The 4-1BB agonist urelumab promotes antigen independent T-cell responses and NK-cell expansion The unexpected propensity of 4-1BB costimulation to promote bystander T-cell activation prompted us to investigate the effects of urelumab, a 4-1BB agonist in our in vitro stimulation system. PBMCs were stimulated with eAPC in the presence of differ- ent concentrations of this antibody. Urelumab enhanced T-cell proliferation in some donors but overall the percentages and numbers of proliferated CD8 Tcells were not significantly higher in our data set (Fig. 5A and B). Importantly, the presence of this antibody reduced the percentage of proliferated CD8 Tcells that stained positive for the pHLA-A2 tetramer pool and signif- icantly increased the numbers of tetramer-negative proliferated CD8 Tcells, indicating proliferation independent of the eAPC expressed antigens (Fig. 5C to E). Significantly higher numbers of tetramer-negative proliferated CD8 Tcells were detected, demon- strating that urelumab induced bystander proliferation (Fig. 5E). These results indicate that the engagement of 4-1BB by 4-1BBL or by urelumab induces bystander activationofTcells. In line, we observed that 4-1BBL and urelumab induced strong NF-kB activation in absence of TCR/CD3 signals in 4-1BB-expressing Jurkat T-cell reporters (Supporting information Fig. S7). Similar to 4-1BBL expressed on eAPC, urelumab promoted a massive expansion of NK cells and the production of cytotoxic effector molecules including IFN- γ, granzyme B, and granlysin (Fig. 5F and G). Moreover, urelumab also induced bystander prolifer- ation upon CD28-costimulation, as it significantly reduced the percentage of pHLA-A2 tetramer positive CFSE low CD8 Tcells in stimulation cultures with eAPC-CD80 (Supporting information Fig. S8).
Myeloid cells reflect a heterogeneous family ofcells comprising granulocytic and phagocytic cells. They are among the earliest immune cells arising in our body and get released into circulation after differentiation has been terminated. Upon pathogen invasion, myeloid cells rapidly enter the local tissue via specific chemokine receptors, where they are activated for phagocytosis. This activation is accompanied by the secretion of inflammatory cytokines, which play important roles in innate immunity (Manz & Boettcher, 2014). Myeloid cells are also known to locally monitor the environment as tissue-resident patrolling sensors, thereby integrating tissue disturbances into the activationof present immune cells (Klose et al., 2017). However, myeloid cells also have the potential to inhibit tissue-resident immunity via inhibitory NCR ligands and cytokines. Since ILC2s are considered as non-migratory cells, these mechanisms regulate their local tissue-specific activation, development and function (Walker et al., 2013). In turn, however, several cytokines derived from ILC2s are effectively signalling back to myeloid cells, thereby controlling their recruitment, functional profile and, ultimately, their survival. Scientists agree that ILC2 cytokine production contributes to tissue protection through innate mechanisms. As previously mentioned, eosinophil homeostasis, i.e. their proliferation, survival and recruitment, is partly controlled by IL-5, which is constitutively expressed by ILC2s (Figure 3). A study with Rag-deficient mice showed that ILC2s are able to promote eosinophilia independently of adaptive immune signals (Kumagai et al., 2016). IL-33, which directly acts on eosinophils, enhances IL-4 production, thus stimulating ILC2s and mediating the crosstalk between the two populations (Bal et al., 2016). Additionally, basophils also promote the proliferation of lung ILC2s by secretion of IL-4 (Motomura et al., 2014).
Antibodies and reagents and cellular treatments. All antibodies used in this study are listed in Supplementary Table 1 . Experiments with Marimastat (Tocris) and Rapamycin (Calbiochem) were performed in the absence (DMSO) or presence of the drug at concentrations of 10 μM and 100 nM, respectively. For transwell invasion experiments upon stem cell co-culture the conventional (continuous) Rapamycin treatment was replaced by a 2 h pulse-treatment with subsequent wash-out from the medium to prevent interference of the drug with the cells in the bottom well. For details of the procedure, see Supplementary Fig. 3 c–g. For immunoblotting experiments involving growth factor stimulation cells were pre-treated with Rapamycin for 30 min. Human recombinant IGF-I and IGF-II (Peprotech) were used at a concentration of 50 ng ml −1 . For immunoblotting experiments involving short-term stimulation with deﬁned growth factors or conditioned medium cells were starved in serum-free medium for 12–16 h and then treated and stimulated as indicated. For neutralisation experiments stem cells in the bottom well were washed twice with basal medium and re-fed with complete growth medium containing an IGF-I neutralising antibody or an equal amount of control-IgG. MMP14-dependent activationof pro-MMP2 was assessed via treatment with Concanavalin A (Sigma, 40 μg ml −1 ) for 6 h. To monitor apoptotic cleavage of caspase-3 in cell and tissue lysates, control cell extracts were prepared by treating cells with 1 μM Staurosporine for 3 h.
Physiologically speaking, extracellular proteins should not lead to the activationof endogenous Tcells in vivo, and the corresponding sequences of TCRs on CD4+ and CD8+ Tcells should be absent due to negative selection. However, although negative selection eliminates autoreactive Tcells in the thymus, it does not completely eliminate all autoreactive Tcells ( Pauken et al., 2015 ; Chiorean, Mahler & Sitaru, 2014 ; Serre, Fazilleau & Guerder, 2015 ). In addition, counter-regulatory mechanisms lead to reduced activationofT lymphocytes when extracellular proteins are added. Alternatively, CD4+ cells may detect the above-mentioned substances, leading directly to reduced activationof both CD4+ and CD8+ Tcells. In the present study, results differed when GAPDH and/or β-actin were added: because both these proteins are intracellular, they were able to activate both CD4+ and CD8 + T lymphocytes, even at low concentrations.
T cell-mediated immune responses require the detection of a foreign antigen by the TCR. The TCR consists of a highly variable α/β heterodimer that recognizes peptide antigens presented by MHC proteins (Murphy et al., 2008). It associates with the CD3 complex, consisting of CD3γ, CD3δ and CD3ε protein chains, and a homodimer of ζ chains to form the TCR complex (Figure 2.2). Both, the CD3 complex and the ζ dimer harbor one or more immunoreceptor tyrosine-based activation motifs (ITAMs), which are crucial for coupling TCR ligation to the activationof intracellular signaling pathways (Love and Hayes, 2010; Malissen, 2008). Engagement of a TCR by an antigen-MHC complex initiates phosphorylation of ITAMs creating a binding platform for molecules involved in proximal TCR signaling (Sharpe and Abbas, 2006). Recognition of antigen-MHC complexes is supported by the co-receptors CD4 or CD8, which bind to MHC class II or MHC class I molecules, respectively (Gascoigne, 2008; Vyas et al., 2008). CD4 and CD8 co-receptors also contribute to ITAM phosphorylation by associating with the tyrosine kinase LCK (lymphocyte-specific protein tyrosine kinase) (Schulze-Luehrmann and Ghosh, 2006). However, productive T cell activation requires a second signal, since TCR stimulation alone leads to apoptosis or a state of unresponsiveness, called anergy (Schmitz and Krappmann, 2006). Therefore, Tcells express co-stimulatory molecules such as CD28 co-receptors, which specifically recognize the proteins B7.1 (CD80) or B7.2 (CD86) expressed on the surface of APCs (Abbas et al., 2014; Chen and Flies, 2013). Co-receptor-mediated signaling augments weak TCR-induced signals to promote efficient T cell activation and a productive immune response (Schmitz and Krappmann, 2006). Collectively, the additional co-stimulatory signal provides a mechanism to prevent autoreactive and aberrant T cell responses (Chen and Flies, 2013).
Abstract: The lack of tumor-reactive Tcells is one reason why immune checkpoint inhibitor therapies still fail in a significant proportion of melanoma patients. A vaccination that induces melanoma-specific Tcells could potentially enhance the efficacy of immune checkpoint inhibitors. Here, we describe a vaccination strategy in which melanoma antigens are targeted to mouse and human CD169 and thereby induce strong melanoma antigen-specific T cell responses. CD169 is a sialic acid receptor expressed on a subset of mouse splenic macrophages that captures antigen from the blood and transfers it to dendritic cells (DCs). In human and mouse spleen, we detected CD169 + cells at an equivalent location using immunofluorescence microscopy. Immunization with melanoma antigens conjugated to antibodies (Abs) specific for mouse CD169 efficiently induced gp100 and Trp2-specific T cell responses in mice. In HLA-A2.1 transgenic mice targeting of the human MART-1 peptide to CD169 induced strong MART-1-specific HLA-A2.1-restricted T cell responses. Human gp100 peptide conjugated to Abs specific for human CD169 bound to CD169-expressing monocyte-derived DCs (MoDCs) and resulted in activationof gp100-specific Tcells. Together, these data indicate that Ab-mediated antigen targeting to CD169 is a potential strategy for the induction of melanoma-specific T cell responses in mice and in humans.
In the present study, it was found that EBV can induce distinct proinsulin-experienced T cell activation, which can be reduced by a CatG inhibitor and, to a lesser extent, by the cysteine protease inhibitor E64d. This suggests EBV infection might trigger the activationof diabetogenic Tcells and treatment with CatG inhibitors might be beneficial to prevent the activationof diabetogenic Tcells, resulting in reduced incidence of T1D as speculated in 4.1. Previous studies have indicated EBV infection to be associated with autoimmune diseases, but no major conclusions have been drawn between EBV infection and T1D yet. Indeed, there is some evidence that EBV may be capable of triggering T1D by molecular mimicry. BOLF1, an 11 amino acid sequence of the EBV protein, was reported to be homologous to residues in the Asp-57 region of the HLA-DQw8 β chain peptide, which is considered to be strongly linked to T1D susceptibility (Sairenji et al. 1991). It was also found that a pentapeptide sequence in the Asp-57 region of the HLA-DQ β chain is successively repeated six times in the EBV-BERF4-encoded epitope (Horn et al. 1988; Parkkonen et al. 1994). On the contrary, there was epidemiological evidence for the protective role of EBV infection, supported by significant lower amount of antibodies against EBV in the sera of T1D patients when compared with healthy controls or their first-degree family members (Krause et al. 2009). Nevertheless, humans encounter more than one virus infection in their lifetime. Therefore, the initiation of autoimmune diseases should integrate more than just one possible environmental triggering factor.
15 transduce signals in the cytoplasm because of the lack of intrinsic enzymatic activity. Therefore, the TCR relies on CD3 ζ-chain chains. The negatively charged transmembrane residues of the CD3 subunits and the ζ chains assemble with the positively charged transmembrane residues of TCRαβ chains to form the TCR/CD3 complex. The distinct feature of CD3 molecules and ζ chains is the presence of immunoreceptor tyrosine-based activation motifs (ITAM) in their cytoplasmic part. Each ITAM consists of two conserved tyrosine motifs YXXL/I (where X represents any amino acid) interspaced by 6-9 amino acids. Each ζ chain contains 3 ITAMs, whereas CD3 subunits contain one ITAM each. Thus, the TCR/CD3 complex contains 10 ITAMs in total (figure 3). TCR triggering brings the ITAMs in close proximity of Lck, a crucial tyrosine kinase that phosphorylates the ITAMs (Figure 4). Phosphorylated ITAMs provide signalling platforms and recruit another tyrosine kinase the zeta chain-associated protein of 70 kDa (ZAP-70). ZAP-70 contains two tandem SH2 domains that can bind to the phosphorylated tyrosines in the ITAMs. Binding of ZAP-70 to the ITAMs induces conformational changes in ZAP-70 which becomes in turn ready for Lck- mediated phosphorylation. ZAP-70 phosphorylation results in its activation. Activated ZAP-70 phosphorylates two scaffold molecules the Linker for ActivationofTcells (LAT) and the SH2 domain-containing leukocyte phosphoprotein of 76kDa (SLP-76) (Figure 4) (Smith-Garvin JE. et al. 2010).
technical support in the laboratory. I am especially grateful to Nicole Iben for teaching me the cell culture technique for N2A cells. I am grateful to all former and current members of the Energy and Semiconductor Research Laboratory, in particular, Dr. J¨ org Ohland, Janet Neerken, Apl. Prof. Dr. Achim Kittel, Dr. Levent G¨ utay, Dr. Stephan Heise, Dr. Devendra Pareek, PD Dr. Leonid Govor, Dr. Annette Hammer, Dr. Mohamed H. Sayed, Dr. Martin Knipper, Dr. Dorothea Scheunemann, Grit Sch¨ urmann and Elzbieta Chojnowski for the friendly working atmosphere and support. I would also like to thank the members of our research training group and all former and current members of both the Neurosensorics and Visual Neu- roscience groups, especially Prof. Dr. Henrik Mouritsen, Prof. Dr. Martin Greschner, Apl. Prof. Dr. Ulrike Janssen-Bienhold, Dr. Christian Puller, Dr. Malte Ahlers, Dr. Jasmin Segelken, Dr. Helena Greb, Dr. Mark Pot- tek, Nicole Iben, Bettina Kewitz, Josef Meyer, Yousef Arzhangnia, Patrick D¨ omer, Lena Nemitz, Sabrina Duda, Elaheh Lotfi, Shubhash Chandra Ya- dav, Stefan Tetenborg, Dr. Arndt Meyer, Anne Depping for the support and providing excellent friendly working conditions. I am grateful to An- gelika Maderitsch for conducting thorough SEM and TEM experiments on our organic photoreceptors and helping us gain a comprehensive insight into our devices. In particular, I am grateful to Dominik H¨ oweling, Dr. Jos´ e Fabio L´ opez Salas, Dr. Matthias Schulz, Majvor Mack, Oliver Kolloge, Dr. Sebastian Str¨ oh, Maj-Britt H¨ olzel, Elena Barykina, Dr. Christopher Krause, Yvonne Zeidler, Angelika Maderitsch and Alexandra Erdt for many nice barbeque and sushi evenings, the moral support and helping me out at all times. I am deeply grateful for my friendship with Dr. Maximilian Hemgesberg who guided me throughout my dissertation and introduced me to the world of industrial chemistry. I will always appreciate the constant support, encouragement, and motivation. I thank Dr. Niklas Struch for our long-time friendship, always being there for me throughout the past years and providing me with good music and sushi.
Figure 6. ATP causes Ca 2+ elevations within a restricted paracrine radius. (A) Low-magnification brightfield image of an intact seminiferous tubule segment dissected from SMMHC-CreER T2 x Ai95D mice and positioned directly in front of the tip of a 250 mm diameter perfusion pencil. ROIs (black lines) are drawn to encompass the area that is directly exposed to fluid flow (ROI 0) as well as adjacent regions (ROIs 1 and 1), respectively. Suction produced by negative pressure (applied through holes in the elastic foil pad beneath the tubule) limits the area of perfusion. (B) Pseudocolor GCaMP6f fluorescence intensity images of the tubule shown in (A) reveals Ca 2+ transients in TPCs in response to ATP. Representative images (rainbow 256 color map) correspond to time points before, during, and after focal ATP exposure (100 mM; 10 s). The area directly challenged with ATP is denoted by the white dotted lines. For clarity, autofluorescence of the perfusion pencil was removed. Note that Ca 2+ elevations are limited to ROI 0. (C) Representative original recordings of changes in GCaMP6f intensity (DF/F) over time from tubule segments of the three different stage groups (I–III). Traces exemplify Ca 2+ signals (or the lack thereof) in ROIs 0, –1, and 1, respectively. Independent of the epithelial cycle stage investigated, ATP-induced [Ca 2+ ]
DNA methylation is the most well studied of all epigenetic modifications. In mammals, only the cytosine residue on CpG dinucleotides of DNA can be methylated via the activity of 2 types of DNA methyltranferase (DNA MTase or Dnmt) family enzymes which catalyze the process of transferring a methyl group to DNA with S-adenosyl methionine (SAM) acting as methyl donor. There are 2 types of Dnmts important for induction of DNA methylation patterns; Dnmt1 and Dnmt3 which has 2 isoforms; Dnmt3a and Dnmt3b (Bheemanaik et al. 2006;Malygin and Hattman 2012). DNA methylation is begun by Dnmt3a and DNMT3b which induce de novo methylation on unmodified CpG sites and then Dnmt1 is upregulated during the S-phase where it is recruited by proliferating cell nuclear antigen (PCNA) and preferentially binds onto hemi-methylated CpG sites on dividing cells where it serves to maintain methylation patterns similar to those of parent cells. Dnmt1 thus serve as maintenance transferases (Okano et al. 1998;Pradhan and Esteve 2003). In general, DNA methylation represses gene activity with the notion that gene silencing correlates with DNA methylation at promoter regions of such genes while hypomethylation at same region is linked to gene activation and expression (Li et al. 2012; Wilson et al. 2005; Wilson et al. 2009). Next to DNA methylation, histone modification of which acetylation is a major player has been implicated and extensively investigated for its role in gene regulation. Reversible acetylation of the amino group of lysine in histone tails by histone acetylases (HATSs)/histone deacetylases (HDACs) is one of the characterized histone modifications and is linked to transcriptional activation/repression respectively. Hyper-acetylated histones are transcriptionally active while hypoacetylated histones are transcriptionally inactive/repressed (Moreira et al. 2003).
A truncated NFAT1 fused to GFP (∆NFAT-GFP), used here as fluorescent reporter of in vivo T cell activation, represents an important technical advance. ∆NFAT-GFP displays qualities rendering it more suitable for two-photon imaging ofT cell activation, than the various previously suggested activation indicators. Among these, TCR complex related genetic markers, such as a CD3zeta-GFP fusion protein (Huppa et al., 2003; Richie et al., 2002; Yudushkin and Vale, 2010), or Linker for ActivationofT-Cells (LAT)-GFP fusion protein (Tanimura et al., 2003), showed T cell activation by clustering at the immunological synapses. However, due to the poor z- resolution (typically, 3-4 μm spacing) of two-photon microscopy, detection of small clusters within three-dimensional volume is not reliably possible. In contrast, ∆NFAT- GFP subcellular localization was detectable in more than 90% of the Tcells during intravital two-photon imaging. Moran and colleagues used GFP expressed from the immediate early gene locus, NUR77, as activation indicator (Moran et al., 2011). This system can distinguish the strength of TCR stimulation according to GFP expression level. However the GFP protein needs to be transcribed and maturated, a process that takes at least few hours, which is too long for real-time imaging ofT cell activation. In sharp contrast, ∆NFAT-GFP nuclear translocation occurs within only few minutes after stimulation. Finally, calcium indicators can be used to monitor T cell activation in vivo (Wei et al., 2007). Small molecular calcium indicator dyes are rapidly pumped out from Tcells and thus fail to label them over extended periods of time. Recently, a genetically encoded Ca 2+ indicator, Twitch-1, has also been developed as a marker of in vivo T cell activation (Mues et al., 2013). However, ∆NFAT-GFP is a robust downstream indicator, as elevated calcium might not always result in full T cell activation. Considering all this, the ∆NFAT-GFP fusion-protein qualifies as a marker to rapidly and reliably detect T cell activation induced by antigen recognition in vivo.
Moreover, we observed an increase in GLUT1 expression and glucose uptake upon tetramer stimulation, which was also been previously described in primary human Tcells (Rathmell et al. 2003; Frauwirth et al. 2002). In contrast to these previous studies, the upregulation of GLUT1 and glucose uptake in OT-I CD8+ Tcells was not AKT-dependent. These contradictory results were also described in a recent study by Macintyre and coworkers (Macintyre et al. 2011), who showed that inhibition of AKT had no effect on glucose uptake after stimulation of P14 TCR tg Tcells with gp33 peptide. Thus, we confirm the results of Macintyre showing that AKT has no impact on the uptake of glucose. However, we additionally show that AKT is required for the upregulation of lactate dehydrogenase expression. Therefore, we suggest that GLUT1 upregulation and glucose uptake are regulated in an AKT-independent manner, whereas lactate production strongly requires the activationof AKT. It was shown that phosphoinositol- dependent protein kinase 1 (PDK1), an upstream activator of AKT, is responsible for the upregulation of glucose uptake independent of the PI3K/AKT pathway (Macintyre et al. 2011). Since the role of PDK1 was assessed in T cell blasts in the presence of high IL-2 concentrations, there is a strong temporal separation from our system. We clearly observe the upregulation of LDH in parallel to the activationof AKT within 48h upon stimulation. This leads to the hypothesis that AKT activation upon stimulation induces an upregulation of LDH, whereas IL-2 production induces an AKT independent upregulation of glucose uptake via PDK1.
or one minute leads to a summation of the CREB activation due to the fact, that the decrease in CREB activation has a much slower time constant than the activation (Fig. 15 A). Thus, after every caclium spike, CREB activation reaches a higher level, resulting in a temporal integration of the incoming signals (Fig. 21 B, C). The question that arises is, what exactly is the physiological effect of the summation of CREB activation. Of course, increasing the amount of activated CREB molecules after a stimulus very fast, should increase the amount of CREB dependent gene expression over the time period these molecules are active. We demonstrated this phenomenon by comparing the integrals beneath the CREB activation curves with five minute interstimulus interval and with one minute interstimulus interval (Fig. 22). Nevertheless, it remains to be shown, that the higher amount of CREB dependent gene expression after the summation of CREB activation really occurs. We assessed this question later in the text (see Fig. 26). Besides the amplitude, the duration and the frequency of the calcium signal, it is crucial to take the location of the stimulation into account. Unfortunately, we were not able to image dendritic calcium and CREB activation in the same cell, so we first showed, that we can elicit spatially confined calcium signals in dendrites (Fig. 23). When we stimulated neurons with high potassium at the distal dendrite, we could obsereve local increases of the calcium concentration. The calcium increase seemed to travel along the dendrite in the direction of the soma, but faded away approximately at half the distance to the soma. An elevation of nuclear calcium was not measureable (Fig. 23 A). In the next step we applied the same stimulus to a dendrite of a neuron and measured CREB activation and nuclear calcium (Fig. 24). We found that an increase of nuclear calcium is absolutely necessary for CREB ativation. Dendritic local calcium signals do not trigger CREB activation in our experimental setup. This is interesting, because controversial ideas concerning nuclear calcium have been discussed (Deisseroth et al., 1998; Hardingham et al., 2001). Deisseroth and colleagues proposed a model where calcium ions activate calmodulin in the cytoplasm, which travels then to the nucleus, where it eventually activates CaMKinases. This process is independent of nuclear calcium. In contrast, Hardingham and colleagues found that nuclear calcium is solely responsible for CREB activation. Our results are clearly in favour for the nuclear calcium hypothesis.
An additional layer of complexity was added by the finding that different metalloprotease- ligand combinations can exist, depending on the cellular context and stimulus (Fischer et al., 2003). In addition, the present study demonstrates within the context of cellular stress signalling situations that different ADAM proteases are able to induce cleavage of a single distinct EGF-like ligand, while other EGF-related growth factors are present. Future studies will be necessary to identify the regulatory mechanisms that determine the substrate specificity of these metalloproteases. The results presented in this study indicate a role for the MAPK p38 in the ligand-dependent EGFR activation upon cellular stress, while the mechanistic details linking p38 to activationof ADAM proteases still have to be elucidated. The data presented here point out the role of EGF-like ligand processing as a mechanistic concept for tumour cells to evade chemotherapy. However, the broad significance of the mechanisms elucidated in this study and the potential relevance towards the development of targeted cancer therapies will need to be addressed in future studies on the basis of both cell culture models and primary tumours. Of special interest is the expression analysis of tumours based on the characterization of the signalling pathways relevant to resistance to chemotherapy. While antibodies directed against the EGFR itself or its relative HER2 are readily used in combination with chemotherapy (Zwick et al., 2001), targeting a distinct growth factor is likely to provide an enhanced selectivity.
Dasatinib is a multi-targeted kinase inhibitor of BCR-ABL and Src family kinases and has greater potency than imatinib due to the rapid clearance of BCR-ABL expression hematopoietic cells. Because several of intracellular signaling molecules triggered by the ABL kinase are also involved in the activation pathways of immune cells (Cwynarski et al. 2004), we sought to identity whether dasatinib may also have the effects on immunity. However, the effects of dasatinib on immunity have not been extensively evaluated, especially for CD8 + T lymphocytes. It is not clear whether some side effects like myelosuppression which is common during treatment with dasatinib may result from inhibiting the function of normal cells such as CD8 + T lymphocytes. This is of importance because some of the therapeutic effects in the treatment of patients with CML are mediated by the induction of leukemia-specific T cell responses (Appel et al. 2005). Furthermore, effects of dasatinib on immune reconstitution and T cell function may be especially relevant in the setting of allogeneic transplantation, where regulation of immune functions in the post transplant period is especially critical and directly impacts morbidity and mortality (Seggewiss et al. 2005).
All TLRs are integral transmembrane glycoproteins that belong to a superfamily called the Toll/IL-1 receptor (TIR) family. The cytoplasmic TIR-domain shares considerable homology with Interleukin-1 receptors (IL-1Rs) and is required for mediating downstream signaling. The extracellular domain of TLRs contains variable leucine-rich repeats (LRR) motifs responsible for ligand recognition [Akira and Takeda, 2004]. TLRs share common and distinct signaling pathways: following LRR domain ligand recognition, TLRs dimerize and undergo conformational changes which is an essential step in the recruitment of cytoplasmic TIR- domain-containing adaptor molecules to the intracellular TIR domain of the activated TLR. Five adaptor molecules have been described yet: the myeloid differentiation primary response gene 88 (MyD88), TIR-containing adapter inducing IFN-β (TRIF), TIR-associated protein (TIRAP)/MyD88-adaptor-like (MAL), TRIF-related adapter molecule (TRAM) and Sterile-alpha and Armadillo motif-containing protein (SARM) [O'Neill et al., 2003; Oshiumi et al., 2003; Kawai and Akira, 2006]. Together these signaling pathways activate the transcription factors NFκB and AP-1, leading to the production of various pro-inflammatory cytokines and chemokines as well as the upregulation of co-stimulatory molecules in order to facilitate an adaptive immune response [Iwasaki and Medzhitov, 2004]. In addition, the inflammatory response is further amplified by recruiting innate immune cells such as monocytes, neutrophils and natural killer (NK) cells to the site of inflammation. They also lead to production of type I interferons via IRF3/7 upon activationof TLRs 3, 4, 7, 8, 9 and RLRs.
Mast cells (MCs) are tissue resident cellsof hemopoietic origin and are critically involved in allergic diseases. MCs bind IgE by means of their high-affinity receptor for IgE (Fc εRI). The FcεRI belongs to a family of multi-chain immune recognition receptors and is activated by cross-linking in response to multivalent antigens (Ags)/allergens. Activationof the Fc εRI results in immediate release of preformed granular substances (e.g. histamine, heparin, and proteases), generation of arachidonic acid metabolites, and production of pro-inflammatory cytokines. The Fc εRI shows a remarkable, bell-shaped dose-response behavior with weak induction of effector responses at both low and high (so-called supra-optimal) Ag concentrations. This is significantly different from many other receptors, which reach a plateau phase in response to high ligand concentrations. To explain this unusual dose-response behavior of the Fc εRI, scientists in the past have drawn parallels to so-called precipitin curves resulting from titration of Ag against a fixed concentration of antibody (Ab) in solution (a.k.a. Heidelberger curves). Thus, for high, supra-optimal Ag concentrations one could assume that every IgE-bound Fc εRI formed a monovalent complex with “its own Ag”, thus resulting in marginal induction of effector functions due to absence of receptor cross-linking. However, this was never proven to be the case. More recently, careful studies of Fc εRI activation and signaling events in MCs in response to supra-optimal Ag concentrations have suggested a molecular explanation for the descending part of this bell-shaped curve. It is obvious now that extensive Fc εRI/IgE/Ag clusters are formed and inhibitory molecules and signalosomes are engaged in response to supra-optimal cross-linking (amongst them the Src family kinase Lyn and the inositol-5 0 -phosphatase SHIP1) and they actively down-regulate MC effector
Based on this finding it was questioned whether ST2L expression was established during starvation to enable IL-33-dependent reactivation of the mTOR pathway. Activationof mTORC1 was indirectly assessed through phosphorylation of the S6K subunit p70, activity of mTORC2 through determination of the mTORC2-specific phosphorylation site Ser473 of Akt (Figure 23). IL-33 and/ or IL-12 stimulation neither re-activated mTORC1, nor enhanced the activation status of mTORC2. It is tempting to speculate that starvation induces IL-33 responsiveness to avoid antigen-unspecific inflammation under conditions limiting fitness and survival of immune cells. It seems likely that re-activationof mTORC1 would lead to an increased metabolic activity and accelerated cell death due to the absence of nutrients. In contrast, mTORC1 re-activation would be required to perform efficient effector functions upon antigen-dependent stimulation related to e.g. viral infection, representing an acute “life-threatening” situation. These findings are supported by an observation in a murine model, in which IL-33 ameliorated inflammation during experimental colitis by facilitating T H 2/ T reg responses. Interestingly, this effect was related
Mast cells (MCs) are tissue resident cellsof hemopoietic origin and are critically involved in allergic diseases. MCs bind IgE by means of their high-affinity receptor for IgE (FcεRI). The FcεRI belongs to a family of multi-chain immune recognition receptors and is activated by cross-linking in response to multivalent antigens (Ags)/allergens. Activationof the FcεRI results in immediate release of preformed granular substances (e.g. histamine, heparin, and proteases), generation of arachidonic acid metabolites, and production of pro-inflammatory cytokines. The FcεRI shows a remarkable, bell-shaped dose-response behavior with weak induction of effector responses at both low and high (so-called supra-optimal) Ag concentrations. This is significantly different from many other receptors, which reach a plateau phase in response to high ligand concentrations. To explain this unusual dose-response behavior of the FcεRI, scientists in the past have drawn parallels to so-called precipitin curves resulting from titration of Ag against a fixed concentration of antibody (Ab) in solution (a.k.a. Heidelberger curves). Thus, for high, supra-optimal Ag concentrations one could assume that every IgE-bound FcεRI formed a monovalent complex with “its own Ag”, thus resulting in marginal induction of effector functions due to absence of receptor cross-linking. However, this was never proven to be the case. More recently, careful studies of FcεRI activation and signaling events in MCs in response to supra-optimal Ag concentrations have suggested a molecular explanation for the descending part of this bell-shaped curve. It is obvious now that extensive FcεRI/IgE/Ag clusters are formed and inhibitory molecules and signalosomes are engaged in response to supra-optimal cross-linking (amongst them the Src family kinase Lyn and the inositol-5 0 -phosphatase SHIP1) and they actively down-regulate MC effector