Cord Blood-derived Dendritic Cells Research Articles

Potency of Peripheral Blood- and Umbilical Cord Blood-derived Dendritic Cells and Their Secretomes as Vaccines for Cancer

Dendritic cell (DC) vaccines, as immunotherapy agents, can gather up and transport cancer-related antigens to T lymphocytes, activating anti-tumor effector responses. After being activated by DC, cytotoxic T lymphocyte cells (CTL) will secrete the cytolytic granzyme B that can effectively induce rapid apoptosis of target cells. On the other hand, DC also secrete several cytokines and a large number of exosomes, which together operate as a whole antigen-presenting entity. The efficacy of the vaccine’s treatment may be affected by the sources used for DC vaccines. Umbilical cord blood (UCB) from healthy donors can be employed when autologous cancer patient’s peripheral blood (PB) cannot be used as a source for isolating DC due to genetic abnormalities. Comparing UCB to other sources, there is a painless method of collecting sources as opposed to PB, which necessitates a venipuncture or leukapheresis procedure to isolate the blood. Many studies related to the use of PB-DC have been carried out, but research on potential comparisons between PB-DC and UCB-DC is still very limited. In this review, the potential of PB- and UCB-derived DC and their secretomes for cancer will be discussed.Keywords: dendritic cells, vaccines, umbilical cord blood, peripheral blood

Gene Editing of Checkpoint Molecules in Cord Blood-Derived Dendritic Cells and CD8+ T Cells Using CRISPR-Cas9.

Immunotherapies targeting checkpoint inhibition and cell therapies are considered breakthroughs for cancer therapy. However, only a part of patients benefit from these treatments and resistance has been observed. Combining both approaches can potentially further enhance their efficacy. With the advent of gene editing techniques, such as clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), the elimination of checkpoint molecules became available as an option in good manufacturing practice conditions to improve persistence and efficacy. However, no data of CRISPR-Cas9 application have been reported in cord blood (CB)-derived immune cells, potentially usable for allogeneic cell therapy purposes. In this article, we describe the optimization of a protocol to deplete checkpoint molecules at the genomic level using CRISPR-Cas9 technology from CB-dendritic cells (DCs) and CB-CD8+ T cells. The protocol is based on the electroporation of a ribonucleoprotein complex, easily translatable to clinical settings. In both cell types, the knock-out (KO) was successful and did not affect cell viability. CB-DCs showed a decrease in expression of the targeted protein ranging from 50% to 95%, while CB-CD8+ T cells showed a reduction in the range of 25-45%. The procedure did not affect the stimulatory function of the CB-DCs or the response of CB-CD8+ T cells (proliferation or TNF-α production). In conclusion, we optimized a protocol to eliminate checkpoint molecules from CB-derived DCs and CD8+ T cells, with the aim to further implement allogeneic cell therapies for cancer.

Clinical Grade Production of Wilms' Tumor-1 Loaded Cord Blood-Derived Dendritic Cells to Prevent Relapse in Pediatric AML After Cord Blood Transplantation.

Hematopoietic cell transplantation (HCT) is a last resort, potentially curative treatment option for pediatric patients with refractory acute myeloid leukemia (AML). Cord blood transplantation (CBT) results in less relapses and less graft-versus-host disease when compared to other sources. Nevertheless, still more than half of the children die from relapses. We therefore designed a strategy to prevent relapses by inducing anti-AML immunity after CBT, using a CB-derived dendritic cell (CBDC) vaccine generated from CD34+ CB cells from the same graft. We here describe the optimization and validation of good manufacturing practice (GMP)-grade production of the CBDC vaccine. We show the feasibility of expanding low amounts of CD34+ cells in a closed bag system to sufficient DCs per patient for at least three rounds of vaccinations. The CBDCs showed upregulated costimulatory molecules after maturation and showed enhanced CCR7-dependent migration toward CCL19 in a trans-well migrations assay. CBDCs expressed Wilms’ tumor 1 (WT1) protein after electroporation with WT1-mRNA, but were not as potent as CBDCs loaded with synthetic long peptides (peptivator). The WT1-peptivator loaded CBDCs were able to stimulate T-cells both in a mixed lymphocyte reaction as well as in an antigen-specific (autologous) setting. The autologous stimulated T-cells lysed not only the WT1+ cell line, but most importantly, also primary pediatric AML cells. Altogether, we provide a GMP-protocol of a highly mature CBDC vaccine, loaded with WT1 peptivator and able to stimulate autologous T-cells in an antigen-specific manner. Finally, these T-cells lysed primary pediatric AML demonstrating the competence of the CBDC vaccine strategy.

In Vitro Generation of Anti-Osteosarcoma Cytotoxic Activity Using Dendritic Cells Loaded with Heat Shock Protein 70-Peptide Complexes

Introduction: This study aimed at evaluating the anti-osteosarcoma activity of cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) pulsed with heat shock protein 70-peptide complexes (Hsp70-PCs). Materials and methods: Human recombinant Hsp70 expression was analyzed using thin layer scanning and Western blot assay. Tumor antigens from Saos-2 cells were extracted to reconstitute Hsp70-PCs. Maturation of cord blood-derived DC was evaluated by alkaline phosphatase-anti-alkaline phosphatase kit and inverted microscope. The anti-osteosarcoma activity of CTLs evoked by DCs loaded with Hsp70-PCs was determined using Thiazolyl Blue Tetrazolium Bromide (MTT) assay. Results: Hsp70 protein level in BL21 (DE3) increased in a time-dependent manner after induction. The expression of surface markers was upregulated and a typical dendritic morphology was observed in mature DCs. Allogeneic CTLs exhibited strong cytotoxic activity against Saos-2 cells. Conclusion: Our in vitro experiment demonstrated the potent induction of cytotoxic activity against osteosarcoma using DC-based vaccine loaded with Hsp70-PCs.

86 - Customized cord blood-derived dendritic cells to get better disease control in refractory AML

86 - Customized cord blood-derived dendritic cells to get better disease control in refractory AML

Efficacy and safety of cord blood-derived dendritic cells plus cytokine-induced killer cells combined with chemotherapy in the treatment of patients with advanced gastric cancer: a randomized Phase II study.

BackgroundCellular immunotherapy has been widely used in the treatment of solid tumors. However, the clinical application of cord blood-derived dendritic cells and cytokine-induced killer cells (CB-DC-CIK) for the treatment of gastric cancer has not been frequently reported. In this study, the efficacy and safety of CB-DC-CIK for the treatment of gastric cancer were evaluated both in vitro and in vivo.MethodsThe phenotypes, cytokines, and cytotoxicity of CB-DC-CIK were detected in vitro. Patients with advanced gastric cancer were divided into the following two groups: the experimental group (CB-DC-CIK combined with chemotherapy) and the control group (chemotherapy alone). The curative effects and immune function were compared between the two groups.ResultsFirst, the results showed that combination therapy significantly increased the overall disease-free survival rate (P=0.0448) compared with chemotherapy alone. The overall survival rate (P=0.0646), overall response rate (P=0.410), and disease control rate (P=0.396) were improved in the experimental group, but these changes did not reach statistical significance. Second, the percentage of T-cell subsets (CD4+, CD3−CD56+, and CD3+CD56+) and the levels of IFN-γ, TNF-α, and IL-2, which reflect immune function, were significantly increased (P<0.05) after immunotherapy. Finally, no serious side effects appeared in patients with gastric cancer after the application of cellular immunotherapy based on CB-DC-CIK.ConclusionCB-DC-CIK combined with chemotherapy is effective and safe for the treatment of patients with advanced gastric cancer.

Dexmedetomidine Inhibits Maturation and Function of Human Cord Blood-Derived Dendritic Cells by Interfering with Synthesis and Secretion of IL-12 and IL-23.

AimsTo investigate the effects and underlying mechanism of dexmedetomidine on the cultured human dendritic cells (DCs).MethodsHuman DCs and cytotoxic T lymphocytes (CTLs) were obtained from human cord blood mononuclear cells by density gradient centrifugation. Cultured DCs were divided into three groups: dexmedetomidine group, dexmedetomidine plus yohimbine (dexmedetomidine inhibitor) group and control group. DCs in the three groups were treated with dexmedetomidine, dexmedetomidine plus yohimbine and culture medium, respectively. After washing, the DCs were co-incubated with cultured CTLs. The maturation degree of DCs was evaluated by detecting (1) the ratios of HLA-DR-, CD86-, and CD80-positive cells (flow cytometry), and (2) expression of IL-12 and IL-23 (PCR and Elisa). The function of DCs was evaluated by detecting the proliferation (MTS assay) and cytotoxicity activity (the Elisa of IFN-γ) of CTLs. In addition, in order to explore the mechanisms of dexmedetomidine modulating DCs, α2-adrenergic receptor and its downstream signals in DCs were also detected.ResultsThe ratios of HLA-DR-, CD86-, and CD80-positive cells to total cells were similar among the three groups (P>0.05). Compared to the control group, the protein levels of IL-12 and IL-23 in the culture medium and the mRNA levels of IL-12 p35, IL-12 p40 and IL-23 p19 in the DCs all decreased in dexmedetomidine group (P<0.05). In addition, the proliferation of CTLs and the secretion of IFN-γ also decreased in the dexmedetomidine group, compared with the control group (P<0.05). Moreover, these changes induced by dexmedetomidine in the dexmedetomidine group were reversed by α2-adrenergic receptor inhibitor yohimbine in the dexmedetomidine plus yohimbine group. It was also found the decrease of mRNA levels of IL-12 p35, IL-12 p40 and IL-23 p19 in the dexmedetomidine group could be reversed by ERK1/2 or AKT inhibitors.ConclusionDexmedetomidine could negatively modulate human immunity by inhibiting the maturation of DCs and then decreasing the proliferation and cytotoxicity activity of CTLs. The α2-adrenergic receptors and its downstream molecules ERK1/2 and AKT are closely involved in the modulation of dexmedetomidine on DCs.

Umbilical cord blood-derived dendritic cells infected by adenovirus for SP17 expression induce antigen-specific cytotoxic T cells against NSCLC cells

Sperm protein 17 (SP17), a cancer/testis antigen, is expressed by non-small cell lung cancer (NSCLC). This study examined whether dendritic cells (DC) from human umbilical cord blood (UCB) could be induced for SP17 expression and induce antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) against NSCLC in vitro. We generated recombinant adenovirus of Ad-SP17 and control Ad-null. Infection with Ad-SP17, but not control, induced higher levels of SP17 expression in UCB-derived DC-Ad-SP17. Infection with Ad-SP17 significantly increased the frequency of CD80+, CD83+, CD86+, and HLA-DR+ DC that produced higher levels of IL-12, but lower IL-10. Co-culture of DC-Ad-SP17 with autologous UCB lymphocytes induced high frequency of IFNγ+ CD8+ CTLs, which had selective cytotoxicity against SP17+ lung cancer CRL-5922 cells in a HLA-I restrictive manner. Thus, UCB-derived DC modulated for SP17 expression induced antigen-specific anti-tumor immunity against SP17+ NSCLC, and SP17 may be a valuable target for development of immunotherapy against SP17+ NSCLC.

Umbilical cord blood-derived dendritic cells loaded with BGC823 tumor antigens and DC-derived exosomes stimulate efficient cytotoxic T-lymphocyte responses and antitumor immunity in vitro and in vivo.

BackgroundUmbilical cord blood (UCB) is a rich source of hematopoietic stem cells and from which a significant number of dendritic cells (DCs) can be produced. But the therapeutic role of DCs and exosomes (EXO) generated from DCs is not fully elucidated.Material and methodsThe UCB-derived DCs were loaded with tumor antigens generated from BGC823 cell line. Exosomes were derived from these DCs by ultracentrifugation. Dendritic cells and DCex were evaluated by light microscope, transmission electron microscope (TEM), flow cytometry, and western blot assay. The therapeutic role of DCs and EXO generated from DCs were then detected in vitro and in vivo.ResultsDendritic cells isolated from umbilical cord blood after loading with tumor antigens generated from BGC823 cell line could express high levels of protein molecules: MHC-I, MHC-II, CD34, CD40, CD80, CD86, CD11c and CD54 and mediate a stronger promotion of T cells proliferation. And, they could also enhance the cytotoxicity effects of the generated CTL in vitro and in vivo. Exosomes isolated from these DCs were 40-90-nm round particles with a complete membrane structure and could also expressed molecules similar to DCs. Exosomes could stimulate T cell proliferation, produce effective cytotoxicity and induce more efficient in vivo antitumor immunity.ConclusionsThese results suggested that tumor antigens loaded DCs derived from unrelated umbilical cord blood or DCex can induce tumor specific cytotoxicity and this may represent a novel immunotherapy for tumors. Because of their advantage of stable, easy to store, DCex have a more brilliant prospects in the tumor immunity.Additional informationWe reported that exosomes derived from umbilical cord blood dendritic cell (UBDC), similar to DCs, can trigger activation of T cells significantly. These data demonstrate that DC-derived exosomes (DCex) can mediate essential adaptive immune functions.

The antitumor effect of human cord blood-derived dendritic cells modified by the livin α gene in lung cancer cell lines

The growth of malignant tumors is associated with mechanisms of immune escape and inhibition of apoptosis. Livin is a novel member of the inhibitors of apoptosis (IAP) protein family that inhibits cell apoptosis. Livin is specifically expressed by the majority of tumor cells, but it is not expressed in normal adult tissues. In this study, we used umbilical cord blood (UCB)-derived dendritic cells (DCs) infected with a recombinant adenovirus encoding the livin gene as a vaccine to activate effector cells such as cytotoxic T lymphocytes (CTLs) to recognize and kill livin-expressing cancer cells in vitro as an improved strategy for overcoming the ability of these cancer cells to escape apoptosis and antitumor immune responses. We employed interferon-γ (IFN-γ) enzyme-linked immunospot assays to confirm that our immunization strategy induced an antigen-specific reaction to livin and flow cytometric analysis of staining with Annexin V and PI to measure the cytotoxic activity of the effector cells against the livin-expressing lung cancer cell lines A549 and H460. Our results show that the recombinant adenovirus was able to promote the maturation of the UCB-derived DCs. This DC vaccine could activate antigen-specific T cells to produce IFN-γ upon recognition of livin peptide in the context of the appropriate HLA molecule. The antigen-specific T cells mediate significant cytotoxicity against the cancer cells, but are unlikely to cause an autoimmune reaction against the human bronchial epithelia cells (HBE), which do not express livin.

Growth hormone influence on functional status of cord blood-derived dendritic cells

To detect the effect of functional status and immunophenotype of dendritic cell modified with growth hormone (GH) from by cord blood. Monocyte was isolated from cord blood, DCs were induced with IL-4 & GM-CSF and modified with different concentration of GH. The morphology, count and growth status of DC were observed appearance. And the immunophenotype of DC was detected with flow cytometry. IL-12 in supernatant was measured with ELISA. The immuno-activity was determined with autologous mixed lymphocyte reaction. Mature DC were induced from adherent lymphomonocyte under the action of IL-4, GM-CSF and GH of different densities (5, 25, 100 ng/ml). GH increased the positive expression of CD83 (31% +/- 4%, 34% +/- 4%, 34% +/- 5%), CD80 (26% +/- 4%, 29% +/- 6%, 30% +/- 6%)and HLA-DR (45% +/- 8%, 49% +/- 6%, 50% +/- 5%) (P < 0.05), enhanced the ability of secreting IL-12 (P < 0.05), and boosted significantly the immuno-activation of DC to autologous lymphocyte (P < 0.05). GH could promote the differentiation, maturation and the expression of costimulatory molecules, increase the IL-12 production in DC and enhanced the ability of DC to stimulate the proliferation of lymphocyte within a certain dose range.

Generation of Influenza HA-Specific Lymphocytes from Autologous Umbilical Cord Blood-Derived Dendritic Cells.

INTRODUCTION: Umbilical cord blood transplantation (CBT) is being performed with increasing frequency for patients lacking HLA-matched donors. CBT is associated with delayed immune reconstitution and high rates of viral infections. Particularly problematic is the reactivation of latent viral infections such as CMV, EBV, and VZV, as is overt infection from common agents like adenovirus and influenza. To address these issues, we proposed to generate influenza-specific CB T-cells ex vivo for potential cell therapy applications, allowing for effective therapeutic interventions until such time that immune reconstitution has occurred.METHODS: Dendritic cells (DC) were generated from both the adherent and the CD14+ CB fractions cultured in AIM-V medium supplemented with human serum, GM-CSF, and IL-4. Antigen loading was performed by incubation with recombinant, purified A/New Caledonia HA antigen. Maturation was performed with the ITIP inflammatory cocktail (IL-1β, TNF-α, IL-6, and PGE2). Autologous CB lymphocytes were co-cultured with mature DC at a ratio of 10:1 for 4 days in IL-12 after which they were expanded in a cytokine cocktail of IL-2, IL-7, IL-12, and IL-15. Lymphocytes were restimulated similarly every 7-10 days. Antigen specificity was demonstrated by IFN-γ ELISpot.RESULTS: Mature DC expressed the appropriate markers including CD80, CD83, CD86, CD209, and HLA-DR. Some DC expressed CD14, others CD11c, an indication of a mixed interstitial/Langerhans myeloid phenotype. We demonstrated a 4–10 fold enhancement of IFN-γ ELISpots when primed lymphocytes were incubated with HA-loaded DC in comparison to autologous unloaded DC (p<0.003) or loaded allogeneic DC (p<0.0001). The CD8 content of the HA-specific lines was typically greater than 50% after two stimulations. At the time of phenotyping CD3+ cells were primarily CD62LposCD197(CCR7)neg and CD127(IL-7Rα)neg, an effector phenotype; however, 6–22% of cells expressed CD127 (effector or central memory), and up to 6% of cells were CD62LposCD197pos (central memory).CONCLUSIONS: The results demonstrate that, despite the generally naïve nature of CB lymphocytes, influenza-specific responses can be generated ex vivo, and could potentially be used clinically in CBT patients who develop infectious complications prior to immune reconstitution.

The Impact of Hyperoxia on the Neonatal and Adult Developing Dendritic Cell

Oxygen is essential therapy for neonates with acute respiratory failure, including those with infections. However, high oxygen levels may be counterproductive for overcoming infections because hyperoxia may kill cells, including dendritic cells that are essential to the emergence of the pulmonary immune system and pivotal in mounting immune responses to infections. We studied the impact of hyperoxia on developing dendritic cells from neonatal cord blood and adult blood monocytes, comparing viability, development of maturation, and endocytic function. Our data suggest that cord blood-derived dendritic cells may be more resistant to hyperoxic-induced cell death than adult blood-derived cells. Moreover, the surviving cells in either group are those that maintain an immature phenotype. This may impair their ability to perform optimal immune function.

Genomic investigation of the metabolic competency of human cord blood-derived dendritic cells

Genomic investigation of the metabolic competency of human cord blood-derived dendritic cells

Anti-Leukemic CTL Generated by RNA Loaded Cord Blood-Derived Dendritic Cells Control Leukaemia in a NOD-SCID Mouse Model.

Acute Lymphocytic Leukaemia (ALL) patients who relapse after transplantation have few therapeutic options. An immunotherapeutic approach that enhances the graft versus leukemia effect may improve their survival. We have developed a method for generating cord blood derived dendritic cells (CBDC) and transfecting them with high efficiency (>90% eGFP positivity) and viability (>90%) (BBMT 2006 12:855–867). We postulate that CTL generated from total ALL RNA-loaded CBDC may be exploited in therapy and therefore tested their ability to control the kinetics of leukaemic growth in a NOD-SCID mouse model of human ALL.CTL induced using total RNA from the pre-B ALL Nalm-6 cell line lysed 49±11% of Nalm-6 cells compared to 7%±1% of autologous and 2.5%±1.3% of allogeneic mononuclear cells (p=0.03 and p=0.022 respectively). Using total RNA from patient derived ALL xenograft, we generated anti-leukemic CTL cultures comprising a mixed population of 69.9±2% CD4, 12.9±2% CD8 and 17.6±1% NK cells. These CTL lysed 63±6% of ALL xenograft targets and 22±7% of autologous MNC (p=0.04) and also lysed autologous survivin mRNA-transfected DC (16±5.4%). Preliminary results suggest that some of the ALL xenograft CTL also recognize the cell surface protein, HM1.24, which is specifically up-regulated in metastatic tumor cells and chemoresistant cancer cells. This suggests that the total ALL RNA generated CTL comprise a polyclonal population, which may prove efficacious in vivo. To test the in vivo efficacy of these anti-leukemic CTL, we established a NOD-SCID mouse model of ALL using patient samples. By varying the inocula of ALL cells, we established a predictable and reproducible model of emerging relapse.Administration of anti-leukaemic CTL 2, 3, 4 and 5 weeks post ALL inoculation, in increasing doses (low - 2.5×106 cells/mouse, medium - 10×106/mouse and high - 20×106/mouse), altered the kinetics of ALL growth in vivo (p=0.00032, see figure). By 8 weeks post transplant, mice given ALL alone had 3.6% human CD45+CD19+ leukemic cells in the blood, whereas recipients of the highest CTL dose had only 0.66% CD45+CD19+ leukemic cells in the blood (p=0.05). After 11 weeks the disease load was the same in mice who received ALL alone and those who received ALL and 4 doses of 20 × 106 CTL. These results suggest that anti-leukemic CTL generated by RNA-loaded CBDC can control leukaemia in a NOD-SCID mouse model and that disease control may be enhanced by antigen-specific restimulation of the anti-leukaemic CTL and/or more refined administration. These results represent an important step forward in the development of clinical immunotherapy protocols for the treatment of ALL and its potential application after cord blood transplantation. [Display omitted]

RNA transfected cord blood-derived dendritic cells generate anti-leukemic cytotoxic T lymphocytes

RNA transfected cord blood-derived dendritic cells generate anti-leukemic cytotoxic T lymphocytes

Significant Decreased in Cord Blood (CB) Versus Adult Peripheral Blood (APB) Monocyte (Mo)-Derived Dendritic Cells (DC) Gene Expression Patterns and T Cell Functional Activation: Insight into Immaturity of CB Cellular Immunity.

It has been recognized that dysfunction of CB immunity is in part due to the immaturity of the neonatal immune system (Cairo, Blood, 1997). However, biological pathways and molecular mechanisms associated with the immaturity of CB immunity are still poorly understood. Recently we have utilized oligonucleotide microarray to examine gene expression profiling of CB versus APB Mo and have demonstrated significant differential gene expression patterns, including surface molecules, cytokines, signaling molecules, transcription factors and apoptotic genes (Jiang/Cairo, et al, J. of Immunol., 2004). We sought to examine whether there are differential expressed genes occurred in Mo-derived CB versus APB DC and their impact on DC mediated T cell activity. Briefly, Mo were purified from fresh CB or APB and cultured for 8 days with GM-CSF and IL-4 (immature DC (iDC)) and LPS for mature DC (mDC). mRNA was isolated and oligonucleotide microarray was carried out (Affymetrix, U133A). Data was analyzed by Microarray Suite Version 5.0 (Affymetrix) and GeneSpring 5.0 software (Silicon Genetics). Selected genes were analyzed by RT-PCR (SuperScript, Invetrogen). We identified gene expression patterns that were significantly lower in CB versus APB DC including surface molecules HLA-DQA1 (4F), HLA-DRB3 (5F), HLA-DRB4 (5.5F), CD80 (3F), CD38 (3.8F); cytokine/chemokine genes IL-1b (2.5F), IL6 (2.9F), IL12B (3.5F), CXCL10 (6.6F); immunoregulatory genes ISG20 (11F), IFI27 (7.6F), TNFSF10 (4.5F), SOCS3 (2.5F). Moreover, several transcription factor genes whose proteins may involve in the activation of expression of these immune regulator genes were also differentially expressed (IRF-5 (3F), IRF7 (3F), MAD (6.3F)). We therefore compared CB versus APB DC antigen presentation activity to APB CD8 T cells by ELISPOT assay for interferon-r (IFNr) production (BD Pharmagen). Briefly, the purified CD8 T cells (MHC HLA A2) were incubated with CB or APB DC that were loaded without or with influenza peptide onto ELISPOT plate (Larsson, et al, J. of Immunol., 2000). The ELISPOT plates were developed, scanned and quantitated by an ELISPOT reader (C.T.L. Technology). Our results demonstrated that, although CB or APB mDC had allogeneic effects, influenza peptide loaded CB mDC was not able to induce CD8 T cells to produce IFNr while APB mDC loaded with influenza peptide strongly induced CD8 T cells to produce IFNr. This stimulatory effect of APB mDC on CD8 T cells to produce IFNr was 3.5 fold greater than that of CB mDC. We further examined DC antigen presentation activity to CD4 T cells and observed that APB-DC had stronger effects on CD4 T cell proliferation (3 fold for mDC vs. iDC) compared with CB-DC (only 1.5 fold for mDC vs. iDC) by CFSE assay (Molecular Probe). We postulate that decreased expression of specific surface molecules and other genes resulting in lower surface protein expression in CB DC may in part be responsible for the lack of initiation of signaling events from cell surface to trigger CB-DC to stimulate activation of CD8 and CD4 T cells. The decreased expression of transcription factor genes may also in part be responsible for the lower expressed surface molecule genes. Furthermore, these decreased expressed genes in other molecular categories in LPS-CB vs. APB DC may also partially be responsible for differential innate and adaptive immune function and properties of CB vs. APB.

Differential Expression of Genes Associated with Toll-Like Receptor-4 (TLR4) Signal Transduction Pathway in Lipopolysaccharide (LPS)-Activated Cord Blood (CB) vs. Adult Peripheral Blood (APB) Monocyte (Mo)-Derived Dendritic Cells (DC).

LPS activates immature DC via TLR4 and induces maturation of DC for initiating antigen presenting activity (Medzhitov; Nat Rev Immunol 2001). We have previously demonstrated decreased gene expression and protein production of IL-12, IL-15, IL-18 in activated CB MNC and decreased DC MLR (Lee/Cairo, Blood 1996; Qian/Cairo, Blood 1997; Wu/Cairo, Blood 100:3668 p51b 2002). Recently, we have identified differential gene expression patterns including differential immunoregulatory and chemokine genes in LPS-CB vs APB Mo by microarray (Jiang/Cairo, J. Immunol 2004). Since the myeloid lineage DC is derived from Mo, we sought to determine in LPS activated CB vs. APB DC, differential expressed genes that associate with TLR4-mediated signaling pathway. Briefly, Mo were purified from fresh CB or APB and cultured for 7 days with GM-CSF & IL-4 [immature DC (iDC)] and LPS [mature DC (mDC)]. Aliquots from iDC and mDC were analyzed for DC immunophenotype, morphology and DC allogeneic antigen activity. mRNA was isolated, reverse transcripted to cDNA, labeled & hybridized to oligonucleotides (Affymetrix, U133A). Data was analyzed by MAS 5.0 (Affymetrix) and GeneSpring 5.0 software (Silicon Genetics). Several genes were analyzed by RT-PCR (One-Step SuperScript, Invitrogen) and protein expression was analyzed by Western Blot (Bio-Rad). Inverted microscopy demonstrated DC mature morphology at day 8 and flow cytometry demonstrated decreased CD14 and increased CD83 expression in CB & APB mDC. We also demonstrated significant increase in the allogeneic stimulatory effects on CD4+ T cells in APB vs. CB mDC. The microarray analysis demonstrated a significant decreased gene expression of TLR4 [3 fold (F)] and CD14 (2.1 F) (p<0.05) in CB vs APB-DC. We further identified LPS significantly induced increased expression of TLR4 downstream signaling molecular genes such as MAPKKK, NF-kB and TANK in APB compared to CB mDC (3–8 F) (p<0.05). There were also significant amplifications of a variety of other gene categories in LPS activated APB vs CB mDC (p<0.05) including cell surface molecule CD80 (3.7F) and IL-2Ra (5.3 F), cytokine IL-23 (3.5F) & IL-12 (13 F), signal transduction STAT1 (3.4F) & IRF-7 (7.7 F), and immunoregulatory TNFSF10 (12F) & ISG20 (39F). Gene expression of NF-kB1, TRAF1 & IRF-7 by RT-PCR demonstrated an increased expression in LPS-APB vs CB mDC and were compatible with microarray. Moreover, Western analysis of IRF-7 demonstrated increased protein expression in LPS-APB vs CB mDC. In summary, we have identified decreased gene expression patterns in LPS-CB vs APB DC, especially those in the TLR4 signal transduction pathway (MAP3K, TRAF, TANK & NF-kB), and suggest these differentially expressed genes may enhance the activation of TLR4 pathway in LPS-APB vs CB DC, resulting in differential regulation of CB vs APB DC antigen presentation capacities. Furthermore, these decreased expressed genes in other molecular categories (e.g.IL-23, IFNg, IL6, CD80, STAT1, IRF-7, SOCS3) in LPS-CB vs APB DC may be partially responsible for differential innate and adaptive immune function of CB vs APB. Moreover, the differential regulated expression of genes may in part help to explain reduced incidence of severe aGVHD, delay in immune reconstitution and/or increased infectious mortality following HLA disparate UCBT.

Efficient transduction and long-term retroviral expression of the melanoma-associated tumor antigen tyrosinase in CD34(+) cord blood-derived dendritic cells.

Differentiation of genetically modified CD34(+) hematopoietic stem cells into dendritic cells (DCs) will contribute to the development of immunotherapeutic anticancer protocols. Retroviral vectors that have been used for the transduction of CD34(+) cells face the problem of gene silencing when integrated into the genome of repopulating stem cells. We reasoned that a high copy number of retroviral DNA sequences might overcome silencing of transgene expression during expansion and differentiation of progenitor cells into functional DCs. To prove this, we utilized a retroviral vector with bicistronic expression of the melanoma-associated antigen tyrosinase and the enhanced green fluorescent protein (EGFP). Human cord blood CD34(+) cells were transduced with vesicular stomatitis virus G-protein (VSV-G) pseudotyped Moloney murine leukemia virus (MoMuLV) particles using 100-150 multiplicity of infection. During expansion of transduced cells with immature phenotype, transgene expression was strongly silenced, but upon differentiation into mature DCs, residual transgene expression was retained. Intracellular processing of the provirally expressed tyrosinase was tested in a chromium release assay utilizing a cytotoxic T cell clone specific for a HLA-A*0201-restricted tyrosinase peptide. We suggest that retroviral transduction of tumor-associated antigens in hematopoietic progenitor cells and subsequent differentiation into DCs is a suitable basis for the development of potent anti-tumor vaccines.

Genomic Organization of the CC Chemokine MIP-3α/CCL20/LARC/EXODUS/SCYA20, Showing Gene Structure, Splice Variants, and Chromosome Localization

We describe the genomic organization of a recently identified CC chemokine, MIP3α/CCL20 (HGMW-approved symbol SCYA20). The MIP-3α/CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISH analysis to 2q35–q36. Two distinct cDNAs were identified, encoding two forms of MIP-3α/CCL20, Ala MIP-3α/CCL20 and Ser MIP-3α/CCL20, that differ by one amino acid at the predicted signal peptide cleavage site. Examination of the sequence around the boundary of intron 1 and exon 2 showed that use of alternative splice acceptor sites could give rise to Ala MIP-3α/CCL20 or Ser MIP-3α/CCL20. Both forms of MIP-3α/CCL20 were chemically synthesized and tested for biological activity. Both flu antigen plus IL-2-activated CD4+ and CD8+ T lymphoblasts and cord blood-derived dendritic cells responded to Ser and Ala MIP-3α/CCL20. T lymphocytes exposed only to IL-2 responded inconsistently, while no response was detected in naive T lymphocytes, monocytes, or neutrophils. The biological activity of Ser MIP-3α/CCL20 and Ala MIP-3α/CCL20 and the tissue-specific preference of different splice acceptor sites are not yet known.