Michal Smida

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.

Cannabinoid receptor type 1- and 2-mediated increase in cyclic AMP inhibits T cell receptor-triggered signaling.

The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to Gi/o proteins, which are associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Δ9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the ζ-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.

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.

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.

A displaced PAG enhances proximal signaling and SDF-1-induced T cell migration

PAG, the phosphoprotein associated with glycosphingolipid‐enriched microdomains (GEM), negatively regulates Src family kinases by recruiting C‐terminal Src kinase (Csk) to the membrane, where Csk phosphorylates the inhibitory tyrosine of the Src kinases. S‐acylation of a CxxC motif juxtaposed to the transmembrane domain within PAG has been proposed to be responsible for targeting PAG to the lipid rafts. Here, we present the characterization of a mutant PAG molecule lacking the palmitoylation motif. We demonstrate that the mutant protein is expressed at the plasma membrane, but does not localize within the GEM. Despite being displaced, the mutant PAG molecule still binds the Src kinase Fyn and the cytoskeletal adaptor ezrin‐radixin‐moesin‐binding phosphoprotein of 50 kDa, becomes tyrosine‐phosphorylated, and recruits Csk to the membrane. Functional characterization of the mutant shows that, unlike WT PAG, it does not block proximal TCR signaling, and surprisingly enhances stromal cell‐derived factor 1 (CXCL12)‐induced migration. The mutant functions by depleting Csk from the GEM fractions, as apparent by changes in the phosphorylation of the inhibitory tyrosines within the Src kinases. Indeed this mechanism is supported by RNA interference of PAG, which results in enhanced migration and Src kinase activity. Our results therefore support a functional role for the compartmentalization of Src kinases within the membrane.

Integrating signals from the T-cell receptor and the interleukin-2 receptor.

T cells orchestrate the adaptive immune response, making them targets for immunotherapy. Although immunosuppressive therapies prevent disease progression, they also leave patients susceptible to opportunistic infections. To identify novel drug targets, we established a logical model describing T-cell receptor (TCR) signaling. However, to have a model that is able to predict new therapeutic approaches, the current drug targets must be included. Therefore, as a next step we generated the interleukin-2 receptor (IL-2R) signaling network and developed a tool to merge logical models. For IL-2R signaling, we show that STAT activation is independent of both Src- and PI3-kinases, while ERK activation depends upon both kinases and additionally requires novel PKCs. In addition, our merged model correctly predicted TCR-induced STAT activation. The combined network also allows information transfer from one receptor to add detail to another, thereby predicting that LAT mediates JNK activation in IL-2R signaling. In summary, the merged model not only enables us to unravel potential cross-talk, but it also suggests new experimental designs and provides a critical step towards designing strategies to reprogram T cells.

PAG/Cbp suppression reveals a contribution of CTLA-4 to setting the activation threshold in T cells

BackgroundPAG/Cbp represents a ubiquitous mechanism for regulating Src family kinases by recruiting Csk to the plasma membrane, thereby controlling cellular activation. Since Src kinases are known oncogenes, we used RNA interference in primary human T cells to test whether the loss of PAG resulted in lymphocyte transformation.ResultsPAG-depletion enhanced Src kinase activity and augmented proximal T-cell receptor signaling; exactly the phenotype expected for loss of this negative regulator. Surprisingly, rather than becoming hyper-proliferative, PAG-suppressed T cells became unresponsive. This was mediated by a Fyn-dependent hyper-phosphorylation of the inhibitory receptor CTLA-4, which recruited the protein tyrosine phosphatase Shp-1 to lipid rafts. Co-suppression of CTLA-4 abrogates this inhibition and restores proliferation to T cells.ConclusionWe have identified a fail-safe mechanism as well as a novel contribution of CTLA-4 to setting the activation threshold in T cells.