Signaling Chain Research Articles

Chimeric Antigen Receptor-Engineered T Cells for Immunotherapy of Cancer

CD4+ and CD8+ T lymphocytes are powerful components of adaptive immunity, which essentially contribute to the elimination of tumors. Due to their cytotoxic capacity, T cells emerged as attractive candidates for specific immunotherapy of cancer. A promising approach is the genetic modification of T cells with chimeric antigen receptors (CARs). First generation CARs consist of a binding moiety specifically recognizing a tumor cell surface antigen and a lymphocyte activating signaling chain. The CAR-mediated recognition induces cytokine production and tumor-directed cytotoxicity of T cells. Second and third generation CARs include signal sequences from various costimulatory molecules resulting in enhanced T-cell persistence and sustained antitumor reaction. Clinical trials revealed that the adoptive transfer of T cells engineered with first generation CARs represents a feasible concept for the induction of clinical responses in some tumor patients. However, further improvement is required, which may be achieved by second or third generation CAR-engrafted T cells.

Inactivation of Pre-B Cell Receptor-Mediated Tumor Suppression by Aberrant Splicing in Ph+ Acute Lymphoblastic Leukemia.

Abstract 579The pre-B cell receptor instructs differentiation of normal pre-B cells and functions as a tumor suppressor in pre-B acute lymphoblastic leukemia (ALL). Its function is impaired in virtually all cases of pre-B ALL carrying the Philadelphia chromosome (Ph), which encodes the BCR-ABL1 kinase. However, genomic deletions in pre-B cell receptor pathway are rare and the mechanism of its inactivation in ALL is not known. Here we report that aberrant splicing of SLP65 (BLNK, BASH) and the pre-B cell receptor-related linker molecules LCP2, SYK and LAT represents a major mechanism of pre-B cell receptor-inactivation, which is consistent with deregulated expression of splice factors in Ph+ ALL. In contrast to normal pre-B cells, BCR-ABL1-transformed pre-B ALL expressed multiple splice variants of the pre-B cell receptor-related linker molecules SLP65, LCP2, SYK and LAT. A detailed sequence analysis of these splice variants revealed that aberrant splicing resulted in functional inactivation of important protein domains in SLP65, LCP2, SYK and LAT transcripts. Of note, the vast majority of SLP65 splice variants lacked a functional SH2 domain, which is required to anchor SLP65 to the Igαa signaling chain of the pre-B cell receptor. We then focused our analysis on the SH2 domain of SLP65 for the following reasons: (1) SLP65 functions as a powerful tumor suppressor downstream of the pre-B cell receptor. (2) Exon 16 which encodes the central part of the SH2 domain was affected in the vast majority of SLP65 splice variants. (3) The SH2 domain of SLP65 is required to link downstream effector molecules (BTK, SYK, PLCγ2) to the Igαa signaling chain of the pre-B cell receptor. We found that deletion of the SH2 domain represents a frequent feature in Ph+ ALL.In reconstitution experiments with pre-B cells from SLP65-/- mice, SLP65 forms lacking the SH2 domain failed to support pre-B cell receptor signaling. By coexpression with full-length SLP65, we demonstrated that SH2-deficient variants have a dominant-negative effect by reducing the Ca2+-release and the leukemia-suppressive function of SLP65. These effects are dose-dependent as demonstrated in a titration experiment, in which SLP65 and SH2-deficient SLP65 were expressed at different ratios. SH2-deficient SLP65 also compromised the leukemia-suppressive function of the pre-B cell receptor in vivo transplantation model: While reconstitution of full-length SLP65 in SLP65-/- BCR-ABL1 ALL cells suppressed leukemia growth and bone marrow infiltration, loss of the SLP65 SH2 domain abolished the tumor suppressor function of the pre-B cell receptor.To address potential mechanisms of aberrant pre-mRNA splicing, we studied expression levels of splicing factors in Ph+ ALL and searched for splice site mutations: Consistent with aberrant splicing of pre-B cell receptor-related linker molecules including SLP65, SYK, LAT and LCP2, we found evidence of splice factor deregulation in Ph+ ALL: Among the 94 pre-mRNA splice factors studied, 24 were differentially expressed between Ph+ ALL (n=15) and normal bone marrow B cell precursors (n=11). Of note, the SRPK1, hnRPC, hnRNP-E2 (PCBP2) and hnRNP-A1 pre-mRNA splice factors that were previously found as BCR-ABL1-regulated, were also identified in our analysis. Sequence analysis of the 3'prime splice site of exon 16 in three Ph+ ALL cell lines and six primary cases of Ph+ ALL revealed one clonal splice site mutation in one primary Ph+ ALL case. In one cell line and one primary Ph+ ALL case, each one additional mutation was found within 2 basepairs distance from the splice site. It is not clear, however, whether and to which extent these mutations affect splice site selection. Interestingly, we found that clones amplified from one primary Ph+ ALL case and one Ph+ ALL cell line harbor mutations encoding premature translation stops close to the end of exon 16. While these mutations do not lead to aberrant splicing of exon 16, they reinforce the notion that Ph+ ALL cells are selected for inactivation of the SLP65 SH2 domain.We conclude that aberrant splicing frequently results in the expression of dominant-negative SH2-deficient SLP65, which is sufficient to compromise the tumor suppressor function of the pre-B cell receptor in Ph+ ALL. Whereas mutations and deletions of the SLP65 gene are rare, we conclude that aberrant splicing of the SLP65 SH2 domain represents a common mechanism in ALL cells to compromise the leukemia-suppressive function of the pre-B cell receptor. Disclosures:Martinelli:Novartis: Research Funding.

Novel Mechanistic Insights into Viral Modulation of Immune Receptor Signaling

To successfully infect, replicate, and persist in the host, viruses have evolved numerous strategies to take control of multiple cellular processes, including those that target transmembrane (TM) signal transduction mediated by immune receptors. Despite tremendous advancement in recent years, the exact molecular mechanisms underlying these critical points in viral pathogenesis remain unknown. In this Opinion, based on a novel model of immune signaling, the Signaling Chain HOmoOLigomerization (SCHOOL) model, I suggest specific mechanisms used by different viruses such as human immunodeficiency virus (HIV), cytomegalovirus (CMV), severe acute respiratory syndrome coronavirus (SARS-CoV), herpesvirus saimiri (HVS), human herpesvirus 6 (HHV-6), etc., to modulate the host immune response mediated by members of the family of multichain immune recognition receptors (MIRRs). I also demonstrate how the SCHOOL model, together with the lessons learned from viral pathogenesis, can be used practically for rational drug design and the development of new therapies for immune disorders.

Pathogenesis of type II diabetes: are there common causes for insulin resistance and secretion failure?

Insulin resistance of major target tissues and insulin secretion abnormalities play a pivotal role in the pathogenesis of type 2-diabetes. Population studies have demonstrated that both insulin resistance and insulin secretion defects are already detected in a prediabetic state and contribute to the development of overt type 2-diabetes. Major factors causing insulin resistance are obesity, physical activity and the genetic background. Since insulin resistance and insulin secretion abnormalities are often confounded, it can be speculated that a common defect exists for both conditions. Several candidate genes have been studied including insulin signaling elements as well as genes which are important for insulin secretion. A link between defects in the insulin signaling chain and insulin secretion processes was suggested by some of these studies.

What is the function of neuroglobin?

For a long time, haemoglobin and myoglobin had been assumed to represent the only globin types of vertebrates. In 2000, however, we discovered a third globin type by mining the genome sequence data. Based on a preferential expression in the nervous system, this globin is referred to as neuroglobin. Despite nine years of research, its function is still uncertain and a number of hypotheses have been put forward. Neuroglobin enhances cell viability under hypoxia and under various types of oxidative stress in transgenic systems, but does not appear to be strongly upregulated in response to stress. A close phylogenetic relationship with invertebrate nerve globins and its positive correlation with the oxidative metabolism and mitochondria suggest a role in O(2) supply. In vitro studies and cell culture experiments imply that neuroglobin may detoxify reactive oxygen or nitric oxide. Still other studies propose neuroglobin as being part of a signalling chain that transmits the redox state of the cell or that inhibits apoptosis. Although some functions are more probable than others, we conclude that it is still too early to definitively decide what may be the physiological role(s) of neuroglobin in vertebrates. Nevertheless, there is no doubt that neuroglobin has an essential, conserved function and is beneficial to neurons.

Precisely timed signal transmission in neocortical networks with reliable intermediate-range projections

The mammalian neocortex has a remarkable ability to precisely reproduce behavioral sequences or to reliably retrieve stored information. In contrast, spiking activity in behaving animals shows a considerable trial-to-trial variability and temporal irregularity. The signal propagation and processing underlying these conflicting observations is based on fundamental neurophysiological processes like synaptic transmission, signal integration within single cells, and spike formation. Each of these steps in the neuronal signaling chain has been studied separately to a great extend, but it has been difficult to judge how they interact and sum up in active sub-networks of neocortical cells. In the present study, we experimentally assessed the precision and reliability of small neocortical networks consisting of trans-columnar, intermediate-range projections (200–1000 μm) on a millisecond time-scale. Employing photo-uncaging of glutamate in acute slices, we activated a number of distant presynaptic cells in a spatio-temporally precisely controlled manner, while monitoring the resulting membrane potential fluctuations of a postsynaptic cell. We found that signal integration in this part of the network is highly reliable and temporally precise. As numerical simulations showed, the residual membrane potential variability can be attributed to amplitude variability in synaptic transmission and may significantly contribute to trial-to-trial output variability of a rate signal. However, it does not impair the temporal accuracy of signal integration. We conclude that signals from intermediate-range projections onto neocortical neurons are propagated and integrated in a highly reliable and precise manner, and may serve as a substrate for temporally precise signal transmission in neocortical networks.

Ly49D Engagement on T Lymphocytes Induces TCR-Independent Activation and CD8 Effector Functions That Control Tumor Growth

Recent data showing expression of activating NK receptors (NKR) by conventional T lymphocytes raise the question of their role in the triggering of TCR-independent responses that could be damaging for the host. Transgenic mice expressing the activating receptor Ly49D/DAP12 offer the opportunity to better understand the relevance of ITAM signaling in the biology of T cells. In vitro experiments showed that Ly49D engagement on T lymphocytes by a cognate MHC class I ligand expressed by Chinese hamster ovary (CHO) cells or by specific Ab triggered cellular activation of both CD4 and CD8 populations with modulation of activation markers and cytokine production. The forced expression of the ITAM signaling chain DAP12 is mandatory for Ly49D-transgenic T cell activation. In addition, Ly49D stimulation induced T lymphocyte proliferation, which was much stronger for CD8 T cells. Phenotypic analysis of anti-Ly49D-stimulated CD8 T cells and their ability to produce high levels of IFN-gamma and to kill target cells indicate that Ly49D ligation generates effector cytotoxic CD8 T cells. Ly49D engagement by itself also triggered cytotoxic activity of activated CD8 T cells. Adoptive transfer experiments confirmed that Ly49D-transgenic CD8 T cells are able to control growth of CHO tumor cells or RMA cells transfected with Hm1-C4, the Ly49D ligand normally expressed by CHO. In conclusion, Ly49D engagement on T cells leads to T cell activation and to a full range of TCR-independent effector functions of CD8 T cells.

Aberrant Splicing of the SLP65 SH2 Domain Enables Pre-B Cell Transformation and Compromises the Leukemia-Suppressive Function of the Pre-B Cell Receptor

The linker molecule SLP65 is critical for pre-B cell receptor-mediated differentiation signals during early B cell development. Germline mutations of the SLP65 gene in mice and humans result in a profound differentiation block at an early pre-B cell stage. Likewise, B cell lineage acute lymphoblast leukemia (ALL) cells are phenotypically arrested at an early pre-B cell stage in the vast majority of cases. In about 2–5% of cases with ALL, mutations and deletions of the SLP65 gene are found (Sprangers et al., 2006; Mullighan et al., 2007). In a comprehensive study of SLP65 function in human ALL cells, we identified numerous aberrant SLP65 splice variants, which all lack one or more exons that encode the C-terminal SLP65 SH2 domain. A splice variant lacking exon 16, which encodes the central part of the SH2 domain was particularly abundant in ALL but not in normal pre-B cells (EMBL/GenBank Accession number AM180337). The SH2 domain may have critical function because it represents the main ligand for the non-ITAM Y204 in the Igα (CD79A) signaling chain of the pre-B cell receptor (Patterson et al., 2006) and thereby connects the pre-B cell receptor to downstream signaling molecules. To examine a potential role of the SLP65 SH2 domain during the process of malignant transformation in ALL, we measured mRNA levels of full-length SLP65 vs SLP65 splice variants, in which the SH2 domain was deleted (SLP65ΔSH2) in normal pre-B cells and primary human ALL cells by quantitative RT-PCR. While normal pre-B cells express full-length SLP65 at high and SLP65ΔSH2 at very low levels, the SLP65/SLP65ΔSH2 ratio was reduced in E2A-PBX1-driven ALL and particularly low in BCR-ABL1-driven ALL. Given that SLP65ΔSH2 is overexpressed in ALL cells at the expense of full-length SLP65, we investigated biochemical properties of SLP65ΔSH2. To this end, we transduced Slp65−/− pre-B cells with either full-length Slp65, SLP65ΔSH2 or a GFP empty vector control. As opposed to full-length SLP65, SLP65ΔSH2 does not interact with Igα and does not confer tyrosine phosphorylation of BTK downstream of the pre-B cell receptor. Also Ca2+-release in response to pre-B cell receptor engagement and induction of differentiation was drastically reduced in Slp65−/− pre-B cells that were reconstituted with SLP65ΔSH2 instead of SLP65. Co-expression experiments with SLP65 showed that SLP65ΔSH2 has a dominant-negative function and impairs pre-B cell receptor signaling. We next transformed Slp65−/− pre-B cells with a retroviral BCR-ABL1 vector and tested whether SLP65 and SLP65ΔSH2 have a different impact on BCR-ABL1-mediated pre-B cell transformation and BCR-ABL1-driven leukemic growth. Reconstitution of SLP65 expression in Slp65−/− BCR-ABL1-transformed pre-B ALL cells resulted in cell death and nine days after transduction more than 90% of the SLP65-transduced cells had undergone apoptosis. Conversely, reconstitution of Slp65−/− BCR-ABL1-transformed pre-B ALL cells with SLP65ΔSH2 had no significant effect on viability of the leukemia cells. In a titration experiment, we reconstituted SLP65−/−BCR-ABL1-driven ALL cells with SLP65 and SLP65ΔSH2 at various ratios and thereby confirmed that SLP65ΔSH2 has a dominant-negative effect and compromises the leukemia-suppressive function of the pre-B cell receptor. We next tested whether aberrant splicing also interferes with the tumor suppressor function of SLP65 in vivo: To this end, we engrafted SLP65−/−BCR-ABL1- driven ALL cells that were transduced with either SLP65-GFP, SLP65ΔSH2 or GFP into sublethally irradiated NOD/SCID mice. After 24 days, mice that were engrafted with ALL cells transduced with SLP65ΔSH2 or GFP became terminally leukemic, whereas mice injected with SLP65-transduced ALL cells showed no signs of disease. At this time, all mice were sacrificed and analyzed. Whereas mice with SLP65ΔSH2- or GFP-transduced ALL cells had enlarged spleens and substantial leukemic infiltration of the bone marrow (79 and 81% leukemia cell content), leukemic infitration was reduced if SLP65-transduced ALL cells were injected (1.8% leukemia infiltration).Whereas mutations and deletions of the SLP65 gene are rare, we conclude that aberrant splicing of the SLP65 SH2 domain represents a common mechanism in ALL cells to compromise the leukemia-suppressive function of the pre-B cell receptor.

Interaction of CheY2 and CheY2‐P with the cognate CheA kinase in the chemosensory‐signalling chain of Sinorhizobium meliloti

An unusual regulatory mechanism involving two response regulators, CheY1 and CheY2, but no CheZ phosphatase, operates in the chemotactic signalling chain of Sinorhizobium meliloti. Active CheY2-P, phosphorylated by the cognate histidine kinase, CheA, is responsible for flagellar motor control. In the absence of any CheZ phosphatase activity, the level of CheY2-P is quickly reset by a phospho-transfer from CheY2-P first back to CheA, and then to CheY1, which acts as a phosphate sink. In studying the mechanism of this phosphate shuttle, we have used GFP fusions to show that CheY2, but not CheY1, associates with CheA at a cell pole. Cross-linking experiments with the purified proteins revealed that both CheY2 and CheY2-P bind to an isolated P2 ligand-binding domain of CheA, but CheY1 does not. The dissociation constants of CheA-CheY2 and CheA-CheY2-P indicated that both ligands bind with similar affinity to CheA. Based on the NMR structures of CheY2 and CheY2-P, their interactions with the purified P2 domain were analysed. The interacting surface of CheY2 comprises its C-terminal beta4-alpha4-beta5-alpha5 structural elements, whereas the interacting surface of CheY2-P is shifted towards the loop connecting beta5 and alpha5. We propose that the distinct CheY2 and CheY2-P surfaces interact with two overlapping sites in the P2 domain that selectively bind either CheY2 or CheY2-P, depending on whether CheA is active or inactive.

Testosterone-inducible Regulator Is a Kinase That Drives Steroid Sensing and Metabolism in Comamonas testosteroni

The mechanism of gene regulation by steroids in bacteria is still a mystery. We use steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) as a reporter system to study steroid signaling in Comamonas testosteroni. In previous investigations we cloned and characterized the 3alpha-HSD/CR-encoding gene, hsdA. In addition, we identified two negative regulator genes (repA and repB) in the vicinity of hsdA, the protein products which repress hsdA expression on the level of transcription and translation, respectively. Recently, a positive regulator of hsdA expression, TeiR (testosterone-inducible regulator), was found by transposon mutagenesis, but the mode of its action remained obscure. In the present work we produced a TeiR-green fluorescent fusion protein and showed that TeiR is a membrane protein with asymmetrical localization at one of the cell poles of C. testosteroni. Knock-out mutants of the teiR gene revealed that TeiR provides swimming and twitching motility of C. testosteroni to the steroid substrate source. TeiR also mediated an induced expression of 3alpha-HSD/CR which was paralleled by an enhanced catabolism of testosterone. We also found that TeiR responds to a variety of different steroids other than testosterone. Biochemical analysis with several deletion mutants of the teiR gene revealed TeiR to consist of three different functional domains, an N-terminal domain important for membrane association, a central steroid binding site, and a C-terminal part mediating TeiR function. Finally, we could demonstrate that TeiR works as a kinase in the steroid signaling chain in C. testosteroni. Overall, we provide evidence that TeiR mediates steroid sensing and metabolism in C. testosteroni via its steroid binding and kinase activity.

Novel mechanistic concept of platelet inhibition

Background: Activation of circulating platelets by exposed vessel wall collagen is a primary step in the pathogenesis of thrombotic diseases such as heart attack and stroke. Drugs that are capable of blocking platelet activation successfully reduce cardiovascular mortality and morbidity. However, despite intensive research efforts in antithrombotic drug discovery and development, uncontrolled hemorrhage still remains the most common side effect associated with antithrombotic drugs that are currently in use. Objective: The selective inhibition of glycoprotein VI (GPVI), the central platelet collagen receptor, and/or its signaling may inhibit thrombosis without affecting hemostatic plug formation. However, the mechanism of GPVI signaling is not known, hindering the further development of this promising antithrombotic strategy. Methods: This review focuses on an innovative mechanistic concept of platelet inhibition. Results/conclusion: A novel model of GPVI signaling, the signaling chain homooligomerization (SCHOOL) model, has revealed new therapeutic targets for GPVI inhibition, resulting in the development of novel antithrombotic pharmacological approaches and the invention of new platelet inhibitors.

Kinetics of G‐protein‐coupled receptor signals in intact cells

G-protein-coupled receptors (GPCRs) are the largest group of cell surface receptors. They are stimulated by a variety of stimuli and signal to different classes of effectors, including several types of ion channels and second messenger-generating enzymes. Recent technical advances, most importantly in the optical recording with energy transfer techniques--fluorescence and bioluminescence resonance energy transfer, FRET and BRET--, have permitted a detailed kinetic analysis of the individual steps of the signalling chain, ranging from ligand binding to the production of second messengers in intact cells. The transfer of information, which is initiated by ligand binding, triggers a signalling cascade that displays various rate-controlling steps at different levels. This review summarizes recent findings illustrating the speed and the complexity of this signalling system.

Role of thylakoid protein kinases in photosynthetic acclimation

Photosynthetic organisms are able to adjust to changes in light quality through state transition, a process which leads to a balancing of the light excitation energy between the antennae systems of photosystem II and photosystem I. A genetic approach has been used in Chlamydomonas with the aim of elucidating the signaling chain involved in state transitions. This has led to the identification of a small family of Ser–Thr protein kinases associated with the thylakoid membrane and conserved in algae and land plants. These kinases appear to be involved both in short and long term adaptations to changes in the light environment.

Arginase activity mediates reversible T cell hyporesponsiveness in human pregnancy.

Complex regulation of T cell functions during pregnancy is required to ensure materno-fetal tolerance. Here we reveal a novel pathway for the temporary suppression of maternal T cell responses in uncomplicated human pregnancies. Our results show that arginase activity is significantly increased in the peripheral blood of pregnant women and remarkably high arginase activities are expressed in term placentae. High enzymatic activity results in high turnover of its substrate l-arginine and concomitant reduction of this amino acid in the microenvironment. Amino acid deprivation is emerging as a regulatory pathway of lymphocyte responses and we assessed the consequences of this enhanced arginase activity on T cell responses. Arginase-mediated l-arginine depletion induces down-regulation of CD3ζ, the main signalling chain of the TCR, and functional T cell hyporesponsiveness. Importantly, this arginase-mediated T cell suppression was reversible, as inhibition of arginase activity or addition of exogenous l-arginine restored CD3ζ chain expression and T cell proliferation. Thus, l-arginine metabolism constitutes a novel physiological mechanism contributing to the temporary suppression of the maternal immune response during human pregnancy.

In the absence of IGF-1 signaling, IFN-γ suppresses human malignant T-cell growth

AbstractSeveral approaches to target insulin-like growth factor-1 (IGF-1) signaling have resulted in the inhibition of the growth of a broad range of tumor cells. Malignant T cells are insensitive to the antiproliferative effects of the interferon-γ (IFN-γ)/signal transducer and activator of transcription 1 (STAT1) pathway because of the IGF-1–dependent internalization of the IFN-γR2 signaling chain. Here we show that human malignant T cells are also resistant to the growth inhibitory effect of both the IGF-1 receptor–specific inhibitor picropodophyllin (PPP) and retrovirus-mediated gene transfer of a dominant negative IGF-1 receptor. However, blockade of IGF-1 receptor perturbs IFN-γR2 internalization and induces its cell surface accumulation in malignant T cells. This allows the reinstatement of the IFN-γ–induced STAT1 activation, a high expression of proapoptotic molecules, and the suppression of malignant T-cell growth both in vitro and in vivo in a severe combined immunodeficiency (SCID) mouse model. These data indicate that the inhibition of IGF-1 signaling combined with IFN-γ administration could be a promising approach to suppress the growth of neoplastic T cells resistant to each treatment on its own.

JAK kinases control IL-5 receptor ubiquitination, degradation, and internalization

IL-5, IL-3, and GM-CSF are related hematopoietic cytokines, which regulate the function of myeloid cells and are mediators of the allergic inflammatory response. These cytokines signal through heteromeric receptors containing a specific alpha chain and a shared signaling chain, betac. Previous studies demonstrated that the ubiquitin (Ub) proteasome degradation pathway was involved in signal termination of the betac-sharing receptors. In this study, the upstream molecular events leading to proteasome degradation of the IL-5 receptor (IL-5R) were examined. By using biochemical and flow cytometric methods, we show that JAK kinase activity is required for betac ubiquitination and proteasome degradation but only partially required for IL-5R internalization. Furthermore, we demonstrate the direct ubiquitination of the betac cytoplasmic domain and identify lysine residues 566 and 603 as sites of betac ubiquitination. Lastly, we show that ubiquitination of the betac cytoplasmic domain begins at the plasma membrane, increases after receptor internalization, and is degraded by the proteasome after IL-5R internalization. We propose an updated working model of IL-5R down-regulation, whereby IL-5 ligation of its receptor activates JAK2/1 kinases, resulting in betac tyrosine phosphorylation, ubiquitination, and IL-5R internalization. Once inside the cell, proteasomes degrade the betac cytoplasmic domain, and the truncated receptor complex is terminally degraded in the lysosomes. These data establish a critical role for JAK kinases and the Ub/proteasome degradation pathway in IL-5R down-regulation.

Interleukin-23 Restores Immunity to Mycobacterium tuberculosis Infection in IL-12p40-Deficient Mice and Is Not Required for the Development of IL-17-Secreting T Cell Responses

Host control of Mycobacterium tuberculosis is dependent on the activation of CD4+ T cells secreting IFN-gamma and their recruitment to the site of infection. The development of more efficient vaccines against tuberculosis requires detailed understanding of the induction and maintenance of T cell immunity. Cytokines important for the development of cell-mediated immunity include IL-12 and IL-23, which share the p40 subunit and the IL-12Rbeta1 signaling chain. To explore the differential effect of IL-12 and IL-23 during M. tuberculosis infection, we used plasmids expressing IL-23 (p2AIL-23) or IL-12 (p2AIL-12) alone in dendritic cells or macrophages from IL-12p40(-/-) mice. In the absence of the IL-12/IL-23 axis, immunization with a DNA vaccine expressing the M. tuberculosis Ag85B induced a limited Ag-specific T cell response and no control of M. tuberculosis infection. Co-delivery of p2AIL-23 or p2AIL-12 with DNA85B induced strong proliferative and IFN-gamma-secreting T cell responses equivalent to those observed in wild-type mice immunized with DNA85B. This response resulted in partial protection against aerosol M. tuberculosis; however, the protective effect was less than in wild-type mice owing to the requirement for IL-12 or IL-23 for the optimal expansion of IFN-gamma-secreting T cells. Interestingly, bacillus Calmette-Guérin immune T cells generated in the absence of IL-12 or IL-23 were deficient in IFN-gamma production, but exhibited a robust IL-17 secretion associated with a degree of protection against pulmonary infection. Therefore, exogenous IL-23 can complement IL-12 deficiency for the initial expansion of Ag-specific T cells and is not essential for the development of potentially protective IL-17-secreting T cells.

The left–right axis is regulated by the interplay of Coco, Xnr1 and derrière in Xenopus embryos

Formation of the left–right axis involves a symmetry-breaking signal originating in the node or its equivalents, which increases TGF-β signaling on the left side of the embryo and ultimately leads to asymmetric patterning of the viscera. DAN domain proteins are extracellular inhibitors of TGF-β ligands, and are involved in regulating the left–right axis in chick, mouse and zebrafish. We find that Coco, a Xenopus DAN family member, and two TGF-β ligands, Xnr1 and derrière, are coexpressed in the posterior paraxial mesoderm at neurula stage. Side-specific protein depletion demonstrated that left–right patterning requires Coco exclusively on the right side, and Xnr1 and derrière exclusively on the left, despite their bilateral expression pattern. In the absence of Coco, the TGF-β signal is bilateral. Interactions among the three proteins show that derrière is required for normal levels of Xnr1 expression, while Coco directly inhibits both ligands. We conclude that derrière, Xnr1, and Coco define a posttranscriptionally regulated signaling center, which is a necessary link in the signaling chain leading to an increased TGF-β signal on the left side of the embryo.

Differential Effects of NMDA and AMPA Glutamate Receptors on Functional Magnetic Resonance Imaging Signals and Evoked Neuronal Activity during Forepaw Stimulation of the Rat

Most of the currently used methods for functional brain imaging do not visualize neuronal activity directly but rather rely on the elicited hemodynamic and/or metabolic responses. Glutamate, the major excitatory neurotransmitter, plays an important role in the neurovascular/neurometabolic coupling, but the specific mechanisms are still poorly understood. To investigate the role of the two major ionotropic glutamate receptors [NMDA receptors (NMDA-Rs) and AMPA receptors (AMPA-Rs)] for the generation of functional magnetic resonance imaging (fMRI) signals, we used fMRI [measurements of blood oxygenation level-dependent (BOLD), perfusion-weighted imaging (PWI), and cerebral blood volume (CBV)] together with recordings of somatosensory evoked potentials (SEPs) during electrical forepaw stimulation in the alpha-chloralose anesthetized rat. Intravenous injection of the NMDA-R antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate] (0.06 mg/kg plus 3.6 microg x kg(-1) x h(-1)) significantly decreased BOLD (-51 +/- 19%; n = 5) and PWI (-57 +/- 26%; n = 5) responses but reduced the SEPs only mildly (approximately -10%). Systemic application of the AMPA-R antagonist GYKI-53655 [1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine] significantly decreased both the hemodynamic response (BOLD, -49 +/- 13 and -65 +/- 15%; PWI, -22 +/- 48 and -68 +/- 4% for 5 and 7 mg/kg, i.v., respectively; CBV, -80 +/- 7% for 7 mg/kg; n = 4) and the SEPs (up to -60%). These data indicate that the interaction of glutamate with its postsynaptic and/or glial receptors is necessary for the generation of blood flow and BOLD responses and illustrate the differential role of NMDA-Rs and AMPA-Rs in the signaling chain leading from increased neuronal activity to the hemodynamic response in the somatosensory cortex.

Immune cell signaling: a novel mechanistic model reveals new therapeutic targets

Multichain immune recognition receptors (MIRRs) represent a family of surface receptors that is expressed on different cells and that transduces extracellular signals, leading to many biological responses. The most intriguing common structural feature of MIRR family members is that the extracellular recognition domains and the intracellular signaling domains are located on separate subunits. It is not clear how extracellular ligand binding triggers MIRRs and initiates intracellular signal-transduction processes. In this article, I suggest that the structural similarity of the MIRRs provides the basis for the similarity in the mechanisms of MIRR-mediated transmembrane signaling. This hypothesis assumes that the therapeutic strategies learned from a novel mechanistic model of MIRR-mediated signal transduction, the signaling chain homo-oligomerization model, are generalized for this entire family and have important implications for the treatment of many disorders that are mediated by immune cells, including HIV.