Rolf König

Direct Inhibition of T-Lymphocyte Activation by Anthrax Toxins In Vivo

ABSTRACT The causative agent of anthrax, Bacillus anthracis, produces two toxins that contribute in part to its virulence. Lethal toxin is a metalloprotease that cleaves upstream mitogen-activated protein kinase kinases. Edema toxin is a calmodulin-dependent adenylate cyclase. Previous studies demonstrated that the anthrax toxins are important immunomodulators that promote immune evasion of the bacterium by suppressing activation of macrophages and dendritic cells. Here we showed that injection of sublethal doses of either lethal or edema toxin into mice directly inhibited the subsequent activation of T lymphocytes by T-cell receptor-mediated stimulation. Lymphocytes were isolated from toxin-injected mice after 1 or 4 days and stimulated with antibodies against CD3 and CD28. Treatment with either toxin inhibited the proliferation of T cells. Injection of lethal toxin also potently inhibited cytokine secretion by stimulated T cells. The effects of edema toxin on cytokine secretion were more complex and were dependent on the length of time between the injection of edema toxin and the isolation of lymphocytes. Treatment with lethal toxin blocked multiple kinase signaling pathways important for T-cell receptor-mediated activation of T cells. Phosphorylation of the extracellular signal-regulated kinase and the stress-activated kinase p38 was significantly decreased. In addition, phosphorylation of the serine/threonine kinase AKT and of glycogen synthase kinase 3 was inhibited in T cells from lethal toxin-injected mice. Thus, anthrax toxins directly act on T lymphocytes in a mouse model. These findings are important for future anthrax vaccine development and treatment.

Interactions between MHC molecules and co-receptors of the TCR

Genetic experiments indicate similarity between binding sites on MHC class I (MHCI) for CD8 and on MHCII for CD4, but the crystal structures of CD8/MHCI and CD4/MHCII complexes suggest critical differences between the interfaces in the two complexes. Biophysical analyses using ectodomains of co-receptors and MHC molecules demonstrate extremely fast kinetics and low-affinity interactions. Experiments with soluble multimeric MHC ligands suggest that CD4 and CD8 may differ in the mechanisms by which they promote the formation of ternary TCR/MHC/co-receptor complexes. Co-receptor-influenced duration of TCR signaling controls thymocyte selection. In naïve T cells, CD4/MHCII interactions may promote T-cell survival. Temporal and spatial analysis of TCR and CD4 co-clustering in the immunological synapse suggests that CD4 recruitment is regulated by the half-life of the initial TCR/MHCII complex. Diverse experimental systems have yielded conflicting data that have helped to formulate revised mechanistic models of co-receptor function.

Effects of heavy metal ions on resting and antigen-activated CD4(+) T cells.

Heavy metal environmental pollutants increase susceptibility of affected individuals to bacterial and viral infections, but the mechanisms responsible for this effect are not known. We established cellular in vitro systems to identify molecular targets for the action of heavy metal ions. We used two model systems to determine the effects of heavy metal ions on antigen-induced T lymphocyte responses. The first system was representative of primary antigen responses and utilized CD4(+) primary T lymphocytes derived from DO.11.10 T cell receptor transgenic mice. The second system represented a memory T cell phenotype and utilized the CD4(+) T helper 1 clone, pGL2. We measured the effects of the four heavy metals cadmium, lead, mercury, and vanadium on cytokine and proliferation responses by purified CD4(+) T cell to antigenic stimulation. Cytokine responses were differentially affected by lead and vanadium at concentrations that did not affect T cell proliferation in response to antigen. We also determined whether the metal ions induced apoptotic cell death. Mercury induced apoptosis at concentrations as low as 0.5 microM, whereas cadmium required a concentration of 100 microM. Lead (maximal concentration tested was 200 microM) and vanadium (100 microM) did not induce apoptosis. The results suggested that the different heavy metal ions differentially affected antigen-stimulated responses in T helper cells. These in vitro systems can now be applied to test whether heavy metal ions alter antigen-induced T cell signal transduction pathways in CD4(+) T helper cells.

LIPID PEROXIDATION-DERIVED ALDEHYDE-PROTEIN ADDUCTS CONTRIBUTE TO TRICHLOROETHENE-MEDIATED AUTOIMMUNITY VIA ACTIVATION OF CD4+ T CELLS

Lipid peroxidation is implicated in the pathogenesis of various autoimmune diseases. Lipid peroxidation-derived aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are highly reactive and bind to proteins, but their role in eliciting an autoimmune response and their contribution to disease pathogenesis remain unclear. To investigate the role of lipid peroxidation in the induction and/or exacerbation of autoimmune response, 6-week-old autoimmune-prone female MRL+/+ mice were treated for 4 weeks with trichloroethene (TCE; 10 mmol/kg, ip, once a week), an environmental contaminant known to induce lipid peroxidation. Sera from TCE-treated mice showed significant levels of antibodies against MDA-and HNE-adducted proteins along with antinuclear antibodies. This suggested that TCE exposure not only caused increased lipid peroxidation, but also accelerated autoimmune responses. Furthermore, stimulation of cultured splenic lymphocytes from both control and TCE-treated mice with MDA-adducted mouse serum albumin (MDA-MSA) or HNE-MSA for 72 h showed significant proliferation of CD4+ T cells in TCE-treated mice as analyzed by flow cytometry. Also, splenic lymphocytes from TCE-treated mice released more IL-2 and IFN-gamma into cultures when stimulated with MDA-MSA or HNE-MSA, suggesting a Th1 cell activation. Thus, our data suggest a role for lipid peroxidation-derived aldehydes in TCE-mediated autoimmune responses and involvement of Th1 cell activation.

Chronic exposure to trichloroethene causes early onset of SLE-like disease in female MRL +/+ mice

Trichloroethene (TCE) exacerbates the development of autoimmune responses in autoimmune-prone MRL +/+ mice. Although TCE-mediated autoimmune responses are associated with an increase in serum immunoglobulins and autoantibodies, the underlying mechanism of autoimmunity is not known. To determine the progression of TCE-mediated immunotoxicity, female MRL +/+ mice were chronically exposed to TCE through the drinking water (0.5 mg/ml of TCE) for various periods of time. Serum concentrations of antinuclear antibodies increased after 36 and 48 weeks of TCE exposure. Histopathological analyses showed lymphocyte infiltration in the livers of MRL +/+ mice exposed to TCE for 36 or 48 weeks. Lymphocyte infiltration was also apparent in the pancreas, lungs, and kidneys of mice exposed to TCE for 48 weeks. Immunoglobulin deposits in kidney glomeruli were found after 48 weeks of exposure to TCE. Our results suggest that chronic exposure to TCE promotes inflammation in the liver, pancreas, lungs, and kidneys, which may lead to SLE-like disease in MRL +/+ mice.

Systems biology approaches to understanding Epithelial Mesenchymal Transition (EMT) in mucosal remodeling and signaling in asthma

A pathological hallmark of asthma is chronic injury and repair, producing dysfunction of the epithelial barrier function. In this setting, increased oxidative stress, growth factor- and cytokine stimulation, together with extracellular matrix contact produces transcriptional reprogramming of the epithelial cell. This process results in epithelial-mesenchymal transition (EMT), a cellular state associated with loss of epithelial polarity, expression of mesenchymal markers, enhanced mobility and extracellular matrix remodeling. As a result, the cellular biology of the EMT state produces characteristic changes seen in severe, refractory asthma: myofibroblast expansion, epithelial trans-differentiation and subepithelial fibrosis. EMT also induces profound changes in epithelial responsiveness that affects innate immune signaling that may have impact on the adaptive immune response and effectiveness of glucocorticoid therapy in severe asthma. We discuss how this complex phenotype is beginning to be understood using systems biology-level approaches through perturbations coupled with high throughput profiling and computational modeling. Understanding the distinct changes induced by EMT at the systems level may provide translational strategies to reverse the altered signaling and physiology of refractory asthma.

CD44 expression positively correlates with Foxp3 expression and suppressive function of CD4+ Treg cells

BackgroundCD4+CD25+ regulatory T (Treg) cells develop in the thymus and can suppress T cell proliferation, modulated by Foxp3 and cytokines; however, the relevance of CD44 in Treg cell development is less clear. To address this issue, we analyzed Foxp3 expression in CD44+ Treg cells by using multiple parameters, measured the levels of the immunoregulatory cytokine interleukin (IL)-10 in various thymocyte subsets, and determined the suppressor activity in different splenic Treg cell populations.ResultsWithin mouse thymocytes, we detected Treg cells with two novel phenotypes, namely the CD4+CD8-CD25+CD44+ and CD4+CD8-CD25+CD44- staining features. Additional multi-parameter analyses at the single-cell and molecular levels suggested to us that CD44 expression positively correlated with Foxp3 expression in thymocytes, the production of IL-10, and Treg activity in splenic CD4+CD25+ T cells. This suppressive effect of Treg cells on T cell proliferation could be blocked by using anti-IL-10 neutralizing antibodies. In addition, CD4+CD25+CD44+ Treg cells expressed higher levels of IL-10 and were more potent in suppressing effector T cell proliferation than were CD4+CD25+CD44- cells.ConclusionThis study indicates the presence of two novel phenotypes of Treg cells in the thymus, the functional relevance of CD44 in defining Treg cell subsets, and the role of both IL-10 and Foxp3 in modulating the function of Treg cells.ReviewersThis article was reviewed by Dr. M. Lenardo, Dr. L. Klein & G. Wirnsberger (nominated by Dr. JC Zungia-Pfluker), and Dr. E.M. Shevach.

CD4+ Th Cells Resembling Regulatory T Cells That Inhibit Chronic Colitis Differentiate in the Absence of Interactions Between CD4 and Class II MHC

Regulatory CD4+ Th cells can prevent many autoimmune diseases; however, the factors selecting for these cells remain poorly defined. In transgenic mice with a mutation in the CD4 binding region on class II MHC, the disruption of CD4-class II interactions selected for CD4+ Th cells that expressed surface markers and cytokines associated with regulatory Th cells. Th cells from these mice were enriched for CD45RBlow as well as CD25+, while they expressed high levels of the transcription factor associated with regulatory T cells, Foxp3, and cytokines, including IL-4, IL-10, and IFN-γ mRNA and protein. These regulatory Th cells inhibited the function of APCs via IL-10 production, and adoptive transfer of these cells prevented weight loss and inflammation in a model of colitis. CD4+ regulatory Th cells emerged only when interactions between CD4 and class II MHC were deficient on cells of nonhemopoietic origin. These data support a novel model controlling the differentiation of regulatory Th cells and suggest that interactions between CD4 and class II MHC may a useful target for re-educating T cells as a treatment for inflammatory diseases.

The role of CD4 in regulating homeostasis of T helper cells

Intrathymic T cell selection and peripheral activation of mature T cells are crucial for self-recognition and the general immune response to viral, bacterial, and tumor antigens. The T cell coreceptors, CD4 and CD8, contribute to the regulation of these processes. The importance of interactions between CD4 and moleculesencoded by the class II major histocompatibility complex (MHC) for thymic T cell selection has been clearly established, however, the role of CD4-MHC class II interactions in T helper (Th) cell differentiation, in the maintenance of homeostasis in the peripheral immune system, and in the generation of memoryTh cells is largely unclear. Here, we present evidence for a role of CD4 in controlling homeostasis in the peripheral immune system. We also demonstrate the importance of CD4-MHC class II interactions in inducing these previously not recognized functions of CD4.

T cell receptor-independent CD4 signalling: CD4-MHC class II interactions regulate intracellular calcium and cyclic AMP.

CD4 is a coreceptor on T helper (Th) cells that interacts with MHC class II molecules (MHCII). The mechanisms mediating the effects of CD4 on responses by T helper cells to stimulation of the antigen-specific T cell receptor (TCR) are still poorly understood. Here, we demonstrate T cell costimulation via CD4 signalling independent of T cell receptor-mediated signals. Incubation of T helper cells with peptide mimetics of the CD4-binding region on the MHC class II beta2 domain caused intracellular calcium mobilization in the absence of antigen or other T cell receptor stimuli. Engagement of CD4 by peptide mimetics or wild-type MHC class II, but not by mutant MHC class II molecules incapable of engaging CD4, inhibited the T cell receptor-mediated increase in cyclic AMP (cAMP) concentrations in T helper cells. CD4-mediated signals activated cyclic AMP phosphodiesterases (PDEs) and inhibited adenylyl cyclase. Full activation and clonal expansion of antigen-stimulated T helper cells required the CD4-mediated regulation of cyclic AMP. Our results suggest a costimulatory mechanism of CD4 function that acts on the second messengers, calcium and cyclic AMP.