S1P Signalling Differentially Affects Migration of Peritoneal B Cell Populations In Vitro and Influences the Production of Intestinal IgA In Vivo.

Introduction: Sphingosine-1-phosphate (S1P) regulates the migration of follicular B cells (B2 cells) and directs the positioning of Marginal zone B cells (MZ B cells) within the spleen. The function of S1P signalling in the third B cell lineage, B1 B cells, mainly present in the pleural and peritoneal cavity, has not yet been determined. Methods: S1P receptor expression was analysed in peritoneal B cells by real-time polymerase chain reaction (qPCR). The chemotactic response to S1P was studied in vitro. The role of S1P signalling was further explored in a s1p4-/- mouse strain. Results: Peritoneal B cells expressed considerable amounts of the S1P receptors 1 and 4 (S1P1 and S1P4, respectively). S1P1 showed differential expression between the distinct peritoneal B cell lineages. While B2 cells showed no chemotactic response to S1P, B1 B cells showed a migration response to S1P. s1p4-/- mice displayed significant alterations in the composition of peritoneal B cell populations, as well as a significant reduction of mucosal immunoglobulin A (IgA) in the gut. Discussion: S1P signalling influences peritoneal B1 B cell migration. S1P4 deficiency alters the composition of peritoneal B cell populations and reduces secretory IgA levels. These findings suggest that S1P signalling may be a target to modulate B cell function in inflammatory intestinal pathologies.

Cytotoxic T cells modulate inflammation and endogenous opioid analgesia in chronic arthritis.

BACKGROUND: This study examined the development of chronic pain, a cardinal symptom of rheumatoid arthritis (RA), in mice with antigen- and collagen-induced arthritis (ACIA). Since the role of CD8+ T cells in arthritis is controversial, we investigated the consequences of CD8-depletion on arthritis development and opioid modulation of pain in this novel model of chronic autoimmune arthritis. METHODS: Disease severity in control and CD8-depleted animals was determined by histological assessment of knee-joint sections and measurement of autoantibody formation. Pain was evaluated by measuring mechanical allodynia and thermal hyperalgesia in von Frey and Hargreaves tests, respectively. The production and release of endogenous opioids and inflammatory cytokines was assessed in immunoassays. RESULTS: In ACIA, mice display persistent mechanical allodynia and thermal hyperalgesia for more than 2 months after induction of arthritis. The blockade of peripheral opioid receptors with naloxone-methiodide (NLXM) transiently increased thermal hyperalgesia, indicating that endogenous opioid peptides were released in the arthritic joint to inhibit pain. CD8+ T cell depletion did not affect autoantibody formation or severity of joint inflammation, but serum levels of the pro-inflammatory cytokines TNFα and IL-17 were increased. The release of opioid peptides from explanted arthritic knee cells and the NLXM effect were significantly reduced in the absence of CD8+ T cells. CONCLUSIONS: We have successfully modeled the development of chronic pain, a hallmark of RA, in ACIA. Furthermore, we detected a yet unknown protective role of CD8+ T cells in chronic ACIA since pro-inflammatory cytokines rose and opioid peptide release decreased in the absence of these cells.

The transcriptional coactivator Bob1 promotes the development of follicular T helper cells via Bcl6.

Follicular T helper (Tfh) cells are key regulators of the germinal center reaction and long-term humoral immunity. Tfh cell differentiation requires the sustained expression of the transcriptional repressor Bcl6; however, its regulation in CD4(+)T cells is incompletely understood. Here, we report that the transcriptional coactivator Bob1, encoded by thePou2af1gene, promotes Bcl6 expression and Tfh cell development. We found that Bob1 together with the octamer transcription factors Oct1/Oct2 can directly bind to and transactivate theBcl6andBtlapromoters. Mixed bone marrow chimeras revealed that Bob1 is required for the expression of normal levels of Bcl6 andBTLA, thereby controlling the pool size and composition of the Tfh compartment in a T cell-intrinsic manner. Our data indicate that T cell-expressed Bob1 is directly involved in Tfh cell differentiation and required for mounting normal T cell-dependent B-cell responses.

MicroRNA-34a promotes genomic instability by a broad suppression of genome maintenance mechanisms downstream of the oncogene KSHV-vGPCR.

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded chemokine receptor vGPCR acts as an oncogene in Kaposi's sarcomagenesis. Until now, the molecular mechanisms by which the vGPCR contributes to tumor development remain incompletely understood. Here, we show that the KSHV-vGPCR contributes to tumor progression through microRNA (miR)-34a-mediated induction of genomic instability. Large-scale analyses on the DNA, gene and protein level of cell lines derived from a mouse model of vGPCR-driven tumorigenesis revealed that a vGPCR-induced upregulation of miR-34a resulted in a broad suppression of genome maintenance genes. A knockdown of either the vGPCR or miR-34a largely restored the expression of these genes and confirmed miR-34a as a downstream effector of the KSHV-vGPCR that compromises genome maintenance mechanisms. This novel, protumorigenic role of miR-34a questions the use of miR-34a mimetics in cancer therapy as they could impair genome stability.

Dysregulated development of IL-17- and IL-21-expressing follicular helper T cells and increased germinal center formation in the absence of RORγt.

Interleukin 17-producing helper T (Th17) cells have been widely defined by the lineage transcription factor retinoid-related orphan receptor (ROR)γt. Pathophysiologically, these cells play a crucial role in autoimmune diseases and have been linked to dysregulated germinal center (GC) reactions and autoantibody production. In this study, we used gene expression and flow cytometric analyses for the characterization of Rorγt(-/-) and Rorγt(-/-)Il21(RFP/+) mice to demonstrate a previously unknown transcriptional flexibility in the development of IL-17-producing Th-cell subsets. We found an accumulation of follicular Th (Tfh) cells by 5.2-fold, spontaneous 13-fold higher GC formation, decreased frequency of follicular Foxp3(+) T-regulatory (Treg) cells (50%), and a 3.4-fold increase in the number of proliferating follicular B cells in RORγt-deficient vs. wild-type mice. Dysregulated B-cell responses were associated with enhanced production of IL-17 (6.4-fold), IL-21 (2.2-fold), and B-cell-activating factor (BAFF) (2-fold) and were partially rescued by adoptive transfer of Treg cells. In an unexpected finding, we detected RORγt-independent IL-17 expression in ICOS(+)CXCR5(+)Tfh and in ICOS(+)CXCR5(-)Th cells. Based on the observed high Irf4 and Batf gene expression, we suggest that CD4(+) T-cell transcription factors other than RORγt can cooperatively induce differentiation of IL-17-producing Th cells, including Th17-like Tfh-cell subsets. We conclude that the occurrence of aberrant Tfh and follicular Treg cells support spontaneous GC formation and dysregulated B-cell responses in RORγt-deficient mice.

Potent anti-tumor response by targeting B cell maturation antigen (BCMA) in a mouse model of multiple myeloma.

Multiple myeloma (MM) is an aggressive incurable plasma cell malignancy with a median life expectancy of less than seven years. Antibody-based therapies have demonstrated substantial clinical benefit for patients with hematological malignancies, particular in B cell Non-Hodgkin's lymphoma. The lack of immunotherapies specifically targeting MM cells led us to develop a human-mouse chimeric antibody directed against the B cell maturation antigen (BCMA), which is almost exclusively expressed on plasma cells and multiple myeloma cells. The high affinity antibody blocks the binding of the native ligands APRIL and BAFF to BCMA. This finding is rationalized by the high resolution crystal structure of the Fab fragment in complex with the extracellular domain of BCMA. Most importantly, the antibody effectively depletes MM cells in vitro and in vivo and substantially prolongs tumor-free survival under therapeutic conditions in a xenograft mouse model. A BCMA-antibody-based therapy is therefore a promising option for the effective treatment of multiple myeloma and autoimmune diseases.

Suppression of Peripheral Pain by Blockade of Voltage-Gated Calcium 2.2 Channels in Nociceptors Induces RANKL and Impairs Recovery From Inflammatory Arthritis in a Mouse Model.

OBJECTIVE: A hallmark of rheumatoid arthritis (RA) is the chronic pain that accompanies inflammation and joint deformation. Patients with RA rate pain relief as the highest priority; however, few studies have addressed the efficacy and safety of therapies directed specifically toward pain pathways. The ω-conotoxin MVIIA (ziconotide) is used in humans to alleviate persistent pain syndromes, because it specifically blocks the voltage-gated calcium 2.2 (CaV 2.2) channel, which mediates the release of neurotransmitters and proinflammatory mediators from peripheral nociceptor nerve terminals. The aims of this study were to investigate whether blockade of CaV 2.2 can suppress arthritis pain, and to examine the progression of induced arthritis during persistent CaV 2.2 blockade. METHODS: Transgenic mice expressing a membrane-tethered form of MVIIA under the control of a nociceptor-specific gene (MVIIA-transgenic mice) were used in the experiments. The mice were subjected to unilateral induction of joint inflammation using a combination of antigen and collagen. RESULTS: CaV 2.2 blockade mediated by tethered MVIIA effectively suppressed arthritis-induced pain; however, in contrast to their wild-type littermates, which ultimately regained use of their injured joint as inflammation subsided, MVIIA-transgenic mice showed continued inflammation, with up-regulation of the osteoclast activator RANKL and concomitant joint and bone destruction. CONCLUSION: Taken together, our results indicate that alleviation of peripheral pain by blockade of CaV 2.2- mediated calcium influx and signaling in nociceptor sensory neurons impairs recovery from induced arthritis and point to the potentially devastating effects of using CaV 2.2 channel blockers as analgesics during inflammation.

The homeostatic chemokine CCL21 predicts mortality in aortic stenosis patients and modulates left ventricular remodeling.

BACKGROUND: CCL21 acting through CCR7, is termed a homeostatic chemokine. Based on its role in concerting immunological responses and its proposed involvement in tissue remodeling, we hypothesized that this chemokine could play a role in myocardial remodeling during left ventricular (LV) pressure overload. METHODS AND RESULTS: Our main findings were: (i) Serum levels of CCL21 were markedly raised in patients with symptomatic aortic stenosis (AS, n = 136) as compared with healthy controls (n = 20). (ii) A CCL21 level in the highest tertile was independently associated with all-cause mortality in these patients. (iii) Immunostaining suggested the presence of CCR7 on macrophages, endothelial cells and fibroblasts within calcified human aortic valves. (iv). Mice exposed to LV pressure overload showed enhanced myocardial expression of CCL21 and CCR7 mRNA, and increased CCL21 protein levels. (v) CCR7-/- mice subjected to three weeks of LV pressure overload had similar heart weights compared to wild type mice, but increased LV dilatation and reduced wall thickness. CONCLUSIONS: Our studies, combining experiments in clinical and experimental LV pressure overload, suggest that CCL21/CCR7 interactions might be involved in the response to pressure overload secondary to AS.

Dendritic cell-mediated survival signals in Eµ-Myc B-cell lymphoma depend on the transcription factor C/EBPß.

The capacity of dendritic cells (DCs) to regulate tumour-specific adaptive immune responses depends on their proper differentiation and homing status. Whereas DC-associated tumour-promoting functions are linked to T-cell tolerance and formation of an inflammatory milieu, DC-mediated direct effects on tumour growth have remained unexplored. Here we show that deletion of DCs substantially delays progression of Myc-driven lymphomas. Lymphoma-exposed DCs upregulate immunomodulatory cytokines, growth factors and the CCAAT/enhancer-binding protein ß (C/EBPß). Moreover, Eµ-Myc lymphomas induce the preferential translation of the LAP/LAP* isoforms of C/EBPß. C/EBPß(-/-) DCs are unresponsive to lymphoma-associated cytokine changes and in contrast to wild-type DCs, they are unable to mediate enhanced Eµ-Myc lymphoma cell survival. Antigen-specific T-cell proliferation in lymphoma-bearing mice is impaired; however, this immune suppression is reverted by the DC-restricted deletion of C/EBPß. Thus, we show that C/EBPß-controlled DC functions are critical steps for the creation of a lymphoma growth-promoting and -immunosuppressive niche.

Access to follicular dendritic cells is a pivotal step in murine chronic lymphocytic leukemia B-cell activation and proliferation.

UNLABELLED: In human chronic lymphocytic leukemia (CLL) pathogenesis, B-cell antigen receptor signaling seems important for leukemia B-cell ontogeny, whereas the microenvironment influences B-cell activation, tumor cell lodging, and provision of antigenic stimuli. Using the murine Eµ-Tcl1 CLL model, we demonstrate that CXCR5-controlled access to follicular dendritic cells confers proliferative stimuli to leukemia B cells. Intravital imaging revealed a marginal zone B cell-like leukemia cell trafficking route. Murine and human CLL cells reciprocally stimulated resident mesenchymal stromal cells through lymphotoxin-ß-receptor activation, resulting in CXCL13 secretion and stromal compartment remodeling. Inhibition of lymphotoxin/lymphotoxin-ß-receptor signaling or of CXCR5 signaling retards leukemia progression. Thus, CXCR5 activity links tumor cell homing, shaping a survival niche, and access to localized proliferation stimuli. SIGNIFICANCE: CLL and other indolent lymphoma are not curable and usually relapse after treatment, a process in which the tumor microenvironment plays a pivotal role. We dissect the consecutive steps of CXCR5-dependent tumor cell lodging and LTßR-dependent stroma-leukemia cell interaction; moreover, we provide therapeutic solutions to interfere with this reciprocal tumor-stroma cross-talk.

Immunotherapy of B-cell non-Hodgkin lymphoma by targeting the chemokine receptor CXCR5 in a preclinical mouse model.

Bispecific antibodies are promising agents for immunotherapy. Here, we describe a quadroma-based trifunctional bispecific antibody binding the chemokine receptor CXCR5 and the T-cell antigen CD3 that efficiently prevents tumor growth in a mouse B-cell lymphoma model. CXCR5 regulates the tissue homeostasis of mature B cells and is highly expressed on B-cell non-Hodgkin and lymphocyte-predominant Hodgkin lymphoma, as well as on a subset of CD4(+) T cells known as follicular T-helper cells. In vitro, the bispecific CXCR5::CD3 antibody efficiently recruited effector T cells to CXCR5 expressing B cells and induced a co-stimulation-independent activation of CD8(+) and CD4(+) T cells as demonstrated by the de novo expression of CD25 and CD69, and secretion of the cytokines IFN-γ, TNF-α, IL-6 and IL-10 by peripheral blood mononuclear cells. Notably, at low antibody concentrations, CXCR5::CD3 displayed a significantly higher cytotoxic activity against autologous B cells than its parental antibodies or rituximab. In vivo imaging revealed that CXCR5::CD3 and its parental CXCR5 antibody efficiently prevent tumor growth in a xenograft model of B-cell lymphoma in mice and prolong their survival. Taken together, our results identify CXCR5 as a promising target for antibody-based therapies in the treatment of B-cell malignancies.

B-cell-intrinsic STAT6 signaling controls germinal center formation.

Infection with helminths and exposure to antigens induce a strong type 2 immune response resulting in the secretion of the cytokines IL-4 and IL-13 by CD4(+) T cells and several innate cell types. IL-4 and IL-13 promote class switch recombination to IgG1 and IgE while their role for germinal center (GC) formation is poorly understood. We found a dramatic reduction in the numbers of GC B cells when investigating different type 2 immune responses in IL-4/IL-13-deficient mice. IL-4/IL-13 from T cells located outside B-cell follicles was sufficient for GC formation. We further revealed that IL-4/IL-13 acts directly on B cells for the formation of a robust GC response. The frequency of apoptotic GC B cells was not altered in the absence of IL-4/IL-13 and proliferation was even enhanced. However, deficiency of signal transducer and activator of transcription 6 signaling in B cells resulted in failure to downregulate the chemotactic receptor Gpr183 (Ebi2) and downregulation of this receptor has been shown to be essential for proper GC B-cell differentiation. Thus, T-cell-derived extrafollicular IL-4/IL-13 and signal transducer and activator of transcription 6-regulated genes in B cells play a critical role for orchestration of the GC response in type 2 immunity.

Follicular regulatory T cells control humoral autoimmunity via NFAT2-regulated CXCR5 expression.

Maturation of high-affinity B lymphocytes is precisely controlled during the germinal center reaction. This is dependent on CD4(+)CXCR5(+) follicular helper T cells (TFH) and inhibited by CD4(+)CXCR5(+)Foxp3(+) follicular regulatory T cells (TFR). Because NFAT2 was found to be highly expressed and activated in follicular T cells, we addressed its function herein. Unexpectedly, ablation of NFAT2 in T cells caused an augmented GC reaction upon immunization. Consistently, however, TFR cells were clearly reduced in the follicular T cell population due to impaired homing to B cell follicles. This was TFR-intrinsic because only in these cells NFAT2 was essential to up-regulate CXCR5. The physiological relevance for humoral (auto-)immunity was corroborated by exacerbated lupuslike disease in the presence of NFAT2-deficient TFR cells.

Increased levels of CCR7 ligands in carotid atherosclerosis: different effects in macrophages and smooth muscle cells.

AIMS: The homeostatic chemokines, CCL19 and CCL21 and their receptor CCR7, have recently been linked to atherogenesis. We investigated the expression of CCL19/CCL21/CCR7 in carotid atherosclerosis as well as the ability of these chemokines to modulate lipid accumulation in macrophages and vascular smooth muscle cell (SMC) phenotype. METHODS AND RESULTS: Our major findings were: (i) patients with carotid atherosclerosis (n = 158) had increased plasma levels of CCL21, but not of CCL19, compared with controls (n = 20), with particularly high levels in symptomatic (n = 99) when compared with asymptomatic (n = 59) disease. (ii) Carotid plaques showed markedly increased mRNA levels of CCL21 and CCL19 in symptomatic (n = 14) when compared with asymptomatic (n = 7) patients, with CCR7 localized to macrophages and vascular SMC (immunohistochemistry). (iii) In vitro, CCL21, but not CCL19, increased the binding of modified LDL and promoted lipid accumulation in THP-1 macrophages. (iv) CCL19, but not CCL21, increased proliferation and release and activity of matrix metalloproteinase (MMP) 1 in vascular SMC. (v) The differential effects of CCL19 and CCL21 in macrophages and SMC seem to be attributable to divergent signalling pathways, with CCL19-mediated activation of AKT in SMC- and CCL21-mediated activation of extracellular signal-regulated kinase 1/2 in macrophages. CONCLUSION: CCL19 and CCL21 are up-regulated in carotid atherosclerosis. The ability of CCL21 to promote lipid accumulation in macrophages and of CCL19 to induce proliferation and MMP-1 expression in vascular SMC could contribute to their pro-atherogenic potential.

Transition from an autoimmune-prone state to fatal autoimmune disease in CCR7 and RORγt double-deficient mice is dependent on gut microbiota.

Autoimmunity is associated with a strong genetic component, but onset and persistence of clinically apparent autoimmune diseases often require an additional environmental trigger. The balance between immunity and tolerance is regulated by numerous molecular factors including nuclear hormone and homeostatic chemokine receptors. The nuclear hormone receptor RORγt and the chemokine receptor CCR7 are both essentially involved in functional lymphoid organogenesis and maintenance of lymphocyte homeostasis. Lack of one or the other impairs thymic T cell development and alters T cell homeostasis. Mice deficient for both, Ccr7(-/-)Rorγt(-/-), succumbed early to acute destructive inflammation, characterized by massive recruitment of inflammatory leukocytes, pro-inflammatory cytokine and autoantibody production, and wasting disease. Antibiotic-treatment of mice before disease onset reduced the overall gut microflora and abrogated the development of fatal mucosal inflammation. Hence, commensal bacteria and a confined tissue-specific inflammatory milieu serve as complementary trigger to initiate the lethal pathophysiologic process in Ccr7(-/-)Rorγt(-/-) mice.

A chronic model of arthritis supported by a strain-specific periarticular lymph node in BALB/c mice.

Current animal models of arthritis only partially reflect the complexity of rheumatoid arthritis and typically lack either chronicity or autoantibody formation. Here we describe a model that combines features of antigen-induced arthritis and collagen-induced arthritis, which can be efficiently induced in BALB/c and C57BL/6 mice. However, BALB/c mice generate significantly higher titres of anticollagen and anticitrullinated peptide antibodies, show a stronger progressive joint destruction, and in the chronic phase the disease spreads between joints. Concomitant to the observation of a more severe pathology, we discovered a previously undescribed small periarticular lymph node in close proximity to the knee joint of BALB/c mice, which acts as the primary draining lymph node for the synovial cavity. Our model more closely reflects the pathology of rheumatoid arthritis than classical models of arthritis and is hence particularly suitable for further studies of disease pathogenesis.

HER2/neu DNA vaccination by intradermal gene delivery in a mouse tumor model: Gene gun is superior to jet injector in inducing CTL responses and protective immunity.

DNA vaccines are potential tools for the induction of immune responses against both infectious disease and cancer. The dermal application of DNA vaccines is of particular interest since the epidermal and dermal layers of the skin are characterized by an abundance of antigen-presenting cells (APCs). The aim of our study was to compare tumor protection as obtained by two different methods of intradermal DNA delivery (gene gun and jet injector) in a well-established HER2/neu mouse tumor model. BALB/c mice were immunized twice with a HER2/neu-coding plasmid by gene gun or jet injector. Mice were then subcutaneously challenged with HER2/neu(+) syngeneic D2F2/E2 tumor cells. Protection against subsequent challenges with tumor cells as well as humoral and T-cell immune responses induced by the vaccine were monitored. Gene gun immunization was far superior to jet injector both in terms of tumor protection and induction of HER2/neu-specific immune responses. After gene gun immunization, 60% of the mice remained tumor-free until day 140 as compared with 25% after jet injector immunization. Furthermore, gene gun vaccination was able to induce both a strong T(H)1-polarized T-cell response with detectable cytotoxic T-lymphocyte (CTL) activity and a humoral immune response against HER2/neu, whereas the jet injector was not. Although the disadvantages that were associated with the use of the jet injector in our model may be overcome with methodological modifications and/or in larger animals, which exhibit a thicker skin and/or subcutaneous muscle tissue, we conclude that gene gun delivery constitutes the method of choice for intradermal DNA delivery in preclinical mouse models and possibly also for the clinical development of DNA-based vaccines.

The role of CCR7 in allergic airway inflammation induced by house dust mite exposure.

House dust mite (HDM), the most common allergen, activate both the IgE-associated and innate immune responses. To clarify the process of sensitization, we investigated the role of the CCL21, CCL19, and CCR7 axis in a mouse model of HDM-induced allergic asthma. HDM inhalation without systemic immunization resulted in a HDM-specific IgE response. CCR7-knockout (CCR7KO) mice exhibited greater airway inflammation and IgE responses compared to wild-type mice. We examined FoxP3 expression in these mice to clarify the contribution of regulatory cells to the responses. FoxP3 expression was higher in the lungs but not in the lymph nodes of CCR7KO mice compared to wild-type mice. In CCR7KO mice, FoxP3-positive cells were found in lung, but we observed higher release of IL-13, IL-5, TGF-ß, IL-17, and HMGB1 in bronchoalveolar lavage fluid. We demonstrate here that immuno-regulation through CCR7 expression in T cells plays a role in HDM-specific sensitization in the airway.