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We investigated the role of two key immunoregulatory molecules, indoleamine dioxygenase (IDO) and inducible costimulator ligand (ICOSL), in determining the function of bone marrow (BM)-derived plasmacytoid (p)DC, which offer the potential for therapy of allograft rejection. pDC generated from BM of wild-type (WT) or IDO knockout (KO) C57BL/6 mice were used to stimulate T-cell proliferation and interferon-gamma (IFN-gamma) production in response to alloantigen (alloAg) via the direct or indirect pathways. In some experiments, pDC were first activated by exposure to CpG +/- CTLA4Ig for IDO induction via B7 ligation. Although IDO KO pDC induced enhanced T-cell responses compared with WT pDC, the use of the IDO inhibitor 1-methyltryptophan (1-MT) demonstrated that the inferior stimulatory capacity of WT pDC was not caused by the production of functional IDO, even under IDO-inducing conditions. The DNAX-activating protein of 12 kDa (DAP12), which inhibits functional IDO expression, was expressed in BM-pDC. DAP12 silencing increased the T-cell stimulatory capacity of WT pDC, but only in the presence of 1-MT. Compared with WT pDC, activated IDO KO DC expressed much lower levels of ICOSL. Moreover, when ICOSL was blocked on WT pDC, T-cell proliferation resembled that induced by IDO KO pDC, and interleukin (IL)-10 secretion in MLR was markedly decreased. These findings implicate ICOSL-induced IL-10, but not IDO in the regulation of BM-derived pDC function.

The role of dendritic cells (DC) in transplantation is often overshadowed by the more prominent roles of T and B cells, which interact directly with and, in the absence of immunosuppressive therapy, destroy the allograft. It has become increasingly recognized, however, that these potent antigen-presenting cells exert control over the immune response and regulate the balance between tolerance and immunity to transplanted organs and tissues. The role that chemokines play in influencing DC function with impact on regulation of immune responses against the graft is only beginning to be understood. This article considers how the manipulation of DC trafficking during an alloimmune response can affect graft outcome.

The mechanisms by which chronic ethanol (EtOH) consumption results in an immune-compromised state have not been fully elucidated. No studies to date have ascertained whether EtOH affects the migratory capacity of dendritic cells (DC), potent immune regulators. We hypothesized that EtOH exposure might affect hepatic and splenic DC trafficking to secondary lymphoid tissues and the resulting immune response. Hepatic DC from EtOH-treated animals migrated in greater numbers to draining lymphoid tissue than controls, whereas spleen DC were unaffected. Moreover, hepatic EtOH-exposed (E) DC induced more vigorous priming of allogeneic T cells in vivo compared with splenic EDC or controls. Altered hepatic EDC migration was independent of either CCR7 or CD11a expression, with no striking changes in surface expression of other adhesion molecules analyzed. The modified trafficking to secondary lymphoid tissue observed for hepatic EDC may play a role in the altered immune response to microbial pathogens in chronic alcohol users.

Purpose of Review: Use or targeting of dendritic cells for therapeutic manipulation of immune responses is being pursued in the areas of cancer, autoimmune disease, and allograft rejection. There is, however, a dearth of information regarding the optimal route of cell delivery or target location for maximal therapeutic effect, particularly in the field of transplantation. Further, little attention has been given to the roles that conventional experimental/immunosuppressive modalities have on the migratory capacity of these important antigen-presenting cells., Recent Findings: Current understanding of the role of dendritic cells in immunologic ignorance, graft rejection, or tolerance to alloantigen suggests their function is influenced by subset, secondary lymphoid tissue location, and the type of organ transplanted. It also has been determined recently that dendritic cell subsets probably utilize distinct migratory routes to secondary lymphoid tissues, further underscoring the importance of understanding dendritic cell trafficking for optimization of dendritic cell therapy protocols., Summary: Increased comprehension of the requirements for dendritic cell-T cell interactions to take place in specific secondary lymphoid tissues for the induction of rejection versus tolerance, with and without antirejection therapy, will facilitate the ease with which cell-based therapy can be designed and implemented in transplant recipients.

In this study, we propagated myeloid dendritic cells (DC) from BALB/c (H2(d)) mouse bone marrow progenitors in IL-10 and TGF-beta, then stimulated the cells with LPS. These "alternatively activated" (AA) DC expressed lower TLR4 transcripts than LPS-stimulated control DC and were resistant to maturation. They expressed comparatively low levels of surface MHC class II, CD40, CD80, CD86, and programmed death-ligand 2 (B7-DC; CD273), whereas programmed death-ligand 1 (B7-H1; CD274) and inducible costimulatory ligand expression were unaffected. AADC secreted much higher levels of IL-10, but lower levels of IL-12p70 compared with activated control DC. Their poor allogeneic (C57BL/10; B10) T cell stimulatory activity and ability to induce alloantigen-specific, hyporesponsive T cell proliferation was not associated with enhanced T cell apoptosis. Increased IL-10 production was induced in the alloreactive T cell population, wherein CD4+Foxp3+ cells were expanded. The AADC-expanded allogeneic CD4+CD25+ T cells showed enhanced suppressive activity for T cell proliferative responses compared with freshly isolated T regulatory cells. In vivo migration of AADC to secondary lymphoid tissue was not impaired. A single infusion of BALB/c AADC to quiescent B10 recipients induced alloantigen-specific hyporesponsive T cell proliferation and prolonged subsequent heart graft survival. This effect was potentiated markedly by CTLA4-Ig, administered 1 day after the AADC. Transfer of CD4+ T cells from recipients of long-surviving grafts (>100 days) that were infiltrated with CD4+Foxp3+ cells, prolonged the survival of donor-strain hearts in naive recipients. These data enhance insight into the regulatory properties of AADC and demonstrate their therapeutic potential in vascularized organ transplantation.

Dendritic cells (DCs) are uniquely well equipped antigen (Ag)-presenting cells. Their classic function was thought to be that of potent initiators of innate and adaptive immunity to infectious organisms and other Ags (including transplanted organs). Evidence has emerged, however, that DCs have a central and crucial role in determining the fate of immune responses toward either immunity or tolerance. This dichotomous function of DCs, coupled with their remarkable plasticity, renders them attractive therapeutic targets for immune modulation. In transplantation, much recent work has focused on the ability of DCs to silence immune reactivity in an Ag-specific manner in the hope of preventing rejection and diminishing reliance on potentially harmful immunosuppressive agents. Experimental strategies have included in vivo targeting of DCs, as well as ex vivo generation of regulatory (or tolerogenic) DCs with subsequent reinfusion (i.e. cell therapy). Different approaches to 'program' DC toward tolerogenic properties include genetic (transgene insertion), biologic (differential culture conditions, anti-inflammatory cytokine exposure) and pharmacologic manipulation. Recent data suggest a promising role for pharmacologic treatment as a means of generating potent regulatory DCs and have further stimulated speculation regarding their potential clinical application. Herein, we discuss evidence that the potential of regulatory DC therapy is considerable and that there are compelling reasons to evaluate it in the setting of organ transplantation in the near future.

Dendritic cells (DC) exhibit remarkable plasticity in terms of their ability to induce and regulate innate and adaptive immune responses. Human and mouse interferon alpha-producing plasmacytoid (p) DC have been found to regulate allogeneic T cell responses in vitro. Evidence is emerging that pDC may also regulate immune reactivity in vivo, including the responses that underlie graft-versus-host disease and organ transplant rejection. These cells may also offer potential for therapy of adverse immune responses and the promotion of tolerance induction.

The pro-drug FTY720 is undergoing phase III clinical trials for prevention of allograft rejection. After phosphorylation, FTY720 targets the G protein-coupled-sphingosine-1-phosphate receptor 1 (S1PR1) on lymphocytes, thereby inhibiting their egress from lymphoid organs and their recirculation to inflammatory sites. Potential effects on dendritic cell (DC) trafficking have not been evaluated. Here, we demonstrate the expression of all five S1PR subtypes (S1PR1-5) by murine DCs. Administration of FTY720 to C57BL/10 mice markedly reduced circulating T and B lymphocytes within 24 h, but not blood-borne DCs, which were enhanced significantly for up to 96 h, while DCs in lymph nodes and spleen were reduced. Numbers of adoptively transferred, fluorochrome-labeled syngeneic or allogeneic DCs in blood were increased significantly in FTY720-treated animals, while donor-derived DCs and allostimulatory activity for host naïve T cells within the spleen were reduced. Administration of the selective S1PR1 agonist SEW2871 significantly enhanced circulating DC numbers. Flow analysis revealed that CD11b, CD31/PECAM-1, CD54/ICAM-1 and CCR7 expression on blood-borne DCs was downregulated following FTY720 administration. Transendothelial migration of FTY720-P-treated immature DCs to the CCR7 ligand CCL19 was reduced. These novel data suggest that modulation of DC trafficking by FTY720 may contribute to its immunosuppressive effects.

CCL19/MIP-3beta and CCL21/SLC are essential for chemotactic recruitment of mature dendritic cells (DC) to T-cell areas of secondary lymphoid tissue. Paucity of lymph node T-cells (plt/plt) mice lack CCL21-serine (ser) and CCL19 expression. We tested plt/plt and wild type (wt) BALB/c (H2d) mice as recipients of heart or skin allografts from C57BL/10J (H2b) donors. Donor DC trafficking to secondary lymphoid tissue was markedly reduced in plt heart but not skin allograft recipients. Heart, but not skin grafts survived significantly longer in plt recipients. Accordingly, T cells from plt heart transplant recipients demonstrated poor anti-donor responses in ex vivo MLR, compared to wt heart or wt and plt skin recipients. Moreover, donor-reactive T cells from plt heart recipients exhibited Th2-skewing in comparison to T cells from wt heart or skin graft recipients. Anti-CXCL9/Mig was administered for 2 weeks post-transplant to determine whether impairment of activated T-cell migration could further prolong cardiac allograft survival in plt recipients. CXCL9-antagonism extended graft survival significantly only in plt mice, likely due, in part, to retention of alloactivated T cells in secondary lymphoid tissue/reduction of graft-infiltrating T cells. Thus, targeting DC and activated T-cell migration concomitantly has additive effects in prolonging heart graft survival with potential for therapeutic application.

Upon Ag uptake and response to maturation stimuli, dendritic cells (DC) are directed through lymphatic or blood vessel endothelium to T cell areas of secondary lymphoid tissues by the constitutively expressed CC chemokines CCL19 and CCL21. We have shown that mature (m) murine CD8alpha+ DC exhibit poorer migratory ability to these chemokines than classic CD8alpha- DC by quantifying their in vitro chemotaxis through unmodified Transwell filters. We hypothesized that lower surface expression (compared to CD8alpha- mDC) of the adhesion molecule CD11b on CD8alpha+ DC might limit their ability to adhere to filter pores in vitro and/or endothelium in vitro/in vivo. To test the role of this and/or other adhesion molecules (CD11a, CD31, CD54 and CD62L) in regulating murine DC subset migration, we used specific mAbs to block their function and quantified their migration through resting or tumour necrosis factor (TNF)-alpha-activated endothelial cell (EC) layered-Transwell filters. Both CD8alpha+ and CD8alpha- subsets migrated through resting EC (albeit less than in the absence of EC) in response to CCL19 and CCL21, and migration through TNF-alpha-activated EC was enhanced. In contrast to reports concerning human DC, transendothelial migration of the murine DC subsets was not dependent on CD11b, CD31, or CD62L expression by these cells. CD54 and CD11a, however, were at least partly involved in DC/EC interactions. This is the first report to examine adhesion molecules involved in transendothelial migration of murine DC subsets.

Background: We investigated the expression and function of CC chemokine receptors (CCR) on highly-purified kidney and blood dendritic cells isolated from mice in which dendritic cells were mobilized with fms-like tyrosine 3 kinase ligand (Flt3L)., Methods: CCR and CC chemokine expression were determined by RNase protection assay or flow cytometry, and dendritic cell migratory responses assayed using Transwell chambers. Chemokine production in renal tissue was detected by immunofluorescence staining. Trafficking of fluorochrome-labeled dendritic cells was monitored in vivo., Results: Freshly-isolated renal dendritic cells expressed mRNA for CCR1, 2, 5, and 7 and CCR1 and 5 protein. They did not migrate to inducible chemokines--CCL3 [macrophage inflammatory protein (MIP)-1alpha], CCL5 [regulated upon activation, normal T cell expressed and secreted (RANTES)], or CCL20 (MIP-3alpha). Following lipopolysaccharide (LPS) stimulation, the dendritic cells down-regulated CCR1, 2, and 5 expression, up-regulated or sustained signals for CCR7, and migrated to the constitutively expressed ligands CCL19 (MIP-3beta) and CCL21 (secondary lymphoid tissue chemokine). Normal kidneys expressed weak message for CCL2, 3, and 4, with stronger signals for CCL5 and 19. Intrarenal CCL5 production was enhanced by Flt3L administration, in association with marked increases in interstitial CD45+ mononuclear cells. Mobilized blood dendritic cells migrated to CCR2 and CCR5 ligands and trafficked to renal intertubular sites following adoptive (intravenous) transfer. Their migration to the CCR5 ligand MIP-1beta (CCL4) and homing to kidneys of Flt3L-treated recipients were inhibited by CCR5 antagonism., Conclusion: These data implicate specific CCR and their ligands in regulation of the dendritic cell constituency of the kidney. CCR5 antagonism inhibits their directed migration and intrarenal accumulation.

Dendritic cell (DC) trafficking is regulated by chemokine receptor expression and responsiveness to chemokines. The authors compared CC chemokine receptor (CCR) expression by mouse liver myeloid, "lymphoid-related," and plasmacytoid DC subsets and their responsiveness to CC chemokines. CCR mRNA expression by liver DC subsets was evaluated by RNase protection assay. In vitro migration of the DC in response to recombinant murine CC chemokines was assayed using Transwell chambers. Immature liver DC did not respond to any CC chemokines tested, despite expression of mRNA encoding appropriate receptors for their ligands. CCR7 expression by each liver DC subset was strongly enhanced in response to maturation. The migratory capacity of liver plasmacytoid DC was similar to that of liver myeloid and lymphoid-related DC. These findings suggest that targeting of CCR7 and its ligands may be a potential approach for manipulation of liver DC trafficking to secondary lymphoid tissue after liver transplantation.

Background: Migratory antigen-presenting cells resident in kidneys may have tolerogenic potential. Difficulties inherent in their isolation have limited their characterization. The authors examined the phenotype and function of murine kidney dendritic cells (DC) mobilized in vivo by systemic administration of fms-like tyrosine 3 kinase ligand (Flt3L)., Methods: Monoclonal antibody staining was used to characterize DC subsets in situ, immediately after their isolation, and after lipopolysaccharide stimulation. Cytokine and CC chemokine receptor (CCR) gene expression was analyzed by RNase protection assay. Mixed leukocyte reactions were performed to assess DC allostimulatory ability and also the function of putative T-regulatory cells. In vivo DC trafficking was monitored by fluorescence imaging of dye-labeled cells and the influence of renal DC on vascularized heart allograft survival was determined., Results: Flt3L induced a marked increase both in CD11cCD8alpha and in CD11cCD8alpha DC within the renal cortex and medulla. Rarer, CD11cB220 (precursor plasmacytoid) DC were also detected. Bulk freshly isolated DC exhibited no interleukin (IL)-12p35 mRNA, low surface co-stimulatory molecule expression, and CCR transcripts, consistent with immaturity. They elicited only weak allogeneic T-cell proliferative responses, and repeated stimulation induced CD4CD25 IL-10 T cells. In vivo, the freshly isolated DC failed to prime T cells of naive allogeneic hosts for anti-donor cytotoxic T-cell responses. When infused systemically, 1 week before organ transplantation, they prolonged graft survival without immunosuppressive therapy., Conclusions: Hematopoietin-mobilized renal DC are functionally immature and exhibit tolerogenic potential. Mobilization of DC within kidneys is likely to affect their antigen-handling capacity, immunogenicity, and tolerogenic ability.

Murine CD11c(+)CD8alpha(-) and CD11c(+)CD8alpha(+) dendritic cells (DCs) differentially regulate T cell responses. Although specific chemokines that recruit immature (i) or mature (m) CD8alpha(-) DCs have been identified, little is known about the influence of chemokines on CD8alpha(+) DCs. iDCs and mDCs isolated from spleens of fms-like tyrosine kinase 3 ligand-treated B10 mice were compared directly for migratory responses to a panel of CC chemokines or following local or systemic administration. In vitro assays were performed using Transwell(R) chambers. iDCs did not respond to any CC chemokines tested. Both subsets of mDCs migrated to CCL19 and CCL21, with consistently lower percentages of CD8alpha(+) DCs migrating. Chemokine receptor mRNA and protein expression were analyzed, but no correlation between expression and function was demonstrated. In vivo trafficking of fluorochrome-labeled DCs (B10; H2(b)) was assessed by immunohistochemistry and by rare-event flow cytometric analysis of allogeneic recipient (BALB/c; H2(d)) draining lymph node (DLN) and spleen cells. Twenty-four hours after intravenous injection, chloromethylfluorescein diacetate-positive CD8alpha(+) and CD8alpha(-) mDCs were detected by immunohistochemistry in spleens in similar numbers (that decreased over time). Following subcutaneous injection, both DC subsets were detected in DLN at 24 h, but only CD8alpha(-) DCs were evident by flow analysis at 48 h. Although CD8alpha(+) DCs migrate from peripheral tissues to T cell areas of (allogeneic) secondary lymphoid organs, they appear to mobilize as mDCs and less efficiently than CD8alpha(-) mDCs.

There are various approaches to the enhancement of dendritic cell (DC) tolerogenicity for the promotion of cell or organ allograft survival. Both pharmacologic and biologic agents, including several commonly used immunosuppressive drugs, and specific anti-inflammatory cytokines inhibit DC maturation, whereas co-stimulation-blocking agents can also promote the induction of antigen-specific T-cell unresponsiveness by DC. Delivery of genes encoding molecules that subvert T-cell responses by various mechanisms, and targeting of DC migration by selective manipulation of chemokine and chemokine receptor expression, represent additional promising strategies. In this short review, the authors consider those approaches that have been used to promote the tolerogenicity of donor-derived DC in experimental models. Whereas most work to date has focused on myeloid DC, manipulation of other DC subsets may also offer potential for improving the outcome of transplantation and enhancing tolerance induction.

Flt3 ligand (FL) administration markedly increases bone marrow (BM) stem cells and immature dendritic cells. We investigated the influence of CD40-CD40Ligand (CD154) pathway blockade on antidonor immunity, cytokine production, microchimerism and heart graft survival in BALB/c (H2d) recipients of fully allogeneic C57BL/10 (H2b) FL-mobilized BM (FL-BM) or normal BM. Anti-CD40L mAb strongly suppressed anti-donor T-cell proliferative responses in recipients of either normal or FL-BM, but was less efficient in inhibiting antidonor cytolytic T-cell (CTL) activity, especially in recipients of FL-BM. Interestingly, CD40L blockade was more effective in recipients of multiple compared with single donor BM infusions. Anti-donor cytokine responses revealed complete impairment of IFN-gamma, IL-4 and IL-10 production in recipients of normal BM and CD40L mAb. By contrast, and in agreement with the CTL data, mice given FL-BM retained ability to produce IFN-gamma CD40-CD40L blockade did not promote microchimerism, as evidenced by immunohistology and real time polymerase chain reaction. Nevertheless, anti-CD40L mAb enhanced heart allograft survival in recipients of FL-BM, but the effect was inferior to that achieved with normal BM. These data provide insight into the influence of growth factor-expanded donor BM and costimulation blockade on antidonor immune reactivity and transplant outcome. The comparatively poor outcome obtained using FL-BM plus anti-CD40L mAb in this model may be ascribed to the failure of effectively interdicting antidonor CTL activity.

Organ transplant rejection is mediated largely by circulating peripheral leukocytes induced to infiltrate the graft by various inflammatory stimuli. Of these, chemotactic cytokines called chemokines, expressed by inflamed graft tissues, as well as by early innate-responding leukocytes that infiltrate the graft, are responsible for the recruitment of alloreactive leukocytes. This report discusses the impact of these leukocyte-directing proteins on transplant outcome and novel therapeutic approaches for antirejection therapy based on targeting of chemokines and/or their receptors.

At the time of organ transplantation, a variety of non-parenchymal cells are transplanted simultaneously with the allograft. Recognition of the importance of these cells as potential immunostimulatory cells lead to the concept of 'passenger leukocytes' as the principal instigators of rejection. Passenger leukocytes include interstitial dendritic cells (DCs) and blood-derived monocytes/macrophages. As investigators have discovered the significance of DCs in influencing graft outcome, so have they begun to determine the best ways to influence DCs themselves. This review discusses the role of DCs in transplantation and then focuses on three different approaches for manipulating DCs to improve allograft survival: (1) targeting of chemokines involved in DC migration, (2) pharmacological arrest of DC maturation, and (3) genetic engineering of DCs.

Although it is known that dendritic cells (DCs) produce cytokines, there is little information about how cytokine synthesis is regulated during DC development. A range of cytokine mRNA/proteins was analyzed in immature (CD86-) or mature (CD86+) murine bone marrow (BM)- derived DCs. Highly purified, flow-sorted, immature DCs exhibited higher amounts of interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor beta1 (TGF-beta1), and macrophage migration inhibitory factor (MIF) mRNA/protein than mature DCs. After differentiation, DC up-regulated the levels of IL-6 and IL-15 mRNA/protein and synthesized de novo mRNA/protein for IL-12p35, IL-12p40, and IL-18. Although immature BM-derived DCs did not stimulate naive allogeneic T cells, mature DCs elicited a mixed population of T helper (Th) 1 (mainly) and Th2 cells in 3d-mixed leukocyte reactions. CD86+ BM DCs switched to different cytokine patterns according to whether they were terminally differentiated by lipopolysaccharide (LPS) or CD40 ligation. Although both stimuli increased IL-6, IL-12p40, IL-15, and TNF-alpha mRNA/protein levels, only LPS up-regulated transcription of IL-1alpha, IL-1beta, IL-12p35, and MIF genes. Although LPS and CD40 cross-linking increased the T-cell allostimulatory function of BM DCs, only LPS stimulation shifted the balance of naive Th differentiation to Th1 cells, a mechanism dependent on the up-regulation of IL-12p35 and not of IL-23. These results demonstrate that, depending on the stimuli used to terminally mature BM DCs, DCs synthesize a different pattern of cytokines and exhibit distinct Th cell-driving potential.