Dendritic Cells Group Research Articles

MECHANISTIC STUDIES ON DC-INDUCED T CELL HYPORESPONSIVENESS USING A TCR TRANGENIC MODEL

P1007 Aims: Two populations of tolerogenic dendritic cells (DC) have been identified in mice; immature myeloid DC express low MHC and costimulatory molecules, while liver-derived B220+ lymphoid DC are phenotypically mature. To better understand the underlying mechanisms, it was attempted to characterize the antigen-specific T cell responses, using TCR transgenic DO11.10 (H2d) mice with CD4+ T cells expressing transgenic TCR specific for chicken oval albumin (OVA) which can be identified by specific mAb KJ1.26. Methods and Results: Immature myeloid and B220+ lymphoid DC were propagated from BALB/c (H-2d) mouse liver in IL-3 plus CD40L, and GM-CSF, respectively (bone marrow-derived mature myeloid DC were used as controls). Cell divisions were analyzed by CFSE dilution. T cells isolated from DO11.10 mouse lymph nodes (LN) were labeled with CFSE and cultured with irradiated DC (T/DC ratio of 10/1) in the present of 0.3 mM OVA. Responses of OVA specific T cells were analyzed in KJ1.26+ cell population. KJ1.26+ cell divisions were mild following stimulation with immature myeloid DC, reaching ∼16% KJ1.26+ cells at the end of culture, suggesting relatively low T cell proliferation. KJ1.26+ T cell division in B220+ DC group was very active, similar to that cultured with mature myeloid DC, and generated final ∼57% and ∼34% KJ1.26+ cells, respectively, indicating distinct responses to immature myeloid and B220+ DC. Combined observations of CFSE dilution and apoptotic death by anti-annexin V revealed that >90% of death occurred in divided KJ1.26+ cells in all groups, suggesting an activation induced cell death (AICD) nature. Annexin V+ cells in divided KJ1.26+ cells was 90% in liver B220+ DC, 66% in mature myeloid DC and 35% in immature myeloid DC group, suggesting the profound capability of B220+ DC to promote AICD in OVA-specific CD4+ T cells. To examine this in vivo, 2.5 x 105 DC pulsed with OVA were injected into footpad of BALB/c mice that had been adoptive transferred with 2.5 x 106 CFSE labeled DO11.10 T cells 24 h before. T cells isolated from the popliteal LN on d3 were analyzed in KJ1.26+ population. Few divided cells were identified in immature myeloid DC group, and only 0.6% cell were KJ1.26+. In contrast, active cell divisions were observed in injection with B220+ DC mice, and KJ1.26+ cells achieved 1.9% associated with elevated apoptosis activity, compared with 1.6% in mature DC injection group with less apoptotic activity. Conclusion: These data suggest that, distinct from immature myeloid DC, B220+ DC induce hyporesponsiveness by promotion of AICD in antigen-specific T cells.

T CELL APOPTOSIS INDUCED BY LIVER B220+ DENDRITIC CELLS: A MECHNISTIC INSIGHT

P984 Aims: Liver allografts are spontaneously accepted in pigs, rats and mice, which is associated with enhanced activated T cell apoptosis. The underlying mechanisms are unclear. We have identified a novel subset of dendritic cells (DC) in mouse livers that were phenotypically mature, but stimulated poor [3H]TdR incorporation in allogeneic T cells due to apoptotic death of activated T cells. The aims of this stuy was to investigate the involved mechanisms. Methods and Results: Liver-derived B220+ DC were propagated from B10 mouse liver NPC in the presence of IL-3 and CD40L. Bone marrow-derived myeloid DC in GM-CSF and Il-4 were used as controls. Cell cycle analysis showed that dividing of T cells stimulated by liver B220+DC was similar to BM-derived myeloid DC. Low thymidine uptake was a result of enhanced activated T cell apoptosis. ∼30% of activated T cells were TUNEL positive in liver B220+ DC group (evenly distributed in CD4+ and CD8+ populations), only <10% in myeloid DC group. In contrast to myeloid DC that exacerbated allograft rejection, administration of B10 (H-2b) liver B220+ DC (2 x 106) dramatically prolonged survival of B10, but not third party (BALB/c; H-2d) heart allografts in C3H (H-2k) recipients (MST 37 days vs. 10 days in control and third party groups). This was associated with a higher incidence of apoptotic cells in draining lymph nodes and spleen. T cell thymidine uptake in a liver B220+DC/T culture was markedly restored by addition of z-VAD-fmk, a common caspase inhibitor peptide, suggesting an involvement of caspase cascades. To determine the involved pathways, proteins were isolated from sorted T cells at different time point of culture, and incubated with substrates (AC-DEVE-AFC for caspase 3, AC-IETD-AFC for caspase 8 and AC-LEHD-AFC for caspase 9). The caspase activity determined by an OD value showed that liver B220+DC, not myeloid DC, activated caspase 3 and caspase 8, but not caspase 9 in T cells. RNase protection assay data delineated that B220+ DC, not myeloid DC, inhibited T cell mRNA expression of Bcl-w and Bfl-1 (anti-apoptosi), but enhanced Bak and Bad (pro-apoptosis) expression. T cell apoptosis induced by gld (FasL deficient) B220+ DC was reduced about 30%, indicating a partial role of Fas ligation. Liver B220+ DC induced similar apoptosis in TNFR (either p55 or p75) deficient T cells, suggesting that TNF and lymphotoxin (LT)a may not be important ligands. We also examined the role of LTβ, encouraged by a RNase protection assay result that expression of LTβ in liver B220+ DC was extraordinary high, and found that liver B220+ DC from LTβ−/− mice were poor apoptosis inducers, and stimulated profound T cell proliferation, suggesting a critical role of LTβ in mediating the apoptosis. Conclusion: The data suggest an involvement of multiple apoptosis pathways.

Augmented regeneration of partial liver allograft induced by nuclear factor-kappaB decoy oligodeoxynucleotides-modified dendritic cells.

To investigate the effect of NF-kappaB decoy oligodeoxynuleotides (ODNs) - modified dendritic cells (DCs) on regeneration of partial liver allograft. Bone marrow (BM)- derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4 to obtain immature DCs or mature DCs, respectively. GM-CSF-propagated DCs were treated with double-strand NF-kappaB decoy ODNs containing two NF-kappaB binding sites or scrambled ODNs. Allogeneic (SD rat to LEW rat) 50% partial liver transplantation was performed. Normal saline (group A), GM-CSF -propagated DCs (group B), GM-CSF+IL-4 - propagated DCs (group C), and GM-CSF+NF-kappaB decoy ODNs (group D) or scrambled ODNs -propagated DCs (group E) were injected intravenously into recipient LEW rats 7 days prior to liver transplantation and immediately after transplantation. DNA synthesis (BrdU labeling) and apoptosis of hepatocytes were detected with immunostaining and TUNEL staining postoperative 24 h, 48 h, 72 h and 84 h, respectively. Liver graft-resident NK cell activity, hepatic IFN-gamma mRNA expression and recipient serum IFN-gamma level at the time of the maximal liver allograft regeneration were measured with (51)Cr release assay, semiquantitative RT-PCR and ELISA, respectively. Regeneration of liver allograft was markedly promoted by NF-kappaB decoy ODNs-modified immature DCs but was significantly suppressed by mature DCs, the DNA synthesis of hepatocytes peaked at postoperative 72 h in group A, group B and group E rats, whereas the DNA synthesis of hepatocytes peaked at postoperative 84 h in group C rats and 48 h in group D rats, respectively. The maximal BrdU labeling index of hepatocytes in group D rats was significantly higher than that in the other groups rats. NF-kappaB decoy ODNs-modified immature DCs markedly suppressed but mature DCs markedly promoted apoptosis of hepatocytes, liver-resident NK cell activity, hepatic IFN-gamma mRNA expression and recipient serum IFN-gamma production. At the time of the maximal regeneration of liver allograft, the minimal apoptosis of hepatocytes, the minimal activity of liver-resident NK cells, the minimal hepatic IFN-gamma mRNA expression and serum IFN-gamma production were detected in group D rats. The apoptotic index of hepatocytes, the activity of liver- resident NK cells, the hepatic IFN-gamma mRNA expression level and the serum IFN-gamma level in group D rats were significantly lower than that in the other groups rats at the time of the maximal regeneration of liver allograft. The data suggest that the augmented regeneration of partial liver allograft induced by NF-kappaB decoy ODNs-modified DCs may be attributable to the reduced apoptotic hepatocytes, the suppressed activity of liver-resident NK cells and the reduced IFN-gamma production.

Effects of dendritic cells transfected with full-length wild-type p53 and stimulated by gastric cancer lysates on immune response.

To investigate the effects of dendritic cells (DCs) transfected with full-length wild-type p53 and stimulated by gastric cancer lysates on immune response. The wild-type p53 was transduced to DCs with adenovirus, and the DCs were stimulated by gastric cancer lysates. The surface molecules (B7-1, B7-2, MHC-I, MHC-II) of all DCs were detected by FACS, and the ability of the DCs to induce efficient and specific immunological response in anti-51Cr-labeled target cells was studied. BALB/c mice injected with DCs and Mk28 were established, and CTL response in mice immunized with Lywt-p53DC was evaluated. Tumor-bearing mice were treated with Lywt-p53DC. The surface molecules of Lywt-p53DC had a high expression of B7-1 (86.70 +/- 0.07%), B7-2 (18.77 +/- 0.08%), MHC-I (87.20 +/- 0.05%) and MHC-II (56.70 +/- 0.07%); T lymphocytes had a specific CTL lysis ability induced by Lywt-p53DC; the CTL lysis rate was as high as 81%. The immune protection of Lywtp-53DC was obvious, the tumor diameter in Lywtp-53DC group was 3.10 +/- 0.31 mm, 2.73 +/- 0.23 mm, 3.70 +/- 0.07 mm on d 13, 16 and 19, respectively, which were smaller than control, DC, wtp53DC and LyDC group (P<0.05). Tumor growth rate in Lywtp53DC group was slower than that in other groups (P<0.05). DCs transfected with wild-type p53 and stimulated by gastric cancer lysates have specific CTL killing activity.

Regulation of mite allergen-pulsed murine dendritic cells by respiratory syncytial virus.

Dendritic cells (DCs) are the only antigen-presenting cells that determine T-cell differentiation and play an important role in both allergy and viral infection. Respiratory syncytial virus (RSV) can infect DCs and affect their functions. The aim of this study was to determine the interaction between RSV infection and Dermatophagoides farinae allergen (D. farinae) sensitization on the development of allergy at the DC level. Murine bone marrow-derived DCs were prepared and treated as: control; D. farinae-pulsed DCs (D. farinae-DCs); ultraviolet-inactivated RSV challenged; RSV-infected, D. farinae-pulsed plus ultraviolet-inactivated RSV-challenged; and D. farinae-pulsed plus RSV-infected. In in vitro experiments, we compared the expression of costimulatory molecules and cytokine production between the six groups of DCs. Another group of naive mice were then intranasally inoculated with these DCs, after which intranasal challenge with D. farinae was performed to develop allergic airway inflammation in vivo. In comparison with D. farinae-DCs, D. farinae-pulsed plus RSV-infected DCs showed helper T cell (Th) 1-favored expression of costimulatory molecules and cytokine production. Allergic airway inflammation induced by intranasal instillation of D. farinae-DCs was abrogated when infected with RSV, which was associated with a concomitant suppression of Th2 response in the lung. Our results indicated that RSV suppresses D. farinae-DCs to induce Th2 response both in vitro and in vivo through regulation of expression of surface markers and production of immunoregulatory cytokines.

CD1a-positive infiltrating-dendritic cell density and 5-year survival from human breast cancer

Infiltrating CD1a+ dendritic cells (DCs) have been associated with increased survival in a number of human cancers. This study investigated DC infiltration within breast cancers and the association with survival. Classical established prognostic factors, of tumour size, lymph node status, histological grade, lympho-vascular invasion, the KI-67 (MIB-1) fraction and the Nottingham Prognostic Index (NPI) were also compared. A total of 48 breast cancer patients were followed from the time of surgery and CD1a density analysis for 5 years or until death. Our data set validated previous studies, which show a relationship between survival and the NPI (P<0.001), tumour size (P<0.01) and lymph node status (P<0.05). Although more patients were alive at the 5-year time point in the group with higher CD1a DC density than the lower CD1a DC group, this failed to reach statistical significance at the P=0.05 level. Analysis at 10 years postsurgery is required to investigate the association further.

Interleukin-10 modified dendritic cells induce allo-hyporesponsiveness and prolong small intestine allograft survival.

To investigate whether IL-10-transduced dendritic cells (DCs) could induce tolerogenicity and prolong allograft survival in rat intestinal transplantation. Spleen-derived DCs were prepared and genetically modified by hIL-10 gene. The level of IL-10 expression was quantitated by ELISA. DC function was assessed by MTT in mixed leukocyte reaction. Allogeneic T-cell apoptosis was examined by flow cytometric analysis. Seven days before heterotopic intestinal transplantation, 2 X 10(6) donor-derived IL-10-DC were injected intravenously, then transplantation was performed between SD donor and Wistar recipient. Compared with untransduced DC, IL-10-DC could suppress allogeneic mixed leukocyte reaction (MLR). The inhibitory effect was the most striking with the stimulator/effector (S/E) ratio of 1:10. The inhibition rate was 33.25%, 41.19% (P<0.01) and 22.92% with the S/E ratio of 1:1, 1:10 and 1:50 respectively. At 48 hours and 72 hours by flow cytometry counting, apoptotic T cells responded to IL-10-DC in MLR were 13.8% and 30.1%, while untransduced group did not undergo significant apoptosis (P<0.05). IL-10-DC pretreated recipients had a moderate survival prolongation with a mean allograft survival of 19.8 days (P<0.01), compared with 7.3+/-2.4 days in control group and 8.3+/-2.9 days in untransduced DC group. Rejection occurred in the control group within three days. The difference between untreated DC group and control group was not significant. IL-10-DC can induce allogenic T-cell hyporesponsiveness in vitro and apoptosis may be involved in it. IL-10-DC pretreatment can prolong intestinal allograft survival in the recipient.

The immunotherapeutic effect of dendritic cells vaccine modified with interleukin-18 gene and tumor cell lysate on mice with pancreatic carcinoma.

To estimate the effect of a therapeutic vaccine against pancreatic carcinoma based on dendritic cell (DC) vaccine modified with tumor lysate and Interleukin-18 gene. The BALB/C mice model of pancreatic carcinoma was induced with DMBA. DC vaccine was constructed through pulsed with tumor lysate and transfected by the recombinant adenoviral vector encoding IL-18 gene. The immunotherapeutic effects of DC vaccine on mice with pancreatic carcinoma were assessed (divided into DC-IL18-Lysate group, DC-Lysate group, DC-IL18 group, DC group, PBS group). After vaccination of the DC vaccine, the concentration of IL-18 and IFN-gamma were 2161+/-439 ng x L(-1) and 435+/-72 ng x L(-1) in DC-IL18-Lysate group and there was significant difference compared with other groups (P<0.01). After vaccination of the DC vaccine, the transplanted tumors were observed on 30 days in DC-Lysate groups, on 16 days in DC-IL18 groups, on 3 days in control group, but mice remained tumor-free for at least 50 days in DC-IL18-Lysate group and there was significant difference between DC-IL18-Lysate group and other groups (P<0.01). The median survival exceeds 62 days in DC-IL18-Lysate group. But the median survival was 48.6 days in DC-Lysate group, 33 days in DC-IL18 group, 17 days in PBS group. The survival period was obviously prolonged in DC-IL18-Lysate group than in other groups (P<0.05, P<0.01). The weight of pancreatic tumor was 0.22+/-0.083 g in DC-IL18-Lysate group, 1.45+/-0.74 g in DC-Lysate group, 1.89+/-1.34 g in DC-IL18 group, 3.0+/-1.6 g in DC group, 2.9+/-2.0 g in PBS group and the weight of tumor obviously reduced in DC-IL18-Lysate group than in other groups (P<0.05, P<0.01). DC vaccine modified with tumor lysate and Interleukin-18 gene can induce a specific and effective immune response against pancreatic carcinoma cell.

Antitumor activities of human dendritic cells derived from peripheral and cord blood.

To observe the biological specialization of human peripheral blood dendritic cells (DC) and cord blood derived DC and its effects on effector cells killing human hepatocarcinoma cell line BEL-7402. in vitro. The DC biological characteristics were detected with immunohistochemical and MTT assay. Two antitumor experiment groups are divided: peripheral blood DC and cord blood DC groups. Peripheral blood DC groups used LAK cells as the effector cells and BEL-7402 as target cells, while cord blood DC groups used CTL induced by tumor antigen twice pulsed DC as effector cells and BEL-7402 as target cells, additional peripheral blood DC and cord blood DC are added to observe its stimulating activities to effector cells. The effector's cytotoxicity to tumor cells were detected with neutral red colorimetric assay at two effector/target ratios of 5:1 and 10:1. Peripheral blood DC and cord blood DC highly expressed HLA-ABC, HLA-DR, HLA-DQ, CD54 and S-100 protein. The stimulating activities to lymphocyte proliferation were compared between experimental groups (DC added) and control group (no DC added), in six experiment subgroups,the DC/lymphocyte ratio was sequentially 0.25:100, 0.5:100, 1:100, 2:100, 4:100 and 8:100.A values were sequentially 0.75396+/-0.009, 0.84916+/-0.010, 0.90894+/-0.012, 0.98371+/-0.007, 1.01299+/-0.006 and 1.20384+/-0.006 in peripheral blood DC groups and 0.77650+/-0.005, 0.83008+/-0.007, 0.92725+/-0.007, 1.05990+/-0.010, 1.15583+/-0.011, 1.22983+/-0.011 in cord blood DC groups. A value was 0.59517+/-0.005 in control group. The stimulating activities were higher in experimental groups than in control group (P<0.01), which were increased when the DC concentration was enlarged (P<0.01). Two differently derived DCs had the same phenotypes and similar stimulating activities (P<0.05). In peripheral blood DC groups, the cytotoxicity of the LD groups (experimental groups) and L groups (control group) was 58.16%+/-2.03% (5:1), 46.18%+/-2.25% (10:1) and 38.13%+/-1.29% (5:1) and 65.40%+/-1.56% (10:1) respectively; in cord blood DC groups, TD groups (experimental groups) and T groups (control groups) were 69.71%+/-2.33 % (5:1), 77.64%+/-1.94% (10:1) and 56.89%+/-1.82% (5:1) and 60.99%+/-1.42% (10:1) respectively.The cytotoxicity activities were enhanced with increased effector/target ratio (P<0.01). At the same effector/target ratio, the cytotoxicity of experimental groups were bigger than that of control groups (P<0.01). The cytotoxicity activities of cord blood DC groups were higher than that of peripheral blood DC groups (P<0.01). Peripheral blood DC and cord blood DC are mature DC in morphology and function, both can enhance the effector cell killing activities to hepatocarcinoma cells. DC pulsed with tumor antigen can induce higher specific CTL activity than unpulsed DC.

Induction of Th2-directed immune responses by IL-4-transduced dendritic cells in mice

Dendritic cell (DC)-based vaccines have been used to generate Th1-mediated, protective immunity against cancers and infectious microorganisms. As an attempt to develop a new vaccine protocol for the induction of Th2-directed responses, we introduced an IL-4 plasmid vector into the XS106 DC line (derived from A/J mice). Although relatively small fractions of XS106 cells exhibited apparent intracellular deposition of IL-4, they secreted biologically relevant amounts of the cytokine. IL-4-transduced XS106 DC and control XS106 DC transfected with vector alone were pulsed with KLH and injected s.c. into A/J mice. The overall magnitude of KLH-specific cellular and humoral responses was comparable between the two animal groups. However, they differed in the isotype profile albeit only transiently, with the IL-4-transduced DC group showing higher IgE and lower IgG2a responses, and in the cytokine profile, with spleen cells isolated from the IL-4-transduced DC group producing higher IL-13 and lower IL-12. Thus, delivery of IL-4 gene to relatively small numbers of DC is sufficient to modify the immunological outcome of DC-based vaccines.

Antigen-presenting cells in nasal allergy.

Antigen-presenting cells in nasal allergy.

Phase I clinical trial: T‐cell therapy for prostate cancer using autologous dendritic cells pulsed with HLA‐A0201‐specific peptides from prostate‐specific membrane antigen

BACKGROUND Conventional treatment for metastatic prostate cancer have failed to demonstrate curative potential in all patients. Investigations involving the role of T-cell immunity in the clearance of neoplastic cells are now available. Development of T-cell immunotherapy may give a new approach to the treatment of advanced metastatic prostate cancer. METHODS A phase I clinical trial assessing the administration of autologous dendritic cells (DC) pulsed with HLA-A0201-specific prostate-specific membrane antigen (PSMA) peptides were conducted. Participants were divided into five groups receiving four or five infusions of peptides alone (PSM-P1 or PSM-P2; groups 1 and 2, respectively), autologous DC (group 3), or DC pulsed with PSM-P1 or P2 (groups 4 and 5, respectively). RESULTS No significant toxicity was observed in all five groups. Cellular response against PSM-P1 and -P2 was observed in HLA-A2+ patients infused with DC pulsed with PSM-P1 or -P2 (groups 4 and 5), respectively. An average decrease in PSA was detected only in group 5. Seven partial responders were identified based on NPCP criteria + PSA. CONCLUSIONS Infusions of test substances were well tolerated by all study participants. Detection of cellular response and decrease in PSA level in some patients who received DC pulsed with PSM-P2 indicate this method's potential in prostate cancer therapy. © 1996 Wiley-Liss, Inc.

Prognostic significance of tumor-host interaction in clinical gastric cancer: relationship between DNA ploidy and dendritic cell infiltration.

DNA ploidy of tumor cells and the degree of infiltration of dendritic cells were determined in 93 gastric cancer tissue specimens, and the mechanisms of tumor-host interaction on the prognosis were investigated. DNA ploidy patterns were grouped into low and high ploidy, and the degree of infiltration of dendritic cells (DC) was graded into marked and slight infiltration. In the low ploidy group, the 5-year survival rates in patients with marked and slight DC infiltration were 80.7% and 61.5%, respectively (P < 0.05). In the high ploidy group, however, there were no significant differences. In cases of low ploidy, the incidence of lymph node metastasis was significantly lower in the marked DC infiltration group compared with findings in the slight DC group. Thus, markedly infiltrating dendritic cells in gastric cancer tissue may lead to prolongation of survival time for patients with a carcinoma of the low ploidy profile, by preventing widespread nodal involvement.

Intraepithelial airway dendritic cells: a distinct subset of pulmonary dendritic cells obtained by microdissection.

Dendritic cells (DC), in general, and pulmonary DC, in particular, are a heterogeneous population of cells, their phenotype and function being dependent on their anatomic location, their state of activation, and the regulatory effect of locally secreted cytokines. Using a novel microdissection technique, the epithelium from the trachea and entire airway system was harvested, and the contained DC isolated at greater than 90% purity. The phenotype and function of these airway DC (ADC) was compared to DC isolated, at greater than 90% purity, from the parenchyma of the same lung. In contrast to lung DC (LDC), ADC did not express intercellular adhesion molecule 1 (ICAM-1) in situ, the amount of immune associated antigen (Ia) expressed was less (as determined by immunoperoxidase staining and immunopanning), and greater than 50% of ADC displayed Fc receptors (FcR). The majority of LDC were ICAM-1+, less than 5% expressed FcR, and all were intensely Ia+. Airway DC were most numerous in tracheal epithelium, but they were also present in small numbers in the epithelium of the most distal airways. Their numbers increased in all segments of the tracheobronchial epithelium in response to the administration of IFN-gamma. ADC were consistently more effective than LDC in presenting soluble (hen egg lysozyme) and particulate (heat-killed Listeria monocytogenes) antigens to antigen-sensitized T cells. By contrast, LDC were significantly more efficient in stimulating the proliferation of nonsensitized T cells in an autologous mixed leukocyte reaction. These data suggest that in normal animals, intraepithelial DC of airways share many attributes with Langerhans cells of the skin. Interstitial LDC, by contrast, reside in an environment where they may be exposed to a different set of regulatory factors and where they have progressed to a more advanced stage of differentiation than ADC. Both groups of DC are, however, heterogeneous, reflecting the continuous turnover that these cells undergo in the lung.