Jonathan A. Lindquist

A methodology for the structural and functional analysis of signaling and regulatory networks

BackgroundStructural analysis of cellular interaction networks contributes to a deeper understanding of network-wide interdependencies, causal relationships, and basic functional capabilities. While the structural analysis of metabolic networks is a well-established field, similar methodologies have been scarcely developed and applied to signaling and regulatory networks.ResultsWe propose formalisms and methods, relying on adapted and partially newly introduced approaches, which facilitate a structural analysis of signaling and regulatory networks with focus on functional aspects. We use two different formalisms to represent and analyze interaction networks: interaction graphs and (logical) interaction hypergraphs. We show that, in interaction graphs, the determination of feedback cycles and of all the signaling paths between any pair of species is equivalent to the computation of elementary modes known from metabolic networks. Knowledge on the set of signaling paths and feedback loops facilitates the computation of intervention strategies and the classification of compounds into activators, inhibitors, ambivalent factors, and non-affecting factors with respect to a certain species. In some cases, qualitative effects induced by perturbations can be unambiguously predicted from the network scheme. Interaction graphs however, are not able to capture AND relationships which do frequently occur in interaction networks. The consequent logical concatenation of all the arcs pointing into a species leads to Boolean networks. For a Boolean representation of cellular interaction networks we propose a formalism based on logical (or signed) interaction hypergraphs, which facilitates in particular a logical steady state analysis (LSSA). LSSA enables studies on the logical processing of signals and the identification of optimal intervention points (targets) in cellular networks. LSSA also reveals network regions whose parametrization and initial states are crucial for the dynamic behavior.We have implemented these methods in our software tool CellNetAnalyzer (successor of FluxAnalyzer) and illustrate their applicability using a logical model of T-Cell receptor signaling providing non-intuitive results regarding feedback loops, essential elements, and (logical) signal processing upon different stimuli.ConclusionThe methods and formalisms we propose herein are another step towards the comprehensive functional analysis of cellular interaction networks. Their potential, shown on a realistic T-cell signaling model, makes them a promising tool.

A logical model provides insights into T cell receptor signaling

Cellular decisions are determined by complex molecular interaction networks. Large-scale signaling networks are currently being reconstructed, but the kinetic parameters and quantitative data that would allow for dynamic modeling are still scarce. Therefore, computational studies based upon the structure of these networks are of great interest. Here, a methodology relying on a logical formalism is applied to the functional analysis of the complex signaling network governing the activation of T cells via the T cell receptor, the CD4/CD8 co-receptors, and the accessory signaling receptor CD28. Our large-scale Boolean model, which comprises 94 nodes and 123 interactions and is based upon well-established qualitative knowledge from primary T cells, reveals important structural features (e.g., feedback loops and network-wide dependencies) and recapitulates the global behavior of this network for an array of published data on T cell activation in wild-type and knock-out conditions. More importantly, the model predicted unexpected signaling events after antibody-mediated perturbation of CD28 and after genetic knockout of the kinase Fyn that were subsequently experimentally validated. Finally, we show that the logical model reveals key elements and potential failure modes in network functioning and provides candidates for missing links. In summary, our large-scale logical model for T cell activation proved to be a promising in silico tool, and it inspires immunologists to ask new questions. We think that it holds valuable potential in foreseeing the effects of drugs and network modifications.

Mechanisms of Opioid-Mediated Inhibition of Human T Cell Receptor Signaling

Opioids are widely used for the treatment of severe pain. However, it is also known that opioids, in particular morphine, cause immunosuppression. Therefore, their use may complicate treatment of persons with an already impaired immune system, e.g., patients suffering from cancer or AIDS. We investigated the mechanisms of opioid-induced immunosuppression in primary human T lymphocytes and the human T cell line Jurkat. We demonstrated that morphine and the endogenous opioid β-endorphin inhibited the transcription of IL-2 in activated human T lymphocytes as well as the activation of the transcription factors AP-1, NFAT, and NF-κB, which transactivate IL-2. In addition, the TCR-induced calcium flux and MAPK activation were inhibited by the opioids, as well as proximal signaling events, such as the phosphorylation of the linker for activation of T cells and Zap70. A more detailed characterization of the mechanism revealed that incubation of T cells with the opioids caused a marked increase in cAMP. This in turn activated protein kinase A, which augmented the kinase activity of C-terminal Src kinase bound to phosphoprotein associated with glycosphingolipid-enrich microdomains, resulting in a further enhancement of the tonic inhibition of the leukocyte-specific protein tyrosine kinase Lck, thereby blocking the initiation of TCR signaling. These effects were mediated by μ opioid receptors. Our findings contribute to the understanding of immunosuppressive side effects of morphine. Since β-endorphin is expressed and secreted by immune effector cells, including T cells, and up-regulated in these cells by various stimuli, our data also suggest an inhibitory role for β-endorphin in the physiological regulation of T cell activation.

Decreased iNOS synthesis mediates dexamethasone‐induced protection of neurons from inflammatory injury in vitro

Brain inflammation is accompanied by transection of axons and death of neurons in the acute lesions of multiple sclerosis. We explored mechanisms of inflammatory damage to neurons in vitro using cocultures of rat embryonal cortical neurons with microglia activated by interferon‐gamma (IFNγ) and lipopolysaccharide (LPS). Previously, we have demonstrated that microglia are highly toxic to neurons and that nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) is necessary and sufficient to mediate this toxicity. Here, we show that addition of dexamethasone (1 µM) to activated cocultures provides effective neuroprotection. We demonstrate that dexamethasone down‐regulates NO production of primary microglia by ≈ 50% and reduces steady‐state iNOS protein and mRNA expression by ≈ 70%. These changes were reversed by the glucocorticoid receptor blocker RU‐486. Furthermore, we analysed the stability of iNOS protein and show that whilst inhibitors of the proteasome blocked iNOS degradation they did not reverse the dexamethasone effect. Our results indicate that the main mechanism of corticosteroid activity on iNOS is reduction in protein synthesis, not destabilization as previously suggested.

Fyn Regulates the Duration of TCR Engagement Needed for Commitment to Effector Function

In naive T cells, engagement of the TCR with agonist peptide:MHC molecules leads to phosphorylation of key intracellular signaling intermediates within seconds and this peaks within minutes. However, the cell does not commit to proliferation and IL-2 cytokine production unless receptor contact is sustained for several hours. The biochemical basis for this transition to full activation may underlie how T cells receive survival signals while maintaining tolerance, and is currently not well understood. We show here that for CD8 T cells commitment to proliferation and cytokine production requires sustained activation of the Src family kinase Lck and is opposed by the action of Fyn. Thus, in the absence of Fyn, commitment to activation occurs more rapidly, the cells produce more IL-2, and undergo more rounds of division. Our data demonstrate a role for Fyn in modulating the response to Ag in primary T cells.

Transmembrane adapters: attractants for cytoplasmic effectors

Transmembrane adapter proteins (TRAPs) are a relatively new and growing family of proteins that include linker for activation of T cells (LAT), phosphoprotein associated with glycosphingolipid‐enriched micro domains (PAG)/C‐terminal Src kinase (Csk) binding protein (Cbp), SHP2‐interacting transmembrane adapter protein (SIT), T cell receptor interacting molecule (TRIM), and the recently identified non‐T cell activation linker (NTAL) and pp30. TRAPs share several common structural features, but more importantly they possess multiple sites of tyrosine phosphorylation, by which they act as scaffolds for recruiting cytosolic adapter and/or effector proteins. The membrane association of TRAPs places them near to the immunoreceptors, a position from which they coordinate and modulate the signals they receive to produce an appropriate cellular response.

A human TAPBP (TAPASIN)-related gene, TAPBP-R

TAPASIN, a V‐C1 (variable‐constant) immunoglobulin superfamily (IgSF) molecule that links MHC class I molecules to the transporter associated with antigen processing (TAP) in the endoplasmic reticulum (ER) is encoded by the TAPBP gene, located near to the MHC at 6p21.3. A related gene was identified at chromosome position 12p13.3 between the CD27 and VAMP1 genes near a group of MHC‐paralogous loci. The gene, which we have called TAPBP‐R (R for related), also encodes a member of the IgSF, TAPASIN‐R. Its putative product contains similar structural motifs to TAPASIN, with some marked differences, especially in the V domain, transmembrane and cytoplasmic regions. By using the mouse ortholog to screen tissue, we revealed that the TAPBP‐R gene was broadly expressed. Sub‐cellular localization showed that the bulk of TAPASIN‐R is located within the ER but biotinylation experiments were consistent with some expression at thecell surface. TAPASIN‐R lacks an obvious ER retention signal. The function of TAPASIN‐R will be of interest in regards to the evolution of the immune system as well as antigen processing.

T Cell Activation Results in Conformational Changes in the Src Family Kinase Lck to Induce Its Activation

T cell activation involves the conformational activation of the tyrosine kinase Lck. Conformational Kinase Activation Lck is a tyrosine kinase that is critical for T cell activation, and its activity is induced by the T cell receptor (TCR). Phosphorylation of Lck at various residues either promotes or inhibits its activity, and Lck exists in various phosphorylated states in a T cell. With fluorescence lifetime imaging microscopic analysis of live human T cells and biochemical analyses, Stirnweiss et al. found that TCR activation produced a conformational change in Lck. In vitro studies showed that this “open” conformation of Lck exhibited enhanced kinase activity. Thus, phosphorylation, location, and conformation all potentially contribute to the regulation of Lck activity. The lymphocyte-specific Src family protein tyrosine kinase p56Lck (Lck) is essential for T cell development and activation and, hence, for adaptive immune responses. The mechanism by which Lck activity is directed toward specific substrates in response to T cell receptor (TCR) activation remains elusive. We used fluorescence lifetime imaging microscopy to assess the activation-dependent spatiotemporal changes in the conformation of Lck in live human T cells. Kinetic analysis of the fluorescence lifetime of Lck biosensors enabled the direct visualization of the dynamic local opening of 20% of the total amount of Lck proteins after activation of T cells with antibody against CD3 or by superantigen-loaded antigen-presenting cells. Parallel biochemical analysis of TCR complexes revealed that the conformational changes in Lck correlated with the induction of Lck enzymatic activity. These data show the dynamic, local activation through conformational change of Lck at sites of TCR engagement.

The balance of pro-inflammatory and trophic factors in multiple sclerosis patients: effects of acute relapse and immunomodulatory treatment

Background: In multiple sclerosis inflammation is primarily injurious to the central nervous system, but its therapeutic suppression might inhibit repair-promoting factors. Objectives: We aimed at better describing the complexity of biological effects during an acute relapse and analysed the effects of intervention with high-dose i.v. glucocorticoids and immunomodulatory treatment with interferon-beta (IFNβ). Methods: We studied the intracellular expression levels of the pro-inflammatory mediators tumour necrosis factor alpha (TNFα) and inducible nitric oxide synthase (iNOS) together with the neurotrophins ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in freshly isolated peripheral blood mononuclear cells of multiple sclerosis patients during an acute relapse, after intervention with i.v. methylprednisolone and at baseline, using a highly quantitative flow-cytometric approach. Results: We demonstrated the expression of CNTF in human leucocytes. We showed that CNTF levels differed in acutely relapsing multiple sclerosis patients compared with controls and increased after corticosteroid treatment. CNTF can counteract the toxicity of TNFα towards oligodendrocytes and we found TNFα increased during acute relapses. Following corticosteroids, neither TNFα nor iNOS expression was reduced. Levels of BDNF were not affected by glucocorticoids, but increased during IFNβ therapy. However, IFNβ also increased the expression of iNOS and major histocompatibility complex class I (MHC-I), underlining its immunomodulatory potential. Conclusions: Multiple sclerosis patients might benefit from reparative, and not solely from anti-inflammatory, effects of glucocorticoids. Interactive effects of glucocorticoid- and IFNβ-treatment need to be considered to improve neuroprotection and remyelination resulting from immunomodulatory treatment.

Control of lymphocyte development and activation by negative regulatory transmembrane adapter proteins

Summary: Signals emanating from antigen receptors critically regulate immune cell activation, survival, and differentiation. Transmembrane adapter proteins (TRAPs), a group of molecules that organize signaling complexes at the plasma membrane, play a pivotal role in propagating and fine‐tuning antigen receptor‐mediated signaling. During the last years, it has been demonstrated that most of the TRAPs possess inhibitory functions, including linker for activation of T cells (LAT), the best characterized adapter that links the T‐cell receptor (TCR) to Ca2+ flux and mitogen‐activated protein kinase activation. Indeed, it appears that LAT may assemble inhibitory complexes that trigger negative feedback loops, thus terminating T‐cell activation. Additionally, recent data demonstrate that SIT [Src homology 2 domain‐containing phosphatase 2 (SHP2)‐interacting TRAP] fine‐tunes TCR‐mediated signaling events and negatively regulates T‐cell development and homeostasis. The experimental evidence suggests that TRAPs play a crucial role also in establishing tolerance. In fact, loss of SIT, LAX, or NTAL (non‐T cell activation linker)/linker for activation of B cells (LAB) resulted in the spontaneous development of autoimmune diseases. Moreover, we recently showed that in addition to the inhibition of Src‐family kinases, PAG (phosphoprotein associated with glycosphingolipid‐enriched domains) is also involved in the negative regulation of Ras activation. Collectively, these data demonstrate that TRAPs are important modulators of immune cell activation and function. Finally, it appears that TRAPs possess redundant yet not completely overlapping functions.