Faculty Bibliography

Signals that emanate from the pre-T cell receptor (pre-TCR) regulate multiple processes required for the development of the alphabeta T cell lineage. In contrast to the gammadelta TCR, the pre-TCR localizes cell-autonomously to membrane rafts, where it appears to signal in a constitutive and ligand-independent manner. We addressed here the role played by structural features specific to the cytoplasmic domain of the pre-TCRalpha chain (pTalpha). More specifically, we examined a COOH-terminal proline-rich sequence that might play a role in signal transduction and a juxtamembrane cysteine residue that could be a target for palmitoylation, thus allowing spontaneous raft localization. Expression of pTalpha mutants in transgenic mice, retrovirally transduced T cell precursors and cell lines showed that the pTalpha cytoplasmic tail, in particular the proline-rich domain, plays a crucial role in pre-TCR signaling and T cell development. In contrast, the pTalpha juxtamembrane cysteine appeared to be dispensable for pre-TCR function

A transgenic reporter mouse strain, which expressed the human CD25 (hCD25) surface marker as a reporter under the control of the pre-T cell receptor alpha(pTalpha) promoter, was used to identify lymphoid precursors that expressed pTalpha intracellularly. The hCD25 reporter marked intra- and extrathymic precursors of lymphocytes but not myeloid cells. The earliest intrathymic precursors were CD4(lo)CD8(-)CD25(-)CD44(+)c-Kit(+) cells that expressed elevated levels of Notch-1 mRNA. Clonogenic assays showed that the extrathymic precursors were common lymphoid progenitors (CLPs) that included CD19(-), B220(+), Thy1(+) and CD4(+) cells. Thus, the pTalpha reporter can be used to trace lymphopoiesis between CLPs and alphabeta T cells. The slower extinction of the hCD25 reporter compared to pTalpha enabled us to define points at which pTalpha(-) lineages branched off

Recent studies provide fresh insight into the mechanisms by which precursor cells are committed to and develop within the T-lymphocyte lineage. Precursor/product studies have identified developmental stages between that of the pluripotent hematopoietic stem cell and thymocytes committed to the T lineage. Specific ligands and signaling pathways interacting with the Notch-1 receptor and its ability to influence commitment within the lymphoid lineage have been described. Although the structural features or putative ligands endowing the pre-TCR with constitutive signaling capacity remain elusive, numerous distal mediators of pre-TCR signaling have been identified. It remains for the future to determine what roles they may have in survival, proliferation, lineage commitment and allelic exclusion of TCR genes. Receptor editing and lineage commitment of alphabeta T cells still represent controversial topics that need further study

Mutation or ablation of T cell factor 1 and lymphocyte enhancer factor 1 indicated involvement of the Wnt pathway in thymocyte development. The central effector of the Wnt pathway is beta-catenin, which undergoes stabilization upon binding of Wnt ligands to frizzled receptors. We report here that conditional stabilization of beta-catenin in immature thymocytes resulted in the generation of single positive T cells that lacked the alpha beta TCR and developed in the absence of pre-TCR signaling and TCR selection. Although active beta-catenin induced differentiation in the absence of TCRs, its action was associated with reduced proliferation and survival when compared to developmental changes induced by the pre-TCR or the alpha beta TCR

Pre-T cell antigen receptor (pre-TCR) signaling plays a crucial role in the development of immature T cells. Although certain aspects of proximal pre-TCR signaling have been studied, the intermediate signal transducers and the distal transcription modulators have been poorly characterized. We report here a correlation between pre-TCR signaling and a biphasic rise in the cytosolic Ca2+ concentration. In addition, we show that constitutive pre-TCR signaling is associated with an increased rate of Ca2+ influx through store-operated plasma membrane Ca2+ channels. We show also that the biphasic nature of the observed pre-TCR-induced rise in cytosolic Ca2+ differentially modulates the activities of the transcription factors NF-kappaB and NFAT in developing T cells

In this report, we examined whether nitric oxide (NO) is involved in early embryo death. We have chosen various experimentally defined embryotoxic stimuli in mice and determined their ability to induce NO production by 2-cell stage embryos. The embryotoxic factors used were interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), 5-Azacytidine (5-AzaC) and the murine embryotoxic antibody DF4. We showed that in all cases the embryotoxic stimuli induced NO production by early stage embryos that correlated with the induction of the inducible and/or endothelial isoforms of NO synthase. This study was also extended to the human system where sera from women who aborted were tested for their ability to act embryotoxically by inducing NO in early mouse embryos and mature murine placenta. The results obtained confirmed the embryotoxic character of NO found in these particular sera leading to the hypothesis that NO plays a potential role in early embryo death

The detection of corticotropin-releasing hormone (CRH) in the pregnant and non-pregnant uterus has driven research to determine the role of this 41 amino acid neuropeptide in the female reproductive system. As concentrations of CRH mRNA and its peptide product are greater in the implantation sites of the early pregnant uterus compared with the regions between implantation sites, CRH has been hypothesised to participate in blastocyst implantation. Using the mouse system as an experimental model, we studied the distribution of CRH in the uterus during the oestrus cycle and early gestational period, and now provide evidence for its involvement in embryo implantation using cell culture techniques. The percentage of CRH-positive uterine cells and the amount of CRH released during anoestrus, pro-oestrus and oestrus were determined by immunofluorescence and ELISA experiments respectively. The highest number of intracellularly CRH-positive cells was obtained during pro-oestrus, whereas the highest CRH concentration in uterine cell culture supernatants was detected during anoestrus. At early stages of gestation, CRH was detected in the endometrium on days 2, 3 and 4 of pregnancy and in the myometrium on days 3 and 4, whereas it was undetectable on day 5. The functional role of CRH during early gestation was evaluated by administering anti-CRH antibody to mice from day 3 to day 8 of pregnancy. This treatment resulted in implantation failure in 60% of the cases, in which implantation sites, although clearly present in the uterus, had failed to host an embryo. These results provide direct evidence about the involvement of CRH in murine embryo implantation and are in agreement with hypotheses postulated in humans

Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4(-)CD8(-) double-negative (DN) thymocytes into CD4(+)CD8(+) double-positive (DP) cells and for TCR-beta allelic exclusion. The adaptor protein SH2 domain-containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76(-/-) mice are arrested at the CD25(+)CD44(-) DN stage. Here we show that SLP-76(-/-) DN thymocytes express the pre-TCR on their surfaces and that introduction of a TCR-alpha/beta transgene into the SLP-76(-/-) background fails to cause expansion of DN thymocytes or developmental progression to the DP stage. Moreover, analysis of TCR-beta rearrangement in SLP-76(-/-) TCR-transgenic mice or in single CD25(+)CD44(-) DN cells from SLP-76(-/-) mice indicates an essential role of SLP-76 in TCR-beta allelic exclusion

We have examined the question of whether there is an additional checkpoint in T cell development that regulates T cell receptor (TCR)-beta expression in CD25+44- thymocytes by mechanisms that are independent of the pre-TCR. Our analysis in various mutant mice indicates that all changes in cytoplasmic TCR-beta expression can be accounted for by pre-TCR-dependent signal mediation, putting into question the function of a putative pro-TCR

In this report, we examined the involvement of the cytokines tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-4, and IL-10 as well as nitric oxide (NO) in the lipopolysaccharide (LPS)-induced experimental abortion model in BALB/c mice. Although in vivo administration of LPS in pregnant mice showed a 72% decrease of serum IL-10, no significant difference in serum TNF-alpha, IFN-gamma, and IL-4 levels, compared to controls, could be detected. At the same time, a correlation of fetal abortion and maternal splenomegaly with an important increase of NO synthesis in the serum was obtained. Simultaneous administration of LPS and aminoguanidine (AG; an inhibitor to NO synthase) rescued the LPS-induced fetal abortion, reduced maternal spleen weight to physiological levels, and decreased serum NO concentration to control levels. In vitro experiments showed that LPS directly induced NO production in primary placental cells and the TPOPHO-1 trophoblast cell line by stimulating the inducible isoform of NO synthase, which ultimately could be blocked by the NO synthase inhibitors AG and L-NAME. The results indicate that LPS, despite its beneficial involvement in intracellular infections, participates in inflammatory/autoimmune damage during pregnancy, leading to embryotoxicity, which is closely linked to the NO pathway