Infusion Of Dendritic Cells Research Articles

Dendritic Cell Vaccine Harboring Inactivated Mycobacteria Induces Immune Protection Against Tuberculosis in Murine Models and is Well Tolerated in Humans

The limited success of tuberculosis (TB) control measures reflects the inadequacy of Bacille Calmette‐Guérin (BCG), the only licensed TB vaccine. There is a recent resurgence of interest in intravenous administration of BCG. However, direct injection of live BCG bacteria into the bloodstream of human beings is not likely to be practical due to safety concerns. In this study, it is showed that debris of BCG‐infected macrophages induces activation and maturation of dendritic cells (DCs) in vitro, and an intravenous DCs vaccine phagocytosing noninfective cell debris induces robust antigen‐specific T‐cell immune responses and immune protection against Mycobacterium tuberculosis infection in murine models. Further, an investigator‐initiated clinical trial shows the safety of a DCs vaccine harboring the noninfective Mycobacterium vaccae vaccine. Infusions of naive DCs and DCs harboring Mycobacterium vaccae are well tolerated and safe in six active TB patients. Tests of the peripheral blood mononuclear cells of a patient who receives two doses of DCs vaccine infusion show enhanced secretion of IFN‐γ, IL‐2, IL‐17, and TNF‐α in both CD4 and CD8 T cells. The study provides evidence that DC‐based vaccines harboring inactivated mycobacteria can expand T‐cell immune responses in TB‐infected mice and are well tolerated in patients with active TB disease.

Tolerogenic CD11c+dendritic cells regulate CD4+Tregs in replacing delayed ischemic preconditioning to alleviate ischemia-reperfusion acute kidney injury.

Ischemia-reperfusion injury (IRI) is one of the primary clinical causes of acute kidney injury (AKI). The key to IRI lies in immune-inflammatory damage, where dendritic cells (DCs) play a central role in eliciting immune responses within the context of inflammation induced by ischemia-reperfusion. Our previous study has confirmed that delayed ischemic preconditioning (DIPC) can reduce the kidney injury by mediating DCs to regulate T-cells. However, the clinical feasibility of DIPC is limited, as pre-clamping of the renal artery is not applicable for the prevention and treatment of ischemia-reperfusion acute kidney injury (I/R-AKI) in clinical patients. Therefore, the infusion of DCs as a substitute for DIPC presents a more viable strategy for preventing renal IRI. In this study, we further evaluated the impact and mechanism of infused tolerogenic CD11c+DCs on the kidneys following IRI by isolating bone marrow-derived dendritic cells and establishing an I/R-AKI model after pre-infusion of DCs. Renal function was significantly improved in the I/R-AKI mouse model after pre-infused with CD11c+DCs. The pro-inflammatory response and oxidative damage were reduced, and the levels of T helper 2 (Th2) cells and related anti-inflammatory cytokines were increased, which was associated with the reduction of autologous DCs maturation mediated by CD11c+DCs and the increase of regulatory T-cells (Tregs). Next, knocking out CD11c+DCs, we found that the reduced immune protection of tolerogenic CD11c+DCs reinfusion was related to the absence of own DCs. Together, pre-infusion of tolerogenic CD11c+DCs can replace the regulatory of DIPC on DCs and T-cells to alleviate I/R-AKI. DC vaccine is expected to be a novel avenue to prevent and treat I/R-AKI.

Donor-derived regulatory dendritic cell infusion modulates effector CD8+ T cell and NK cell responses after liver transplantation.

Immune cell-based therapies are promising strategies to facilitate immunosuppression withdrawal after organ transplantation. Regulatory dendritic cells (DCreg) are innate immune cells that down-regulate alloimmune responses in preclinical models. Here, we performed clinical monitoring and comprehensive assessment of peripheral and allograft tissue immune cell populations in DCreg-infused live-donor liver transplant (LDLT) recipients up to 12 months (M) after transplant. Thirteen patients were given a single infusion of donor-derived DCreg 1 week before transplant (STUDY) and were compared with 40 propensity-matched standard-of-care (SOC) patients. Donor-derived DCreg infusion was well tolerated in all STUDY patients. There were no differences in postoperative complications or biopsy-confirmed acute rejection compared with SOC patients up to 12M. DCreg administration was associated with lower frequencies of effector T-bet+Eomes+CD8+ T cells and CD16bright natural killer (NK) cells and an increase in putative tolerogenic CD141+CD163+ DCs compared with SOC at 12M. Antidonor proliferative capacity of interferon-γ+ (IFN-γ+) CD4+ and CD8+ T cells was lower compared with antithird party responses in STUDY participants, but not in SOC patients, at 12M. In addition, lower circulating concentrations of interleukin-12p40 (IL-12p40), IFN-γ, and CXCL10 were detected in STUDY participants compared with SOC patients at 12M. Analysis of 12M allograft biopsies revealed lower frequencies of graft-infiltrating CD8+ T cells, as well as attenuation of cytolytic TH1 effector genes and pathways among intragraft CD8+ T cells and NK cells, in DCreg-infused patients. These reductions may be conducive to reduced dependence on immunosuppressive drug therapy or immunosuppression withdrawal.

A randomised phase II clinical trial of low-dose cyclophosphamide and transarterial chemoembolization (TACE) with or without vaccination with dendritic cells (DC) pulsed with HepG2 lysate ex vivo in patients with hepatocellular carcinoma (HCC): The ImmunoTACE trial.

4012 Background: A previous study by our group using autologous monocyte-derived DC pulsed ex vivo with HepG2 cell lysate showed some clinical benefit with evidence of antigen-specific T-cell responses in patients with advanced HCC. The current trial reports the activity of this vaccine in combination with TACE in patients with HCC. All patients also received low-dose cyclophosphamide to deplete regulatory T cells and thereby enhance vaccination. Methods: Patients with intermediate stage HCC (performance status 0-2, Child Pugh A/B7) were randomised 1:1 to TACE plus low-dose cyclophosphamide (Group 1) or TACE plus low-dose cyclophosphamide plus dendritic cell vaccination (Group 2). Cyclophosphamide was administered on Day 1 and 29 followed by TACE on Day 31 (+/- DC infusion), with further cyclophosphamide on Days 60, 90 and 120 (+/- additional DC infusions on Days 62, 92 and 122). The primary endpoint was progression free survival (PFS) by RECIST v1.1. Secondary endpoints included radiological response by RECIST v1.1, PFS and radiological response according to modified (m) RECIST, overall survival (OS), immune response and toxicity. Target recruitment was 48 evaluable patients (24 patients in each arm) to detect a 20% increase in PFS rate at 1 year (30% vs 50%) with a relaxed one-sided statistical significance level of 20% and 80% power using a logrank test. Results: Between March 2016 and October 2019, 55 patients from 3 UK centres were randomised of whom 48 were evaluable (24 each arm). Median PFS by RECIST criteria was significantly longer in Group 2 compared to Group 1 (18.6 vs 10.4 months: hazard ratio (HR) 0.43, 80% CI -∞-0.59; one-sided p = 0.02). Median PFS using mRECIST criteria showed a similar magnitude of benefit (18.6 vs 10.8 months: HR 0.48, 95% CI 0.22-1.02). Median OS was 25.7 months in Group 2 vs 21.5 months in Group 1 (HR 0.61, 95% CI 0.27-1.38). Group 2 showed a higher overall response rate (complete and partial response) by RECIST (54% vs 29%) and mRECIST (75% vs 54%) and a higher disease control rate (complete and partial response and stable disease) by RECIST (92% vs 67%) and mRECIST (88% vs 67%). Treatment with DC infusions was well tolerated; the most common adverse events were chills (30%), fatigue (22%) and nausea (22%), all of which were low grade. Immune response analyses are currently ongoing. Conclusions: The addition of tumour lysate pulsed DC infusions to treatment with TACE plus low-dose cyclophosphamide significantly increased PFS in patients with HCC. To the best of our knowledge, this is the first randomised study to demonstrate efficacy using DC in HCC. Further investigation of the role of DC infusions in the treatment of HCC are warranted but will need to take into account the current evolving immunotherapy landscape. Clinical trial information: 11889464.

Rapamycin Alternatively Modifies Mitochondrial Dynamics in Dendritic Cells to Reduce Kidney Ischemic Reperfusion Injury.

Dendritic cells (DCs) are unique immune cells that can link innate and adaptive immune responses and Immunometabolism greatly impacts their phenotype. Rapamycin is a macrolide compound that has immunosuppressant functions and is used to prevent graft loss in kidney transplantation. The current study evaluated the therapeutic potential of ex-vivo rapamycin treated DCs to protect kidneys in a mouse model of acute kidney injury (AKI). For the rapamycin single (S) treatment (Rapa-S-DC), Veh-DCs were treated with rapamycin (10 ng/mL) for 1 h before LPS. In contrast, rapamycin multiple (M) treatment (Rapa-M-DC) were exposed to 3 treatments over 7 days. Only multiple ex-vivo rapamycin treatments of DCs induced a persistent reprogramming of mitochondrial metabolism. These DCs had 18-fold more mitochondria, had almost 4-fold higher oxygen consumption rates, and produced more ATP compared to Veh-DCs (Veh treated control DCs). Pathway analysis showed IL10 signaling as a major contributing pathway to the altered immunophenotype after Rapamycin treatment compared to vehicle with significantly lower cytokines Tnfa, Il1b, and Il6, while regulators of mitochondrial content Pgc1a, Tfam, and Ho1 remained elevated. Critically, adoptive transfer of rapamycin-treated DCs to WT recipients 24 h before bilateral kidney ischemia significantly protected the kidneys from injury with a significant 3-fold improvement in kidney function. Last, the infusion of DCs containing higher mitochondria numbers (treated ex-vivo with healthy isolated mitochondria (10 µg/mL) one day before) also partially protected the kidneys from IRI. These studies demonstrate that pre-emptive infusion of ex-vivo reprogrammed DCs that have higher mitochondria content has therapeutic capacity to induce an anti-inflammatory regulatory phenotype to protect kidneys from injury.

Modeling Dendritic Cell Pulsed Immunotherapy for Mice with Melanoma—Protocols for Success and Recurrence

Nowadays, immunotherapy has become an important alternative to fight cancer. One way in which biologists and medics use immunotherapy is by injecting antigen-incubated Dendritic Cells (DCs) into mice to stimulate an immune response. The DCs optimal quantities and infusion times for a successful cancer eradication are often unknown to the therapists; usually, these quantities are obtained by testing various protocols. The article shows a model of five differential equations which represents some interactions between some cells of the immune system and tumor cells which is used to test different infusion protocols of Dendritic Cells. This study aims to find operation ranges to DCs quantities and injection times for which the therapy reduces the tumor significantly. To that end, an exhaustive search of operative protocols is performed using simulations of a mathematical model. Furthermore, nonlinear analysis of the model reveals that without the DC therapy tumor cells cannot stay under non-lethal bounds. Finally, we show that a pulsed periodic therapy can prevent tumor relapsing when the doses and period times lie within a certain range.

Donor-derived regulatory dendritic cell infusion results in host cell cross-dressing and T cell subset changes in prospective living donor liver transplant recipients.

Regulatory dendritic cells (DCreg) promote transplant tolerance following their adoptive transfer in experimental animals. We investigated the feasibility, safety, fate, and impact on host T cells of donor monocyte-derived DCreg infused into prospective, living donor liver transplant patients, 7days before transplantation. The DCreg expressed a tolerogenic gene transcriptional profile, high cell surface programed death ligand-1 (PD-L1):CD86 ratios, high IL-10/no IL-12 productivity and poor ability to stimulate allogeneic T cell proliferation. Target DCreg doses (range 2.5-10×106 cells/kg) were achieved in all but 1 of 15 recipients, with no infusion reactions. Following DCreg infusion, transiently elevated levels of donor HLA and immunoregulatory PD-L1, CD39, and CD73 were detected in circulating small extracellular vesicles. At the same time, flow and advanced image stream analysis revealed intact DCreg and "cross-dressing" of host DCs in blood and lymph nodes. PD-L1 co-localization with donor HLA was observed at higher levels than with recipient HLA. Between DCreg infusion and transplantation, T-bethi Eomeshi memory CD8+ T cells decreased, whereas regulatory (CD25hi CD127- Foxp3+ ): T-bethi Eomeshi CD8+ T cell ratios increased. Thus, donor-derived DCreg infusion may induce systemic changes in host antigen-presenting cells and T cells potentially conducive to modulated anti-donor immune reactivity at the time of transplant.

Safety and Long-Term Outcome of Intratumoral Injection of OK432-Stimulated Dendritic Cells for Hepatocellular Carcinomas After Radiofrequency Ablation

Dendritic cell (DC)–based immunotherapies are believed to help eradicate residual tumor cells, including hepatocellular carcinoma (HCC). Here, we assessed the safety and clinical response to OK432-stimulated monocyte-derived DCs (MoDCs) in treating HCC after radiofrequency ablation (RFA). MoDCs were derived from 30 HCC patients in the presence of interleukin-4 and granulocyte-macrophage colony stimulating factor for 5 days and then cultured for 2 more days in the medium (basic protocol) or stimulated with OK432. On day 7, DCs were harvested and percutaneously injected into HCC tumors after RFA. We observed no grade 3 or 4 National Cancer Institute Common Toxicity Criteria adverse events. Kaplan-Meier analysis indicated that patients treated with RFA + OK432-stimulated DCs transfer had longer recurrence-free survival than those treated with RFA + basic-protocol DCs (median: 24.8 vs 13.0 months; P = .003). RFA with DC infusion can enhance various tumor-associated antigen (TAA)–specific T-cell responses. Additionally, the 5-year RFS rate for patients with significantly increased TAA-specific T-cell responses was much higher than for other patients (50.0% vs. 7.7%; P = .030). Our study provides useful information for development of HCC immunotherapies (trial registration: UMIN000001701).

FLT3/FLT3L-mediated CD103+ dendritic cells alleviates hepatic ischemia-reperfusion injury in mice via activation of treg cells

BackgroundThis study was conducted to investigate the protective effect of Fms-like tyrosine kinase 3 (FLT3)/FLT3 ligand (FLT3L)-dependent CD103+ dendritic cells (DCs) on hepatic ischemia-reperfusion injury (IRI). MethodsA mouse model of hepatic IRI and cellular model following hypoxia-reperfusion (H/R) treatment were established. Peripheral blood and liver tissues were obtained and analyzed by flow cytometer in terms of percentage of CD103+DCs and regulatory T (Treg) cells. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were determined to assess liver function. Moreover, pro-inflammatory cytokines levels including tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 were measured using enzyme-linked immunosorbent assay (ELISA). The histological morphology of liver tissues was examined with hematoxylin and eosin (HE) staining. The apoptosis was detected by terminal deoxynucleotidyl transferase (TdT) dUTP Nick End Labeling (TUNEL) assay. Treg-associated cytokines transforming growth factor (TGF)-β and IL-10 expressions were measured using quantitative real time polymerase chain reaction (qRT-PCR). ResultsCD103+ DCs were significantly decreased in peripheral blood and liver tissues of mouse model of hepatic IRI. In vivo experiments indicated that CD103+ DCs infusion ameliorated IRI-induced liver damage and Treg inhibition. Further investigations demonstrated that FLT3/FLT3L-dependent CD103+ DCs suppressed hepatocyte apoptosis via activation of Treg cells in vitro. ConclusionFLT3/FLT3L-induced CD103+ DCs alleviated hepatic IRI through activating Treg cells.

Combined therapy of infusion of DC from rats with higher expression of IDO and CD40L on rejection post heart transplantation.

Indoleamine 2, 3-dioxygenase (IDO) can inhibit rejection of graft via inducing T cell apoptosis. CD40L monoclonal antibody (mAb) inhibits T cell activation. However, the effects of the combination of infusion of dendritic cell (DC) from IDO over-expressed donor mice and CD40L mAb on the treatment of graft rejection after heart transplantation have not been reported. Allogeneic heart transplantation mouse model was established. Recipient mice were divided into three groups, including control group, IDO group (in which DC donors received adenoviral vector of IDO) and combined therapy group (which received both IDO over-expressed DC infusion and CD40L mAb injection post transplantation). Survival time and cardiac function were observed, with IDO expression being quantified. Flow cytometry (FCM) was used to analyze T cell apoptosis, while enzyme linked immunosorbent assay (ELISA) was adopted to test the levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-10 (IL-10) and interleukin-6 (IL-6). IDO expression was significantly elevated in both IDO and combined therapy groups, with enhanced T cell apoptosis compared to control group (p < 0.05). Both groups had better survival time and cardiac functions compared to control group, along with increased IL-10/IL-6 expression and suppressed INF-γ and IL-2 expression (p < 0.05). However, combined therapy had a better efficiency compared to IDO group (p < 0.05). Combined therapy of high IDO expressed mouse DC perfusion with CD40L mAb can elongate the survival time of recipient heart and inhibit rejection reaction via facilitating T cell apoptosis. Meanwhile, combined therapy could also regulate the expression of some immune suppressant factors and mediate the Th1/Th2 cytokine balance.

Donor-Derived Regulatory Dendritic Cell Infusion Maintains Donor-Reactive CD4+CTLA4hi T Cells in Non-Human Primate Renal Allograft Recipients Treated with CD28 Co-Stimulation Blockade.

Donor-derived regulatory dendritic cell (DCreg) infusion before transplantation, significantly prolongs renal allograft survival in non-human primates. This is associated with enhanced expression of the immunoregulatory molecules cytotoxic T-lymphocyte-associated antigen (Ag) 4 (CTLA4) and programmed cell death protein 1 (PD1) by host donor-reactive T cells. In rodents and humans, CD28 co-stimulatory pathway blockade with the fusion protein CTLA4:Ig (CTLA4Ig) is associated with reduced differentiation and development of regulatory T cells (Treg). We hypothesized that upregulation of CTLA4 by donor-reactive CD4+ T cells in DCreg-infused recipients treated with CTLA4Ig, might be associated with higher incidences of donor-reactive CD4+ T cells with a Treg phenotype. In normal rhesus monkeys, allo-stimulated CD4+CTLA4hi, but not CD4+CTLA4med/lo T cells exhibited a regulatory phenotype, irrespective of PD1 expression. CTLA4Ig significantly reduced the incidence of CD4+CTLA4hi, but not CD4+CTLA4med/lo T cells following allo-stimulation, associated with a significant reduction in the CD4+CTLA4hi/CD4+CTLA4med/lo T cell ratio. In CTLA4Ig-treated renal allograft recipient monkeys, there was a marked reduction in circulating donor-reactive CD4+CTLA4hi T cells. In contrast, in CTLA4Ig-treated monkeys with DCreg infusion, no such reduction was observed. In parallel, the donor-reactive CD4+CTLA4hi/CD4+CTLA4med/lo T cell ratio was reduced significantly in graft recipients without DCreg infusion, but increased in those given DCreg. These observations suggest that pre-transplant DCreg infusion promotes and maintains donor-reactive CD4+CTLA4hi T cells with a regulatory phenotype after transplantation, even in the presence of CD28 co-stimulation blockade.

Pre-transplant infusion of donor-derived dendritic cells maintained at the immature stage by sinomenine increases splenic Foxp3+ Tregs in recipient rats after renal allotransplantation

ObjectiveThe immunosuppressive mechanism of sinomenine in organ allotransplantation was investigated, especially its effect of blocking dendritic cell (DC) maturation, which might influence the frequency of regulatory T cells (Tregs). MethodsBone marrow cells from male donor Wistar rats were induced to differentiate into DCs in vitro in the presence or absence of sinomenine, and characterized by flow cytometry. These two groups of DCs were respectively injected into male recipient Sprague-Dawley rats via the tail vein, at both high and low doses. Sprague-Dawley rats receiving saline injection were used as controls. Seven days later, renal transplantation was performed from donor Wistar rats to the recipient Sprague-Dawley rats. Seven days after transplantation, spleens were collected from the recipients. The proportions of Tregs and Foxp3+ Tregs to CD4+ T cells were determined using flow cytometry. ResultsWith sinomenine treatment, the frequency of mature DCs was reduced, as indicated by lower expression of the surface markers CD80, CD86, and RT1B. In recipient Sprague-Dawley rats that received sinomenine-treated DCs before renal allotransplantation, the proportions of splenic Tregs and Foxp3+ Tregs were significantly higher than in control recipients receiving saline or DCs without sinomenine treatment (all p<0.05). A high dose of sinomenine-treated DCs (106 cells) had a more obvious effect in increasing Tregs than the low dose (105 cells) (p<0.05). ConclusionPre-transplant infusion of donor-derived sinomenine-induced maturation arrested DCs could result in the increase of Foxp3+ Tregs in the spleens of recipients after renal allotransplantation.

Intratumoral Delivery of Immunotherapy-Act Locally, Think Globally.

Immune mechanisms have evolved to cope with local entry of microbes acting in a confined fashion but eventually inducing systemic immune memory. Indeed, in situ delivery of a number of agents into tumors can mimic in the malignant tissue the phenomena that control intracellular infection leading to the killing of infected cells. Vascular endothelium activation and lymphocyte attraction, together with dendritic cell-mediated cross-priming, are the key elements. Intratumoral therapy with pathogen-associated molecular patterns or recombinant viruses is being tested in the clinic. Cell therapies can be also delivered intratumorally, including infusion of autologous dendritic cells and even tumor-reactive T lymphocytes. Intralesional virotherapy with an HSV vector expressing GM-CSF has been recently approved by the Food and Drug Administration for the treatment of unresectable melanoma. Immunomodulatory monoclonal Abs have also been successfully applied intratumorally in animal models. Local delivery means less systemic toxicity while focusing the immune response on the malignancy and the affected draining lymph nodes.

Prolonged intralymphatic delivery of dendritic cells through implantable lymphatic ports in patients with advanced cancer.

BackgroundThe currently-used modes of administration of immunotherapeutic agents result in their limited delivery to the lymph nodes and/or require repetitive ultrasound-guided nodal injections or microsurgical lymphatic injections, limiting their feasibility. Here, we report on the feasibility and safety of a new method of long-term repetitive intralymphatic (IL) infusion of immune cells, using implantable delivery ports.MethodsNine patients with stage IV recurrent colorectal cancer underwent complete resection and received autologous dendritic cells (DCs) loaded with killed autologous tumor cells, KLH and PADRE, for up to four monthly cycles. Leg lymphatic vessels were cannulated, connected to 6.6Fr low-profile implantable subcutaneous delivery ports, and used to infuse 12 doses of DC over each 72 h-long cycle (every 6 h), followed by heparin flushes of the cannula-port system (one 72 h-long cycle per month). The patients who opted for alternative route of vaccine administration (2 patients) or whose ports became non-functional between cycles, continued treatment via intranodal (one injection/cycle) or intradermal (four injections/cycle) routes.ResultsA total of nine lymphatic cannulations and implantations of subcutaneous delivery ports were attempted in seven patients, with a success rate of eight out of nine (89 %). The average patency of the IL delivery system was 7.5 (±3.2) weeks. All six patients with IL ports successfully completed at least one complete 72 h-long DC infusion cycle (12 injections). Five patients (56 %) completed two full IL cycles (24 IL injections). No patients received more than two IL cycles without replacement of the IL port, due to catheter occlusion and/or local side effects: cellulitis and hematoma. Intranodal and intradermal backup options were used in, respectively, one and two patients. Overall cohort survival was >28 (±25) months. One patient with aggressive recurrent carcinomatosis, who received DC vaccines by intranodal route is alive at > 90 months, without evidence of disease.ConclusionsWe conclude that an intermediate-duration IL delivery of multiple doses of immunotherapeutic factors using implantable delivery ports is feasible, highly-tolerable and can be reproducibly performed in cancer patients to administer immune cells, or potentially, other immune factors. However, long-term IL port placement (>7.5 weeks), is not a currently-feasible option.Trial registrationNCT00558051, registered Nov. 13, 2007.

Eomesodermin(lo) CTLA4(hi) Alloreactive CD8+ Memory T Cells Are Associated With Prolonged Renal Transplant Survival Induced by Regulatory Dendritic Cell Infusion in CTLA4 Immunoglobulin-Treated Nonhuman Primates.

Memory T cells (Tmem), particularly those resistant to costimulation blockade (CB), are a major barrier to transplant tolerance. The transcription factor Eomesodermin (Eomes) is critical for Tmem development and maintenance, but its expression by alloactivated T cells has not been examined in nonhuman primates. We evaluated Eomes and coinhibitory cytotoxic T lymphocyte antigen-4 (CTLA4) expression by alloactivated rhesus monkey T cells in the presence of CTLA4 immunoglobulin, both in vitro and in renal allograft recipients treated with CTLA4Ig, with or without regulatory dendritic cell (DCreg) infusion. In normal monkeys, CD8+ T cells expressed significantly more Eomes than CD4+ T cells. By contrast, CD8+ T cells displayed minimal CTLA4. Among T cell subsets, central Tmem (Tcm) expressed the highest levels of Eomes. Notably, Eomes(lo)CTLA4(hi) cells displayed higher levels of CD25 and Foxp3 than Eomes(hi)CTLA4(lo) CD8+ T cells. After allostimulation, distinct proliferating Eomes(lo)CTLA4(hi) and Eomes(hi)CTLA4(lo) CD8+ T cell populations were identified, with a high proportion of Tcm being Eomes(lo)CTLA4(hi). CB with CTLA4Ig during allostimulation of CD8+ T cells reduced CTLA4 but not Eomes expression, significantly reducing Eomes(lo)CTLA4(hi) cells. After transplantation with CB and rapamycin, donor-reactive Eomes(lo)CTLA4(hi) CD8+ T cells were reduced. However, in monkeys also given DCreg, absolute numbers of these cells were elevated significantly. Low Eomes and high CTLA4 expression by donor-reactive CD8+ Tmem is associated with prolonged renal allograft survival induced by DCreg infusion in CTLA4Ig-treated monkeys. Prolonged allograft survival associated with DCreg infusion may be related to maintenance of donor-reactive Eomes(lo)CTLA4(hi) Tcm.

Infusion of Dendritic Cells Carrying Donor Lymphocytes Treated With 8-Methoxypsoralen and Ultraviolet A Light Induces CD19+IL-10+ Regulatory B cells and Promotes Skin Allograft Survival

PUVA-SP DCs are immature dendritic cells (imDCs) that have taken up donor splenic lymphocytes treated with 8-methoxypsoralen and ultraviolet A light (PUVA-SPs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient imDCs, and infusion of PUVA-SP DCs can induce CD4+CD25highFoxp3+ regulatory T cells (Treg). However, the regulatory effect of PUVA-SP DCs on B cells is poorly understood. In this study, we compared PUVA-SP DCs with imDCs in terms of lipopolysaccharide (LPS) responsiveness and flow cytometric analysis of interleukin (IL) 10 expression in splenic CD19+B cells. Our results demonstrate that PUVA-SP DCs can suppress subsequent LPS-induced DC maturation and that infusion of PUVA-SP DCs, in the absence of an immunosuppressant, significantly promotes skin allograft survival. This effect was associated with up-regulation of circulating regulatory B cells exhibiting preferential IL-10 secretion. Our results suggest that effective treatments involving infusion of PUVA-SP DCs is likely related to the modulation not only of T-cell and Treg functions but also of B-cell and regulatory B-cell (Breg) functions.

Adoptive infusion of tolerance dendritic cells prolongs survival of small intestine allografts in rats: systematic review and meta-analysis

Postoperative infections and rejection are the main limiting factors of small intestine allograft survival. In this study, we performed a systematic review and meta-analysis to review rat small intestine allograft survival following infusion of tolerance dendritic cells (Tol-DCs) induced by different methods. Relevant publications were searched from PubMed database and EMbase database. Meta-analysis was performed using RevMan 5.0 software. We chose allograft survival, mixed leukocyte reaction, Th1/Th2 differentiation, Treg induction, and cytotoxic T lymphocyte activity as the outcomes by which to examine possible mechanisms that promote survival. Eleven suitable articles were identified and assessed. Tol-DCs induced by four methods all prolonged allograft survival. The difference in survival time between the Tol-DC group and the control group was indicated by SMD as follows: drug intervention (SMD = 3.02, 95% CI 1.16 to 4.88, P = 0.001), gene modification (SMD = 2.43, 95% CI 1.77 to 3.10, P < 0.00001), imDC (SMD = 1.76, 95% CI 0.90 to 2.62, P < 0.0001), cytokine induction (SMD = 1.68, 95% CI 0.40 to 2.96, P = 0.01). Tol-DCs were also synergistic with immunosuppressive drugs or costimulation inhibitors, but no immune tolerance was observed. A single-dose intravenous injection of 5×10(6) to 6×10(6) Tol-DCs showed the highest allograft survival. Possible mechanisms included donor-specific T-cell hyporesponsiveness and Th2 differentiation. Our results demonstrated that Tol-DCs induced by four methods prolong rat small intestine allograft survival. Intravenous infusion of 5×10(6) to 6×10(6) Tol-DCs was the optimum dose in rat small intestine transplantation. Immunosuppressive or costimulatory blockade was synergistic with Tol-DC on graft survival. Additional high-quality studies with larger sample sizes are needed to better investigate small intestinal graft longer term survival.

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 × 106/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.

The protective effects of CD4+ CD25+ Treg cell on the allograft after infusion of dendritic cells with low expression of CD40 from donor in mouse heart transplantation

Objective To explore the effects of CD4+CD25+Treg cell on the allograft after infusion of dendritic cells (DCs) with low expression of CD40 from donor in mouse heart transplantation.Methods In vitro,mouse bone marrow-derived DCs were infected by CD40-RNAi lentiviral vector,and tolerogenic DCs (Tol-DCs) with low expression of CD40 were prepared.A heterotopic abdominal heart transplantation model was established in mice,and the other three groups that were control group,noninfected DC group and lentivirus infected DC group were designed correspondingly.Cardiac allograft survival time was recorded and pathological grading for acute rejection was assessed on the 7 d after heterotopic abdominal heart transplantation.Concentrations of CD4+CD25+Treg cells in peripheral blood were analyzed before and after transplantation by flow cytometry.Results After 48 h infection of DCs by CD40-RNAi lentiviral vector in vitro,the expression of CD40 mRNA was down-regulated significantly,whose inhibition rate was 80.9％.The expression of CD40 was decreased from 74.37％ ±4.08％ to 40.07％ ± 4.03％ (P＜0.05) after 48 h infection.Compared with the control group and the noninfected DC group,the cardiac allograft survival time was significantly prolonged in the CD40-RNAi lentivirus infected DC group,which was (14 ± 4) d(P＜0.01) ; concentrations of CD4+CD25+Treg cells in peripheral blood were increased both on the 3 d and the 7 d after transplantation (P＜0.05) ; the pathological grading for acute rejection was decreased on the 7 d after transplantation (P＜0.05).Conclusion The CD4+CD25+Treg cell in peripheral blood was protective to cardiac allograft in prolonging its survival time in mouse heart transplantation. Key words: Regulatory T cell; Costimulatory pathway; Dendritic cell; CD40; Heart transplantation

Roles of dendritic cells in murine hepatic warm and liver transplantation-induced cold ischemia/reperfusion injury

Dendritic cells (DCs) induce and regulate both innate and adaptive immune responses; however, their in vivo functional importance in hepatic ischemia/reperfusion (IR) injury is perplexing. We hypothesized that liver-resident DC and locally recruited blood-borne DC might have distinctive roles in hepatic IR injury. We tested this hypothesis by using DC-deficient, fms-like tyrosine kinase 3 ligand (Flt3L) knockout (KO) mice in hepatic warm (70% partial clamping for 60 minutes) and cold IR injury (liver transplant [LTx] with 24-hour cold storage). Flt3L KO liver and lymphoid organs contained virtually no CD11c+ F4/80- DC. Hepatic warm IR injury was significantly lower in Flt3L KO than in wildtype (WT) mice with lower alanine aminotransferase (ALT) levels, reduced hepatic necrosis, and lower neutrophil infiltration. Hepatic messenger RNA (mRNA) and protein levels for inflammatory cytokines (tumor necrosis factor alpha [TNFα], interleukin [IL]-6) and chemokines (CCL2, CXCL2) were also significantly lower in Flt3L KO than in WT mice, indicating that lack of both liver-resident and blood-borne DC ameliorated hepatic warm IR injury. Adoptive transfer of splenic or hepatic WT DC into Flt3L KO or WT mice increased hepatic warm IR injury, suggesting injurious roles of DC infusion. When Flt3L KO liver was transplanted into WT mice, ALT levels were significantly higher than in WT to WT LTx, with enhanced hepatic necrosis and neutrophil infiltration, indicating a protective role of liver-resident DC. Using both warm and cold hepatic IR models, this study suggests differential roles of liver-resident versus blood-borne DC, and points to the importance of the local microenvironment in determining DC function during hepatic IR injury.