Bridget L. Colvin

“Alternatively Activated” Dendritic Cells Preferentially Secrete IL-10, Expand Foxp3+CD4+ T Cells, and Induce Long-Term Organ Allograft Survival in Combination with CTLA4-Ig

In this study, we propagated myeloid dendritic cells (DC) from BALB/c (H2d) mouse bone marrow progenitors in IL-10 and TGF-β, 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.

The Sphingosine-1-Phosphate Receptor Agonist FTY720 Modulates Dendritic Cell Trafficking In Vivo

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.

Regulatory dendritic cell therapy in 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.

Chemokines, their receptors, and transplant outcome

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.

Pharmacologic, biologic, and genetic engineering approaches to potentiation of donorderived dendritic cell tolerogenicity1

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.

CXCL9 Antagonism Further Extends Prolonged Cardiac Allograft Survival in CCL19/CCL21-Deficient Mice

CCL19/MIP‐3β 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.

Comparative evaluation of CC chemokine-induced migration of murine CD8α+ and CD8α- dendritic cells and their in vivo trafficking

Murine CD11c+CD8α− and CD11c+CD8α+ dendritic cells (DCs) differentially regulate T cell responses. Although specific chemokines that recruit immature (i) or mature (m) CD8α− DCs have been identified, little is known about the influence of chemokines on CD8α+ 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® chambers. iDCs did not respond to any CC chemokines tested. Both subsets of mDCs migrated to CCL19 and CCL21, with consistently lower percentages of CD8α+ 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; H2b) was assessed by immunohistochemistry and by rare‐event flow cytometric analysis of allogeneic recipient (BALB/c; H2d) draining lymph node (DLN) and spleen cells. Twenty‐four hours after intravenous injection, chloromethylfluorescein diacetate‐positive CD8α+ and CD8α− 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 CD8α− DCs were evident by flow analysis at 48 h. Although CD8α+ DCs migrate from peripheral tissues to T cell areas of (allogeneic) secondary lymphoid organs, they appear to mobilize as mDCs and less efficiently than CD8α− mDCs.

Allostimulatory activity of bone marrow-derived plasmacytoid dendritic cells is independent of indoleamine dioxygenase but regulated by inducible costimulator ligand expression

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.

Disparate ability of murine CD8α− and CD8α+ dendritic cell subsets to traverse endothelium is not determined by differential CD11b expression

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 CD8α+ DC exhibit poorer migratory ability to these chemokines than classic CD8α– DC by quantifying their in vitro chemotaxis through unmodified Transwell® filters. We hypothesized that lower surface expression (compared to CD8α– mDC) of the adhesion molecule CD11b on CD8α+ 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)‐α‐activated endothelial cell (EC) layered‐Transwell® filters. Both CD8α+ and CD8α– subsets migrated through resting EC (albeit less than in the absence of EC) in response to CCL19 and CCL21, and migration through TNF‐α‐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.

Dendritic cells and chemokine-directed migration in transplantation: where are we headed?

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.