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In rodents, low doses of CD28-specific superagonistic monoclonal antibodies (CD28 superagonists, CD28SA) selectively activate regulatory T cells (Treg). This observation has recently been extended to humans, suggesting an option for the treatment of autoimmune and inflammatory diseases. However, a mechanistic explanation for this phenomenon is still lacking. Given that CD28SA amplify T cell receptor (TCR) signals, we tested the hypothesis that the weak tonic TCR signals received by conventional CD4\(^{+}\) T cells (Tconv) in the absence of cognate antigen require more CD28 signaling input for full activation than the stronger TCR signals received by self-reactive Treg. We report that in vitro, the response of mouse Treg and Tconv to CD28SA strongly depends on MHC class II expression by antigen-presenting cells. To separate the effect of tonic TCR signals from self-peptide recognition, we compared the response of wild-type Treg and Tconv to low and high CD28SA doses upon transfer into wild-type or H-2M knockout mice, which lack a self-peptide repertoire. We found that the superior response of Treg to low CD28SA doses was lost in the absence of self-peptide presentation. We also tested if potentially pathogenic autoreactive Tconv would benefit from self-recognition-induced sensitivity to CD28SA stimulation by transferring TCR transgenic OVA-specific Tconv into OVA-expressing mice and found that low-dose CD28SA application inhibited, rather than supported, their expansion, presumably due to the massive concomitant activation of Treg. Finally, we report that also in the in vitro response of human peripheral blood mononuclear cells to CD28SA, HLA II blockade interferes with the expansion of Treg by low-dose CD28SA stimulation. These results provide a rational basis for the further development of low-dose CD28SA therapy for the improvement of Treg activity.
Breakdown of sphingomyelin as catalyzed by the activity of sphingomyelinases profoundly affects biophysical properties of cellular membranes which is particularly important with regard to compartmentalization of surface receptors and their signaling relay. As it is activated both upon TCR ligation and co-stimulation in a spatiotemporally controlled manner, the neutral sphingomyelinase (NSM) has proven to be important in T cell activation, where it appears to play a particularly important role in cytoskeletal reorganization and cell polarization. Because these are important parameters in directional T cell migration and motility in tissues, we analyzed the role of the NSM in these processes. Pharmacological inhibition of NSM interfered with early lymph node homing of T cells in vivo indicating that the enzyme impacts on endothelial adhesion, transendothelial migration, sensing of chemokine gradients or, at a cellular level, acquisition of a polarized phenotype. NSM inhibition reduced adhesion of T cells to TNF-α/IFN-γ activated, but not resting endothelial cells, most likely via inhibiting high-affinity LFA-1 clustering. NSM activity proved to be highly important in directional T cell motility in response to SDF1-α, indicating that their ability to sense and translate chemokine gradients might be NSM dependent. In fact, pharmacological or genetic NSM ablation interfered with T cell polarization both at an overall morphological level and redistribution of CXCR4 and pERM proteins on endothelial cells or fibronectin, as well as with F-actin polymerization in response to SDF1-α stimulation, indicating that efficient directional perception and signaling relay depend on NSM activity. Altogether, these data support a central role of the NSM in T cell recruitment and migration both under homeostatic and inflamed conditions by regulating polarized redistribution of receptors and their coupling to the cytoskeleton.
Dendritic cells (DCs) and macrophages (Mph) share many characteristics as components of the innate immune system. The criteria to classify the multitude of subsets within the mononuclear phagocyte system are currently phenotype, ontogeny, transcription patterns, epigenetic adaptations, and function. More recently, ontogenetic, transcriptional, and proteomic research approaches uncovered major developmental differences between Flt3L-dependent conventional DCs as compared with Mphs and monocyte-derived DCs (MoDCs), the latter mainly generated in vitro from murine bone marrow-derived DCs (BM-DCs) or human CD14\(^{+}\) peripheral blood monocytes. Conversely, in vitro GM-CSF-dependent monocyte-derived Mphs largely resemble MoDCs whereas tissue-resident Mphs show a common embryonic origin from yolk sac and fetal liver with Langerhans cells (LCs). The novel ontogenetic findings opened discussions on the terminology of DCs versus Mphs. Here, we bring forward arguments to facilitate definitions of BM-DCs, MoDCs, and LCs. We propose a group model of terminology for all DC subsets that attempts to encompass both ontogeny and function.
Acute graft-versus-host disease (aGvHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell plus T cell transplantation (allo-HSCT). In this study, we investigated the requirement for CD28 co-stimulation of donor CD4\(^{+}\) conventional (CD4\(^{+}\)CD25\(^{-}\)Foxp3\(^{-}\), Tconv) and regulatory (CD4\(^{+}\)CD25\(^{+}\)Foxp3\(^{+}\), Treg) T cells in aGvHD using tamoxifen-inducible CD28 knockout (iCD28KO) or wild-type (wt) littermates as donors of CD4\(^{+}\) Tconv and Treg. In the highly inflammatory C57BL/6 into BALB/c allo-HSCT transplantation model, CD28 depletion on donor CD4\(^{+}\) Tconv reduced clinical signs of aGvHD, but did not significantly prolong survival of the recipient mice. Selective depletion of CD28 on donor Treg did not abrogate protection of recipient mice from aGvHD until about day 20 after allo-HSCT. Later, however, the pool of CD28-depleted Treg drastically declined as compared to wt Treg. Consequently, only wt, but not CD28-deficient, Treg were able to continuously suppress aGvHD and induce long-term survival of the recipient mice. To our knowledge, this is the first study that specifically evaluates the impact of CD28 expression on donor Treg in aGvHD. Moreover, the delayed kinetics of aGvHD lethality after transplantation of iCD28KO Treg provides a novel animal model for similar disease courses found in patients after allo-HSCT.
Opportunistic infections with the saprophytic yeast Candida albicans are a major cause of morbidity in immunocompromised patients. While the interaction of cells and molecules of innate immunity with C. albicans has been studied to great depth, comparatively little is known about the modulation of adaptive immunity by C. albicans. In particular, direct interaction of proteins secreted by C. albicans with CD4\(^{+}\) T cells has not been studied in detail. In a first screening approach, we identified the pH-regulated antigen 1 (Pra1) as a molecule capable of directly binding to mouse CD4\(^{+}\) T cells in vitro. Binding of Pra1 to the T cell surface was enhanced by extracellular Zn\(^{2+}\) ions which Pra1 is known to scavenge from the host in order to supply the fungus with Zn\(^{2+}\). In vitro stimulation assays using highly purified mouse CD4\(^{+}\) T cells showed that Pra1 increased proliferation of CD4\(^{+}\) T cells in the presence of plate-bound anti-CD3 monoclonal antibody. In contrast, secretion of effector cytokines such as IFNγ and TNF by CD4\(^{+}\) T cells upon anti-CD3/ anti-CD28 mAb as well as cognate antigen stimulation was reduced in the presence of Pra1. By secreting Pra1 C. albicans, thus, directly modulates and partially controls CD4\(^{+}\) T cell responses as shown in our in vitro assays.
Immature or semi-mature dendritic cells (DCs) represent tolerogenic maturation stages that can convert naive T cells into Foxp3\(^{+}\) induced regulatory T cells (iTreg). Here we found that murine bone marrow-derived DCs (BM-DCs) treated with cholera toxin (CT) matured by up-regulating MHC-II and costimulatory molecules using either high or low doses of CT (CT\(^{hi}\), CT\(^{lo}\)) or with cAMP, a known mediator CT signals. However, all three conditions also induced mRNA of both isoforms of the tolerogenic molecule cytotoxic T lymphocyte antigen 2 (CTLA-2α and CTLA-2β). Only DCs matured under CT\(^{hi}\) conditions secreted IL-1β, IL-6 and IL-23 leading to the instruction of Th17 cell polarization. In contrast, CT\(^{lo}\)- or cAMP-DCs resembled semi-mature DCs and enhanced TGF-β-dependent Foxp3\(^{+}\) iTreg conversion. iTreg conversion could be reduced using siRNA blocking of CTLA-2 and reversely, addition of recombinant CTLA-2α increased iTreg conversion in vitro. Injection of CT\(^{lo}\)- or cAMP-DCs exerted MOG peptide-specific protective effects in experimental autoimmune encephalomyelitis (EAE) by inducing Foxp3\(^{+}\) Tregs and reducing Th17 responses. Together, we identified CTLA-2 production by DCs as a novel tolerogenic mediator of TGF-β-mediated iTreg induction in vitro and in vivo. The CT-induced and cAMP-mediated up-regulation of CTLA-2 also may point to a novel immune evasion mechanism of Vibrio cholerae.
Thymus-derived natural Foxp3\(^{+}\) CD4\(^{+}\) regulatory T cells (nTregs) play a key role in maintaining immune tolerance and preventing autoimmune disease. Several studies indicate that dendritic cells (DCs) are critically involved in the maintenance and proliferation of nTregs. However, the mechanisms how DCs manage to keep the peripheral pool at constant levels remain poorly understood. Here, we describe that the NF-κB/Rel family transcription factor RelB controls the frequencies of steady-state migratory DCs (ssmDCs) in peripheral lymph nodes and their numbers control peripheral nTreg homeostasis. DC-specific RelB depletion was investigated in CD11c-Cre × RelB\(^{fl/fl}\) mice (RelB\(^{DCko}\)), which showed normal frequencies of resident DCs in lymph nodes and spleen while the subsets of CD103\(^{-}\) Langerin\(^{-}\) dermal DCs (dDCs) and Langerhans cells but not CD103\(^{+}\) Langerin\(^{+}\) dDC of the ssmDCs in skin-draining lymph nodes were increased. Enhanced frequencies and proliferation rates were also observed for nTregs and a small population of CD4\(^{+}\) CD44\(^{high}\) CD25\(^{low}\) memory-like T cells (Tml). Interestingly, only the Tml but not DCs showed an increase in IL-2-producing capacity in lymph nodes of RelB\(^{DCko}\) mice. Blocking of IL-2 in vivo reduced the frequency of nTregs but increased the Tml frequencies, followed by a recovery of nTregs. Taken together, by employing RelB\(^{DCko}\) mice with increased frequencies of ssmDCs our data indicate a critical role for specific ssmDC subsets for the peripheral nTreg and IL-2\(^{+}\) Tml frequencies during homeostasis.
Background:
The foamy viral genome encodes four central purine-rich elements localized in the integrase-coding region of pol. Previously, we have shown that the first two of these RNA elements (A and B) are required for protease dimerization and activation. The D element functions as internal polypurine tract during reverse transcription. Peters et al., described the third element (C) as essential for gag expression suggesting that it might serve as an RNA export element for the unspliced genomic transcript.
Results:
Here, we analysed env splicing and demonstrate that the described C element composed of three GAA repeats known to bind SR proteins regulates env splicing, thus balancing the amount of gag/pol mRNAs. Deletion of the C element effectively promotes a splice site switch from a newly identified env splice acceptor to the intrinsically strong downstream localised env 3′ splice acceptor permitting complete splicing of almost all LTR derived transcripts. We provide evidence that repression of this env splice acceptor is a prerequisite for gag expression. This repression is achieved by the C element, resulting in impaired branch point recognition and SF1/mBBP binding. Separating the branch point from the overlapping purine-rich C element, by insertion of only 20 nucleotides, liberated repression and fully restored splicing to the intrinsically strong env 3′ splice site. This indicated that the cis-acting element might repress splicing by blocking the recognition of essential splice site signals.
Conclusions:
The foamy viral purine-rich C element regulates splicing by suppressing the branch point recognition of the strongest env splice acceptor. It is essential for the formation of unspliced gag and singly spliced pol transcripts.
Measles Virus Glycoprotein-Based Lentiviral Targeting Vectors That Avoid Neutralizing Antibodies
(2012)
Lentiviral vectors (LVs) are potent gene transfer vehicles frequently applied in research and recently also in clinical trials. Retargeting LV entry to cell types of interest is a key issue to improve gene transfer safety and efficacy. Recently, we have developed a targeting method for LVs by incorporating engineered measles virus (MV) glycoproteins, the hemagglutinin (H), responsible for receptor recognition, and the fusion protein into their envelope. The H protein displays a single-chain antibody (scFv) specific for the target receptor and is ablated for recognition of the MV receptors CD46 and SLAM by point mutations in its ectodomain. A potential hindrance to systemic administration in humans is pre-existing MV-specific immunity due to vaccination or natural infection. We compared transduction of targeting vectors and non-targeting vectors pseudotyped with MV glycoproteins unmodified in their ectodomains (MV-LV) in presence of \(\alpha\)-MV antibody-positive human plasma. At plasma dilution 1: 160 MV-LV was almost completely neutralized, whereas targeting vectors showed relative transduction efficiencies from 60% to 90%. Furthermore, at plasma dilution 1: 80 an at least 4-times higher multiplicity of infection (MOI) of MV-LV had to be applied to obtain similar transduction efficiencies as with targeting vectors. Also when the vectors were normalized to their p24 values, targeting vectors showed partial protection against \(\alpha\)-MV antibodies in human plasma. Furthermore, the monoclonal neutralizing antibody K71 with a putative epitope close to the receptor binding sites of H, did not neutralize the targeting vectors, but did neutralize MV-LV. The observed escape from neutralization may be due to the point mutations in the H ectodomain that might have destroyed antibody binding sites. Furthermore, scFv mediated cell entry via the target receptor may proceed in presence of a-MV antibodies interfering with entry via the natural MV receptors. These results are promising for in vivo applications of targeting vectors in humans.
The RNA-binding protein RC3H1 (also known as ROQUIN) promotes TNF\(\alpha\) mRNA decay via a 3'UTR constitutive decay element (CDE). Here we applied PAR-CLIP to human RC3H1 to identify ~3,800 mRNA targets with >16,000 binding sites. A large number of sites are distinct from the consensus CDE and revealed a structure-sequence motif with U-rich sequences embedded in hairpins. RC3H1 binds preferentially short-lived and DNA damage-induced mRNAs, indicating a role of this RNA-binding protein in the post-transcriptional regulation of the DNA damage response. Intriguingly, RC3H1 affects expression of the NF-\(\kappa\)B pathway regulators such as I\(\kappa\)B\(\alpha\) and A20. RC3H1 uses ROQ and Zn-finger domains to contact a binding site in the A20 3'UTR, demonstrating a not yet recognized mode of RC3H1 binding. Knockdown of RC3H1 resulted in increased A20 protein expression, thereby interfering with I\(\kappa\)B kinase and NF-\(\kappa\)B activities, demonstrating that RC3H1 can modulate the activity of the IKK/NF-\(\kappa\)B pathway.