By continuing to browse this site you agree to us using cookies as described in About Cookies
Notice: Due to essential maintenance the subscribe/renew pages will be unavailable on Wednesday 26 October between 02:00- 08:00 BST/ 09:00 – 15:00 SGT/ 21:00- 03:00 EDT. Apologies for the inconvenience.
G. Shi, MD, PhD, Rheu-matology, State Key Laboratory, West China Hospital/West China Medical School, Sichuan University, Chengdu, Sichuan 610041, China. E-mail: email@example.com
Dendritic cells (DC) are key factors in regulating immune responses, and they induce immune response or tolerance depends on its maturation states. Previous studies demonstrated that blocking IKK2 in bone marrow-derived dendritic cells (BMDC) by adenoviral transfection with a kinase-defective dominant negative form of IKK2 (IKK2dn) could inhibit NF-κB activation and impair DC maturation. Here, we transfected IKK2dn into recipient rat (Lewis) BMDC by adenovirus vector (Adv-IKK2dn-DC) and found that Adv-IKK2dn-DC had reduced B7-2 and B7-1 expression under alloantigen stimulation. Their ability to induce allogeneic T-cell proliferation was markedly reduced in comparison with uninfected DC. A higher IL-10 secretion and a lower IFN-γ secretion were detected in Adv-IKK2dn-DC-stimulated allogenic T cells. Furthermore, we showed that Adv-IKK2dn-DC pulsed with BN (Brown Norway rats) splenocyte lysates markedly prolonged the survival of renal allografts in an antigen-specific manner. These findings suggested that Adv-IKK2dn-DC loaded with BN antigen could suppress anti-alloimmune response and induce tolerance to allografts, which provided an experimental base for immune tolerance induction by recipient DC loaded with donor antigens. Our finding may provide a more feasible strategy for deceased-donor renal transplantation.
The greatest barrier in allotransplantation is the anti-alloimmune rejection. Dendritic cells (DC) have been proposed as the first initiator of allograft rejection. DC are the most potent professional antigen-presenting cells and play crucial roles in innate and adopted immune responses. Studies indicated that the maturation states of DC are related with their ability to induce immune response or tolerance [1–3]. The mature DC with high levels of cell surface class II major histocompatibility complex (MHC-II) and costimulatory molecules including CD80 (B7-1), CD86 (B7-2), and CD40 induce immune response, while immature DC characterized by low expression of both MHC class II and costimulatory molecules are capable of inducing tolerance [1–4]. Mechanisms of immature DC-inducing tolerance include T-cell anergy, immune deviation, promotion of activated T-cell apoptosis, and formation of regulatory T cells [3–5].
Tolerogenic immature DC can be generated in several different ways, including conditioning the cells with immunological or pharmacological reagents [4–6] genetic engineering with different genes [7–11]. It was reported that the nuclear factor-kappa B plays a critical role in dendritic cell maturation and tolerance induction [12–14]. Further study indicated that IKK2 plays essential role in DC antigen presentation . Treatment of murine bone marrow-derived DC with double-stranded oligodeoxyribonucleotides (ODN), which contains binding sites for NF-κB, generated DC with a significantly reduced CD80 and CD86 expression when compared with untreated cells. ODN-treated DC exhibited an impaired allostimulatory capacity in vitro and prolonged heart allograft survival when infused in MHC-mismatched mice . Blocking IKK2 in human monocyte-derived DC by adenoviral transfection with a kinase-defective dominant negative form of IKK2 (IKK2dn) generated DC with impaired allostimulatory capacity, which failed to increase MHC-II antigens and costimulatory molecules in response to CD40 engagement . Using adenoviral vector encoding for IKK2dn to block NF-κB of rat bone marrow-derived DC results in blocking DC maturation, and IKK2-blocked donor DC treatment prolonged kidney allograft survival in rat by inducing regulatory T-cell generation . Those results indicated that NF-κB inhibition is capable of blocking DC maturation and inducing allogenic tolerance, while those studies are transferring donor’s DC into recipients. In a practical view, transfer of donor’s DC into recipients will be a potential donor’s DC activating host anti-alloimmune response, and in most of the time recipient’s DC is relatively easy to get than donor’s DC, because the majority of transplanted kidneys are from dead donors. Therefore, it is important to address whether transfer of immature recipient’s DC loaded with donor antigens could also induce anti-allotolerance or even working better than directly transfer donor’s DC. In this study, we generated recipient’s immature DC by adenoviral infection with a kinase-defective dominant negative form of IKK2 (IKK2dn), then loaded with donor antigens and tested their ability to induce anti-allotolerance in an allo-kidney graft rat model. Our results indicated that IKK2dn-transfected DC are capable of inducing tolerance and significantly prolonged transplanted allograft survival by reducing B7-1 and B7-2 expression, increasing IL-10 and decrease IFN-γ production.
Materials and methods
Animals and reagents. Male Lewis (LEW/CrlBR), Brown Norway (BN/CrlBR), and Wistar (Crl:(WI)BR) rats, 8–10 weeks, body weight around 180–200 g, were purchased from Charles River (Vital River, Beijing, China) and maintained in the university animal facility. Procedures involving animals and their care were conducted in accordance with the institutional guidelines that are in compliance with national and international laws and policies. The recombinant rrGM-CSF and rrIL-4 were purchased from Peprotech (Rocky Hill, CT, USA). FITC or PE-conjugated mouse anti-Rat CD80, CD86, and MHC-II antibodies were purchased from Serotec (Oxford, UK); IL-2, IL-10, IFN-γ ELISA kits are from R&D (Minneapolis, MN, USA). All other reagents are from Sigma (St. Louis, MO, USA). The replication-deficient adenovirus encoding a kinase-defective dominant negative form of human IKK2 plasmid, pACCMVpLpASR(+)-IKK2dn, was a kind gift from Dr Rain D Martin (University of Vienna, Vienna, Austria). pAdxsi-GFP-IKK2dn and pAdxsi-GFP-0 were constructed by SinoGenoMax Company, Beijing.
DC preparation and gene transfer. Bone marrow cells were obtained from the tibias and femurs of Lewis rats. Bone marrow cells (2 × 106/ml) were cultured in 6-well plates (Becton Dickinson, Heidelberg, Germany) with recombinant rat granulocyte macrophage-colony stimulating factor (GM-CSF) (5 ng/ml), in combination with recombinant rat IL-4 (5 ng/ml). On day 3 and every other day after, half of the medium containing the appropriate cytokines was replaced.
Recombinant, replication-deficient adenoviral vectors encoding IKK2dn and Adv-0 were constructed as follows: IKK2dn cDNA was cloned into adenovirus transfer vector pShuttle-CMV-GFP(−)TEMP (Sinogenomax, Beijing, China) and analysed by restriction endonuclease KpnI & HindIII digestion. Then, the obtained plasmid, pShuttle-CMV-GFP(−)TEMP-IKK2dn was transferred into pAdxsi vector to construct pAdxsi-GFP-IKK2dn plasmid and was identified by XhoI digestion. The correct adenoviral recombinant was then cleaved with Pac I and transfected into 293 cells to produce and purify viral particles. The virus was then infected into Hela cells, and the infection titre was monitored by green fluorescence protein (GFP) expression, and IKK2dn gene was identified by RT-PCR. All viruses belong to the Ad5 serotype.
On day 7, cultured DC were harvested, replaced at 1 × 106 cells/ml in serum-free RPMI 1640, and infected with adenoviruses at different multiplicities of infection (MOI) for 2 h (10, 25, 50, 100, and 200 MOI). Three hours later, complete RPMI 1640 were restored, and cells were cultured for another 2 days. DC were then washed twice with complete medium before experiments. For pulsing with donor antigens, BN spleen cell lysate that was prepared by repeat freezing (5 min in dry ice–ethanol bath) and thawing (10 min in 37 °C warm bath) for 5 times, and added at 1/5 of DC/spleen cell (used to prepare lysate) ratio for the last 48 h of DC culture. Then, cells were harvested, analysed by flow cytometer, and used as stimulators for mixed leucocyte reaction (MLR). Uninfected and Adv-0-DC served as control.
To analyse gene expression of IKK2dn, RNA from AdV-0-DC and Adv-IKK2dn-DC was treated with DNase and reversely transcribed to cDNA. For IKK2dn polymerase chain reaction (PCR) analysis, the following primers were used: sense, 5′-GGCCTTTGAGTGCATCAC-3′ and antisense, 5′-CTCTAGGTCGTCCAGCGT-3′. All samples were run in triplicate. To assess the overall cDNA content, glyceraldehyde phosphate dehydrogenase (GAPDH) served as a housekeeping gene control. The following pair of primers was used for GAPDH: sense, 5′-GGAAGGTGAAGGTCGGAGTC-3′ and antisense, 5′-GTAGAGGCAGGGATGATGTTC-3′; The PCR was performed in a GeneAmp PCR System 2700 (Applied Biosystems Inc, Foster City, CA, USA) thermal cycler by 30 cycles of denaturization (94 °C, 30 s), annealing (55 °C, 30 s), and extension (72 °C, 1 min).
Flow cytometry. Expression of DC surface antigens was analysed by EPICS ELITE flow cytometer (Beckman-Coulter, Fullerton, CA, USA). Cell staining was performed as previously reported ; briefly, cells were stained with FITC or PE-conjugated mouse monoclonal antibodies anti-rat MHC class II, CD80 or CD86 after blocking non-specific binding with 10% vol/vol normal serum. FITC- or PE-conjugated isotype-matched irrelevant mAbs were used as negative controls (all from Serotec Corp).
Mixed lymphocyte reaction. To maintain immature condition of DC, 7-day-cultured Lewis DC were infected with 25-100 MOI of AdV-IKK2dn. Adv-0-infected DC were used as control. To determine the antigen-presenting capacity of DC in vitro, MLR was performed with mitomycin C (MMC, 25 mg/ml for 30 min)-inactivated DC from different MOI groups as stimulators and nylon wool-purified Lewis or NB splenic T cells as responders. In Lewis T cell as responder cell experiments, Lewis DC pulsed with BN spleen cell lysate were used as stimulators, and DC not pulsed with alloantigen were used as control. The stimulator used was 3 × 102, 1 × 103, 3 × 103, and 1 × 104. Cultures were established in triplicate in 96-well round-bottom microculture plates (200 ul/well with 1 × 106 T cells) and maintained in complete medium for 72 h in 5% CO2 at 37 °C. MTT (0.1 mg/well) was added for the final 4 h of culture. Results were presented as Stimulation Index according to the formula: SI = (MLR well optical density (OD) – blank well OD)/(T cell alone well OD – blank well OD). The optical density was measured at 490 nm.
Cytokine secretion. The levels of cytokines IL-10 and IFN-γ in cell culture supernatants and IL-2, IFN-γ in recipient rats serum were detected by ELISA kits (R& D Systems, Minneapolis, MN, USA) as described before , according to the manufacturer’s protocols. Standard curve was generated for each assay.
Renal transplantation. Renal transplantation was performed as previously described . Lewis recipient rats were administered an intravenous injection of 1 × 107 syngeneic Adv-IKK2dn-DC, AdV-0-DC or uninfected immature DC 7 days before allotransplantation. The Adv-IKK2dn-DC-treated third part donators (Wistar rats) group was served as control. Graft survival was monitored daily by abdominal palpation, and rejection was confirmed by histological examination.
Statistical analysis. Data are presented as mean ± SD and were analysed by general linear model anova. Survival curves were established by the Kaplan–Meier method. Graft survival between groups of transplanted animals was analysed with a log-rank test. And values of P < 0.05 were considered statistically significant.
Assessment of the DC transfection efficiency
To investigate the transfection efficiency of DC by adenovirus, DC were infected with AdV-IKK2dn at 10, 25, 50, 100, and 200 MOI. At day 9, the infection was monitored by GFP expression (Fig. 1A). At 200 and 100 MOI infections, almost all of DC were GFP positive. At 50 MOI, the GFP-positive cell percentage was approximately 96%. At 25 and 10 MOI infection, the GFP-positive percentages were lower, approximately 62% and 33% individually (Fig. 1A). However, a high percentage of cell death was found in 200-MOI-infected DC, as demonstrated by MTT assay (85% cell death). Therefore, it is indicated that blocking NF-κB by IKK2dn could cause cellular damage in DC. Cell death rate was lower in 100-MOI-infected DC (45% cell death); the cell death rate was markedly reduced at 50 MOI (18% cell death). Meanwhile, the percentages of cell death at 25 and 10 MOI were much lower (Fig. 1B). The infection rate and live cell percentages in WT virus (Adv-0) infection are similar to those in Adv-IKK2dn infection at different MOIs (Fig. 1B). These results suggested that 50 MOI Adv-IKK2dn infection may be a suitable dose.
To further confirm the infection, we detected the IKK2dn expression by RT-PCR in Adv-IKK2dn and Adv-0-infected DC (Fig. 1C, lines 1 and 2). The PCR results were run on gel, the expression of GAPDH in Adv-IKK2 and Adv-0 infected DC (Fig. 1c, lines 3 and 4) was used as control. A specific 1060-kb band was detected in Adv-IKK2dn-infected DC, but no signal was detected in the same molecular weight in control Adv (AdV-0)-infected DC (Fig. 1C, lines 1 and 2). Those results indicated that the dominant negative form of human IKK2 was successfully transfected by adenoviral vector and expressed in infected DC.
IKK2dn transfection impair alloantigen stimulated DC B7-2 and B7-1 expression
To investigate the effect of IKK2dn on DC maturation, first we analysed the MHC class II, B7-1 and B7-2 expression on the surface of Adv-IKK2dn-infected, control virus-infected and -uninfected Lewis DC by fluorochrome-labelled antibody staining followed by flow cytometry analysis. Then, the surface expression of MHC-II, B7-1 and B7-2 expression on alloantigen stimulated IKK2dn-transfected and uninfected DC were tested with the same methods. In accordance with published data , our results showed that MHC-II, CD80, and CD86 are up-regulated by control virus infection. In agreement with published data (15), Adv-IKK2dn infection suppressed those costimulatory molecule up-regulation in different MOIs (Fig. 2A,B). The expression levels of CD86 in 50 MOI Adv-Ikk2dn-infected group are significantly lower compared with wild type (Adv-0) virus-infected group (P < 0.01), but there is no significant difference compared with all other groups including uninfected group. The expression levels of CD80 in 50-MOI Adv-Ikk2dn-infected group are much lower in comparison with Adv-0 group and 25-MOI Adv-Ikk2dn-infected groups (P < 0.01), and there are no statistic differences compared with 100 MOI and uninfected groups. The MHC-II expression in 50-MOI Adv-Ikk2-infected group is reduced compared with Adv-0-infected group and slightly higher than uninfected and 100-MOI Adv-Ikk2dn-infected groups but no statistic significance (Fig. 2A, B). Results also suggested that 50 MOI Adv-IKK2dn infections produced a reasonable DC maturation suppression without inducing significant cell death as indicated in Fig. 1B. The MHC-II, B7-1 and B7-2 molecules were slightly increased in Adv-IKK2dn-DC in the presence of alloantigen (BN Ag) compared with no BN Ag present, but there are no statistic significances (Fig. 2C). By contrast, MHC-II, B7-1 and B7-2 expression were significantly increased in uninfected immature DC after BN Ag stimulation (Fig. 2C) (P < 0.01). In Adv-IKK2dn-transfected DC with alloantigen stimulation group, their MHC-II expression was increased compared with uninfected DC without alloantigen stimulation (P < 0.05), but there are no statistical differences compared with uninfected DC stimulation with alloantigen. The B7-1 and B7-2 expression in Adv-IKK2dn-infected DC stimulated with alloantigen is reduced in comparison with uninfected DC stimulated with alloantigen, but there are no differences compared with all other groups (Fig. 2C). These results indicated that BN antigen-loaded uninfected DC and IKK2dn-transfected DC have similar MHC-II expression, so as to their antigen-presenting ability. Alloantigen stimulation significantly increased the costimulatory molecule B7-2 and B7-2 expression in uninfected DC but not in IKK2dn-transfected DC. Those results indicated that IKK2dn transfection could affect DC maturation by interrupting their costimulatory molecule upgrade in response to alloantigen stimulation.
Optimal T-cell response requires two signals, the TCR signal provided by antigen-MHC complex as well as costimulatory signals provided by costimulatory molecules expression on APC. To investigate the antigen-presenting function of IKK2dn-transfected DC, a mixed lymphocyte reaction was preformed by co-culturing different number of MMC-treated Adv-IKK2dn-infected Lewis DC and fixed number (1 × 106) of BN T cells, using MMC-treated uninfected Lewis DC and control virus-infected Lewis DC as controls. T-cell proliferation was measured by MTT assay, and results are presented as stimulation index. Results indicated that different Adv-IKK2 infection could significantly suppress Lewis DC-induced BN T-cell proliferation (Fig. 3A). DC infected by over 50 MOI Adv-IKK2 are compatible with uninfected immature DC in terms of their capacity to stimulate allogenic T-cell proliferation. These results also indicated that 50 MOI Adv-IKK2 infection is sufficient to inhibit DC maturation and suppress their ability to stimulate alloreactive T-cell proliferation. Further, we used 50 MOI Adv-IKK2dn-infected Lewis DC loaded with BN antigen and studied their ability to stimulate Lewis T-cell proliferation, without alloantigen-loaded IKK2dn-transfected DC, uninfected immature DC with or without alloantigen loaded were used as controls. Results indicated that IKK2dn transfection significantly suppressed the ability of alloantigen-loaded DC-induced syngeneic T-cell proliferation (Fig. 3B).
IKK2dn-DC induces IL-10 but not IFN-r production
To understand the mechanism of IKK2dn transfection suppressed alloreactive T-cell proliferation, we tested the cytokine production in the supernatant of the mixed lymphocyte cultures. We found that the IL-10 production was markedly increased in Adv-IKK2dn-DC co-cultured group in comparison with uninfected and control virus-infected DC co-cultured groups. In contrast, the IFNγ production was significantly lower in Adv-IKK2dn-infected DC and uninfected DC co-cultured groups than control virus-infected group; there is no statistical difference between Adv-IKK2dn-DC and uninfected immature DC groups in terms of their IFNγ production (Fig. 3C,D).
Prolonged allo-kidney graft survival in Adv-IKK2dn-DC-treated rats
In vitro studies indicated that Adv-IKK2dn-infected DC have the potential to suppress anti-alloimmune response. To investigate whether IKK2dn-DC had a tolerogenic potential in vivo, 1 × 107 uninfected immature DC, Adv-IKK2dn-DC, and AdV-0-DC from LW rats loaded with BN antigen were infused into naive LW rats 7 days before kidney transplantation, and no immunosuppressive drugs were used during the study. Their survival was monitored everyday after transplantation. Results indicated that in Adv-IKK2dn-DC-treated group the survival time was prolonged significantly in comparison with untreated, uninfected DC treated, and Adv-0-DC treated, as well as Wister groups (Fig. 4). The detailed rat number and survival time in each group were described in Table 1. These results indicated that Adv-IKK2dn modified DC loaded with donor antigens did suppress anti-alloimmune response and prolong allograft survival in a DC-load alloantigen-specific manner.
Table 1. Number of rats in each group and individual survival time of kidney transplanted rats.
Number of rats (n)
Mean survival time
aP > 0.01, Compare with group 1. bP > 0.01, Compare with group 2. cP > 0.01, Compare with group 3. dP > 0.01, Compare with group 4.
6, 7(×4), 8(×2)
7.1 ± 0.26
9, 11, 12(×3), 14, 16, 17(×2), 18
13.8 ± 0.96a
4(×3), 5(×2), 6(×2)
4.9 ± 0.34b
20, 22(×2), 25, 27(×2), 29, 33, 36
26.8 ± 1.76a,b,c
6(×3), 7(×2), 8, 9
7.0 ± 0.44b,d
IKK2 silence decreases allograft-induced Th1 cytokine production in vivo
To understand the in vivo immune regulation of the IKK2dn-transfected DC, the serum levels of IL-2, IFN,γ and IL-10 in different groups were tested on day 5 and day 14 post-renal transplantation. On day 5 after transplantation, in untreated control, Adv-0 and Wistar kidney transplanted groups, the levels of IL-2 and IFN-γ were significantly increased in comparison with the levels of IL-2 and IFN-γ in Adv-IKK2dn-DC loaded with BN antigens-treated group and uninfected immature DC-treated group (P < 0.01). In contrast, IL-10 levels are significantly higher in Adv-IKK2dn-DC-treated group and uninfected DC-treated groups compared with all other groups (Fig. 5A–C). There are no differences in terms of the IL-2 and IFNγ as well as IL-10 levels in uninfected immature DC and Adv-IKK2dn-DC-treated group (Fig. 5A–C). However, by day 14, in uninfected immature DC-treated group, the IL-2 and IFNγ levels are getting higher, and the Adv-IKK2dn-DC-treated group still has low serum IL-2 and IFNγ levels (Fig. 5D). There are significant statistical differences between these two groups (P < 0.001). The IL-10 levels in Adv-IKK2dn-DC-treated group are significantly higher compared with uninfected DC-treated group (P < 0.001). Taking together, Adv-IKK2dn-DC loaded with BN antigen treatment reduced IL-2 and IFN-γ production and increased IL-10 production. It also indicated that donor antigen-loaded DC could prolong allograft survival by suppressing anti-allograft Th1 immune response and enhancing Th2 response in vivo.
In this study, we presented further evidence that IKK2 inhibition could impair DC maturation and antigen-presenting function ; we also showed that IKK2 inhibition was able to inhibit alloantigen stimulated DC CD86 and CD80 upgrading but not MHC class II (Fig. 2). IKK2dn-transfected DC loaded with alloantigen could inhibit syngeneic T-cell proliferation and IFNγ production but increase IL-10 secretion (Fig. 3). Finally, we have demonstrated in vivo that host DC transfected with IKK2dn and loaded with donor antigen prolonged allo-kidney survival by reducing Th1 immune response and enhancing Th2 immune response towards transplanted graft (Figs 4 and 5, Table 1).
As previously shown, IKK2 inhibition could impair DC maturation . IKK2dn-transfected DC could induce regulatory T (Treg) cell generation [7, 20], and donor IKK2dn-transfected DC therapy prolonged allograft survival . However, those studies are based on LPS stimulation or donor’s DC, as most of the organ transplantation is using dead donors, and donor’s DC are not easy to get; thus, it is important to know whether recipient tolerogenic DC loaded with donor antigen could induce tolerance to allograft. Our results showed that Lewis DC transfected with IKK2dn and loaded with BN antigen treatment significantly prolonged transplanted BN kidney survival, but not transplanted Wistar kidney (Fig. 4). Results suggested that tolerogenic recipient DC generated by silencing IKK2 loaded with BN antigen could induce specific tolerance to BN graft. Therefore, using recipient tolerogenic DC loaded with donor antigen could be a feasible way to induce donor graft-specific tolerance.
In vitro study indicated that IKK2dn transfection could significantly suppress alloantigen stimulated DC CD86 and CD80 expression, but not MHC class II expression. These results indicated that IKK2dn-transfected DC have normal antigen-presenting function but there is also a lack of costimulation, which were important in inducing tolerance. It also indicated that those DC induce antigen-specific tolerance by lack of costimulation.
Regulatory T cells play critical roles in transplanted allograft tolerance induction [21–25], and it is broadly accepted that immature stage dendritic cells (also called tolerogenic DC) could induce tolerance [26–29]. Although the underlying mechanisms of how tolerogenic DC induce transplant tolerance is still not very clear, the regulatory T cells induction of tolerogenic DC is believed as one of the mechanisms [4, 21, 30, 31]. It was reported that inhibit IKK2 could produce tolerogenic DC and those DC were able to induce regulatory T-cell production [7, 20]. To understand the mechanisms of how recipient Adv-IKK2dn-DC loaded with donor antigen induced transplant tolerance, we tested the cytokine production, which is important in immune response and regulatory T-cell induction. In accordance with published data, we found in MLR assay, the IFNγ production was significantly lower. Meanwhile, IL-10 production was markedly higher in Adv-IKK2dn-DC group in comparison with controls. In vivo studies indicated that Adv-IKK2dn-DC-treated group had significantly reduced IL-2 and IFNγ levels and increased IL-10 levels, in the serum of allo-kidney transplanted rats. These indicated that recipient Adv-IKK2-DC loaded with donor antigen prolongs allograft survival by suppressing anti-alloimmune response, and inducing regulatory T-cell generation may be one of the mechanisms.
It was broadly accepted that immature DC could induce tolerance instead of inducing immune response [1–4]. In accordance with this concept, our data showed that the survival of transplanted allo-kidney in BN Ag-loaded immature host DC-treated Lewis rats was prolonged in some extent and led to low levels of IL-2 and INFγ and high levels of IL-10 in early time point. There are no differences between those serum cytokines between immature host DC loaded with donor antigen-treated group and Adv-IKK2-DC-treated groups when matured in day 5 after transplantation (Fig. 5A–C). However, in day 14 after transplantation, the IL-2 and INFγ levels are significantly higher and the IL-10 levels are significantly lower in DC-treated group than Adv-IKK2dn-DC-treated group (P < 0.001) (Fig. 5D). These results indicated that immature DC may not maintain their immature state in vivo for a prolonged time and lost their tolerance-inducing function. Meanwhile, Adv-IKK2dn transduction inhibited DC maturation and kept their immature states for a longer time.
This work was supported by Jiangsu Province Department of Health, grants RC2007080, H200610, and H200714 to Dr Ouyang. Chinese Education Ministry start-up grants for overseas return scholar 20098-8-6 to Dr Shi.