CD28 costimulation is essential for human T regulatory expansion and function

The costimulatory requirements required for peripheral blood T regulatory cells (Tregs) are unclear. Using cell-based artificial APCs we found that CD28 but not ICOS, OX40, 4-1BB, CD27, or CD40 ligand costimulation maintained high levels of Foxp3 expression and in vitro suppressive function. Only CD28 costimulation in the presence of rapamycin consistently generated Tregs that consistently suppressed xenogeneic graft-vs-host disease in immunodeficient mice. Restimulation of Tregs after 8-12 days of culture with CD28 costimulation in the presence of rapamycin resulted in >1000-fold expansion of Tregs in <3 wk. Next, we determined whether other costimulatory pathways could augment the replicative potential of CD28-costimulated Tregs. We observed that while OX40 costimulation augmented the proliferative capacity of CD28-costimulated Tregs, Foxp3 expression and suppressive function were diminished. These studies indicate that the costimulatory requirements for expanding Tregs differ from those for T effector cells and, furthermore, they extend findings from mouse Tregs to demonstrate that human postthymic Tregs require CD28 costimulation to expand and maintain potent suppressive function in vivo.     

An indispensable role for the chemokine receptor CCR10 in IgA antibody-secreting cell accumulation

The differential expression of chemokines and chemokine receptors, by tissues and leukocytes, respectively, contributes to the specific accumulation of leukocyte subsets to different tissues. CCR10/CCL28 interactions are thought to contribute to the accumulation of IgA Ab-secreting cells (ASC) to mucosal surfaces, such as the gastrointestinal tract and the lactating mammary gland. Although the role of CCL28 in lymphocyte homing is well established, direct in vivo evidence for CCR10 involvement in this process has not been previously shown. In this study, we describe the generation of a CCR10-deficient mouse model. Using this model, we demonstrate that CCR10 is critical for efficient localization and accumulation of IgA ASC to the lactating mammary gland. Surprisingly, IgA ASC accumulation to the gastrointestinal tract is minimally impacted in CCR10-deficient mice. These results provide the first direct evidence of CCR10 involvement in lymphocyte homing and accumulation in vivo, and demonstrate that reliance on CCR10-mediated recruitment of IgA ASC varies dramatically within mucosal tissues.     

Ex vivo rapamycin generates apoptosis-resistant donor Th2 cells that persist in vivo and prevent hemopoietic stem cell graft rejection

Because ex vivo rapamycin generates murine Th2 cells that prevent Graft-versus-host disease more potently than control Th2 cells, we hypothesized that rapamycin would generate Th2/Tc2 cells (Th2/Tc2.R cells) that abrogate fully MHC-disparate hemopoietic stem cell rejection more effectively than control Th2/Tc2 cells. In a B6-into-BALB/c graft rejection model, donor Th2/Tc2.R cells were indeed enriched in their capacity to prevent rejection; importantly, highly purified CD4+ Th2.R cells were also highly efficacious for preventing rejection. Rapamycin-generated Th2/Tc2 cells were less likely to die after adoptive transfer, accumulated in vivo at advanced proliferative cycles, and were present in 10-fold higher numbers than control Th2/Tc2 cells. Th2.R cells had a multifaceted, apoptosis-resistant phenotype, including: 1) reduced apoptosis after staurosporine addition, serum starvation, or CD3/CD28 costimulation; 2) reduced activation of caspases 3 and 9; and 3) increased anti-apoptotic Bcl-xL expression and reduced proapoptotic Bim and Bid expression. Using host-versus-graft reactivity as an immune correlate of graft rejection, we found that the in vivo efficacy of Th2/Tc2.R cells 1) did not require Th2/Tc2.R cell expression of IL-4, IL-10, perforin, or Fas ligand; 2) could not be reversed by IL-2, IL-7, or IL-15 posttransplant therapy; and 3) was intact after therapy with Th2.R cells relatively devoid of Foxp3 expression. We conclude that ex vivo rapamycin generates Th2 cells that are resistant to apoptosis, persist in vivo, and effectively prevent rejection by a mechanism that may be distinct from previously described graft-facilitating T cells.     

Structural analysis of the PP2C phosphatase tPphA from Thermosynechococcus elongatus: a flexible flap subdomain controls access to the catalytic site

The homologue of the phosphoprotein PII phosphatase PphA from Thermosynechococcus elongatus, termed tPphA, was identified and its structure was resolved in two different space groups, C222₁ and P4₁2₁2, at a resolution of 1.28 and 3.05 Å, respectively. tPphA belongs to a large and widely distributed subfamily of Mg²⁺/Mn²⁺-dependent phosphatases of the PPM superfamily characterized by the lack of catalytic and regulatory domains. The core structure of tPphA shows a high degree of similarity to the two PPM structures identified so far. In contrast to human PP2C, but similar to Mycobacterium tuberculosis phosphatase PstP, the catalytic centre exhibits a third metal ion in addition to the dinuclear metal centre universally conserved in all PPM members. The fact that the third metal is only liganded by amino acids, which are universally conserved in all PPM members, implies that the third metal could be general for all members of this family. As a specific feature of tPphA, a flexible subdomain, previously recognized as a flap domain, could be revealed. Comparison of different structural isomers of tPphA as well as site-specific mutagenesis implied that the flap domain is involved in substrate binding and catalytic activity. The structural arrangement of the flap domain was accompanied by a large side-chain movement of an Arg residue (Arg169) at the basis of the flap. Mutation of this residue strongly impaired protein stability as well as catalytic activity, emphasizing the importance of this amino acid for the regional polysterism of the flap subdomain and confirming the assumption that flap domain flexibility is involved in catalysis.     

Short- and long-term effects of (+)-methamphetamine and (+/-)-3,4-methylenedioxymethamphetamine on monoamine and corticosterone levels in the neonatal rat following multiple days of treatment

Rats treated with (±)-3,4-methylenedioxymethamphetamine (MDMA) or (+)-methamphetamine (MA) neonatally exhibit long-lasting learning impairments (i.e., after treatment on postnatal days (P)11–15 or P11–20). Although both drugs are substituted amphetamines, they each produce a unique profile of cognitive deficits (i.e., spatial vs. path integration learning and severity of deficits) which may be the result of differential early neurochemical changes. We previously showed that MA and MDMA increase corticosterone (CORT) and MDMA reduces levels of serotonin (5-HT) 24 h after treatment on P11, however, learning deficits are seen after 5 or 10 days of drug treatment, not just 1 day. Accordingly, in the present experiment, rats were treated with MA or MDMA starting on P11 for 5 or 10 days (P11–15 or P11–20) and tissues collected on P16, P21, or P30. Five-day MA administration dramatically increased CORT on P16, whereas MDMA did not. Both drugs decreased hippocampal 5-HT on P16 and P21, although MDMA produced larger reductions. Ten-day treatment with either drug increased dopamine utilization in the neostriatum on P21, whereas 5-day treatment had no effect. No CORT or brain 5-HT or dopamine changes were found with either drug on P30. Although the monoamine changes are transient, they may alter developing neural circuits sufficiently to permanently disrupt later learning and memory abilities.     

Sweet taste receptor interacting protein CIB1 is a general inhibitor of InsP3-dependent Ca2+ release in vivo

In a search for sweet taste receptor interacting proteins, we have identified the calcium- and integrin-binding protein 1 (CIB1) as specific binding partner of the intracellular carboxyterminal domain of the rat sweet taste receptor subunit Tas1r2. In heterologous human embryonic kidney 293 (HEK293) cells, the G protein chimeras Gα_16gust44 and Gα_15i3 link the sweet taste receptor dimer TAS1R2/TAS1R3 to an inositol 1,4,5-trisphosphate (InsP₃)-dependent Ca²⁺ release pathway. To demonstrate the influence of CIB1 on the cytosolic Ca²⁺ concentration, we used sweet and umami compounds as well as other InsP₃-generating ligands in FURA-2-based Ca²⁺ assays in wild-type HEK293 cells and HEK293 cells expressing functional human sweet and umami taste receptor dimers. Stable and transient depletion of CIB1 by short-hairpin RNA increased the Ca²⁺ response of HEK293 cells to the InsP₃-generating ligands ATP, UTP and carbachol. Over-expression of CIB1 had the opposite effect as shown for the sweet ligand saccharin, the umami receptor ligand monosodium glutamate and UTP. The CIB1 effect was dependent on the thapsigargin-sensitive Ca²⁺ store of the endoplasmic reticulum (ER) and independent of extracellular Ca²⁺. The function of CIB1 on InsP₃-evoked Ca²⁺ release from the ER is most likely mediated by its interaction with the InsP₃ receptor. Thus, CIB1 seems to be an inhibitor of InsP₃-dependent Ca²⁺ release in vivo .     

High temporal resolution of amino acid levels in rat nucleus accumbens during operant ethanol self-administration: involvement of elevated glycine in anticipation

Capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF) provides 15-s temporal resolution of amino acid levels in microdialysate, which, for the first time, allows almost real time measurement of changes during episodes of behavior. We trained Sprague-Dawley rats to self-administer either 10% ethanol-containing gelatin or non-alcoholic gelatin in a typical operant chamber. After rats reached stable daily levels of responding, microdialysis probes were inserted into nucleus accumbens and samples were collected before, during and after operant sessions with on-line analysis via CE-LIF. During the first 15 min of the operant session, there was a significant increase in taurine that correlated with the amount of ethanol consumed ( R ² = 0.81) but no change in rats responding for plain gel. There were large, consistent increases in glycine in both the ethanol and plain gel groups which correlated with the amount of gel consumed. A smaller increase was observed in rats with free non-operant access to plain gel compared to the increase seen with the same amount of gel consumed under operant conditions. When rats were given a time out after each delivery of gel in the operant protocol, the greatest increase of glycine was obtained with the longest time out period. Thus, increases in glycine in nucleus accumbens appear to be related to anticipation of reinforcement.     

Terminal deoxynucleotidyltransferase is required for the establishment of private virus-specific CD8+ TCR repertoires and facilitates optimal CTL responses

Virus-immune CD8(+) TCR repertoires specific for particular peptide-MHC class I complexes may be substantially shared between (public), or unique to, individuals (private). Because public TCRs can show reduced TdT-mediated N-region additions, we analyzed how TdT shapes the heavily public (to D(b)NP(366)) and essentially private (to D(b)PA(224)) CTL repertoires generated following influenza A virus infection of C57BL/6 (B6, H2(b)) mice. The D(b)NP(366)-specific CTL response was virtually clonal in TdT(-/-) B6 animals, with one of the three public clonotypes prominent in the wild-type (wt) response consistently dominating the TdT(-/-) set. Furthermore, this massive narrowing of TCR selection for D(b)NP(366) reduced the magnitude of D(b)NP(366)-specific CTL response in the virus-infected lung. Conversely, the D(b)PA(224)-specific responses remained comparable in both magnitude and TCR diversity within individual TdT(-/-) and wt mice. However, the extent of TCR diversity across the total population was significantly reduced, with the consequence that the normally private wt D(b)PA(224)-specific repertoire was now substantially public across the TdT(-/-) mouse population. The key finding is thus that the role of TdT in ensuring enhanced diversity and the selection of private TCR repertoires promotes optimal CD8(+) T cell immunity, both within individuals and across the species as a whole.     

Neutropenia with impaired immune response to Streptococcus pneumoniae in ceramide kinase-deficient mice

In mammals, ceramide kinase (CerK)-mediated phosphorylation of ceramide is the only known pathway to ceramide-1-phosphate (C1P), a recently identified signaling sphingolipid metabolite. To help delineate the roles of CerK and C1P, we knocked out the gene of CerK in BALB/c mice by homologous recombination. All in vitro as well as cell-based assays indicated that CerK activity is completely abolished in Cerk-/- mice. Labeling with radioactive orthophosphate showed a profound reduction in the levels of de novo C1P formed in Cerk-/- macrophages. Consistently, mass spectrometry analysis revealed a major contribution of CerK to the formation of C16-C1P. However, the significant residual C1P levels in Cerk-/- animals indicate that alternative routes to C1P exist. Furthermore, serum levels of proapoptotic ceramide in these animals were significantly increased while levels of dihydroceramide as the biosynthetic precursor were reduced. Previous literature pointed to a role of CerK or C1P in innate immune cell function. Using a variety of mechanistic and disease models, as well as primary cells, we found that macrophage- and mast cell-dependent readouts are barely affected in the absence of CerK. However, the number of neutrophils was strikingly reduced in blood and spleen of Cerk-/- animals. When tested in a model of fulminant pneumonia, Cerk-/- animals developed a more severe disease, lending support to a defect in neutrophil homeostasis following CerK ablation. These results identify ceramide kinase as a key regulator of C1P, dihydroceramide and ceramide levels, with important implications for neutrophil homeostasis and innate immunity regulation.