Renal Allograft Survival in Nonhuman Primates Infused With Donor Antigen-Pulsed Autologous Regulatory Dendritic Cells.

Systemic administration of autologous regulatory dendritic cells (DCreg; unpulsed or pulsed with donor antigen [Ag]), prolongs allograft survival and promotes transplant tolerance in rodents. Here, we demonstrate that nonhuman primate (NHP) monocyte-derived DCreg preloaded with cell membrane vesicles from allogeneic peripheral blood mononuclear cells induce T cell hyporesponsiveness to donor alloantigen (alloAg) in vitro. These donor alloAg-pulsed autologous DCreg (1.4-3.6 × 106 /kg) were administered intravenously, 1 day before MHC-mismatched renal transplantation to rhesus monkeys treated with costimulation blockade (cytotoxic T lymphocyte Ag 4 immunoglobulin [CTLA4] Ig) and tapered rapamycin. Prolongation of graft median survival time from 39.5 days (no DCreg infusion; n = 6 historical controls) and 29 days with control unpulsed DCreg (n = 2), to 56 days with donor Ag-pulsed DCreg (n = 5) was associated with evidence of modulated host CD4+ and CD8+ T cell responses to donor Ag and attenuation of systemic IL-17 production. Circulating anti-donor antibody (Ab) was not detected until CTLA4 Ig withdrawal. One monkey treated with donor Ag-pulsed DCreg rejected its graft in association with progressively elevated anti-donor Ab, 525 days posttransplant (160 days after withdrawal of immunosuppression). These findings indicate a modest but not statistically significant beneficial effect of donor Ag-pulsed autologous DCreg infusion on NHP graft survival when administered with a minimal immunosuppressive drug regimen.

Clinical implications of basic science discoveries: nociceptive neurons as targets to control immunity--potential relevance for transplantation.

Increasing evidence indicates the existence of a complex cross-regulation between the most important biosensors of the human body: The immune and nervous systems. Cytokines control body temperature and trigger autoimmune disorders in the central nervous system, whereas neuropeptides released in peripheral tissues and lymphoid organs modulate inflammatory (innate) and adaptive immune responses. Surprisingly, the effects of nerve fibers and the antidromic release of its pro-inflammatory neuropeptides on the leukocytes of the immune system that mediate graft rejection are practically unknown. In the transplantation field, such area of research remains practically unexplored. A recent study by Riol-Blanco et al has revealed new details on how nociceptive nerves regulate the pro-inflammatory function of leukocytes in peripheral tissues. Although the mechanism(s) by which neuroinflammation affects the immune response against the allograft remains unknown, recent data suggest that this new area of research is worth exploring for potential development of novel complementary therapies for prevention/treatment of graft rejection.

Regulatory dendritic cell infusion prolongs kidney allograft survival in nonhuman primates.

We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine-mobilized donor blood monocytes in vitamin D3 and IL-10, on renal allograft survival in a clinically relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand-1 (B7-H1), and were resistant to pro-inflammatory cytokine-induced maturation. They were infused intravenously (3.5-10 × 10(6) /kg), together with the B7-CD28 costimulation blocking agent CTLA4Ig, 7 days before renal transplantation. CTLA4Ig was given for up to 8 weeks and rapamycin, started on Day -2, was maintained with tapering of blood levels until full withdrawal at 6 months. Median graft survival time was 39.5 days in control monkeys (no DC infusion; n = 6) and 113.5 days (p < 0.05) in DCreg-treated animals (n = 6). No adverse events were associated with DCreg infusion, and there was no evidence of induction of host sensitization based on circulating donor-specific alloantibody levels. Immunologic monitoring also revealed regulation of donor-reactive memory CD95(+) T cells and reduced memory/regulatory T cell ratios in DCreg-treated monkeys compared with controls. Termination allograft histology showed moderate combined T cell- and Ab-mediated rejection in both groups. These findings justify further preclinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation.

Dendritic cell therapies in transplantation revisited: deletion of recipient DCs deters the effect of therapeutic DCs.

A critical goal in transplantation is the achievement of donor-specific tolerance, minimizing the use of immunosuppressants. Dendritic cells (DCs) are antigen (Ag) presenting cells (APCs) with capability to promote immunity or tolerance. The immune-regulatory properties of DCs have been exploited for generation of tolerogenic/immunosuppressive (IS) DCs that, when transfer systemically, prolong allograft survival in murine models. Surprisingly, the in vivo mechanisms of therapies based on (donor- or recipient-derived) ISDCs in transplantation remain unknown, given that previous studies investigated their effects in vitro, or ex vivo after transplantation. Since once injected, ISDCs are short-lived and transfer Ag to recipient APCs, we assessed the role of recipient DCs by depleting them at the time of ISDC-therapy in a mouse model of cardiac transplantation. The results indicate that, contrary to the accepted paradigm, systemically administered ISDCs reduce the alloresponse and prolong allograft survival, not by themselves, but through conventional DCs (cDCs) of the recipient. These findings raise doubts on the advantages of the currently used ISDC-therapies, since the immune-regulatory properties of the injected ISDC do not seem to be functionally relevant in vivo, and the quiescent/pro-tolerogenic status of cDCs may be compromised in patients with end-stage diseases that require transplantation.

The expression profile of C19MC microRNAs in primary human trophoblast cells and exosomes.

The largest gene cluster of human microRNAs (miRNAs), the chromosome 19 miRNA cluster (C19MC), is exclusively expressed in the placenta and in undifferentiated cells. The precise expression pattern and function of C19MC members are unknown. We sought to profile the relative expression of C19MC miRNAs in primary human trophoblast (PHT) cells and exosomes. Using high-throughput profiling, confirmed by PCR, we found that C19MC miRNAs are among the most abundant miRNAs in term human trophoblasts. Hypoxic stress selectively reduced miR-520c-3p expression at certain time-points with no effect on other C19MC miRNAs. Similarly, differentiation in vitro had a negligible effect on C19MC miRNAs. We found that C19MC miRNAs are the predominant miRNA species expressed in exosomes released from PHT, resembling the profile of trophoblastic cellular miRNA. Predictably, we detected the similar levels of circulating C19MC miRNAs in the serum of healthy pregnant women at term and in women with pregnancies complicated by fetal growth restriction. Our data define the relative expression levels of C19MC miRNAs in trophoblasts and exosomes, and suggest that C19MC miRNAs function in placental-maternal signaling.

EBV-specific CD8+ T cell reactivation in transplant patients results in expansion of CD8+ type-1 regulatory T cells.

Posttransplantation lymphoproliferative disorders (PTLD) are life-threatening complications of solid organ transplantation, triggered by EBV infection in chronically immunosuppressed (IS) patients. Our goal is to establish DC-based protocols for adoptive immunotherapy of refractory PTLD, while understanding how the immunosuppressive drug environment may subvert DC-EBV-specific T cell interactions. Type-1 CD8(+) T cells are critical for efficient immune surveillance and control of EBV infection, whereas type-2 or Treg/type-3 responses may provide an environment conductive to disease progression. We have recently reported that chronic IS inhibits DC function in transplant patients. Here, we have analyzed the comparative ability of mature, type-1 polarized DCs (i.e. DC1) generated from quiescent transplant patients or healthy controls, to boost type-1 EBV-specific CD8(+) T cells in vitro. Our results show that unlike healthy controls, where DC1 loaded with MHC class I EBV peptides preferentially reactivate specific type-1 CD8(+) T cells, DC1 generated from transplant patients reactivate EBV-specific CD8(+) T cells that produce both IFN-gamma and IL-10, up-regulate FOXP3 mRNA, and suppress noncognate CD4(+) T-cell proliferation via cell-cell contact. These data support a novel regulatory pathway for anti-EBV T-cell-mediated responses in IS transplant patients, with implications for the design of adoptive immunotherapies in this setting.

Use of the inhibitory effect of apoptotic cells on dendritic cells for graft survival via T-cell deletion and regulatory T cells.

Tolerance induction against donor allo-antigens (allo-Ag) remains one of the most challenging aspects of transplant immunology. The ability of dendritic cells (DC) to participate in immunity and tolerance makes them an excellent tool for tolerance induction. Here, we employed the immunosuppressive properties of apoptotic cells to deliver simultaneously an inhibitory signal and donor allo-Ag to recipient DC for treatment of allograft rejection. DC that captured apoptotic cells remained immature and activated deficiently anti-donor CD4(+) T cells that were unable to upregulate T-cell activation markers, to secrete IL-2 and IFN-gamma and to survive under homeostatic conditions due to low expression of Bcl-X(L), IL-7R and IL-15R. Administration of donor apoptotic cells decreased the systemic anti-donor T- and B-cell response and prolonged cardiac allograft survival in mice. The effect was donor specific and required the interaction of donor apoptotic cells with recipient quiescent CD8alpha(+) DC. When combined with CD40-CD154-blockade, administration of donor apoptotic cells resulted in indefinite graft survival mediated by generation of regulatory T cells. The use of the inhibitory effects of apoptotic cells on the anti-donor response provides a new approach to treat transplant rejection.

The immune regulatory effect of apoptotic cells and exosomes on dendritic cells: its impact on transplantation.

Dendritic cells (DC) are professional antigen (Ag) presenting cells (APC) that trigger the anti-donor T-cell response that causes allograft rejection. During the past decade several laboratories have employed in vitro generated DC with tolerogenic potential for prolongation of allograft survival. This minireview describes the development of a second-generation of DC-based strategies for transplantation tolerance based on the delivery in situ of donor allogeneic (allo)-Ag to quiescent DC of graft recipients by means of donor-derived apoptotic cells or exosomes. Donor leukocytes in early apoptosis are rich in allo-Ag, are internalized efficiently by recipient DC in vivo and deliver immunosuppressive signals to DC. Administration (i.v.) of donor apoptotic leukocytes prolongs bone marrow engraftment and cardiac allografts survival in mice by exerting a profound down-regulatory effect on the anti-donor T-cell response. Exosomes are nanovesicles (<100 nm) produced by different cell types, including APC. DC-derived exosomes are rich in major histocompatibility complex (MHC) molecules that can be employed to target DC in situ. Once i.v. injected, exosomes carrying donor MHC molecules are captured by recipient's DC and prolong allograft survival in rodents. The use of the regulatory functions of apoptotic cells and exosomes may be useful tools to develop new strategies for transplantation tolerance.

Dendritic cells genetically engineered to express IL-4 exhibit enhanced IL-12p70 production in response to CD40 ligation and accelerate organ allograft rejection.

C57BL/10 (B10; H2(b)) bone marrow-derived myeloid dendritic cells (DC) propagated in GM-CSF + IL-4 were transduced with r adenoviral (Ad) vectors encoding either control neomycin-resistance gene (Ad-Neo) or murine IL-4 (Ad-IL-4) on day 5 of culture following CD11c immunomagnetic bead purification. Both Ad-Neo- and Ad-IL-4-transduced DC displayed upregulated surface MHC class II and costimulatory molecules (CD40, CD80, CD86). Ad-IL-4 DC secreted higher levels of bioactive IL-12p70 after CD40 ligation or LPS stimulation than either Ad-Neo or unmodified DC. Only Ad-IL-4 DC produced IL-12p70 in primary MLR, in which they induced augmented proliferative responses of naïve allogeneic C3H/HeJ (C3H; H2(k)) T-cells. Compared with Ad-Neo DC, Ad-IL-4 DC were also more effective in priming naïve allogeneic recipients to exhibit specifically enhanced anti-donor T-cell proliferative and CTL responses. T-cells primed in vivo 7 days previously with Ad-IL-4 DC displayed enhanced secretion of Th2 (IL-4, IL-10) but also higher Th1 cytokine (IFNgamma) production following ex vivo challenge with donor alloAg. Moreover, pretreatment of vascularized heart graft recipients with i.v. Ad-IL-4 DC, 1 week before transplant, significantly accelerated rejection and antagonized the therapeutic effect of anti-CD40L (CD154) mAb. These data contrast markedly with recently reported inhibitory effects of autologous Ad-IL-4 DC on autoimmune inflammatory disease.

Dermal-resident CD14+ cells differentiate into Langerhans cells.

Epidermal Langerhans cells (LCs) show extraordinary immunostimulatory capacity and play a key role in the initiation and regulation of immune responses. Studies of LC biology are currently the focus of efforts to engineer immune responses and to better understand the immunopathology of cutaneous diseases. Here we identified and characterized a population of LC precursors that were resident in human skin. These immediate precursors expressed CD14, langerin and functional CCR6. When cultured with transforming growth factor-beta1 alone, they had the potential to differentiate into epidermal LCs; when cultured in the presence of granulocyte macrophage-colony-stimulating factor and interleukin 4 they differentiated into functionally mature dendritic cells. Identification and characterization of these LC precursors provided insight into LC biology and the mechanism(s) through which LCs repopulate the epidermis.

Potential of tolerogenic dendritic cells for transplantation.

Dendritic cells (DC) are professional antigen (Ag)-presenting cells considered traditionally as the passenger leukocytes that, after migration from transplanted tissues, stimulate allospecific naive T cell responses and trigger acute rejection. However, there is recent evidence that, besides their role in central T lymphocyte deletion in the thymus, DC perform a crucial function to induce/maintain peripheral T cell tolerance. This paper outlines conceptual models that try to explain how DC may induce/maintain tolerance. It also considers how such ideas have been implemented recently in an effort to generate tolerogenic DC to induce donor Ag-specific tolerance/ immunosuppression and prolonged allograft survival. These approaches include genetic engineering of donor- or recipient-derived DC to express molecules capable of promoting tolerance to alloAg.

Cytokine production by mouse myeloid dendritic cells in relation to differentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation.

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.

Designer dendritic cells for tolerance induction: guided not misguided missiles.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play crucial roles as initiators and modulators of adaptive immune responses. Although DC-based vaccines have been utilized successfully to generate cytolytic T-cell activity against tumor antigens (Ags), evidence has accumulated that DCs also have potent capabilities to tolerize T cells in an Ag-specific manner. DCs cultured in the laboratory can suppress auto- or alloimmunity. Current and prospective strategies to promote this inherent tolerogenic potential of DCs might prove to be important for the therapy of transplant rejection and autoimmune diseases.

Aspirin inhibits in vitro maturation and in vivo immunostimulatory function of murine myeloid dendritic cells.

Aspirin is the most commonly used analgesic and antiinflammatory agent. In this study, at physiological concentrations, it profoundly inhibited CD40, CD80, CD86, and MHC class II expression on murine, GM-CSF + IL-4 stimulated, bone marrow-derived myeloid dendritic cells (DC). CD11c and MHC class I expression were unaffected. The inhibitory action was dose dependent and was evident at concentrations higher than those necessary to inhibit PG synthesis. Experiments with indomethacin revealed that the effects of aspirin on DC maturation were cyclooxygenase independent. Nuclear extracts of purified, aspirin-treated DC revealed a decreased NF-kappaB DNA-binding activity, whereas Ab supershift analysis indicated that aspirin targeted primarily NF-kappaB p50. Unexpectedly, aspirin promoted the generation of CD11c+ DC, due to apparent suppression of granulocyte development. The morphological and ultrastructural appearance of aspirin-treated cells was consistent with immaturity. Aspirin-treated DC were highly efficient at Ag capture, via both mannose receptor-mediated endocytosis and macropinocytosis. By contrast, they were poor stimulators of naive allogeneic T cell proliferation and induced lower levels of IL-2 in responding T cells. They also exhibited impaired IL-12 expression and did not produce IL-10 after LPS stimulation. Assessment of the in vivo function of aspirin-treated DC, pulsed with the hapten trinitrobenzenesulfonic acid, revealed an inability to induce normal cell-mediated contact hypersensitivity, despite the ability of the cells to migrate to T cell areas of draining lymphoid tissue. These data provide new insight into the immunopharmacology of aspirin and suggest a novel approach to the manipulation of DC for therapeutic application.

Mammalian and viral IL-10 enhance C-C chemokine receptor 5 but down-regulate C-C chemokine receptor 7 expression by myeloid dendritic cells: impact on chemotactic responses and in vivo homing ability.

The immunosuppressive and anti-inflammatory cytokine IL-10 inhibits the phenotypic and functional maturation of dendritic cells (DC) and has been reported to confer tolerogenic properties on these important professional APC. Here, we exposed murine bone marrow-derived myeloid DC to either mouse (m) or viral (v) IL-10 early during their in vitro generation in response to GM-CSF and IL-4. Both mIL-10 and vIL-10 down-regulated the expression of CCR7 mRNA determined by RT-PCR, while mIL-10 up-regulated the expression of CCR5 transcripts. These changes in CCR7 and CCR5 expression were associated with inhibition and augmentation, respectively, of DC chemotaxis toward their respective agonists, macrophage inflammatory proteins 3beta and 1alpha, while in vivo homing of DC from peripheral s.c. sites to secondary lymphoid tissue of syngeneic or allogeneic recipients was significantly impaired. Anti-mIL-10R mAb reversed the effects of mIL-10 on CCR expression and restored DC homing ability. Retroviral transduction of mIL-10- and vIL-10-treated DC to overexpress transgenic CCR7 partially restored the cells' lymphoid tissue homing ability in allogeneic recipients. However, CCR7 gene transfer did not reinstate the capacity of IL-10-treated DC to prime host naive T cells for ex vivo proliferative responses or Th1 cytokine (IFN-gamma) production in response to rechallenge with (donor) alloantigen. These findings suggest that in addition to their capacity to subvert DC maturation/function and confer tolerogenic potential on these cells, mIL-10 and vIL-10 regulate DC migratory responses via modulation of CCR expression.

Role of dendritic cells in the immune response against allografts.

Graft-derived 'passenger' dendritic cells have been classically considered as the instigators of acute organ rejection. However, recent advances have revealed that dendritic cells are also involved in the induction/maintenance of peripheral tolerance. This paper briefly reviews the most recent knowledge of the role of donor and recipient dendritic cells during the immune response against allografts, and of the clinical potential of 'tolerogenic' dendritic cells.

Cell-type-specific and regulatable transgenesis in the adult brain: adenovirus-encoded combined transcriptional targeting and inducible transgene expression.

To achieve transient transgenesis within specific areas or cell populations in the adult central nervous system (CNS), we have developed a dual adenoviral vector system encoding for cell-type-specific and regulatable transcription units. To achieve combined cell-type-specific transcriptional targeting and inducible expression, we have engineered the expression of the tetracycline-dependent transcriptional elements (1) to be under the transcriptional control of either the astrocyte-specific, glial fibrillary acidic protein (GFAP) (2) or the neuronal specific enolase (NSE) promoter (3) within a dual adenoviral vector system. Cell-type specificity, inducibility, and levels of transgene expression were characterized in vitro in cell lines, and primary neocortical cultures and in the central nervous system (CNS) in vivo, and compared to a powerful pancellular beta-actin/CMV promoter. We demonstrate that the GFAP promoter is able to restrict tetracycline-dependent transgene expression to glial cells in cell lines, primary cultures, and in the CNS in vivo. However, although the NSE promoter did not show neuronal restricted transgene expression in vitro, it did so in the CNS in vivo. Our dual viral system also has provided evidence that an excess of transactivator is needed to achieve maximal transgene expression. Administration of doxycycline completely abrogated transgene expression both in vitro and in vivo. Consequently, our strategy demonstrates that combined cell-type specificity and simultaneous regulation of transgene expression can be obtained in the brain using adenoviral vectors.