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.

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.

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.

CCL19 (ELC) improves TH1-polarized immune responses and protective immunity in a murine Her2/neu DNA vaccination model.

BACKGROUND: DNA vaccination is an attractive approach for tumor vaccination because plasmid DNA (pDNA) can be used as a 'general vaccine' across major histocompatibility complex barriers. Coexpression of immunomodulatory molecules can help to amplify the immunogenicity of DNA vaccines. CCL19 (ELC) is a CC chemokine with immunoregulatory properties, binding to the chemokine receptor CCR7 that is expressed on dendritic cells (DCs) and T cells. In vivo, CCL19 is a key regulator for the interactions between DCs and T cells in regional lymph nodes. METHODS: pDNA encoding Her2/neu and CCL19 was used as an intramuscular vaccine. Vaccination was performed in BALB/c mice, which were subsequently challenged with syngeneic Her2/neu(+) tumor cells. Groups of mice were immunized with pDNA(Her2/neu) plus pDNA(CCL19), pDNA(Her2/neu) plus pDNA(CCL19) plus pDNA(GM-CSF), pDNA(Her2/neu) plus pDNA(GM-CSF), pDNA(Her2/neu), pDNA(CCL19), pDNA(GM-CSF) or mock vector. Tumor protection by the vaccine and immune responses were monitored. RESULTS: Coadministration of pDNA(Her2/neu) and pDNA(CCL19) led to substantial improvement of tumor protection by the vaccine and induced a TH1-polarized, Her2/neu-specific immune response. Forty-seven days after the tumor challenge, 58% of the mice coinjected with pDNA(Her2/neu) and pDNA(CCL19) remained tumor-free compared to 22% after vaccination with pDNA(Her2/neu) alone. Additional administration of pDNA(GM-CSF) led to further improvement of tumor protection and an amplification of Her2/neu-specific immune responses. CONCLUSIONS: CCL19 is able to induce a TH-1 polarization of the anti-Her2/neu immune response, which can be further amplified by granulocyte macrophage-colony-stimulating factor (GM-CSF). Clinical use of a pDNA(Her2/neu-CCL19 ± GM-CSF) vaccine might be promising in Her2/neu + breast cancer in the clinical situation of minimal residual disease.

CCR9+ macrophages are required for acute liver inflammation in mouse models of hepatitis.

BACKGROUND & AIMS: Antigen-presenting cells (APCs) are involved in the induction of liver inflammation. We investigated the roles of specific APCs in the pathogenesis of acute liver injury in mice. METHODS: We used concanavalin A (con A) or carbon tetrachloride to induce acute liver inflammation in mice and studied the roles of macrophages that express CCR9. RESULTS: After injection of con A, we detected CCR9(+)CD11b(+)CD11c(-) macrophages that express tumor necrosis factor (TNF)-α in livers of mice, whereas CCR9(+)Siglec-H(+)CD11b(-)CD11c(low) plasmacytoid DCs (pDCs), which are abundant in normal livers, disappeared. The CCR9(+) macrophages were also detected in the livers of RAG-2(-/-) mice, which lack lymphocytes and natural killer T cells, after injection of con A. Under inflammatory conditions, CCR9(+) macrophages induced naive CD4(+) T cells to become interferon gamma-producing Th1 cells in vivo and in vitro. CCR9(-/-) mice injected with con A did not develop hepatitis unless they also received CCR9(+) macrophages from mice that received con A; more CCR9(+) macrophages accumulated in their inflamed livers than CCR9(+) pDCs, CCR9(-) pDCs, or CCR9(-) macrophages isolated from mice that had received injections of con A. Levels of CCL25 messenger RNA increased in livers after injection of con A; neutralizing antibodies against CCL25 reduced the induction of hepatitis by con A by blocking the migration of CCR9(+) macrophages and their production of TNF-α. Peripheral blood samples from patients with acute hepatitis had greater numbers of TNF-α-producing CCR9(+)CD14(+)CD16(high) monocytes than controls. CONCLUSIONS: CCR9(+) macrophages contribute to the induction of acute liver inflammation in mouse models of hepatitis.

Coordinated regulation of lymph node vascular-stromal growth first by CD11c+ cells and then by T and B cells.

Lymph node blood vessels play important roles in the support and trafficking of immune cells. The blood vasculature is a component of the vascular-stromal compartment that also includes the lymphatic vasculature and fibroblastic reticular cells (FRCs). During immune responses as lymph nodes swell, the blood vasculature undergoes a rapid proliferative growth that is initially dependent on CD11c(+) cells and vascular endothelial growth factor (VEGF) but is independent of lymphocytes. The lymphatic vasculature grows with similar kinetics and VEGF dependence, suggesting coregulation of blood and lymphatic vascular growth, but lymphatic growth has been shown to be B cell dependent. In this article, we show that blood vascular, lymphatic, and FRC growth are coordinately regulated and identify two distinct phases of vascular-stromal growth--an initiation phase, characterized by upregulated vascular-stromal proliferation, and a subsequent expansion phase. The initiation phase is CD11c(+) cell dependent and T/B cell independent, whereas the expansion phase is dependent on B and T cells together. Using CCR7(-/-) mice and selective depletion of migratory skin dendritic cells, we show that endogenous skin-derived dendritic cells are not important during the initiation phase and uncover a modest regulatory role for CCR7. Finally, we show that FRC VEGF expression is upregulated during initiation and that dendritic cells can stimulate increased fibroblastic VEGF, suggesting the scenario that lymph node-resident CD11c(+) cells orchestrate the initiation of blood and lymphatic vascular growth in part by stimulating FRCs to upregulate VEGF. These results illustrate how the lymph node microenvironment is shaped by the cells it supports.

Manifestation of spontaneous and early autoimmune gastritis in CCR7-deficient mice.

Autoimmune gastritis is a common autoimmune disorder characterized by chronic inflammatory cell infiltrates, atrophy of the corpus and fundus, and the occurrence of autoantibodies to parietal cell antigen. In CCR7-deficient mice, autoimmune gastritis developed spontaneously and was accompanied by metaplasia of the gastric mucosa and by the formation of tertiary lymphoid organs at gastric mucosal sites. T cells of CCR7-deficient mice showed an activated phenotype in the gastric mucosa, mesenteric lymph nodes, and peripheral blood. In addition, elevated serum IgG levels specific to gastric parietal cell antigen were detected. Because the role of organized lymphocytic aggregates at this inflammatory site is not completely understood, we first analyzed the cellular requirements for the formation of these structures. Autoreactive CD4(+) T cells were pivotal for tertiary lymphoid follicle formation, most likely in cooperation with dendritic cells, macrophages, and B cells. Second, we analyzed the necessity of secondary lymph nodes and tertiary lymphoid organs for the development of autoimmune gastritis using CCR7 single- and CCR7/lymphotoxin α double-deficient mice. Strikingly, manifestation of autoimmune gastritis was observed in the absence of secondary lymph nodes and preceded the development of tertiary lymphoid organs. Taken together, these findings identify an inflammatory process where gastric autoreactive T cells independent of organized tertiary lymphoid organs and classic lymph nodes can induce and maintain autoimmune gastritis.

Sphingosine-1-phospate receptor 4 (S1P4) deficiency profoundly affects dendritic cell function and TH17-cell differentiation in a murine model.

Although predominantly expressed on lymphocytic and hematopoietic cells, the role of sphingosine-1-phospate receptor 4 (S1P(4)) in immune homeostasis is still poorly understood. In this report, we used a S1P(4)-deficient murine model to characterize the biological role of S1P(4)-mediated S1P signaling in the immune system. S1p(4)(-/-) animals showed normal peripheral lymphocyte numbers and a regular architecture of secondary lymphoid organs. Interestingly, S1P(4) only marginally affects T-cell function in vivo. In contrast, dendritic cell (DC) migration and cytokine secretion are profoundly affected by S1P(4) deficiency. Lack of S1P(4) expression on DCs significantly reduces T(H)17 differentiation of T(H) cells. Furthermore, in various in vivo models of T(H)1- or T(H)2-dominated immune reactions, S1P(4) deficiency consistently increased the amplitude of T(H)2-dominated immune responses, while those depending on T(H)1-dominated mechanisms were diminished. Finally, S1p(4)(-/-) mice showed decreased pathology in a model of dextran sulfate sodium-induced colitis. In summary, for the first time, we show that S1P(4) signaling is involved in the regulation of DC function and T(H)17 T-cell differentiation. S1P(4)-mediated S1P signaling also modifies the course of various immune diseases in a murine model. We propose that S1P(4) may constitute an interesting target to influence the course of various autoimmune pathologies.

Cooperative function of CCR7 and lymphotoxin in the formation of a lymphoma-permissive niche within murine secondary lymphoid organs.

Lymphoma cell survival and progression are putatively dependent on a specific microanatomic localization within secondary lymphoid organs. Despite compelling data correlating homeostatic chemokine receptor expression and human lymphoma pathogenesis, genetic models that either mimic lymphoma dissemination or dissect a crosstalk of lymphoma and stromal cells are missing. Applying the genetically tractable Eµ-Myc transgenic mouse model, we show that the chemokine receptor CCR7 regulates Eµ-Myc lymphoma homing to lymph nodes and distinctive microanatomic sites of the spleen. CCR7-controlled access of lymphoma cells to the splenic T-cell zone led to a significant survival advantage compared with CCR7-deficient lymphoma cells, which were excluded from this zone. Within the niche, lymphoma cells stimulated a reciprocal cross-talk with gp38(+) fibroblastic reticular cells. This reciprocal cooperation program was mediated by lymphoma B cell-presented lymphotoxin, which acted on lymphotoxin-ß-receptor-bearing stromal cells followed by alteration of stromal cellular composition. Cross-talk inhibition by lymphotoxin-α deletion and using a lymphotoxin-ß receptor-immunoglobulin fusion protein impaired lymphoma growth. Thus, abrogation of CCR7-governed migration and of sustained lymphotoxin signaling could provide new targets in lymphoma therapy.

Steady state migratory RelB+ langerin+ dermal dendritic cells mediate peripheral induction of antigen-specific CD4+ CD25+ Foxp3+ regulatory T cells.

Tolerance to self-antigens expressed in peripheral organs is maintained by CD4(+) CD25(+) Foxp3(+) Treg cells, which are generated as a result of thymic selection or peripheral induction. Here, we demonstrate that steady-state migratory DCs from the skin mediated Treg conversion in draining lymph nodes of mice. These DCs displayed a partially mature MHC II(int) CD86(int) CD40(hi) CCR7(+) phenotype, used endogenous TGF-ß for conversion and showed nuclear RelB translocation. Deficiency of the alternative NF-κB signaling pathway (RelB/p52) reduced steady-state migration of DCs. These DCs transported and directly presented soluble OVA provided by s.c. implanted osmotic minipumps, as well as cell-associated epidermal OVA in transgenic K5-mOVA mice to CD4(+) OVA-specific TCR-transgenic OT-II T cells. The langerin(+) dermal DC subset, but not epidermal Langerhans cells, mediated conversion of naive OT-II×RAG-1(-/-) T cells into proliferating CD4(+) CD25(+) Foxp3(+) Tregs. Thus, our data suggest that steady-state migratory RelB(+) TGF-ß(+) langerin(+) dermal DCs mediate peripheral Treg conversion in response to epidermal antigen in skin-draining lymph nodes.

Shaping of terminal megakaryocyte differentiation and proplatelet development by sphingosine-1-phosphate receptor S1P4.

Megakaryocytes, which mature from hematopoietic progenitors in the bone marrow, further differentiate by reorganizing their cytoplasm into long proplatelet extensions that release platelets into the circulation. The molecular mechanisms underlying this highly dynamic cytoplasmic and cytoskeletal remodeling process are only poorly understood. Here we report that sphingosine 1-phosphate receptor 4 (S1P(4)) is specifically up-regulated during the development of human megakaryocytes from progenitor cells and is expressed in mature murine megakaryocytes. Megakaryocytes generated from S1P(4)-deficient murine bone marrow showed atypical and reduced formation of proplatelets in vitro. The recovery of platelet numbers after experimental thrombocytopenia was significantly delayed in S1p4(-/-) mice. Remarkably, overexpression and stimulation of S1P(4) in human erythroleukemia HEL cells promoted endomitosis, formation of cytoplasmic extensions, and subsequent release of platelet-like particles. These observations indicate that S1P(4) is involved in shaping the terminal differentiation of megakaryocytes.