Survival Of Dendritic Cells Research Articles

Immunoregulatory properties of the cytokine IL-34.

Interleukin-34 is a cytokine with only partially understood functions, described for the first time in 2008. Although IL-34 shares very little homology with CSF-1 (CSF1, M-CSF), they share a common receptor CSF-1R (CSF-1R) and IL-34 has also two distinct receptors (PTP-ζ) and CD138 (syndecan-1). To make the situation more complex, IL-34 has also been shown as pairing with CSF-1 to form a heterodimer. Until now, studies have demonstrated that this cytokine is released by some tissues that differ to those where CSF-1 is expressed and is involved in the differentiation and survival of macrophages, monocytes, and dendritic cells in response to inflammation. The involvement of IL-34 has been shown in areas as diverse as neuronal protection, autoimmune diseases, infection, cancer, and transplantation. Our recent work has demonstrated a new and possible therapeutic role for IL-34 as a Foxp3+ Treg-secreted cytokine mediator of transplant tolerance. In this review, we recapitulate most recent findings on IL-34 and its controversial effects on immune responses and address its immunoregulatory properties and the potential of targeting this cytokine in human.

Cathepsin E Deficiency Ameliorates Graft-versus-Host Disease and Modifies Dendritic Cell Motility.

Microbial products influence immunity after allogeneic hematopoietic stem cell transplantation (allo-SCT). In this context, the role of cathepsin E (Ctse), an aspartate protease known to cleave bacterial peptides for antigen presentation in dendritic cells (DCs), has not been studied. During experimental acute graft-versus-host disease (GVHD), we found infiltration by Ctse-positive immune cells leading to higher Ctse RNA- and protein levels in target organs. In Ctse-deficient allo-SCT recipients, we found ameliorated GVHD, improved survival, and lower numbers of tissue-infiltrating DCs. Donor T cell proliferation was not different in Ctse-deficient vs. wild-type allo-SCT recipients in MHC-matched and MHC-mismatched models. Furthermore, Ctse-deficient DCs had an intact ability to induce allogeneic T cell proliferation, suggesting that its role in antigen presentation may not be the main mechanism how Ctse impacts GVHD. We found that Ctse deficiency significantly decreases DC motility in vivo, reduces adhesion to extracellular matrix (ECM), and diminishes invasion through ECM. We conclude that Ctse has a previously unrecognized role in regulating DC motility that possibly contributes to reduced DC counts and ameliorated inflammation in GVHD target organs of Ctse-deficient allo-SCT recipients. However, our data do not provide definite proof that the observed effect of Ctse−/− deficiency is exclusively mediated by DCs. A contribution of Ctse−/−-mediated functions in other recipient cell types, e.g., macrophages, cannot be excluded.

Tsc1 expression by dendritic cells is required to preserve T-cell homeostasis and response

Dendritic cells (DCs) are pivotal to the induction of adaptive T-cell immune responses. Recent evidence highlights a critical role of tuberous sclerosis complex 1 (Tsc1), a primarily upstream negative regulator of mammalian target of rapamycin (mTOR), in DC development, but whether and how Tsc1 directly regulate mature DC function in vivo remains elusive. Here we show that selective disruption of Tsc1 in DCs results in a lymphoproliferative disorder with the spontaneous activation of T cells. Tsc1 deficiency results in the activation of mTORC1-PPARγ pathway, which leads to the upregulation of neuropilin-1 (Nrp1) expression on DCs to stimulate naive T-cell proliferation. However, Tsc1-deficient DCs have defects in the ability to induce antigen-specific T-cell responses in vitro and in vivo owing to impaired survival during antigen transportation and presentation. Indeed, Tsc1 promotes DC survival through restraining independent mTORC1 and ROS-Bim pathways. Our study identifies Tsc1 as a crucial signaling checkpoint in DCs essential for preserving T-cell homeostasis and response.

Role of Fas and RANKL Signaling in Peripheral Immune Tolerance

The death receptor, Fas, has been well-characterized and is a critical factor in apoptosis in immune cells. Fas also has an important role in maintaining immune tolerance as demonstrated in the autoimmune-prone MRL/lpr mouse strain which carries a defect in Fas-mediated apoptosis of T cells. However, the role of Fas-independent apoptosis remains to be characterized in autoimmune diseases. In dendritic cells (DCs), binding of receptor activator of nuclear factor-κB ligand (RANKL) to RANK perpetuates the survival of mature DCs. However, cross-talk between the RANK/RANKL pathway and Fas-mediated signaling during the function or activation of DCs has not been well-studied. This short communication review describes a mechanism involving interactions between activated DCs and T cells in the autoimmune response of MRL/lpr mice and a novel Fas-independent apoptosis pathway in T cells that maintains peripheral tolerance, and controls autoimmunity in MRL/lpr mice.

Nuclear Translocation of SGPP-1 and Decrease of SGPL-1 Activity Contribute to Sphingolipid Rheostat Regulation of Inflammatory Dendritic Cells.

A balanced sphingolipid rheostat is indispensable for dendritic cell function and survival and thus initiation of an immune response. Sphingolipid levels are dynamically maintained by the action of sphingolipid enzymes of which sphingosine kinases, S1P phosphatases (SGPP-1/2) and S1P lyase (SGPL-1), are pivotal in the balance of S1P and sphingosine levels. In this study, we present that SGPP-1 and SGPL-1 are regulated in inflammatory dendritic cells and contribute to S1P fate. TLR-dependent activation caused SGPL-1 protein downregulation with subsequent decrease of enzymatic activity by two-thirds. In parallel, confocal fluorescence microscopy revealed that endogenous SGPP-1 was expressed in nuclei of naive dendritic cells and was translocated into the cytoplasmatic compartment upon inflammatory stimulation resulting in dephosphorylation of S1P. Mass spectrometric determination showed that a part of the resulting sphingosine was released from the cell, increasing extracellular levels. Another route of diminishing intracellular S1P was possibly taken by its export via ATP-binding cassette transporter C1 which was upregulated in array analysis, while the S1P transporter, spinster homolog 2, was not relevant in dendritic cells. These investigations newly describe the sequential expression and localization of the endogenous S1P regulators SGPP-1 and SGPL-1 and highlight their contribution to the sphingolipid rheostat in inflammation.

Src-like Adaptor Protein Promotes the CD103+ dendritic Cell Homeostasis in the Lung

Dendritic cells (DCs) along with mast cells function as sentinels for the innate immune system and perform as antigen presenting cells (APCs) to mount an adaptive immune response against invading pathogen. FLT3 receptor tyrosine kinase signaling has been shown to regulate the homeostatic mechanisms of subsets of DCs particularly, CD103+DCs compared to CD11b+DCs. CD103+DCs are regarded as APCs with superior capabilities to mount an effective immune response, thus understanding their homeostasis mechanism(s)/function is of paramount importance to devise effective therapeutics including DC vaccines. The Src-like adapter protein (SLAP) has been shown to dampen the signaling downstream of receptor tyrosine kinases including FLT3, cKit, and immune cell receptors including T cell receptor, B cell receptor, and Granulocyte-monocyte colony stimulating factor receptor via by recruiting c-Cbl, an ubiquitin ligase.Here, we report that SLAP deficient mice (KO) have reduced numbers of CD103+DC in lung while equal numbers in liver and kidney compared to control mice. To further confirm reduced CD103+DC in the lung, efferocytosis assays that are dependent upon CD+103 DC in lung epithelium to cleanse the apoptotic cells were performed. Flow cytometric quantification of CD103+DCs that uptake fluorescently labeled apoptotic cells administered via intranasal route and migrate to mediastinal lymph nodes confirmed reduced number of CD103+DCs in SLAP KO mice. Further analysis of DC progenitor populations showed reduced pre-DC progenitor in the lung in SLAP KO mice while bone marrow compartment showed equal progenitor populations including pre-DC and common dendritic progenitors suggesting the role of SLAP in localized FLT3 signaling in the lung. Consistently, DCs in lymphoid compartment including spleen, thymus, inguinal and popliteal lymph node did not show any defects. Upon further dissecting the cellular mechanism, SLAP KO DCs showed increased apoptosis while having similar proliferation potential in vivo at steady state.Bone marrow progenitors from SLAP KO mice failed to generate mature DCs in the presence of FLT3 ligand in vitrodue to enhanced apoptosis at early time points. Also, submaximal inhibition of FLT3 with an inhibitor, quizartinib partially rescues the apoptotic phenotype of SLAP KO bone marrow progenitors suggesting a cell-intrinsic role of SLAP in the survival of DCs. Biochemical analysis revealed that SLAP is directly recruited to the juxta-membrane residues of the FLT3 receptor in an inducible manner suggesting a direct role of SLAP in the regulation of FLT3 signaling. Phosphoflow analysis of DCs generated in the combined presence of GMCSF and FLT3 ligands showed that SLAP promotes the signaling to SHP2 while perturbs signaling to the mTOR pathway. Together these results suggest that SLAP is a critical regulator of CD103+DCs homeostasis in selective peripheral organs including the lung. DisclosuresNo relevant conflicts of interest to declare.

Anti-GITR therapy promotes immunity against malignant glioma in a murine model.

Regulatory T cells (Tregs) are potently immunosuppressive cells that accumulate within the glioma microenvironment. The reduction in their function and/or trafficking has been previously shown to enhance survival in preclinical models of glioma. Glucocorticoid-induced TNFR-related protein (GITR) is a tumor necrosis factor superfamily receptor enriched on Tregs that has shown promise as a target for immunotherapy. An agonistic antibody against GITR has been demonstrated to inhibit Tregs in a number of models and has only been recently addressed in glioma. In this study, we examined the modality of the antibody function at the tumor site as opposed to the periphery as the blood-brain barrier prevents efficient antibody delivery to brain tumors. Mice harboring established GL261 tumors were treated with anti-GITR monotherapy and were shown to have a significant increase in overall survival (p<0.01) when antibodies were injected directly into the glioma core, whereas peripheral antibody treatment only had a modest effect. Peripheral treatment resulted in a significant decrease in granzyme B (GrB) expression by Tregs, whereas intratumoral treatment resulted in both a decrease in GrB expression by Tregs and their selective depletion, which was largely mediated by FcγR-mediated destruction. We also discovered that anti-GITR treatment results in the enhanced survival and functionality of dendritic cells (DCs)-a previously unreported effect of this immunotherapy. In effect, this study demonstrates that the targeting of GITR is a feasible and noteworthy treatment option for glioma, but is largely dependent on the anatomical location in which the antibodies are delivered.

Plasmacytoid Dendritic Cells Die by the CD8 T Cell-Dependent Perforin Pathway during Acute Nonviral Inflammation.

Regulation of the inflammatory response involves the control of dendritic cell survival. To our knowledge, nothing is known about the survival of plasmacytoid dendritic cells (pDC) in such situation. pDC are specialized in type I IFN (IFN-I) secretion to control viral infections, and IFN-I also negatively regulate pDC survival during the course of viral infections. In this study, we asked about pDC behavior in the setting of virus-free inflammation. We report that pDC survival was profoundly reduced during different nonviral inflammatory situations in the mouse, through a mechanism independent of IFN-I and TLR signaling. Indeed, we demonstrated that during inflammation, CD8(+) T cells induced pDC apoptosis through the perforin pathway. The data suggest, therefore, that pDC have to be turned down during ongoing acute inflammation to not initiate autoimmunity. Manipulating CD8(+) T cell response may therefore represent a new therapeutic opportunity for the treatment of pDC-associated autoimmune diseases, such as lupus or psoriasis.

Inhibition of SIRPα in dendritic cells potentiates potent antitumor immunity

ABSTRACTDespite their central function in tumor immunity, dendritic cells (DCs) can respond to inhibitory signals and become tolerogenic, curtailing T cell responses in vivo. Here, we provide the evidence for an inhibitory function of signal regulatory protein (SIRP) α in DC survival and activation. In tumors from human liver cancer patients, infiltrative DCs expressed elevated levels of SIRPα, which is correlated with the induction of immune tolerance within the tumors. Silencing of SIRPα resulted in a significant increase in the longevity of antigen-pulsed DCs in the draining lymph nodes. In addition, SIRPα controls the activation and output of DCs. Silencing of DC-expressed SIRPα induced spontaneous and enhanced production of IL12 and costimulatory molecules, resulting in more potent cytotoxic T lymphocyte responses, including the eradication of previously established solid tumors. SIRPα exerted such effects, at least in part, via the association and sequestration of p85 subunit of PI3K. Thus, SIRPα is a critical regulator of DC lifespan and activity, and its inhibition might improve the clinical efficacy of DC-based tumor vaccines.

Leishmania infantum and Leishmania braziliensis: Differences and Similarities to Evade the Innate Immune System.

Visceral leishmaniasis is a severe form of the disease, caused by Leishmania infantum in the New World. Patients present an anergic immune response that favors parasite establishment and spreading through tissues like bone marrow and liver. On the other hand, Leishmania braziliensis causes localized cutaneous lesions, which can be self-healing in some individuals. Interactions between host and parasite are essential to understand disease pathogenesis and progression. In this context, dendritic cells (DCs) act as essential bridges that connect innate and adaptive immune responses. In this way, the aim of this study was to compare the effects of these two Leishmania species, in some aspects of human DCs’ biology for better understanding of the evasion mechanisms of Leishmania from host innate immune response. To do so, DCs were obtained from monocytes from whole peripheral blood of healthy volunteer donors and from those infected with L. infantum or L. braziliensis for 24 h. We observed similar rates of infection (around 40%) as well as parasite burden for both Leishmania species. Concerning surface molecules, we observed that both parasites induced CD86 expression when DCs were infected for 24 h. On the other hand, we detected a lower surface expression of CD209 in the presence of both L. braziliensis and L. infantum, but only the last one promoted the survival of DCs after 24 h. Therefore, DCs infected by both Leishmania species showed a higher expression of CD86 and a decrease of CD209 expression, suggesting that both enter DCs through CD209 molecule. However, only L. infantum had the ability to inhibit DC apoptotic death, as an evasion mechanism that enables its spreading to organs like bone marrow and liver. Lastly, L. braziliensis was more silent parasite, once it did not inhibit DC apoptosis in our in vitro model.

Lack of TAK1 in dendritic cells inhibits the contact hypersensitivity response induced by trichloroethylene in local lymph node assay

Trichloroethylene (TCE) is a ubiquitous environmental contaminant. Occupational TCE exposure has been associated with severe, generalized contact hypersensitivity (CHS) skin disorder. The development of CHS depends on innate and adaptive immune functions. Transforming growth factor-β activated kinase-1 (TAK1) controls the survival of dendritic cells (DCs) that affect the immune system homeostasis. We aimed to investigate the role of TAK1 activity in DC on TCE-induced CHS response. Control mice and DC-specific TAK1 deletion mice were treated with 80% (v/v) TCE using local lymph node assay (LLNA) to establish a TCE-induced CHS model. The draining lymph nodes (DLNs) were excised and the lymphocytes were measure for proliferation by BrdU-ELISA, T-cell phenotype analysis by flow cytometry and signaling pathway activation by western blot. The ears were harvested for histopathological analysis. Control mice in the 80% TCE group displayed an inflammatory response in the ears, increased lymphocyte proliferation, elevated regulatory T-cell and activated T-cell percentages, and more IFN-γ producing CD8+ T cells in DLNs. In contrast to control mice, DC-specific TAK1 deletion mice in the 80% TCE group showed an abolished CHS response and this was associated with defective T-cell expansion, activation and IFN-γ production. This effect may occur through Jnk and NF-κB signaling pathways. Overall, this study demonstrates a pivotal role of TAK1 in DCs in controlling TCE-induced CHS response and suggests that targeting TAK1 function in DCs may be a viable approach to preventing and treating TCE-related occupational health hazards.

Rapamycin increases RSV RNA levels and survival of RSV-infected dendritic cell depending on T cell contact

The macrolide rapamycin inhibits mTOR (mechanist target of rapamycin) function and has been broadly used to unveil the role of mTOR in immune responses. Inhibition of mTOR on dendritic cells (DC) can influence cellular immune response and the survival of DC. RSV is the most common cause of hospitalization in infants and is a high priority candidate to vaccine development. In this study we showed that rapamycin treatment on RSV-infected murine bone marrow-derived DC (BMDC) decreases the frequency of CD8+CD44high T cells. However, inhibition of mTOR on RSV-infected BMDC did not modify the activation phenotype of these cells. RSV-RNA levels increase when infected BMDC were treated with rapamycin. Moreover, we observed that rapamycin diminishes apoptosis cell death of RSV-infected BMDC co-culture with T cells and this effect was abolished when the cells were co-cultured in a transwell system that prevents cell-to-cell contact or migration. Taken together, these data indicate that rapamycin treatment present a toxic effect on RSV-infected BMDC increasing RSV-RNA levels, affecting partially CD8 T cell differentiation and also increasing BMDC survival in a mechanism dependent on T cell contact.

Apolipoproteins L control cell death triggered by TLR3/TRIF signaling in dendritic cells.

Apolipoproteins L (ApoLs) are Bcl-2-like proteins expressed under inflammatory conditions in myeloid and endothelial cells. We found that Toll-like receptor (TLR) stimuli, particularly the viral mimetic polyinosinic:polycytidylic acid (poly(I:C)), specifically induce ApoLs7/11 subfamilies in murine CD8α(+) dendritic cells (DCs). This induction requires the TLR3/TRIF (where TRIF is TIR domain containing adapter-inducing interferon β) signaling pathway and is dependent on IFN-β in all ApoLs subfamilies except for ApoL7c. Poly(I:C) treatment of DCs is also associated with induction of both cell death and autophagy. ApoLs expression is related to promotion of DC death by poly(I:C), as ApoLs7/11 knockdown increases DC survival and ApoLs7 are associated with the anti-apoptotic protein Bcl-xL (where Bcl-xL is B-cell lymphoma extra large). Similarly, in human monocyte-derived DCs poly(I:C) induces both cell death and the expression of ApoLs, principally ApoL3. Finally, the BH3-like peptide of ApoLs appears to be involved in the DC death-promoting activity. We would like to propose that ApoLs are involved in cell death linked to activation of DCs by viral stimuli.

Activation-induced cell death in GM-CSF differentiated dendritic cells is dependent on Sphingosine kinase 1

Abstract Sphingosine 1-phosphate (S1P) is an immune modulatory lipid mediator and has been implicated in numerous pathophysiological processes. S1P is produced by sphingosine kinase 1 (Sphk1) and Sphk2. Dendritic cells are central for the direction of immune responses and crucially involved in autoimmunity and cancerogenesis. In this study we examined the function and survival of dendritic cells under long-term inflammatory stimulation. We observed that GM-CSF differentiated dendritic cells undergo activation-induced cell death upon LPS stimulation with an increased metabolic activity after short-term stimulation followed by a rapid activation of caspase 3 and subsequent augmented apoptosis. Importantly, we highlighted a profound role of Sphk1 in secretion of inflammatory cytokines and survival of dendritic cells that might be mediated by a change in sphingolipid level as well as by a change in STAT3 expression. Cell expansion in Sphk1-deficient cells treated with the functional S1P receptor antagonist FTYP was reduced. Importantly, in dendritic cells we failed to find a compensatory upregulation of Sphk2 mRNA in SphK1-deficient cells. Instead, we observed a massive increase in Sphk1 mRNA level upon long-term stimulation with LPS in wild type cells that might function as an attempt to rescue from inflammation caused cell death. Taken together, in this investigation we describe details of a crucial involvement of sphingosine, S1P and Sphk1 in activation-induced cell death during long-term immunogenic activity of dendritic cells.

TAK1 deficiency in dendritic cells inhibits adaptive immunity in SRBC-immunized C57BL/6 mice.

Dendritic cells (DCs) are important in the initiation of primary T‐cell responses, while transforming growth factor‐β (TGF‐β)‐activated kinase 1 (TAK1) is a critical regulator of DC survival and homeostasis. This study evaluated the T‐cell dependent antibody response (TDAR) to sheep red blood cells (SRBC) on a DC‐specific TAK1‐deficient mice model. The results showed that TAK1 deficiency in DCs significantly suppressed the humoral and cellular immune response in mice. DC‐specific TAK1 deletion impaired splenic T‐cell population and conventional DCs, abolished the cytokine production of splenic T cells and down‐regulated some functional gene expression in the spleen. Collectively, this study suggests that TAK1 plays an essential role in the development of the humoral immune response.

Activation-Induced Cell Death of Dendritic Cells Is Dependent on Sphingosine Kinase 1.

Sphingosine 1-phosphate (S1P) is an immune modulatory lipid mediator and has been implicated in numerous pathophysiological processes. S1P is produced by sphingosine kinase 1 (Sphk1) and Sphk2. Dendritic cells (DCs) are central for the direction of immune responses and crucially involved in autoimmunity and cancerogenesis. In this study we examined the function and survival of bone marrow-derived DCs under long-term inflammatory stimulation. We observed that differentiated cells undergo activation-induced cell death (AICD) upon LPS stimulation with an increased metabolic activity shortly after stimulation, followed by a rapid activation of caspase 3 and subsequent augmented apoptosis. Importantly, we highlight a profound role of Sphk1 in secretion of inflammatory cytokines and survival of dendritic cells that might be mediated by a change in sphingolipid levels as well as by a change in STAT3 expression. Cell growth during differentiation of Sphk1-deficient cells treated with the functional S1P receptor antagonist FTYP was reduced. Importantly, in dendritic cells we did not observe a compensatory regulation of Sphk2 mRNA in Sphk1-deficient cells. Instead, we discovered a massive increase in Sphk1 mRNA concentration upon long-term stimulation with LPS in wild type cells that might function as an attempt to rescue from inflammation-caused cell death. Taken together, in this investigation we describe details of a crucial involvement of sphingolipids and Sphk1 in AICD during long-term immunogenic activity of DCs that might play an important role in autoimmunity and might explain the differences in immune response observed in in vivo studies of Sphk1 modulation.

Antitumor effect of dendritic cells transfected with prostate-specific membrane antigen recombinant adenovirus on prostate cancer: An in vitro study.

The aim of the present study was to construct a dendritic cell (DC) vaccine transfected with a prostate-specific membrane antigen (PSMA) recombinant adenovirus and to observe the ability of the recombinant DCs in eliciting a PSMA-directed T‑cell response to prostate cancer (PCa) in vitro. Replication‑defective adenoviral vectors, were constructed using the Adxsi system. The virus titer was measured by TCID50 assay, and the expression of the PSMA gene was identified by polymerase chain reaction (PCR) generation of peripheral blood mononuclear cell (PBMC) derived DCs in vitro. In addition, a PE‑7AAD apoptosis and necrosis kit was applied to detect the survival of DCs at different MOI values to determine the optimal MOI. Morphological changes of DC were observed under a fluorescence microscope, expression of the PSMA protein was detected by western blotting 48 h after transfection, the expression of DC markers prior to and following transfection was detected by direct immunofluorescence, and the interleukin (IL)-12 concentration in the culture supernatant of the three groups was measured by ELISA. The antitumor effect of DC-stimulated cytotoxic T lymphocytes (CTLs) on different PCa cell lines was analyzed using a Cell Counting Kit‑8 assay. The optimal MOI value was determined to be 200. The PSMA protein was expressed in DCs, and the recombinant adenovirus did not impact the maturation and morphological changes of the DCs. The expression levels of the co-stimulatory molecules, CD80, CD83, CD86 and HLA‑DR, were significantly higher in the Ad‑PSMA‑DC group than in the other two groups (P<0.05). The concentration of IL‑12 in the supernatant of the Ad‑PSMA‑DC group (79.51±1.60 pg) was comparable with that of the Ad‑GFP‑DC group (not significantly different), and the two were significantly higher than the non‑transfection group (P<0.05). The optimal effector to target (E:T) ratio was determined to be 40:1. However, at the same E:T ratio, the cytotoxic activity of PSMA‑DC‑T against the LNCap cells was markedly stronger than its activity against the other target cells (DU145 and 22RV; P<0.05); furthermore, the the cytotoxic activity of PSMA-DC-T against the LNCap cells was significantly higher than the anti‑LNCap effect of DC‑T cells in other groups (P<0.05). In vitro experiments indicated that mature DCs transfected with Ad‑PSMA secreted high concentrations of IL‑12 and elicited potent antitumor immune responses targeting PSMA‑expressing cells by stimulating the cytotoxic activity of CTLs.

Ixazomib Impairs Dendritic Cell Function and T Cell Proliferation and Affects the Development of GvHD in a Schedule-Dependent Fashion

Background: Targeting T cells alone has yielded limited success in the prevention of graft-versus-host disease (GvHD) following allogeneic blood and marrow transplantation (BMT). Dendritic cells (DCs) play a central role in alloreactivity and therefore represent a suitable target. Proteasome inhibitors (PI), with their ability to inhibit the function and maturation of DC, have prompted investigators to examine their potential role in the prevention of GvHD. The investigational PI, ixazomib (IXZ), dissociates rapidly from 20S and is therefore truly reversible. It is also orally bioavailable. Our aim in this study was to explore its effect on healthy volunteer peripheral blood dendritic and T cells and in a pre-clinical GvHD mouse model.Methods: To characterize the effects of IXZ on healthy volunteer peripheral blood DCs, DCs were isolated using EasySep Pan-DC Pre-Enrichment Cocktail with purity over 90% (STEMCELL Technologies). DCs were then treated with IXZ at different concentrations (10-40nM) for 4 hrs and then stimulated with lipopolysaccharide (LPS) for 16 hrs. After this treatment, DCs were surface stained with antibodies against maturation markers and analyzed by flow cytometry. DC survival was evaluated with 7AAD staining and FACS analysis. To assess the effect of IXZ on the production of pro-inflammatory cytokines, DCs were incubated with IXZ at increasing concentration before or after the addition of LPS. Total pro-inflammatory cytokines in the supernatant of tissue culture were measured using EMD Millipore cytokine arrays. Standard mixed lymphocyte reaction and T cell proliferation assays were used to evaluate T cell function. At a minimum, all experiments were done in triplicate. Unpaired t test was used for statistical analysis. A p-value < 0.05 was considered significant. The B6 → BALB/c pre-clinical GvHD model was adopted to evaluate the effect of IXZ on GvHD development. Mice were transplanted in 3 groups. Group 1 received a lethal dose of total body irradiation (TBI), donor bone marrow (BM) cells, and IXZ, group 2 received TBI, donor BM cells donor splenocytes, and a vehicle, and group 3 received TBI, donor BM cells, donor splenocytes, and IXZ. The dose of BM cells and splenocytes was 5 X 106 each. IXZ was given at 1.5 mg/kg subcutaneously. Two dosing schedules were tested in 2 separate experiments: day-1 and +2 or day +1 and +4.Results: IXZ inhibited the expression of 6 DC maturation markers including CD40, CD54, CD80, CD83, CD86 and CD197 (CCR-7). The inhibition started at a concentration of 10nM and was dose-related. IXZ also decreased the percentage of total DCs simultaneously expressing multiple markers. DCs viability remained unchanged in comparison to control at a concentration of 10nM and dropped to 68% and 43%, on average with concentrations of 20nM and 40nM, respectively. IXZ significantly decreased DC production of IL-6, IL-12, and IL-23 starting at the concentration of 20nM. IL-1β was decreased at the concentration of 40 nM. Importantly, there was no significant change in the cytokine production by DCs when IXZ was added 4 hrs after LPS except for IL-1β which increased at 30nM. Starting at the concentration of 10nM, IXZ dose-dependently inhibited T cell proliferation. At 40nM IXZ abolished T cells. In our in vivo study IXZ improved GvHD scores on days +7 and +11 in group 3 in comparison to group 2 when it was given on days -1 and +2. Conversely, when IXZ was given on day +1 and +4, group 3 mice had higher scores of GvHD and worse survival outcomes when compared to group 2. There was no noticeable drug toxicity in group 1 mice.Conclusion: In summary: 1) IXZ inhibits DC maturation with relative preservation of cell viability and inhibits pro-inflammatory cytokine production in DCs when added before LPS stimulation; 2) IXZ inhibits T-cell proliferation; 3) IXZ affects GvHD development in a schedule-dependent fashion with early administration improving and late administration worsening GvHD. Additional analysis of tissue and serum samples is in progress. These results provide background for careful design of clinical trials using IXZ for the prevention of GvHD. DisclosuresAl-Homsi:Millennium Pharmaceuticals: Research Funding.

Phase 1 Study of Carfilzomib for the Prevention of Relapse and Graft-Versus-Host Disease in Allogeneic Hematopoietic Cell Transplantation for High-Risk Hematologic Malignancies

Allogeneic hematopoietic cell transplant (HCT) remains the primary curative option for patients (pts) with poor-risk hematologic malignancies, but graft-versus-host disease (GVHD) and relapse remain major obstacles. Of note, GVHD therapy with systemic steroid and more potent immunosuppression abrogates graft-versus-tumor effect and further increases relapse. The NF-κB signaling pathway has a pivotal role in maintaining malignant cell survival and in mediating inflammatory and immune response by promoting activated T and dendritic cell function and survival. Inhibition of proteasome function results in down-regulation of the NF-κB signaling pathway, thus selective apoptosis of malignant cells, in which this pathway is up-regulated, and of allo-reactive T and dendritic cells. The reversible proteasome inhibitor bortezomib may abrogate GVHD and relapse based on pre-clinical studies and a phase 1/2 clinical trial when added to high-risk mismatched unrelated donor HCTs for pts with hematologic malignancies. (Koreth J, et al. J Clin Oncol 2012;30(26):3202-8.) Carfilzomib is a second-generation irreversible immunoproteasome inhibitor, with potentially increased efficacy and specificity to the immune and hematologic malignancy cells. It has a less off-target toxicity profile, especially myelosuppression, neuropathy and diarrhea. We are exploring the addition of carfilzomib to fludarabine-based conditioning regimens and GVHD prophylaxis with tacrolimus and methotrexate.Primary Objective: To identify the maximal tolerated dose (MTD) of carfilzomib when administered on day +1, +2, +6, +7.Methods: The phase 1 design is standard 3+3. Eligible conditioning regimens include fludarabine/busulfan and fludarabine/melphalan. Tacrolimus is started on day -3 and tapered off by day +180 if ≥grade II acute GVHD do not occur. Methotrexate 5 mg/m2 is given IV on day +1, +3, +6, +11. Carfilzomib is administered IV on days +1, +2 at 20 mg/m2/day fixed dose and on day +6, +7 at 4 dose levels: 20, 27, 36, 45 mg/m2/day. Premedication with 4 mg dexamethasone IV is administered prior to each dose of carfilzomib 20 and 27 mg/m2 and with 8 mg prior to each dose of 36 and 45 mg/m2, respectively. Carfilzomib is given after IV methotrexate on day +1 and +6. Subcutaneous filgrastim 5 μg/kg/day is started on day +1 until engraftment. Dose-limiting toxicities (DLTs) are defined as any ≥grade 3 non-hematologic National Cancer Institute Common Terminology Criteria for Adverse Events version 4 toxicities which occur within 28 days of carfilzomib treatment (day +35) and are directly attributed to carfilzomib.Results: Since October 2014, 10 pts (Male: Female, 5:5) have been enrolled. Nine pts received all planned 4 doses and 1 female received 3 doses only before being removed due to unrelated infectious adverse events. Among 9 evaluable pts, the median age was 55 (range, 29-61) years; all donors were 8/8 matched (7 sibling, 2 unrelated). Stem cell sources were peripheral blood in 8 and bone marrow in 1 unrelated donor. Primary diseases included FLT3-ITD+ acute myeloid leukemia in 2, acute bi-phenotypic leukemia in 1, BCR/ABL+ acute lymphoblastic leukemia in 1, myelodysplastic syndrome in 2, lymphoma in 2, and multiple myeloma in 1. All pts received the myeloablative reduced-toxicity fludarabine and busulfan x 4 (FluBu4), with busulfan kinetics targeting the concentration at steady state at 600-900 ng/mL. One pt with diffuse large B cell lymphoma received rituximab-FluBu4. All 9 pts engrafted neutrophil and platelet at a median time of 10 (range, 10-15) and 12 (9-14) days, respectively. A pt of dose level 2 (20/27 mg/m2) who received a bone marrow stem cell dose of 1.3 x 106 CD34+ cells/kg unexpectedly engrafted as early as day +15. Expected common toxicities related to the FluBu4 treatment was oral mucositis. Median CD33+ and CD3+ donor chimerisms were 100% and 88% at day 30, respectively, and were 100% and 91% at day 100, respectively. As of 8/4/15, no DLTs occurred in the 8 pts who had passed day +35, with the median transplant day of day +172 (range, 65-292). The 9th pt of dose level 3 (20/36 mg/m2) is day +14 and has engrafted.Conclusion: Adding the irreversible immunoproteasome inhibitor carfilzomib to fludarabine-based conditioning HCT appears safe and feasible. Dose escalation to level 4 (20/45 mg/m2) is ongoing and the results will be updated at the meeting. DisclosuresPawarode:Onyx Pharmaceuticals: Research Funding. Off Label Use: Carfilzomib is an irreversible proteasome inhibitor and is FDA approved for treament of relapsed refractory multiple myeloma. It is here being investigated for its immunomodulatory and anti-tumor properties in the prevention of graft-versus-host disease (GVHD) and disease relapse when incorporated into fludarabine based conditioning and GVHD prophylaxis regimens for allogeneic hematopoietic cell transplantation in patients with high-risk hematologic malignancy.. Levine:Novartis: Consultancy.

Control of Homeostasis and Dendritic Cell Survival by the GTPase RhoA.

Tissues accommodate defined numbers of dendritic cells (DCs) in highly specific niches where different intrinsic and environmental stimuli control DC life span and numbers. DC homeostasis in tissues is important, because experimental changes in DC numbers influence immunity and tolerance toward various immune catastrophes and inflammation. However, the precise molecular mechanisms regulating DC life span and homeostasis are unclear. We report that the GTPase RhoA controls homeostatic proliferation, cytokinesis, survival, and turnover of cDCs. Deletion of RhoA strongly decreased the numbers of CD11b(-)CD8(+) and CD11b(+)Esam(hi) DC subsets, whereas CD11b(+)Esam(lo) DCs were not affected in conditional RhoA-deficient mice. Proteome analyses revealed a defective prosurvival pathway via PI3K/protein kinase B (Akt1)/Bcl-2-associated death promoter in the absence of RhoA. Taken together, our findings identify RhoA as a central regulator of DC homeostasis, and its deletion decreases DC numbers below critical thresholds for immune protection and homeostasis, causing aberrant compensatory DC proliferation.