negative selection of immature thymocytes. To more completely characterize a role for calcineurin in T cell development in vivo, we have generated transgenic mice that express an activated form of this enzyme in thymocytes and peripheral T cells. We find that the transgene causes a block in early thymic development, resulting in a reduction in the steady-state number of CD4 and CD8 double positives, but not on the number of mature T cells. We also find that thymocytes and mature T cells expressing this transgene are more sensitive to signals through their TCR. In thymocytes this sensitivity difference is manifested as an increase in positive selection, although negative selection seems to remain unaffected. Therefore, these studies confirm and extend past reports that suggested a role for calcineurin in thymic development and selection.     

Acquisition of functional MHC class II/peptide complexes by T cells during thymic development and CNS-directed pathogenesis

This study provides evidence that both rat and mouse thymic and splenic T cells express significant levels of MHC class II glycoproteins (MHCII) in vivo. Derivation of rat and mouse chimeras revealed that a major source of MHCII on thymic T cells was acquired from radioresistant host APC. Expression of MHC on thymic T cells appeared physiologically relevant because presentation of rat myelin basic protein (RMBP) by nonadherent, radiosensitive thymic T cells was associated with the adoptive transfer of tolerance. Mature MBP-specific effector T cells isolated from the CNS in both rat and mouse models of EAE also expressed significant levels of MHCII. Adoptive transfer of activated B10.PL MBP/I-A(u)-restricted TCR transgenic T cells into F1(C57BL/6 x B10.PL) mice revealed acquisition of allogeneic I-A(b) on encephalitogenic CNS-derived T cells. Overall, this study indicates that immature and mature T cells in rats and mice acquire functional MHCII in vivo during thymic development and pathogenic inflammation.     

Unexpected thymic hyperplasia in transgenic mice harboring a neuronal promoter fused with simian virus 40 large T antigen.

The hypothalamic peptide growth hormone-releasing factor (GRF) regulates the secretion and production of growth hormone from the anterior pituitary (M. C. Gelato and G. R. Merriam, Annu. Rev. Physiol. 48:569-591). To study GRF gene regulation, transgenic mice were generated that harbor the human GRF promoter fused to the coding sequences from the simian virus 40 early region. These mice had normal hypothalamic functions but unexpectedly suffered from severe thymic hyperplasia. Immunohistochemical analysis revealed that large T antigen was expressed in the thymic epithelial cells. These cells have endocrine properties and are known to produce thymic hormones [corrected]. The thymic hyperplasia was the apparent consequence of inappropriate production of T-cell maturation factors by epithelial cells and could involve increased self renewal of apparently normal T stem cells in the thymus.     

Severe defect in thymic development in an insertional mutant mouse model

Transgenic mice were generated expressing NK1.1, an NK cell-associated receptor, under control of the human CD2 promoter. Unexpectedly, one of the founder lines, Tg66, showed a marked defect in thymic development characterized by disorganized architecture and small size. Mapping of the transgene insertion by fluorescence in situ hybridization revealed integration in chromosome 2, band G. Already from postnatal day 3, the thymic architecture was disturbed with a preferential loss of cortical thymic epithelial cells, a feature that became more pronounced over time. Compared with wild-type mice, total thymic cell numbers decreased dramatically between 10 and 20 days of age. Thymocytes isolated from adult Tg66 mice were predominantly immature double-negative cells, indicating a block in thymic development at an early stage of differentiation. Consequently, Tg66 mice had reduced numbers of peripheral CD4(+) and CD8(+) T cells. Bone marrow from Tg66 mice readily reconstituted thymi of irradiated wild-type as well as RAG-deficient mice. This indicates that the primary defect in Tg66 mice resided in nonhemopoietic stromal cells of the thymus. The phenotype is observed in mice heterozygous for the insertion and does not resemble any known mutations affecting thymic development. Preliminary studies in mice homozygous for transgene insertion reveal a more accelerated and pronounced phenotype suggesting a semidominant effect. The Tg66 mice may serve as a useful model to identify genes regulating thymic epithelial cell differentiation, thymic development, and function.     

NZB mice exhibit a primary T cell defect in fetal thymic organ culture

Defects in T cell development have been suggested to be a factor in the development of systemic autoimmunity in NZB mice. However, the suggestion of a primary T cell defect has often been by extrapolation, and few direct observations of T cell precursors in NZB mice have been performed. Moreover, the capacity of NZB bone marrow T cell precursors to colonize the thymus and the ability of the NZB thymic microenvironment to support T lymphopoiesis have not been analyzed. To address this important issue, we employed the fetal thymic organ culture system to examine NZB T cell development. Our data demonstrated that NZB bone marrow cells were less efficient at colonizing fetal thymic lobes than those of control BALB/c or C57BL/6 mice. In addition, NZB bone marrow cells did not differentiate into mature T cells as efficiently as bone marrow cells from BALB/c or C57BL/6 mice. Further analysis revealed that this defect resulted from an intrinsic deficiency in the NZB Lin-Sca-1+c-kit+ bone marrow stem cell pool to differentiate into T cells in fetal thymic organ culture. Taken together, the data document heretofore unappreciated deficiencies in T cell development that may contribute to the development of the autoimmune phenotype in NZB mice.     

Deficiency of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) causes enrichment of CD4+CD25+ regulatory T cells

A subpopulation of T cells, named regulatory T cells (T(reg) cells), has been shown to play a key role in tolerance and the prevention of autoimmunity. It is not known how changes in TCR signal strength during thymic T cell development affect the generation of a T(reg) population. In this study, we took two different strategies to modulate the TCR signal strength: an intrinsic approach, where signaling was enhanced by the loss of a negative regulator, and an extrinsic approach, where signaling strength was altered through variations in the concentrations of the selecting peptide. The tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a known negative regulator of TCR-mediated signaling. motheaten mice, lacking expression of SHP-1, showed a 2- to 3-fold increase in the percentage of CD4(+)CD25(+) T(reg) cells within the CD4(+) T cells. Similarly, the percentage of T(reg) cells was heightened in fetal thymic organ cultures (FTOCs) derived from motheaten mice compared with wild-type FTOCs, thus establishing the thymic origin of these T(reg) cells. Using FTOCs derived from DO11.10 TCR transgenic mice, we demonstrated that exposure to increasing concentrations of the cognate OVA peptide favored the appearance of T(reg) cells. Our data suggest that the development of CD4(+)CD25(+) T(reg) cells is intrinsically different from non-T(reg) cells and that T(reg) cells are selectively enriched under conditions of enhanced negative selection. Our data also reveal a key role for the SHP-1-mediated regulation of TCR signal strength in influencing the ratio of T(reg) vs non-T(reg) cells.     

BMP signaling is required for normal thymus development

The microenvironment of the thymus fosters the generation of a diverse and self-tolerant T cell repertoire from a pool of essentially random specificities. Epithelial as well as mesenchymal cells contribute to the thymic stroma, but little is known about the factors that allow for communication between the two cells types that shape the thymic microenvironment. In this study, we investigated the role of bone morphogenetic protein (BMP) signaling in thymus development. Transgenic expression of the BMP antagonist Noggin in thymic epithelial cells under the control of a Foxn1 promoter in the mouse leads to dysplastic thymic lobes of drastically reduced size that are ectopically located in the neck at the level of the hyoid bone. Interestingly, the small number of thymocytes in these thymic lobes develops with normal kinetics and shows a wild-type phenotype. Organ initiation of the embryonic thymic anlage in these Noggin transgenic mice occurs as in wild-type mice, but the tight temporal and spatial regulation of BMP4 expression is abrogated in subsequent differentiation stages. We show that transgenic Noggin blocks BMP signaling in epithelial as well as mesenchymal cells of the thymic anlage. Our data demonstrate that BMP signaling is crucial for thymus development and that it is the thymic stroma rather than developing thymocytes that depends on BMP signals.