Flt3 ligand therapy for recipients of allogeneic bone marrow transplants expands host CD8 alpha(+) dendritic cells and reduces experimental acute graft-versus-host disease

Recent evidence suggests that dendritic cells (DCs) can regulate and amplify immune responses. Flt3 ligand (FL)-derived DC function was tested as a stimulator of allogeneic lymphocytes in vitro and in vivo. Treatment of mice with FL dramatically expanded DC number, but DCs isolated from FL-treated mice (FL DCs) were poor stimulators of allogeneic T-cell responses in vitro. Further activation of FL DCs did not restore their stimulatory ability, and FL DCs did not suppress the stimulation of the allogeneic T cells by normal DCs. FL treatment significantly increased the CD8 alpha(+) DC subset, which appeared to be the reason for their poor stimulatory capacity. These observations were confirmed in vivo using a mouse model of acute graft-versus-host disease (GVHD) wherein host DCs play a critical role. FL treatment of recipients before allogeneic bone marrow transplantation dramatically suppressed donor T-cell responses to host antigens, thereby reducing GVHD mortality (P <.01). These data represent a novel strategy that alters host DCs and reduces acute GVHD.     

Plasmacytoid dendritic cells efficiently cross-prime naive T cells in vivo after TLR activation

Cross-presentation is a crucial mechanism in tumoral and microbial immunity because it allows internalized cell associated or exogenous antigens (Ags) to be delivered into the major histocompatibility complex I pathway. This pathway is important for the development of CD8(+) T-cell responses and for the induction of tolerance. In mice, cross-presentation is considered to be a unique property of CD8alpha+ conventional dendritic cells (DCs). Here we show that splenic plasmacytoid DCs (pDCs) efficiently capture exogenous Ags in vivo but are not able to cross-present these Ags at steady state. However, in vitro and in vivo stimulation by Toll-like receptor-7, or -9 or viruses licenses pDCs to cross-present soluble or particulate Ags by a transporter associated with antigen processing-dependent mechanism. Induction of cross-presentation confers to pDCs the ability to generate efficient effector CD8+ T-cell responses against exogenous Ags in vivo, showing that pDCs may play a crucial role in induction of adaptive immune responses against pathogens that do not infect tissues of hemopoietic origin. This study provides the first evidence for an in vivo role of splenic pDCs in Ag cross-presentation and T-cell cross-priming and suggests that pDCs may constitute an attractive target to boost the efficacy of vaccines based on cytotoxic T lymphocyte induction.     

Distinct antifibrogenic effects of erlotinib,sunitinib and sorafenib on rat pancreatic stellate cells

AIM:To study if three clinically available small molecule kinase inhibitors(SMI),erlotinib,sunitinib and sorafenib,exert antifibrogenic effects on pancreatic stellate cells(PSC)and analyze the basis of their action.METHODS:Cultured rat PSC were exposed to SMI.Cell proliferation and viability were assessed employing 5-bromo-2’-deoxyuridine incorporation assay and flow cytometry,respectively.2-Deoxy-2-[18F]fluoroglucose(18F-FDG)uptake was measured to study metabolic activity.Exhibition of the myofibroblastic PSC phenotype was monitored by immunofluorescence analysis ofα-smooth muscle actin(α-SMA)expression.Levels of mRNA were determined by real-time PCR,while protein expression and phosphorylation were analyzed by immunoblotting.Transforming growth factor-β1 (TGF-β1)levels in culture supernatants were quantified by ELISA.RESULTS:All three SMI inhibited cell proliferation and18F-FDG uptake in a dose-dependent manner and without significant cytotoxic effects.Furthermore,additive effects of the drugs were observed.Immunoblot analysis showed that sorafenib and sunitib,but not erlotinib,efficiently blocked activation of the AKT pathway,while all three drugs displayed little effect on phosphorylation of ERK1/2.Cells treated with sorafenib or sunitinib expressed less interleukin-6 mRNA as well as less collagen type 1 mRNA and protein.Sorafenib was the only drug that also upregulated the expression of matrix metalloproteinase-2 and reduced the secretion of TGF-β1 protein.All three drugs showed insignificant or discordant effects on the mRNA and protein levels ofα-SMA.CONCLUSION:The tested SMI,especially sorafenib,exert inhibitory effects on activated PSC,which should be further evaluated in preclinical studies.     

Selective Rac1 inhibition in dendritic cells diminishes apoptotic cell uptake and cross-presentation in vivo

To better understand the influence of cytoskeletal regulation on dendritic cell (DC) function in vivo, the Rho guanosine triphosphatase (GTPase) Rac1 was selectively inhibited in DCs in transgenic (Tg) mice. Although transgene expression did not interfere with the migratory capacities of DC in vivo, a decreased uptake of fluorescent probes was observed. Interestingly, the absence of full Rac1 function most strongly affected the development and function of CD8+ DCs. Apoptotic cell uptake was severely reduced in Tg mice, impairing subsequent DC-mediated cross-presentation and priming of bacteria-specific T-cell responses. These findings highlight a special role for Rac1 in the capacity of CD8+ DCs to endocytose apoptotic cells and prime T cells via cross-presentation.     

Immunotherapy directed against alpha-fetoprotein results in autoimmune liver disease during liver regeneration in mice

BACKGROUND & AIMS: Priming immune responses against alpha-fetoprotein (AFP) highly expressed in the majority of hepatocellular carcinomas results in significant antitumoral T-cell responses. Liver regeneration in humans and mice, however, is also associated with increased AFP expression. Therefore, we evaluated the risk of AFP-directed immunotherapeutic approaches to induce autoimmunity against the regenerating liver. METHODS: Mice were immunized with DNA encoding mouse AFP. For induction of liver regeneration, partial hepatectomy was performed and mice were monitored by serial histopathologic examinations and measurements of serum ALT activities (U/L), and by determination of the kinetics of AFP-specific T-cell responses. RESULTS: Livers of AFP immune mice without partial hepatectomy were characterized by minor lymphocytic infiltrations without transaminase elevations. By contrast, a significant hepatocyte damage was observed in regenerating liver that correlated well with the number of AFP-specific CD8(+) T cells, the activity of liver regeneration, and the level of AFP synthesis. Autoimmune liver damage was mediated by CD4(+) T cell-dependent CD8(+) cytotoxic T lymphocytes. CONCLUSIONS: These results show that priming of T-cell responses against shared tumor-specific self antigens may be accompanied by induction of autoimmunity dependent on the level of expression of the self antigen and have important implications for the development of antitumoral vaccines targeted against antigens that are not strictly tumor-specific.     

Intranodal injection of semimature monocyte-derived dendritic cells induces T helper type 1 responses to protein neoantigen

Dendritic cells (DCs) represent the most potent antigen-presenting cells of the immune system capable of initiating primary immune responses to neoantigens. Here we characterize the primary CD4 T-cell immune response to protein keyhole limpet hemocyanin (KLH) in 5 metastatic melanoma patients undergoing a tumor peptide-based dendritic cell vaccination trial. Monocyte-derived dendritic cells displaying a semimature phenotype, as defined by surface markers, were loaded ex vivo with antigen and injected intranodally at weekly intervals for 4 weeks. All patients developed a strong and long-lasting delayed-type hypersensitivity reactivity to KLH, which correlated with the induction of KLH-dependent proliferation of CD4 T cells in vitro. Secondary in vitro stimulation with KLH showed significant increase in interferon-gamma and interleukin-2 (IL-2) but not IL-4, IL-5, nor IL-10 secretion by bulk T cells. On the single-cell level, most TH1 cells among in vitro-generated KLH-specific T-cell lines confirmed the preferential induction of a KLH-specific type 1 T helper immune response. Furthermore, the induction of KLH-specific antibodies of the IgG2 subtype may reflect the induction of a type 1 cytokine profile in vivo after vaccination. Our results indicate that intranodal vaccination with semimature DCs can prime strong, long-lasting CD4 T-cell responses with a TH1-type cytokine profile in cancer patients.     

Antiangiogenic therapy promoted metastasis of hepatocellular carcinoma by suppressing host-derived interleukin-12b in mouse models

Antiangiogenic therapy, specially sorafenib, has become the standard of care for patients with advanced hepatocellular carcinoma (HCC), however, the improvement in survival time is not satisfactory. Previous studies have found that, in some circumstances, antiangiogenic therapy promoted tumor metastasis and the mechanistic studies were mainly focus on cancer-cell-autonomous manners. In two experimental metastasis models with tail-vein injection with hepatoma cells and an orthotopic HCC mouse model, we found that pretreatment with two vascular endothelial growth factor receptor (VEGFR) inhibitors, sunitinib and sorafenib, facilitated tumor cell survival in blood stream and promoted lung metastasis from tumors that were subsequently incubated after drug discontinuation, indicating that host response joined into the pro-metastatic effects. An antibody microarray identified that interleukin (IL)-12b was decreased in the peripheral blood of the mice treated with the two VEGFR inhibitors. IL-12b suppression in macrophages and dendritic cells from host organs was found to play a crucial role in treatment-induced metastasis. Supplement with recombinant mouse IL-12b or restoration of IL-12b expression in the host by zoledronic acid, which was previously reported to enhance IL-12 expression in vitro and in vivo, alleviated the metastasis-promoting effects of sunitinib and sorafenib. These studies suggest that host response to VEGFR inhibitors facilitates HCC metastasis and restoration of IL-12b expression could translate into clinical benefits.     

Depletion of CD25⁺ T cells from hematopoietic stem cell grafts increases posttransplantation vaccine-induced immunity to neuroblastoma

A multifaceted immunotherapeutic strategy that includes hematopoietic stem cell (HSC) transplantation, T-cell adoptive transfer, and tumor vaccination can effectively eliminate established neuroblastoma tumors in mice. In vivo depletion of CD4? T cells in HSC transplantation recipients results in increased antitumor immunity when adoptively transferred T cells are presensitized, but development of T-cell memory is severely compromised. Because increased percentages of regulatory T (Treg) cells are seen in HSC transplantation recipients, here we hypothesized that the inhibitory effect of CD4? T cells is primarily because of the presence of expanded Treg cells. Remarkably, adoptive transfer of presensitized CD25-depleted T cells increased tumor vaccine efficacy. The enhanced antitumor effect achieved by ex vivo depletion of CD25? Treg cells was similar to that achieved by in vivo depletion of all CD4? T cells. Depletion of CD25? Treg cells resulted in elevated frequencies of tumor-reactive CD8 and CD4? T cells and increased CD8-to-Treg cell ratios inside tumor masses. All mice given presensitized CD25-depleted T cells survived a tumor rechallenge, indicating the development of long-term CD8? T-cell memory to tumor antigens. These observations should aid in the future design of immunotherapeutic approaches that promote the generation of both acute and long-term antitumor immunity.     

Induction of tolerance to immunogenic tumor antigens associated with lymphomagenesis in HOX11 transgenic mice

Transgenic mice expressing human HOX11 in B lymphocytes die prematurely from lymphomas that initiate in the spleen and frequently disseminate to distant sites. Preneoplastic hematopoiesis in these mice is unperturbed. We now report that expression of the HOX11 transgene does not affect the ability of dendritic cells (DCs) to process and present foreign peptides and activate antigen-specific T cell responses. We also show that nontransgenic DCs presenting peptides derived from the human HOX11 protein are highly efficient stimulators of autologous T cells, whereas transgenic T cells are nonresponsive to peptides derived from the HOX11 transgene and the murine Meis1 protein. HOX11 transgenic mice thus show normal development of tolerance to immunogenic antigens expressed throughout B cell maturation. DCs pulsed with cell lysates prepared from lymphomas, obtained from HOX11 transgenic mice with terminal lymphoma, activate T cells from nontransgenic and premalignant transgenic mice, whereas T cells isolated from lymphomatous transgenic mice are nonresponsive to autologous tumor cell antigens. These data indicate that HOX11 lymphoma cells express tumor-rejection antigens that are recognized as foreign in healthy transgenic mice and that lymphomagenesis is associated with the induction of anergy to tumor antigen-specific T cells. These findings are highly relevant for the development of immunotherapeutic protocols for the treatment of lymphoma.     

CD11c(+) dendritic cells maintain antigen processing, presentation capabilities, and CD4(+) T-cell priming efficacy under hypercholesterolemic conditions associated with atherosclerosis

Recent reports suggest dyslipidemia impairs dendritic cell (DC) function and adaptive immunity. This study aimed to characterize the effect of hypercholesterolemia on antigen-presenting cell function of DCs and DC-dependent CD4(+) T-cell responses. DCs incubated in vitro with acetylated low-density lipoprotein cholesterol with or without an acyl-coenzyme A:cholesterol acyl-transferase inhibitor maintained their ability to prime CD4(+) T cells. Analysis of T-cell proliferation and interferon-gamma and tumor necrosis factor-alpha production after ex vivo coculture of na?ve CD4(+) T cells with splenic, inguinal, or iliac DCs from low-density lipoprotein receptor-deficient (LDLR(-/-)) or apolipoprotein E-deficient (ApoE(-/-)) mice fed an atherogenic diet highlighted DC efficacy in effector T-cell generation under hypercholesterolemic conditions. Adoptive transfer of carboxyfluorescein diacetate, succinimidyl ester (CFSE)-labeled na?ve CD4(+) T cells in LDLR(-/-) recipients and subsequent immunization demonstrated effective priming of na?ve T cells in hypercholesterolemic mice. CFSE dilution analyses revealed that hypercholesterolemic DCs were equipotent in na?ve CD4(+) T-cell priming efficacy with normocholesterolemic DCs. Quantitative real-time PCR and flow cytometric analyses demonstrated that DC expression of multiple molecules involved in antigen processing, presentation, and T-cell stimulation remained unaltered by dyslipidemia. Finally, endogenous antigen-primed CD4(+) T cells responded equivalently to a secondary ex vivo antigenic challenge, regardless of whether they were primed in vivo under hypercholesterolemic or control conditions, demonstrating that all essential steps in CD4(+) T-cell responses remain intact under atherogenic conditions. This study affirms that the adaptive immune response prevails under the hypercholesterolemic conditions present in atherosclerosis. In particular, DCs remain functional antigen-presenting cells and maintain their ability to prime CD4(+) T cells even when cholesterol-loaded.     

Activation of the aryl hydrocarbon receptor promotes allograft-specific tolerance through direct and dendritic cell-mediated effects on regulatory T cells

VAF347 is a low-molecular-weight compound, which activates the aryl hydrocarbon receptor (AhR). Herein, we report that oral administration of a water-soluble derivative of VAF347 (VAG539) promotes long-term graft acceptance and active tolerance in Balb/c mice that receive a transplant of MHC-mismatched pancreatic islet allografts. In vivo VAG539 treatment results in increased frequency of splenic CD4(+) T cells expressing CD25 and Foxp3, markers associated with regulatory T (Tr) cells, and in vitro VAF347 treatment of splenic CD4(+) T cells improved CD4(+)CD25(+)Foxp3(+) T-cell survival. Interestingly, transfer of CD11c(+) dendritic cells (DCs), but not of CD4(+) T or CD19(+) B cells, from VAG539-treated long-term tolerant hosts into mice that recently underwent transplantation resulted in donor (C57Bl/6)-specific graft acceptance and in a significantly higher frequency of splenic CD4(+)CD25(+)Foxp3(+) Tr cells. Furthermore, the transfer of CD4(+)CD25(+) T cells from these mice into mice that recently underwent transplantation promoted graft acceptance. Similarly, cell therapy with in vitro VAF347-treated bone marrow-derived mature DCs prevented islet graft rejection, and reduced OVA-specific T-cell responses in OVA-immunized mice. Collectively, our data indicate that AhR activation induces islet allograft-specific tolerance through direct as well as DC-mediated effects on Tr-cell survival and function.     

Therapeutic effect of tolerogenic dendritic cells in established collagen‐induced arthritis is associated with a reduction in Th17 responses

Objective

Tolerogenic dendritic cells (DCs) are antigen‐presenting cells with an immunosuppressive function. They are a promising immunotherapeutic tool for the attenuation of pathogenic T cell responses in autoimmune arthritis. The aims of this study were to determine the therapeutic action of tolerogenic DCs in a type II collagen–induced arthritis model and to investigate their effects on Th17 cells and other T cell subsets in mice with established arthritis.

Methods

Tolerogenic DCs were generated by treating bone marrow–derived DCs with dexamethasone and vitamin D₃ during lipopolysaccharide‐induced maturation. Mice with established arthritis received 3 intravenous injections of tolerogenic DCs, mature DCs, or saline. Arthritis severity was monitored for up to 4 weeks after treatment. Fluorescence‐labeled tolerogenic DCs were used for in vivo trafficking studies. The in vivo effect of tolerogenic DCs on splenic T cell populations was determined by intracellular cytokine staining and flow cytometry.

Results

Tolerogenic DCs displayed a semi‐mature phenotype, produced low levels of inflammatory cytokines, and exhibited low T cell stimulatory capacity. Upon intravenous injection into arthritic mice, tolerogenic DCs migrated to the spleen, liver, lung, feet, and draining lymph nodes. Treatment of arthritic mice with type II collagen–pulsed tolerogenic DCs, but not unpulsed tolerogenic DCs or mature DCs, significantly inhibited disease severity and progression. This improvement coincided with a significant decrease in the number of Th17 cells and an increase in the number of interleukin‐10–producing CD4+ T cells, whereas tolerogenic DC treatment had no detectable effect on Th1 cells or interleukin‐17–producing γ/δ T cells.

Conclusion

Treatment with type II collagen–pulsed tolerogenic DCs decreases the proportion of Th17 cells in arthritic mice and simultaneously reduces the severity and progression of arthritis.

     

B7-H1 (PD-L1) on T cells is required for T-cell-mediated conditioning of dendritic cell maturation

Studies have shown that T-cell-dendritic cell (DC) interaction is required for efficient DC maturation. However, the identities of the molecules that mediate the interaction in vivo are largely unknown. Here, we show that maturation of DCs as well as CD8 T-cell responses were impaired in B7-H1-deficient (B7-H1^−/−) mice to influenza virus infection. Both defects were restored by transferring B7-H1-expressing naïve T cells into B7-H1^−/− mice. Similarly, transferring DCs from wild-type mice or from RAG1^−/− mice that had been injected with B7-H1-expressing naïve T cells also restored CD8 T-cell responses in B7-H1^−/− mice. These results demonstrate that B7-H1 on naïve T cells is required to condition immature DCs to undergo efficient maturation when they encounter microbial infection. In return, the mature DCs stimulate a robust T-cell reponse against the infecting pathogen.     

Immunomodulatory drug costimulates T cells via the B7-CD28 pathway

Although thalidomide (Thal) does not directly induce T-cell activation, it increases proliferation of T cells following CD3 activation. In this study, we examined the immunomodulatory effects of a more potent analog of Thal, immunomodulatory drug (IMiD), on T cells. Although IMiD3 does not directly stimulate proliferation of normal donor CD3+ T cells, it significantly costimulates proliferation of CD3+ T cells induced by CD3 ligation (stimulation index [SI], 2.4), immature dendritic cells (DCs; SI, 2.1), and mature DCs (SI, 2.6). T-cell proliferation triggered by DCs was abrogated by cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA-4-Ig), and IMiD3 partially overcomes this inhibitory effect. IMiD3 also overcomes the inhibitory effects of CTLA-4-Ig on Epstein-Barr virus (EBV) and influenza (Flu)-specific CD4 and CD8 T-cell responses, as measured by cytokine capture and enzyme-linked immunosorbent spot (ELISPOT) assay. IMiD3 did not induce up-regulation of CD28 expression on T cells, or of CD80-CD86 expression on dendritic cells. Importantly, IMiD3 triggers tyrosine phosphorylation of CD28 on T cells, followed by activation of nuclear factor kappaB (NF-kappaB), a known downstream target of CD28 signaling. These results therefore define the costimulatory mechanism whereby IMiD3 induces T-cell activation and provide the cellular and molecular basis for use of IMiD3 as an adjuvant in immunotherapeutic treatment strategies for multiple myeloma.     

Sensitivity toward sorafenib and sunitinib varies between different activating and drug-resistant FLT3-ITD mutations

OBJECTIVE: Activating mutations in FLT3 are known to be a frequent transforming event in acute myeloid leukemia. Small molecule-inhibitor therapy targeting the FLT3 kinase is, therefore, an attractive strategy. FLT3 kinase inhibitors, such as PKC412, have already entered clinical trials. Even though results are encouraging, emergence of primary and secondary resistance does occur in the majority of patients. Thus, it will be crucial to carefully characterize the activity of every single compound against different activating and resistance FLT3-internal tandem duplication (ITD) mutations. Here we tested the efficacy of sunitinib and sorafenib to inhibit primary FLT3 activating mutations (ITD and D835Y) and of secondary resistance mutations. METHODS: Ba/F3 cell lines stably expressing oncogenic FLT3 mutations were used to calculate cellular IC(50) values for sunitinib and sorafenib using cell proliferation assays. Differential IC(50) values for sorafenib toward FLT3-ITD and FLT3-D835Y were confirmed by Western blotting. Cell death was measured by propidium-iodide staining and flow cytometry. RESULTS: Sorafenib inhibits FLT3-ITD more potent than FLT3-D835Y, while sunitinib is equally effective against both mutant forms of FLT3. Importantly, sensitivity toward sorafenib and sunitinib varies between the different secondary FLT3-ITD resistance mutations. CONCLUSIONS: These results establish sensitivity profiles for the FLT3 inhibitors sunitinib and sorafenib. This may help to develop rational treatment strategies for acute myeloid leukemia with these compounds.     

Platelet function is disturbed by the angiogenesis inhibitors sunitinib and sorafenib,but unaffected by bevacizumab

Introduction

At the clinical introduction of antiangiogenic agents as anticancer agents, no major toxicities were expected as merely just endothelial cells (ECs) in tumors would be affected. However, several (serious) toxicities became apparent, of which underlying mechanisms are largely unknown. We investigated to what extent sunitinib (multitargeted antiangiogenic tyrosine kinase inhibitor (TKI)), sorafenib (TKI) and bevacizumab [specific antibody against vascular endothelial growth factor (VEGF)] may impair platelet function, which might explain treatment-related bleedings.

Materials and methods

In vitro, the influence of sunitinib, sorafenib, and bevacizumab on platelet aggregation, P-selectin expression and fibrinogen binding, platelet–EC interaction, and tyrosine phosphorylation of c-Src was studied by optical aggregation, flow cytometry, real-time perfusion, and western blotting. Ex vivo, platelet aggregation was analyzed in 25 patients upon sunitinib or bevacizumab treatment. Concentrations of sunitinib, VEGF, and platelet and EC activation markers were measured by LC–MS/MS and ELISA.

Results

In vitro, sunitinib and sorafenib significantly inhibited platelet aggregation (20 μM sunitinib: 71.3%, p < 0.001; 25 μM sorafenib: 55.8%, p = 0.042). Sorafenib and sunitinib significantly inhibited P-selectin expression on platelets. Exposure to both TKIs resulted in a reduced tyrosine phosphorylation of c-Src. Ex vivo, within 24 h sunitinib impaired platelet aggregation (83.0%, p = 0.001, N = 8). Plasma concentrations of sunitinib, VEGF, and platelet/EC activation markers were not correlated with disturbed aggregation. In contrast, bevacizumab only significantly impaired platelet aggregation in vitro at high concentrations, but not ex vivo.

Conclusion

Sunitinib significantly inhibits platelet aggregation in patients already after 24 h of first administration, whereas bevacizumab had no effect on aggregation. These findings may explain the clinically observed bleedings during treatment with antiangiogenic TKIs.

     

DIgR2, dendritic cell-derived immunoglobulin receptor 2, is one representative of a family of IgSF inhibitory receptors and mediates negative regulation of dendritic cell-initiated antigen-specific T-cell responses

Dendritic cells (DCs) are specialized antigen-presenting cells that play crucial roles in the initiation and regulation of immune responses. Maturation and activation of DCs are controlled by a balance of the inhibitory and activating signals transduced through distinct surface receptors. Many inhibitory receptors expressed by DCs have been identified, whereas the new members and their functions need further investigation. In this study, we functionally characterized DC-derived immunoglobulin receptor 2 (DIgR2) as a novel representative of a family of inhibitory receptors belonging to the immunoglobulin superfamily. We show that DIgR2 contains 2 immunoreceptor tyrosine-based inhibitory motifs (ITIMs) within its cytoplasmic region and that DIgR2 associates with Src homology-2 domain-containing protein tyrosine phosphatases-1 (SHP-1). Blockade of DIgR2 on DCs by pretreatment with DIgR2-Ig fusion protein or by silencing with specific small interfering RNA enhances DC-initiated T-cell proliferation and antigen-specific T-cell responses both in vitro and in vivo. Furthermore, immunization of mice with antigen-pulsed, DIgR2-silenced DCs elicits more potent antigen-specific CD4+ and CD8+ T-cell responses, thus protecting the vaccinated mice from tumor challenge more effectively. Our data suggest that DIgR2 is a functionally inhibitory receptor and can mediate negative signaling to regulate DC-initiated antigen-specific T-cell responses.     

Inhibition of Bcl-2 antiapoptotic members by obatoclax potently enhances sorafenib-induced apoptosis in human myeloid leukemia cells through a Bim-dependent process

Interactions between the multikinase inhibitor sorafenib and the BH3-mimetic obatoclax (GX15-070) were examined in human acute myeloid leukemia (AML) cells. Treatment with sorafenib/obatoclax induced pronounced apoptosis in and reduced the clonogenic growth of multiple AML lines and primary AML cells but not normal CD34(+) cells. Sorafenib triggered rapid and pronounced Mcl-1 down-regulation accompanied by enhanced binding of Bim to Bcl-2 and Bcl-xL, effects that were abolished by obatoclax coadministration. Notably, shRNA knockdown of Bim, Bak, or Bax, but not Noxa, significantly attenuated obatoclax/sorafenib lethality, whereas ectopic expression of Mcl-1 exerted a protective effect. Furthermore, exposure of leukemia cells to sorafenib and obatoclax markedly induced autophagy, reflected by rapid and pronounced LC3 processing and LC3-green fluorescent protein (GFP) punctate formation. Multiple autophagy inhibitors or VPS34 knockdown, significantly potentiated sorafenib/obatoclax lethality, indicating a cytoprotective role for autophagy in this setting. Finally, studies in a xenograft mouse model revealed that combined sorafenib/obatoclax treatment markedly reduced tumor growth and significantly prolonged survival in association with Mcl-1 down-regulation and apoptosis induction, whereas agents administered individually had only modest effects. These findings suggest that combining sorafenib with agents that inhibit Mcl-1 and Bcl-2/Bcl-xL such as obatoclax may represent a novel and potentially effective strategy in AML.     

Killer artificial antigen-presenting cells: a novel strategy to delete specific T cells

Several cell-based immunotherapy strategies have been developed to specifically modulate T cell–mediated immune responses. These methods frequently rely on the utilization of tolerogenic cell–based antigen-presenting cells (APCs). However, APCs are highly sensitive to cytotoxic T-cell responses, thus limiting their therapeutic capacity. Here, we describe a novel bead-based approach to modulate T-cell responses in an antigen-specific fashion. We have generated killer artificial APCs (aAPCs) by coupling an apoptosis-inducing -Fas (CD95) IgM mAb together with HLA-A₂ Ig molecules onto beads. These aAPCs deplete targeted antigen-specific T cells in a Fas/Fas ligand (FasL)–dependent fashion. T-cell depletion in cocultures is rapidly initiated (30 minutes), dependent on the amount of aAPCs and independent of activation-induced cell death (AICD). aAPCs represent a novel technology that can control T cell–mediated immune responses, and therefore has potential for use in treatment of autoimmune diseases and allograft rejection.     

Conditional ablation of CD205+ conventional dendritic cells impacts the regulation of T-cell immunity and homeostasis in vivo

Dendritic cells (DCs) are composed of multiple subsets that play a dual role in inducing immunity and tolerance. However, it is unclear how CD205(+) conventional DCs (cDCs) control immune responses in vivo. Here we generated knock-in mice with the selective conditional ablation of CD205(+) cDCs. CD205(+) cDCs contributed to antigen-specific priming of CD4(+) T cells under steady-state conditions, whereas they were dispensable for antigen-specific CD4(+) T-cell responses under inflammatory conditions. In contrast, CD205(+) cDCs were required for antigen-specific priming of CD8(+) T cells to generate cytotoxic T lymphocytes (CTLs) mediated through cross-presentation. Although CD205(+) cDCs were involved in the thymic generation of CD4(+) regulatory T cells (Tregs), they maintained the homeostasis of CD4(+) Tregs and CD4(+) effector T cells in peripheral and mucosal tissues. On the other hand, CD205(+) cDCs were involved in the inflammation triggered by Toll-like receptor ligand as well as bacterial and viral infections. Upon microbial infections, CD205(+) cDCs contributed to the cross-priming of CD8(+) T cells for generating antimicrobial CTLs to efficiently eliminate pathogens, whereas they suppressed antimicrobial CD4(+) T-cell responses. Thus, these findings reveal a critical role for CD205(+) cDCs in the regulation of T-cell immunity and homeostasis in vivo.