Human progesterone receptor A and B isoforms in CHO cells. II. Comparison of binding, transactivation and ED50 values of several synthetic (anti)progestagens in vitro in CHO and MCF-7 cells and in vivo in rabbits and rats

Presented here is a cell-suspension model for positive selection using thymocytes from alphabeta-TCR (H-2Db-restricted) transgenic mice specific to the lymphocytic choriomeningitis virus (LCMV) on a nonselecting MHC background (H-2d or TAP-1 -/-), cocultured with freshly isolated adult thymus stromal cells of the selecting MHC type. The thymic stromal cells alone induced positive selection of functional CD4- CD8+ cells whose kinetics and efficiency were enhanced by nominal peptide. Fibroblasts expressing the selecting MHC alone did not induce positive selection; however, together with nonselecting stroma and nominal peptide, there was inefficient positive. These results suggest multiple signaling in positive selection with selection events able to occur on multiple-cell types. The ease with which this model can be manipulated should greatly facilitate the resolution of the mechanisms of positive selection in normal and pathological states.     

Thymic cortical epithelium is sufficient for the development of mature T cells in relB-deficient mice

Thymic development of T lymphocytes progresses as a consequence of both TCR-mediated and non-TCR-mediated interactions between thymocytes and stromal cells. As relB-deficient mice appear to lack thymic medullary epithelium and mature dendritic cells, we studied the effect of this "cortex-only" thymus on T cell development. Two major consequences were observed. First, in both relB mutant and TCR transgenic/relB mutant mice, positive selection of both TCR alpha beta and delta gamma T cells appeared to proceed normally, with export of fully functional T cells to the periphery, suggesting that the thymic medullary stromal cells are not required for full maturation of T cells nor is an organized medullary compartment required for accumulation of mature single positive CD4 and CD8 T cells. Second, thymic negative selection was impaired, as evidenced by significant autoreactive proliferative responses to normal spleen stimulators. Peripheral T cells in relB mutant mice showed an unusually high proportion of CD69+ and CD44high cells. While some of these cells may be autoreactive T cells, most of the cells appeared to be activated by cytokines produced by relB mutant nonlymphoid cells, as the effect is minimized in relB mutant bone marrow chimeras. In sum, while the TCR-mediated steps in T cell maturation require both thymic cortex and medulla (epithelium and dendritic cells) for normal positive and negative selection of the repertoire, non-TCR-mediated interactions in the thymic cortex alone are sufficient to generate mature functional T cells.     

Development of a method of thymocyte differentiation of bone marrow-enriched CD34+CD38- cells in postnatal allogeneic cultured thymic epithelia to evaluate immunodeficiency disorders

An in vitro model of CD34+CD38- stem cell (SC) differentiation in postnatal cultured thymic epithelia fragment (CTEF) cocultures is described. Sequential phenotypic analysis of the progeny of the SC-CTEF demonstrated predominantly thymocytes and minor populations of promyelocytes, monocytes and natural killer cells. Triple-positive CD3+CD4+CD8+, double-positive CD4+CD8+, and mature single-positive CD4+ and CD8+ T cells, which were TCR alpha beta+, were identified indicating normal thymocyte maturation. In kinetic studies, mature single-positive CD4+ T cells increased from 29% of total cells at one week to 54% at four weeks of coculture. These findings demonstrate that coculture of bone marrow-derived SC and allogeneic cultured thymic epithelia in vitro results in continuous normal predominantly thymocyte differentiation. The SC-CTEF cocultures were then infected with two different strains of human immunodeficiency virus. CD4+ thymocytes were markedly decreased. However, inhibition of early thymocyte maturation steps was also suggested by the presence of increased triple-negative and CD44+CD25-CD3-thymocytes and decreased CD44+CD25+ thymocytes. This model system of thymocyte maturation will be useful in the evaluation of primary T cell immunodeficiency disorders, gene therapy of SC and pharmacological augmentation of thymic function.     

In vitro positive selection and anergy induction of class II-restricted TCR transgenic thymocytes by a cortical thymic epithelial cell line

Thymic epithelial cell lines isolated from hyperplastic thymi of transgenic mice over-expressing human papilloma viral oncogenes E6 and E7 constitutively displayed a phenotype consistent with a cortical origin. Exposure to IFN-gamma induced class II MHC and ICAM-1 expression, and up-regulated expression of VCAM-1 and class I MHC molecules. CD40 expression was maximally induced by a combination of IFN-gamma and IL-1, with lower levels of induction observed with a mixture of IFN-gamma and tumor necrosis factor (TNF)-alpha or TNF-alpha alone. B7-1 or B7-2 was not expressed constitutively or in response to cytokines. These stromal cells supported the development of CD4 single-positive (SP) cells in reaggregate co-cultures with CD4+ CD8+ thymocytes from TCR transgenic mice, but did not stimulate class II MHC-restricted, moth cytochrome c (MCC)-reactive T cells in vitro. The behavior of the culture system was consistent with positive selection, i.e. increased numbers of CD4 SP cells, gain of antigen responsiveness, and requirement for epithelial class II MHC products. Some variants of these stromal cell lines required exogenous MCC peptide in the reaggregation cultures (RC) for positive selection to occur. While a low concentration of MCC peptide (0.01-0.1 microM) significantly enhanced the accumulation of CD4 SP cells, higher concentrations of peptide (1-10 microM) resulted in recovery of predominantly CD4- CD8- and CD4(low) CD8- cells. Thymocytes recovered from RC containing low, but not high concentrations of peptide responded to MCC peptide in secondary cultures with splenic antigen-presenting cells.     

Timing and casting for actors of thymic negative selection

We have recently proposed a new model for the differentiation pathway of alphabeta TCR thymocytes, with the CD4 and CD8 coreceptors undergoing an unexpectedly complex series of expression changes. Taking into account this new insight, we reinvestigated the timing of thymic negative selection. We found that, although endogenous superantigen-driven thymic negative selection could occur at different steps during double-positive/single-positive cell transition, this event was never observed among CD4lowCD8low TCRint CD69+ thymocytes, i.e., within the first subset to be generated upon TCR-mediated activation of immature double-positive cells. We confirm a role for CD40/CD40L interaction, and the absence of involvement of CD28 costimulation, in thymic deletion in vivo. Surprisingly, we found that thymic negative selection was impaired in the absence of Fas, but not FasL, molecule expression. Finally, we show involvement in opposing directions for p59fyn and SHP-1 molecules in signaling for thymic negative selection.     

Comparisons of endothelial cell G- and F-actin distribution in situ and in vitro

Precursor of T lymphocytes undergo proliferation and maturation under the influence of the thymic microenvironment. In our study, we have attempted to determine the distribution of human postnatal thymocytes in division according to their stage of differentiation. Our data show that about 11.5% of all thymic cells are in S/G2/M phases, and that a subset of the cortical and precortical subpopulations contains most of the dividing cells. Rate of cell division is maintained at high levels from the prethymocyte precursor along the successive stages of differentiation represented by CD1+CD3-CD4-CD8- and CD1+CD3-CD4+CD8- cells. The percentage of dividing cells is maximal in an intermediate subset of CD1+CD3-CD4+CD8-CD45RO+ cells defined by the distinct expression of class I HLAdim/high molecules, which could contain cells in transit from prethymocytes to double-positive cortical cells. The CD3- fraction of the double-positive cortical cells contains most of the dividing thymocytes, although the rate of division within this subset is much less than that of the precursor CD1+CD3-CD4+CD8- cells. In a linear scheme of differentiation, cell division stops at or near the point of initiation of CD3 expression. These results suggest that in human thymus cell expansion takes place before the initiation of the positive selection process. According to this view the stringency of the selection process would require the previous generation of a large number of precursors to permit the production of sufficient numbers of mature T cells.     

Children''s narrative representations of mothers: their development and associations with child and mother adaptation

To investigate the role of antigen receptor-mediated interactions in lymphomagenesis we have analyzed the influence of alpha beta TCR-mediated selection on the development of spontaneous thymic lymphomas, which appear with a high (up to 50%) frequency in mice expressing a transgenic TCR specific for the male antigen (HY) in the context of H-2Db molecules. To this end we compared the kinetics and the incidence of thymic lymphomas developing in females and males with selecting (H-2b) and non-selecting (H-2k) MHC molecules. The kinetics of development of thymic lymphomas was similar in positively selecting (H-2b females) and non-selecting (H-2k females and males) environments but significantly slower (P < 0.01) in the negatively selecting environment (H-2b male). Injection of lymphoma cells derived from a H-2b female into the thymus of a H-2b male resulted in strong, antigen-specific inhibition of growth, indicating that the slower kinetics of lymphomagenesis in H-2b males could be due, at least partially, to the sensitivity of oncogenically transformed thymocytes to TCR-mediated negative selection. Phenotypic and functional analysis of lymphoma cells indicated that they originated from the stage of pre-TCR-dependent transition of immature CD4-CD8- to CD4+ CD8+ thymocytes, which in H-2b females and males developed into tumors under different environmental pressures. These results failed to provide convincing evidence for the role of positive selection but provided a strong indication that self antigen-induced negative selection, in addition to its well established role in self tolerance, can occasionally act as a tumor surveillance mechanism by eliminating or suppressing growth of thymocytes undergoing oncogenic transformation.     

An alternate pathway for T cell development supported by the bone marrow microenvironment: recapitulation of thymic maturation

In the principal pathway of alpha/beta T cell maturation, T cell precursors from the bone marrow migrate to the thymus and proceed through several well-characterized developmental stages into mature CD4+ and CD8+ T cells. This study demonstrates an alternative pathway in which the bone marrow microenvironment also supports the differentiation of T cell precursors into CD4+ and CD8+ T cells. The marrow pathway recapitulates developmental stages of thymic maturation including a CD4+CD8+ intermediary cell and positive and negative selection, and is strongly inhibited by the presence of mature T cells. The contribution of the marrow pathway in vivo requires further study in mice with normal and deficient thymic or immune function.     

Ovine brain natriuretic peptide in cardiac tissues and plasma: effects of cardiac hypertrophy and heart failure on tissue concentration and molecular forms

It is well established that lymphoid dendritic cells (DC) play an important role in the immune system. Beside their role as potent inducers of primary T cell responses, DC seem to play a crucial part as major histocompatibility complex (MHC) class II+ "interdigitating cells" in the thymus during thymocyte development. Thymic DC have been implicated in tolerance induction and also by some authors in inducing major histocompatibility complex restriction of thymocytes. Most of our knowledge about thymic DC was obtained using highly invasive and manipulatory experimental protocols such as thymus reaggregation cultures, suspension cultures, thymus grafting, and bone marrow reconstitution experiments. The DC used in those studies had to go through extensive isolation procedures or were cultured with recombinant growth factors. Since the functions of DC after these in vitro manipulations have been reported to be not identical to those of DC in vivo, we intended to establish a system that would allow us to investigate DC function avoiding artificial interferences due to handling. Here we present a transgenic mouse model in which we targeted gene expression specifically to DC. Using the CD 11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types. We report that I-E expression on thymic DC is sufficient to negatively select I-E reactive CD4+ T cells, and to a less complete extent, CD8+ T cells. In contrast, it only DC expressed I-E in a class II-deficient background, positive selection of CD4+ T cells could not be observed. Thus negative, but not positive, selection events can be induced by DC in vivo.     

Dual MHC class I and class II restriction of a single T cell receptor: distinct modes of tolerance induction by two classes of autoantigens

In the final stages of thymic development, immature T cells undergo three distinct processes (positive selection, negative selection, and lineage commitment) that all depend on interactions of thymocyte TCRs with MHC molecules. It is currently thought that TCRs are preferentially restricted by either MHC class I or class II molecules. In this report, we present direct evidence that the TCR previously described as H-Y/H-2Db specific cross-reacts with H-2IAb if expressed in CD4+ cells. We also demonstrate an increase in thymocyte numbers in H-Y TCR-trangenic mice deficient in MHC class II, suggesting a relatively discrete form of negative selection by MHC class II compared with that induced by H-Y/H-2Db. We propose that inability to generate CD4+ T cells expressing H-Y TCR in different experimental settings may be due to tolerance to self-MHC class II. These results, therefore, support an intriguing possibility that tolerance to self may influence and/or interfere with the outcome of the lineage commitment.     

Regulated expression of gp130 and IL-6 receptor alpha chain in T cell maturation and activation

The functional receptor for the inflammatory cytokine IL-6 is composed of the ligand binding IL-6 receptor alpha chain (IL-6R alpha) and the signal transducing chain gp130, which is a shared component of multiple cytokine receptors. We analyzed the surface expression of gp130 and IL-6R alpha in thymocytes and peripheral T cells. While all thymocytes expressed gp130 throughout thymic maturation, they gained expression of IL-6R alpha at the CD4 or CD8 single-positive stage. Approximately 10-30% of the CD4-CD8+ and 40-50% of the CD4+CD8- thymocytes expressed IL-6R alpha. Within the CD4+CD8- population, the IL-6R alpha- subpopulation was cortisone sensitive, appeared immature according to the cell surface markers expressed and failed to proliferate after TCR cross-linking. Peripheral T cells were predominantly gp130+ and IL-6R alpha+, but down-regulated gp130 and IL-6R alpha expression upon TCR engagement in vitro and in vivo. Peripheral gp130low/-IL-6R alphalow/- T cells expressed surface markers characteristic of memory T cells. We show that gp130 and IL-6R alpha are expressed in a regulated manner in T cells, depending on the developmental and functional stage.     

Abnormal thymocyte subset distribution and differential reduction of CD4+ and CD8+ T cell subsets during peripheral maturation in diabetes-prone BioBreeding rats

In this study we quantified CD8+ and CD4+ T cells in T lymphocytopenic BB rats as compared with control rats at given stages along the maturational pathway from immature thymocytes to mature peripheral T cells. Our results show that BB rats exhibit abnormal thymocyte subset distribution. Numbers of mature TCRhigh/CD4-8+ thymocytes, and also their TCRhigh/CD4+8+ precursors were decreased, as were levels of CD8 expression on all thymocyte subsets investigated. By analogy with mouse thymocyte development, these findings suggest a decreased efficiency for positive selection of CD8 precursors in BB rats. Furthermore, as related to the number of available mature TCRhigh single positive thymocytes, numbers of CD4+ and CD8+ T cells most recently migrated from the thymus were severely decreased in BB blood, indicating either reduced thymic output or rapid cell death after migration. Subsequently, in peripheral blood and cervical lymph nodes, a 95% decrease of CD8+ and a 50 to 80% decrease of CD4+ T cells were demonstrated upon maturation from recent thymic migrants to mature peripheral T cells, leaving the BB rat with a severely reduced T cell population, consisting of CD4+ T cells and a minute population of CD8+ T cells. The vast majority of the latter was found to have an immature peripheral phenotype. Possible consequences of our findings for the generation of autoreactive CD8+ T cells are discussed.     

Critical role for the common cytokine receptor gamma chain in intrathymic and peripheral T cell selection

The common cytokine receptor gamma chain (gammac), which is a functional subunit of the receptors for interleukins (IL)-2, -4, -7, -9, and -15, plays an important role in lymphoid development. Inactivation of this molecule in mice leads to abnormal T cell lymphopoiesis characterized by thymic hypoplasia and reduced numbers of peripheral T cells. To determine whether T cell development in the absence of gammac is associated with alterations of intrathymic and peripheral T cell selection, we have analyzed gammac-deficient mice made transgenic for the male-specific T cell receptor (TCR) HY (HY/gammac- mice). In HY/gammac- male mice, negative selection of autoreactive thymocytes was not diminished; however, peripheral T cells expressing transgenic TCR-alpha and -beta chains (TCR-alphaT/betaT) were absent, and extrathymic T cell development was completely abrogated. In HY/gammac- female mice, the expression of the transgenic TCR partially reversed the profound thymic hypoplasia observed in nontransgenic gammac- mice, generating increased numbers of thymocytes in all subsets, particularly the TCR-alphaT/betaT CD8+ single-positive thymocytes. Despite efficient positive selection, however, naive CD8+ TCR-alphaT/betaT T cells were severely reduced in the peripheral lymphoid organs of HY/gammac- female mice. These results not only underscore the indispensible role of gammac in thymocyte development, but also demonstrate the critical role of gammac in the maintenance and/or expansion of peripheral T cell pools.     

Differential thymic selection outcomes stimulated by focal structural alteration in peptide/major histocompatibility complex ligands

The T lineage repertoire is shaped by T cell receptor (TCR)-dependent positive and negative thymic selection processes. Using TCR-transgenic (N15tg) beta2-microglobulin-deficient (beta2m-/-) RAG-2(-/-) H-2(b) mice specific for the VSV8 (RGYVYQGL) octapeptide bound to Kb, we identified a single weak agonist peptide variant V4L (L4) inducing phenotypic and functional T cell maturation. The cognate VSV8 peptide, in contrast, triggers negative selection. The crystal structure of L4/Kb was determined and refined to 2.1 A for comparison with the VSV8/Kb structure at similar resolution. Aside from changes on the p4 side chain of L4 and the resulting alteration of the exposed Kb Lys-66 side chain, these two structures are essentially identical. Hence, a given TCR recognizes subtle distinctions between highly related ligands, resulting in dramatically different selection outcomes. Based on these finding and the recent structural elucidation of the N15-VSV8/Kb complex, moreover, it appears that the germ-line Valpha repertoire contributes in a significant way to positive selection.     

A role for calcium influx in setting the threshold for CD4+CD8+ thymocyte negative selection

We have applied an in vitro system that mimics thymic negative selection to investigate signaling pathways that may be important for the removal of autoreactive cells from the thymus. We sought to more precisely determine the contribution of calcium-dependent pathways to CD4+CD8+ thymocyte deletion that is mediated by either an antigenic peptide or a peptide analogue. We show that the requirement for external calcium influx is dependent upon the strength of the deleting ligand. Furthermore, these results correlate well with a requirement, under certain circumstances, for signaling through the calcium/calmodulin-dependent phosphatase calcineurin. The use of suboptimal stimuli may, therefore, be useful in revealing biochemical pathways important for CD4+CD8+ thymocyte negative selection.     

Rhesus thymic/liver xenografts in severe combined immunodeficient mice: immunologic reconstitution and intrathymic infection with simian immunodeficiency virus

By serving as host recipients of xenografts from both humans and animals, severe combined immunodeficient (SCID) mice have become valuable to many laboratories interested in examining the pathophysiology of different diseases. To gain insight into the usefulness of the SCID mutation in retrovirus research, rhesus monkey fetal hematolymphoid tissues (liver and thymus) were used to construct a SCID-rhesus chimeric mouse (SCID-rh) and were engrafted in the renal capsule. The size and maturation of the thymic engrafts were monitored grossly, histologically, and immunologically. SCID mice were tolerant to rhesus tissues, and thymic engrafts contained thymocytes at different stages of maturation and differentiation that had morphologic features similar to age-matched rhesus thymus. Mature single positive CD2+, CD4+, and CD8+ T lymphocytes that were phenotypically similar to rhesus T lymphocytes were present at low levels (2% to 5%) in the peripheral blood and at moderately higher levels (7% to 15%) in the spleens of SCID-rh mice obtained between 12 and 15 weeks after thymus/liver engraftment. Within 3 weeks after engraftment, > 85% of the thymocytes in the thymic engrafts were immature double positive CD4+CD8+ T cells. The highest number of positive cells were seen in thymic engrafts obtained at 12 to 18 weeks. During these weeks, > 90% of the cells were double positive (CD2+CD4+, CD2+CD8+, and CD4+CD8+). After infection of the engrafted thymus tissue with simian immonodeficiency virus (SIVmac239), PCR analysis revealed successful viral infection of engrafts at 2 and 4 weeks after infection. No significant histopathologic and flow cytometric changes were observed in the thymic engrafts at 2 and 4 weeks after infection. An unrelated lesion of thymic lymphomas involving the SCID host thymus was seen in 12% of the mice. The data presented herein suggest that the SCID-rh is a valuable model for specific studies related to thymus-retrovirus interaction and that it could be used for further studies. The results are discussed in relation to current knowledge of thymus involvement during simian and human immunodeficiency virus infection.     

The JNK pathway regulates the In vivo deletion of immature CD4(+)CD8(+) thymocytes

The extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase pathways are triggered upon ligation of the antigen-specific T cell receptor (TCR). During the development of T cells in the thymus, the ERK pathway is required for differentiation of CD4(-)CD8(-) into CD4(+)CD8(+) double positive (DP) thymocytes, positive selection of DP cells, and their maturation into CD4(+) cells. However, the ERK pathway is not required for negative selection. Here, we show that JNK is activated in DP thymocytes in vivo in response to signals that initiate negative selection. The activation of JNK in these cells appears to be mediated by the MAP kinase kinase MKK7 since high levels of MKK7 and low levels of Sek-1/MKK4 gene expression were detected in thymocytes. Using dominant negative JNK transgenic mice, we show that inhibition of the JNK pathway reduces the in vivo deletion of DP thymocytes. In addition, the increased resistance of DP thymocytes to cell death in these mice produces an accelerated reconstitution of normal thymic populations upon in vivo DP elimination. Together, these data indicate that the JNK pathway contributes to the deletion of DP thymocytes by apoptosis in response to TCR-derived and other thymic environment- mediated signals.     

Obstructive sleep apnea syndrome and bronchial hyperreactivity

Cyclosporin A (CsA) inhibits the development of mature thymocytes from their CD4+ CD8+ precursors, but may allow autoreactive cells to mature. Using 3-color flow cytometry, we have followed the progressive development of thymocytes, including potentially autoreactive cells, during CsA treatment. Numbers of CD4+ CD8+ CD3high thymocytes dropped immediately, suggesting that the generation of these mature thymocyte precursors, normally dependent upon positive selection, was inhibited by CsA. Numbers of CD4+ CD8- thymocytes also declined rapidly, but CD4 - CD8+ thymocytes were unaffected for 2 days, suggesting that the mature single-positive subsets are not symmetrically derived from a common GsA-sensitive precursor. An exceptional subset of CD8 SP thymocytes, expressing CD45RA, did not respond to CsA for about 10 days, indicating that they are distantly derived from a CsA-sensitive precursor. Apoptosis of TCR-V beta 3 + thymocytes caused by Mtv-6, quantified according to the down-regulation of CD4 and CD8 on immature thymocytes, was partially inhibited by CsA, to maximal effect within 24 hours. This did not, however, facilitate their development into mature thymocytes.     

A role for Fas in negative selection of thymocytes in vivo

To seek information on the role of Fas in negative selection, we examined subsets of thymocytes from normal neonatal mice versus Fas-deficient lpr/lpr mice injected with graded doses of antigen. In normal mice, injection of 1-100 microg of staphylococcal enterotoxin B (SEB) induced clonal elimination of SEB-reactive Vbeta8+ cells at the level of the semi-mature population of HSAhi CD4+ 8- cells found in the thymic medulla; deletion of CD4+ 8+ cells was minimal. SEB injection also caused marked elimination of Vbeta8+ HSAhi CD4+ 8- thymocytes in lpr/lpr mice. Paradoxically, however, elimination of these cells in lpr/lpr mice was induced by low-to-moderate doses of SEB (相似文献