Neurodevelopmental and neuropsychiatric behaviour defects arise from 14-3-3ζ deficiency.

Complex neuropsychiatric disorders are believed to arise from multiple synergistic deficiencies within connected biological networks controlling neuronal migration, axonal pathfinding and synapse formation. Here, we show that deletion of 14-3-3ζ causes neurodevelopmental anomalies similar to those seen in neuropsychiatric disorders such as schizophrenia, autism spectrum disorder and bipolar disorder. 14-3-3ζ-deficient mice displayed striking behavioural and cognitive deficiencies including a reduced capacity to learn and remember, hyperactivity and disrupted sensorimotor gating. These deficits are accompanied by subtle developmental abnormalities of the hippocampus that are underpinned by aberrant neuronal migration. Significantly, 14-3-3ζ-deficient mice exhibited abnormal mossy fibre navigation and glutamatergic synapse formation. The molecular basis of these defects involves the schizophrenia risk factor, DISC1, which interacts isoform specifically with 14-3-3ζ. Our data provide the first evidence of a direct role for 14-3-3ζ deficiency in the aetiology of neurodevelopmental disorders and identifies 14-3-3ζ as a central risk factor in the schizophrenia protein interaction network.

Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts.

While CD28 functions as the major T cell costimulatory receptor, a number of other T cell molecules have also been described to induce T cell costimulation. Here, we investigated the mechanisms by which costimulatory molecules other than CD28 contribute to T cell activation. Non-CD28 costimulatory molecules such as CD5, CD9, CD2, and CD44 were present in the detergent-insoluble glycolipid-enriched (DIG) fraction/raft of the T cell surface, which is rich in TCR signaling molecules and generates a TCR signal upon recruitment of the TCR complex. Compared with CD3 ligation, coligation of CD3 and CD5 as an example of DIG-resident costimulatory molecules led to an enhanced association of CD3 and DIG. Such a DIG redistribution markedly up-regulated TCR signaling as observed by ZAP-70/LAT activation and Ca2+ influx. Disruption of DIG structure using an agent capable of altering cholesterol organization potently diminished Ca2+ mobilization induced by the coligation of CD3 and CD5. This was associated with the inhibition of the redistribution of DIG although the association of CD3 and CD5 was not affected. Thus, the DIG-resident costimulatory molecules exert their costimulatory effects by contributing to an enhanced association of TCR/CD3 and DIG.

CD5 costimulation up-regulates the signaling to extracellular signal-regulated kinase activation in CD4+CD8+ thymocytes and supports their differentiation to the CD4 lineage.

CD5 positively costimulates TCR-stimulated mature T cells, whereas this molecule has been suggested to negatively regulate the activation of TCR-triggered thymocytes. We investigated the effect of CD5 costimulation on the differentiation of CD4+CD8+ thymocytes. Coligation of thymocytes with anti-CD3 and anti-CD5 induced enhanced tyrosine phosphorylation of LAT (linker for activation of T cells) and phospholipase C-gamma (PLC-gamma) compared with ligation with anti-CD3 alone. Despite increased phosphorylation of PLC-gamma, this treatment down-regulated Ca2+ influx. In contrast, the phosphorylation of LAT and enhanced association with Grb2 led to activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase. When CD3 and CD5 on CD4+CD8+ thymocytes in culture were coligated, they lost CD8, down-regulated CD4 expression, and induced CD69 expression, yielding a CD4+(dull)CD8-CD69+ population. An ERK inhibitor, PD98059, inhibited the generation of this population. The reduction of generation of CD4+CD8- cells resulted from decreased survival of these differentiating thymocytes. Consistent with this, PD98059 inhibited the anti-CD3/CD5-mediated Bcl-2 induction. These results indicate that CD5 down-regulates a branch of TCR signaling, whereas this molecule functions to support the differentiation of CD4+CD8+ thymocytes by up-regulating another branch of TCR signaling that leads to ERK activation.

Association of a tetraspanin CD9 with CD5 on the T cell surface: role of particular transmembrane domains in the association.

CD9 is a member of the tetraspanin superfamily which is characterized by four transmembrane (TM) domains and associates with other surface molecules. This tetraspanin was recently found to be expressed on mature T cells. Here, we investigated which molecules associate with CD9 on T cells and which CD9 domains are required for the association. Immunoprecipitation of T cell lysates with anti-CD9 mAb followed by immunoblotting with mAb against various T cell molecules showed the association of CD9 with CD3, CD4, CD5, CD2, CD29 and CD44. Because association with CD5 was most prominent, we determined the role of CD9 TM or extracellular (EC) domains in the association with CD5. CD9 mutant genes lacking each domain were constructed and introduced into EL4 thymoma cells deficient in CD9 but expressing CD5. Among various types of stable EL4 transfectants, EL4 transfected with the mutant gene lacking TM domains (TM2/TM3) between two EC domains expressed a small amount of the relevant protein without showing association with CD5. CD9(-)CD5(-) monkey COS-7 cells transfected with this mutant gene and the CD5 gene expressed both transfected gene products, but the association of these was not detected. EL4 cells transfected with a CD9/CD81 chimera gene (the CD9 gene containing TM2/TM3 of CD81) expressed the chimeric protein on the cell surface and showed association with CD5. These results suggest an essential role of particular CD9 TM domains in the surface expression of the CD9 molecule as well as the association with CD5.

Differential involvement of a Fas-CPP32-like protease pathway in apoptosis of TCR/CD9-costimulated, naive T cells and TCR-restimulated, activated T cells.

Our previous study showed that CD9 costimulation of TCR-triggered naive T cells elicits activation ([3H]TdR incorporation) that is similar to CD28 costimulation; however, unlike CD28 costimulation, CD9 costimulation results in apoptosis of these previously activated T cells. Here, we investigated whether the apoptosis occurring after TCR/CD9 stimulation is associated with a death pathway involving Fas stimulation and Fas-mediated caspase activation as observed in activation-induced cell death (AICD). In contrast to AICD, the apoptosis resulting from TCR/CD9 stimulation in C57BL/6 T cells was independent of Fas, because this form of apoptosis was not prevented by anti-Fas ligand mAb and was also induced in MRL/lpr T cells. AICD was observed at 12 h after the restimulation of activated T cells with anti-CD3 and reached a peak level at 24 h after this restimulation. CPP32-like protease activity was detected during AICD. Although TCR/CD9 stimulation-associated apoptosis was observed at 24 h after the stimulation of naive T cells and reached a peak level at 36 h after this stimulation, CPP32-like protease activity in these T cells was only marginal at all time points. Nevertheless, both forms of apoptosis were prevented similarly by two different peptide-based caspase inhibitors. These results indicate that the apoptosis that follows the T cell activation which is induced as a result of CD9 costimulation does not involve a Fas-CPP32-like protease pathway, but suggest that different caspase members are likely to be critical in this form of apoptosis.

A fundamental difference in the capacity to induce proliferation of naive T cells between CD28 and other co-stimulatory molecules.

T cell activation requires two signals: a signal from the TCR and a co-stimulatory signal provided by antigen-presenting cells (APC). In addition to CD28, multiple molecules on the T cell have been described to deliver co-stimulatory signals. Here, we investigated whether there exist quantitative or qualitative differences in the co-stimulatory capacity between CD28 and other molecules. Anti-CD28 monoclonal antibody (mAb) and mAb against CD5, CD9, CD2, CD44 or CD11a all induced activation of naive T cells in the absence of APC when co-immobilized with a submitogenic dose of anti-CD3 mAb. [3H]Thymidine incorporation determined 2 days after co-stimulation was all comparable. In contrast to progressive T cell proliferation induced by CD28 co-stimulation, co-stimulation by other T cell molecules led to a decrease in viable cell recovery along with the induction of apoptosis of once activated T cells. This was associated with a striking difference in IL-2 production; CD28 co-stimulation induced progressively increasing IL-2 production, whereas co-stimulation by other molecules produced limited amounts of IL-2. Addition of recombinant IL-2 to the latter cultures corrected the induction of apoptosis, resulting in levels of cellular proliferation comparable to those observed for CD28 co-stimulation. These results indicate that a fundamental difference exists in the nature of co-stimulation between CD28 and other molecules, which can be evaluated by the levels of IL-2 production, but not simply by [3H]thymidine incorporation.

Suppression of allograft responses induced by interleukin-6, which selectively modulates interferon-gamma but not interleukin-2 production.

BACKGROUND: Interferon (IFN)-gamma produced by activated T cells represents an important effector cytokine in mediating an inflammatory response. METHODS: The present study investigated the modulation of allograft responses by inhibiting IFN-gamma production. C57BL/6 (B6) lymph node cells were stimulated with class II H2-disparate B6-C-H-2bm12 (bm12) spleen cells. RESULTS: Addition of interleukin (IL)-6 to the primary B6 anti-bm12 mixed lymphocyte reaction (MLR) inhibited neither proliferative responses nor IL-2 production. However, IL-6 induced a dose-dependent suppression of IFN-gamma production in the same MLR cultures. B6 mice were engrafted with bm12 skin grafts, and IL-6 was given to bm12 skin graft recipients every other day. T cells from these recipient mice produced significantly less IFN-gamma in secondary B6 anti-bm12 MLR than those from bm12 skin graft recipients that had not received IL-6 injections. IFN-gamma production by these T cells was suppressed more strongly when the secondary MLR was conducted in the presence of IL-6. In addition to suppression of IFN-gamma expression, IL-6 injections resulted in prolongation of bm12 skin graft survival. The critical involvement of IFN-gamma in anti-bm12 rejection responses was substantiated by evidence that administration of anti-IFN-gamma monoclonal antibody strikingly prolonged bm12 skin graft survival. The prolongation of graft survival by in vivo treatment with either IL-6 or anti-IFN-gamma monoclonal antibody was found to be induced without blocking cellular infiltration of the grafts. CONCLUSIONS: These results indicate that IFN-gamma acts as a key cytokine in a B6 anti-bm12 allograft response and that IL-6 may down-regulate this response by inhibiting IFN-gamma production of alloreactive T cells.

Synergy between CD28 and CD9 costimulation for naive T-cell activation.

Our previous study demonstrated that CD9 is expressed on most mature naive T-cells and delivers a potent costimulatory signal that functions independently of CD28. Here, we investigated whether this CD9-mediated signal is different from the CD28-mediated signal in the mode of costimulation and whether both signals function synergistically for T-cell activation. Anti-CD9 or anti-CD28 monoclonal antibody (mAb) increased [3H]TdR incorporation of naive T-cells in the absence of antigen-presenting cells (APC) when coimmobilized with submitogenic doses of anti-CD3 mAb. The levels of costimulation induced by ligation of CD9 and CD28 were comparable. However, the costimulatory effect differed between soluble anti-CD9 and CD28 mAb. A soluble form of anti-CD28 mAb could costimulate anti-CD3-triggered T-cells, whereas soluble anti-CD9 mAb failed to costimulate. Although anti-CD28 costimulated naive T-cells treated with phorbol myristate acetate (PMA) instead of anti-CD3 mAb, a combination of PMA plus anti-CD9 mAb could not induce T-cell activation. The combined costimulation of anti-CD3-triggered T-cells with anti-CD9 and anti-CD28 mAbs resulted in strikingly enhanced [3H]TdR uptake and lymphokine (IL-2 and IFN-gamma) production when compared to those induced by each costimulation. These results suggest that CD9 and CD28 induce T-cell costimulation using different signaling pathways, thereby inducing synergy in T-cell activation.

Expression of an inducible type of nitric oxide (NO) synthase in the thymus and involvement of NO in deletion of TCR-stimulated double-positive thymocytes.

The present study investigates the role of nitric oxide (NO) in the deletion of TCR-stimulated double-positive (DP) thymocytes. Fetal thymi expressed mRNA for an inducible type of NO synthase (iNOS). The levels of iNOS mRNA became maximal around gestation day 18 with a decline after birth. Administration of anti-CD3 mAb to fetal thymus organ culture (FTOC) or young mice resulted in enhanced expression of mRNAs for iNOS as well as IFN-gamma. Immunohistochemical analyses revealed that iNOS was produced in the corticomedullary junction and medulla. The effects of iNOS-induced NO on anti-CD3-unstimulated or anti-CD3-stimulated thymocytes were examined by culturing them in the presence or absence of a NO-generating compound. Stimulation of DP thymocytes with anti-CD3 alone induced the generation of CD4(low)CD8(low) thymocytes. The subsequent exposure of these anti-CD3-stimulated thymocytes to NO promoted down-regulation of CD4 and CD8 expression. The recovery of viable DP cells was considerably reduced compared with stimulation with anti-CD3 or NO alone. Even in a viable DP population, high incidences of DNA strand breaks were detected in the CD4(low)CD8(low) compartment. In contrast to DP cells, the recovery of viable single-positive cells was not decreased but rather slightly enhanced by treatment with anti-CD3 and/or NO. The recovery of anti-CD3-stimulated thymocytes were also reduced when cultured on the thymic stromal monolayer with the capacity to produce NO upon IFN-gamma stimulation. These results indicate that NO, which is generated in association with TCR stimulation in the thymus, functions to induce deletion of DP thymocytes, especially when their TCR is stimulated.

IL-12 produced by antigen-presenting cells induces IL-2-independent proliferation of T helper cell clones.

We investigated the role of IL-12 in proliferation of various Th cell clones (class II-alloreactive (4-86 and 4-55) and keyhole limpet hemocyanin + self I-Ek-reactive (9-16)) following stimulation with Ag on APCs. These clones proliferated in response to stimulation with rIL-2, rIL-12, or Ag/APC. The proliferation induced by Ag/APC stimulation was not affected by anti-IL-2 Ab but was markedly inhibited by anti-IL-12 Abs. Consistent with this finding was the absence of detectable IL-2 activity in culture supernatants 12 to 48 h after Ag/APC stimulation, and the detection of significant levels of IL-12 in an Ab-capture bioassay. IL-12 was produced within 12 h after Ag/APC stimulation, reaching a peak after 18 to 24 h. The production of IL-12 in cultures of Th clones and APC contrasted with the production of IL-2 but not IL-12 upon allostimulation of primary T cells and the inhibition of their proliferation exclusively by anti-IL-2 Abs. Analysis of the expression of IL-12-binding sites on Th cells revealed low levels of IL-12 receptors in resting Th clones but high IL-12R levels 2 to 3 days after Ag/APC stimulation, declining gradually thereafter. The changes in IL-12R expression levels correlated closely with the IL-12 responsiveness of Th populations at various times after Ag/APC stimulation; Th populations obtained 3 and 10 days after Ag/APC stimulation exhibited very high and weak or marginal responsiveness to rIL-12, respectively, whereas the responses to rIL-2 were comparable in both Th populations. These results indicate that the Ag/APC-stimulated proliferation of terminally differentiated Th clones, in contrast to naive T cells, depends on the production of IL-12 by APC and on the simultaneous up-regulation of IL-12R on Th cells rather than on an IL-2 autocrine mechanism.

Suppression of allograft responses by combining alloantigen-specific i.v. pre-sensitization with suboptimal doses of rapamycin.

C57BL/6 (B6) mice were injected i.v. with class I H-2-disparate B10.QBR spleen cells (10(7)/mouse). This regimen, termed donor alloantigen-specific i.v. pre-sensitization (DSP), induced almost complete reduction of the anti-B10.QBR mixed lymphocyte reaction (MLR) that has been regarded to represent a cytotoxic T lymphocyte (CTL)-independent graft rejection pathway. Because the DSP regimen failed to affect the generation of CTL responses, it did not prolong graft survival. Repeated (four or 11 times) administration in vivo (during 5 or 18 days) of rapamycin at suboptimal doses (0.5-2.0 mg/kg/day) failed to eliminate the capacities to exhibit MLR as well as to generate CTL responses. The suppression of CTL responses was achieved only through the combination of these two treatments. It was also shown that prolongation of skin graft survival was not induced by either of a single DSP or rapamycin treatment alone, but by the combination of these. Potent suppression of CTL responses was also induced when a single DSP was combined with repeated injection of a suboptimal dose (0.75 mg/kg/day) of another immunosuppressive drug, FK506, instead of rapamycin. However, there was a substantial difference in cellular mechanisms underlying the suppression of CTL responses by the above two different combinations. Under conditions in which lymphoid cells from mice receiving the treatments with DSP plus FK506 failed to generate CTL responses, the addition of recombinant IL-2 (rIL-2) to cultures restored the CTL generation, suggesting that CTL precursors themselves are not attenuated by the combined treatment.(ABSTRACT TRUNCATED AT 250 WORDS)