TCR signalling network organization at the immunological synapses of murine regulatory T cells.

Regulatory T (Treg) cells require T-cell receptor (TCR) signalling to exert their immunosuppressive activity, but the precise organization of the TCR signalling network compared to conventional T (Tconv) cells remains elusive. By using accurate mass spectrometry and multi-epitope ligand cartography (MELC) we characterized TCR signalling and recruitment of TCR signalling components to the immunological synapse (IS) in Treg cells and Tconv cells. With the exception of Themis which we detected in lower amounts in Treg cells, other major TCR signalling components were found equally abundant, however, their phosphorylation-status notably discriminates Treg cells from Tconv cells. Overall, this study identified 121 Treg cell-specific phosphorylations. Short-term triggering of T cell subsets via CD3 and CD28 widely harmonized these variations with the exception of eleven TCR signalling components that mainly regulate cytoskeleton dynamics and molecular transport. Accordingly, conjugation with B cells indeed caused variant cellular morphology and revealed a Treg cell-specific recruitment of TCR signalling components such as PKCθ, PLCγ1 and ZAP70 as well as B cell-derived CD86 into the IS. Together, results from this study support the existence of a Treg cell-specific IS and suggest Treg cell-specific cytoskeleton dynamics as a novel determinant for the unique functional properties of Treg cells.

Insights into the key roles of proteoglycans in breast cancer biology and translational medicine.

Proteoglycans control numerous normal and pathological processes, among which are morphogenesis, tissue repair, inflammation, vascularization and cancer metastasis. During tumor development and growth, proteoglycan expression is markedly modified in the tumor microenvironment. Altered expression of proteoglycans on tumor and stromal cell membranes affects cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Despite the high complexity and heterogeneity of breast cancer, the rapid evolution in our knowledge that proteoglycans are among the key players in the breast tumor microenvironment suggests their potential as pharmacological targets in this type of cancer. It has been recently suggested that pharmacological treatment may target proteoglycan metabolism, their utilization as targets for immunotherapy or their direct use as therapeutic agents. The diversity inherent in the proteoglycans that will be presented herein provides the potential for multiple layers of regulation of breast tumor behavior. This review summarizes recent developments concerning the biology of selected proteoglycans in breast cancer, and presents potential targeted therapeutic approaches based on their novel key roles in breast cancer.

IgG1 anti-epidermal growth factor receptor antibodies induce CD8-dependent antitumor activity.

Anti-EGFR monoclonal antibodies (mAb) like Cetuximab are commonly used for treatment of EGFR+ solid tumors mainly by exerting their therapeutic effect through inhibition of signal transduction. Additionally, IgG1 is a potent mediator of antibody-dependent cytotoxicity (ADCC). In case of the IgG1, Cetuximab induction of ADCC in vivo is controversially discussed. In our study, we investigated the efficiency of Cetuximab-mediated ADCC in a humanized mouse tumor model in vivo and analyzed the contribution of immunologic processes toward antitumor activity. Therefore, we used immunodeficient NOD/Scid mice transgenic for human MHC class I molecule HLA-A2 and adoptively transferred human HLA-A2+ PBMC after engraftment of human epidermoid cell carcinoma A431. Here, we show that high doses of anti-EGFR mAb induced strong tumor regression independent of the immune system. However, tumor regression by low doses of anti-EGFR mAb treatment was ADCC dependent and mediated by tumor infiltrating CD8+ T effector cells. This novel mechanism of ADCC conducted by CD8+ T effector cells was restricted to IgG1 anti-EGFR mAb, dependent of binding to CD16 on T cells and could be inhibited after EGFR blockade on tumor cells. Furthermore, CD8+ T effector cell-mediated ADCC was enhanced in the presence of IL-15 and strongly improved after glycosylation of anti-EGFR mAb indicating the potential of glycoengineered therapeutic mAb as efficient biologicals in cancer therapy.

Modulation of dendritic cell properties by laquinimod as a mechanism for modulating multiple sclerosis.

Laquinimod is an orally administered compound that is under investigation in relapsing-remitting multiple sclerosis. To understand the mechanism by which laquinimod exerts its clinical effects, we have performed human and murine studies assessing its immunomodulatory properties. In experimental autoimmune encephalomyelitis, the therapeutic administration of laquinimod beginning during the recovery of SJL mice, prevented further relapses as expected and strongly reduced infiltration of CD4+ and CD8+ T cells in the central nervous system. We hypothesized that this beneficial effect was mediated by dendritic cells, since we and others found a modulation of different dendritic cell subsets under treatment. According to the findings on antigen-presenting cells in the murine system, we found a reduced capacity of human monocyte-derived dendritic cells treated with therapeutic concentrations of laquinimod, upon maturation with lipopolysaccharide, to induce CD4+ T cell proliferation and secretion of pro-inflammatory cytokines. Furthermore, laquinimod treatment of mature dendritic cells resulted in a decreased chemokine production by both murine and human dendritic cells, associated with a decreased monocyte chemo-attraction. In laquinimod-treated patients with multiple sclerosis we consistently found reduced chemokine and cytokine secretion by conventional CD1c+ dendritic cells upon lipopolysaccharide stimulation. Similarly to the animal model of relapsing-remitting multiple sclerosis, dendritic cell subsets were altered in patients upon laquinimod treatment, as the number of conventional CD1c+ and plasmacytoid CD303+ dendritic cells were decreased within peripheral blood mononuclear cells. Moreover, laquinimod treatment in patients with multiple sclerosis and mice modified the maturation of dendritic cells demonstrated by an upregulation of CD86 expression in vivo. Our data suggest that inhibition of the NF-κB pathway is responsible for the changes observed in dendritic cell maturation and functions. These findings indicate that laquinimod exhibits its disease-modulating activity in multiple sclerosis by downregulating immunogenicity of dendritic cell responses. We suggest that monitoring dendritic cell properties in multiple sclerosis should be implemented in future therapeutic trials.

The role of ICOS in directing T cell responses: ICOS-dependent induction of T cell anergy by tolerogenic dendritic cells.

Tolerogenic dendritic cells (DC) play an important role in maintaining peripheral T cell tolerance in steady-state conditions through induction of anergic, IL-10-producing T cells with suppressive properties. ICOS, an activation-induced member of the CD28 family on T cells, is involved in the induction of IL-10, which itself could contribute to induction of anergy and development of suppressive T cells. Therefore, we analyzed the functional role of ICOS in the differentiation process of human CD4(+) T cells upon their interaction with tolerogenic DC. We compared the functional properties of CD4(+) T cells from healthy volunteers and ICOS-deficient patients after stimulation with tolerogenic DC. We report that induction of T cell anergy and suppressive capacity is completely blocked after knockdown of ICOS expression in T cells as well as after blocking of ICOS-ICOS ligand interaction in DC/T cell cocultures. Moreover, CD4(+) T cells from ICOS-deficient patients were completely resistant to anergy induction and differentiation into suppressive T cells even after supplementation of IL-10. Furthermore, ICOS/ICOS ligand interaction stabilizes IL-10R expression on T cells and thus renders them sensitive to IL-10 effects. Taken together, these results indicate a crucial role for ICOS in the induction of peripheral tolerance maintained by tolerogenic DC mediated mostly via an IL-10-independent mechanism.

Calcipotriol/betamethasone dipropionate aerosol foam for the treatment of psoriasis vulgaris: a review of real-world evidence (RWE).

BACKGROUND: Randomized controlled trials (RCTs) demonstrated the advantages of an aerosol foam formulation of calcipotriol/betamethasone dipropionate (Cal/BD) in patients with psoriasis. In this review, we investigated whether such benefits could also be obtained in real-world clinical practice. METHODS: PubMed was searched for studies reporting real-world data in patients with psoriasis treated with Cal/BD aerosol foam. RESULTS: Three large real-world studies with Cal/BD aerosol foam were identified: a prospective non-interventional study from Germany, a medical chart review from the US, and a retrospective non-interventional study from Spain. Some key findings included the following: high levels of adherence to treatment (82-93%); after 4 weeks, about 50% of patients achieved complete/almost complete responses; at final assessment, 85-95% of patients were extremely satisfied/very satisfied/satisfied with Cal/BD aerosol foam; all healthcare providers were satisfied or somewhat satisfied with the efficacy of Cal/BD foam and they reported similar findings for symptom improvements (itch, pain, erythema/redness, flaking, and plaque thickness). Global ratings by investigators/healthcare providers for symptom control, overall efficacy and the emotional status of patients were also very high (75-100%). CONCLUSION: The results from real-world studies undertaken in diverse healthcare systems reinforce the positive findings reported in RCTs with Cal/BD aerosol foam in patients with psoriasis.

Cladribine exerts an immunomodulatory effect on human and murine dendritic cells.

Cladribine is a purine nucleoside analog developed to treat lymphoid malignancies. Reported therapeutic benefits for the autoimmune disease multiple sclerosis indicate additional immunomodulatory effects beyond the well-characterized cytotoxic activity causing lymphopenia. Here, we demonstrate that cladribine reduces the secretion of inflammatory cytokines and chemokines by murine and human dendritic cells, the most potent antigen-presenting cells. This compound also modulates the expression of the activation markers CD86 and MHC II. Furthermore, cladribine affects the T cell priming capacity of dendritic cells, resulting in reduced induction of interferon-γ- and tumor necrosis factor-α-producing T cells and increased induction of interleukin-10-producing T cells. These effects, observed at cladribine concentrations in the therapeutically relevant range of serum steady-state concentrations for leukemia and multiple sclerosis, confirm the immunomodulatory activity of cladribine.

Biglycan-triggered TLR-2- and TLR-4-signaling exacerbates the pathophysiology of ischemic acute kidney injury.

Exacerbated inflammation in renal ischemia-reperfusion injury, the major cause of intrinsic acute renal failure, is a key trigger of kidney damage. During disease endogenous danger signals stimulate innate immune cells via Toll-like receptors (TLR)-2 and -4 and accelerate inflammatory responses. Here we show that production of soluble biglycan, a small leucine-rich proteoglycan, is induced during reperfusion and that it functions as endogenous agonist of TLR-2/4. Biglycan-mediated activation of TLR-2/4 initiates an inflammatory response in native kidneys, which is marked by the release of cytokines and chemokines and recruitment of inflammatory cells. Overexpression of soluble circulating biglycan before ischemic reperfusion enhanced plasma and renal levels of TNF-α, CXCL1, CCL2 and CCL5, caused influx of neutrophils, macrophages and T cells and overall worsened renal function in wild type mice. We provide robust genetic evidence for TLR-2/4 requirement insofar as biglycan biological effects were markedly dampened in mice deficient in both innate immune receptors, Tlr2(-/-);Tlr4(-/-) mice. Thus, signaling of soluble biglycan via TLR-2/4 could represent a novel therapeutic target for the prevention and possible treatment of patients with acute renal ischemia-reperfusion injury.

Costimulatory molecules on immunogenic versus tolerogenic human dendritic cells.

Dendritic cells (DC) are sentinels of immunity, essential for homeostasis of T cell-dependent immune responses. Both functions of DC, initiation of antigen-specific T cell immunity and maintenance of tissue-specific tolerance originate from distinct stages of differentiation, immunogenic versus tolerogenic. Dependent on local micro milieu and inflammatory stimuli, tissue resident immature DC with functional plasticity differentiate into tolerogenic or immunogenic DC with stable phenotypes. They efficiently link innate and adaptive immunity and are ideally positioned to modify T cell-mediated immune responses. Since the T cell stimulatory properties of DC are significantly influenced by their expression of signal II ligands, it is critical to understand the impact of distinct costimulatory pathways on DC function. This review gives an overview of functional different human DC subsets with unique profiles of costimulatory molecules and outlines how different costimulatory pathways together with the immunosuppressive cytokine IL-10 bias immunogenic versus tolerogenic DC functions. Furthermore, we exemplarily describe protocols for the generation of two well-defined monocyte-derived DC subsets for their clinical use, immunogenic versus tolerogenic.

Synthesis of maleimide-functionalyzed HPMA-copolymers and in vitro characterization of the aRAGE- and human immunoglobulin (huIgG)-polymer conjugates.

Herein the synthesis of antibody-polymer conjugates, with a quite narrow dispersity based on the polymer HPMA, are reported. These conjugates are synthesized by coupling antibodies to maleimide-functionalized poly(N-(2-hydroxypropyl)-methacrylamide) (poly-HPMA) copolymers derived through reversible addition-fragmentation chain transfer (RAFT) polymerization of pentafluorophenyl methacrylate via the intermediate step of an activated ester polymer. We develop a protocol that allows the attachment of two different model antibodies, monoclonal anti-RAGE (receptor for advanced glycation end-products) antibody, and polyclonal human immunoglobulin (huIgG). Modification of the antibody and conjugation is monitored by SDS-PAGE electrophoresis. Preserved affinity is demonstrated by Western Blott and cell-uptake analysis, for example, to cells of the immune system.

Plasmacytoid dendritic cells are inefficient in activation of human regulatory T cells.

BACKGROUND: Dendritic cells (DC) play a key role in initiation and regulation of immune responses. Plasmacytoid DC (pDC), a small subset of DC, characterized as type-I interferon producing cells, are critically involved in anti-viral immune responses, but also mediate tolerance by induction of regulatory T cells (Treg). In this study, we compared the capacity of human pDC and conventional DC (cDC) to modulate T cell activity in presence of Foxp3(+) Treg. PRINCIPAL FINDINGS: In coculture of T effector cells (Teff) and Treg, activated cDC overcome Treg anergy, abrogate their suppressive function and induce Teff proliferation. In contrast, pDC do not break Treg anergy but induce Teff proliferation even in coculture with Treg. Lack of Treg-mediated suppression is independent of proinflammatory cytokines like IFN-α, IL-1, IL-6 and TNF-α. Phenotyping of pDC-stimulated Treg reveals a reduced expression of Treg activation markers GARP and CTLA-4. Additional stimulation by anti-CD3 antibodies enhances surface expression of GARP and CTLA-4 on Treg and consequently reconstitutes their suppressive function, while increased costimulation with anti-CD28 antibodies is ineffective. CONCLUSIONS/SIGNIFICANCE: Our data show that activated pDC induce Teff proliferation, but are insufficient for functional Treg activation and, therefore, allow expansion of Teff also in presence of Treg.