Standard Cytokine Cocktail Research Articles

Evaluation of quality parameters in thawed dendritic cell vaccine

Background & Aim Manufacturing of Dendritic Cell (DC) vaccine for cancer, a type of Advanced Therapy Medicinal Products, is regulated by cGMP rules. Our experience of DC vaccine preparation started in 2001 and was tested in many clinical trials. Methods, Results & Conclusion DC vaccines were prepared from adherent fraction of Peripheral Blood Mononuclear Cells obtained from leukapheresis, by differentiation and maturation with standard cytokine cocktail after pulsing with autologous tumor homogenate. Aliquots of DC vaccine were frozen by automatic freezing in autologous plasma with 10% DMSO and stored in nitrogen vapors until their use. These aliquots were thawed and resuspended in sterile saline at a final concentration of 5 × 106 cells/ml, packed in two syringes for a total of 10 million that represent the thawed final product. To ensure the maintaining of compliance with release parameters of the thawed final product, three representative batches, stored at 2-8°C for 8 hours, were analyzed using the following tests: Mycoplasma Detection (Method EP. 2.6.7), Bacterial Endotoxin levels (Method EP 2.6.14), Sterility (Method EP 2.6.27) Viable Cell Count (Method EP 2.7.29) Phenotype analysis (internal method) Potency test (ELISPOT COSTIM assay was performed to establish the potency of DCs. We modified the COSTIM assay, which uses IFN-gamma ELISPOT as a read-out system. In this assay DCs provide responder allogeneic T cells, sub-optimally pre stimulated with OKT3, with the costimulatory signals required for full activation). The results and the acceptance criteria are listed in Table 1. Manufacturing of Dendritic Cell (DC) vaccine for cancer, a type of Advanced Therapy Medicinal Products, is regulated by cGMP rules. Our experience of DC vaccine preparation started in 2001 and was tested in many clinical trials. DC vaccines were prepared from adherent fraction of Peripheral Blood Mononuclear Cells obtained from leukapheresis, by differentiation and maturation with standard cytokine cocktail after pulsing with autologous tumor homogenate. Aliquots of DC vaccine were frozen by automatic freezing in autologous plasma with 10% DMSO and stored in nitrogen vapors until their use.

NF-κB activation triggers NK-cell stimulation by monocyte-derived dendritic cells.

Background:In therapeutic cancer vaccination, monocyte-derived dendritic cells (moDCs) efficiently activate specific T-cell responses; however, optimizing the activation of innate immune cells could support and improve the antitumor effects. A major disadvantage of moDCs matured with the standard cytokine cocktail (consisting of IL-1β, IL-6, TNFα, and PGE2) is their inability to secrete IL-12p70. IL-12 prominently activates natural killer (NK) cells, which are crucial in innate antitumor immunity, as they act as helper cells for the induction of a cytotoxic T lymphocyte (CTL) response and are also able to directly kill the tumor.Methods:Previously we have shown that triggering the NF-κB pathway in moDCs by transfection of mRNA encoding constitutively active IKKβ (caIKKβ) led to IL-12p70 secretion and improved the dendritic cells’ capability to activate and expand CTLs with a memory-like phenotype. In this study, we examined whether such dendritic cells could activate autologous NK cells.Results:moDCs matured with the standard cytokine cocktail followed by transfection with the caIKKβ-RNA were able to activate autologous NK cells, detected by the upregulation of CD54, CD69, and CD25 on the NK cells, their ability to secrete IFNγ, and their high lytic activity. Moreover, the ability of NK-cell activation was not diminished by simultaneous T-cell activation.Conclusion:The capacity of caIKKβ-DCs to activate both the adaptive and innate immune response indicates an enhanced potential for clinical efficacy.

Inducing Effect of Modified Cytokine Cocktail on Dendritic Cells

To investigate the inducing effect of 'modified' cytokine cocktail on the dendritic cell maturation and migration capability. PBMNC were isolated from human peripheral blood stem cell (PBSC) by using density gradient centrifugation, the immature DC (imDC) were induced by using GM-CSF and IL-4 in vitro. Total A549 RNA was transfected into imDC by using electroporation, which was stimulated to matuation by the "gold standard" cytokine cocktail and "modified" cytokine cocktail, respectively. The expression of DC surface markers (CD11c, HLA-DR, CD80, CD83 and CD86) and chemokine receptor (CCR5, CCR7 and CXCR4) were detected by flow cytometry; the mRNA expression levels of DC chemokine receptor (CCR2, CCR5, CCR7, CXCR3 and CXCR4) and chemokine (CCL2, CCL3, CCL5, CCL19, CCL21, CXCL10 and CXCL12) were detected by RT-PCR. As compared with "gold standard cytokine cocktail", the "modified" cytokine cocktail-induced DC expressed higher levels of surface markers (CD11c, HLA-DR, CD80, CD83 and CD86), chemokine receptors (CXCR4) and chemokine (CCL2, CCL3, CCL5, CCL19, CCL21, CXCL10 and CXCL12). The "modified" cytokine cocktail can more effectively induce the DC maturation, enhace the migratory capability of DC and more generate the immunostimulatory DC, when compared with the "gold standard" cytokine cocktail effect.

Cytokine-enhanced maturation and migration to the lymph nodes of a human dying melanoma cell-loaded dendritic cell vaccine.

Dendritic cells (DCs) are professional APCs used for the development of cancer vaccines because of their ability to activate adaptive immune responses. Previously, we designed the DC/Apo-Nec vaccine using human DCs loaded with dying melanoma cells that primed Ag-specific cytotoxic T cells. Here, we evaluate the effect of a standard pro-inflammatory cytokine cocktail (CC) and adjuvants on DC/Apo-Nec maturation and migration. CC addition to the vaccine coculture allowed efficient Ag uptake while attaining strong vaccine maturation with an immunostimulatory profile. The use of CC not only increased CCR7 expression and the vaccine chemokine responsiveness but also upregulated matrix metalloproteinase-9 secretion, which regulated its invasive migration in vitro. Neither IL-6 nor prostaglandin E2 had a negative effect on vaccine preparation. In fact, all CC components were necessary for complete vaccine maturation. Subcutaneously injected DC/Apo-Nec vaccine migrated rapidly to draining LNs in nude mice, accumulating regionally after 48 h. The migrating cells of the CC-matured vaccine augmented in proportion and range of distribution, an effect that increased further with the topical administration of imiquimod cream. The migrating proportion of human DCs was detected in draining LNs for at least 9 days after injection. The addition of CC during DC/Apo-Nec preparation enhanced vaccine performance by improving maturation and response to LN signals and by conferring a motile and invasive vaccine phenotype both in vitro and in vivo. More importantly, the vaccine could be combined with different adjuvants. Therefore, this DC-based vaccine design shows great potential value for clinical translation.

Low-dose insulin-like growth factor binding proteins 1 and 2 and angiopoietin-like protein 3 coordinately stimulate ex vivo expansion of human umbilical cord blood hematopoietic stem cells as assayed in NOD/SCID gamma null mice

IntroductionInsulin-like growth factors (IGFs), IGF binding proteins (IGFBPs) and angiopoietin-like proteins (ANGPTLs) can enhance the ex vivo expansion of hematopoietic stem cells (HSCs) when used with a standard cytokine cocktail of stem cell factor (SCF), thrombopoietin (TPO) and FLT3 ligand (FL). In order to determine the optimal dose and combination of IGFs, IGFBPs and ANGPTLs, serial dilution and full permutation of IGFBP1, IGFBP2, IGF2 and ANGPTL3 were applied on a cryopreserved umbilical cord blood mononuclear cell (UCB-MNC) ex vivo expansion system.MethodsIn this system, 4 × 105 cells/ml of UCB-MNCs were inoculated in serum-free Stemspan® medium (Stemcell technologies, vancouver, BC, Canada) supplied with standard basal cytokine combination of 100 ng/ml SCF, 50 ng/ml FL and 100 ng/ml TPO and supported by a bone marrow mesenchymal stromal cell layer.ResultsParadoxically, experiment results showed that the highest expansion of CD34+CD38−CD90+ primitive progenitor was stimulated by cytokine combination of SCF + TPO + FL + IGFBP1 + IGFBP2 + ANGPTL3 at a low dose of 15 ng/ml IGFBP1 and 20 ng/ml IGFBP2 and ANGPTL3. This ex vivo expansion was further validated in 8-week-old to 10-week-old nonobese diabetic/severe combined immunodeficiency interleukin 2 gamma chain null (NOD/SCID-IL2Rγ−/−) mice. Limiting dilution assay showed excellent correlation between the HSC ex vivo surface marker of CD34+CD38−CD90+ and the in vivo competitive repopulating unit (CRU) functional assay.ConclusionIGFBP1, IGFBP2, IGF2 and ANGPTL3 can stimulate the expansion of CD34+CD38−CD90+ primitive progenitor at low dose. The optimal combination comprises IGFBP1, IGFBP2 and ANGPTL3 together with the standard cytokine cocktail of SCF, FL and TPO. The CD34+CD38−CD90+ phenotype can serve as a surrogate ex vivo surface marker for HSCs due to consistency with the in vivo CRU functional assay.

MP28-03 INVASIVE HUMAN BLADDER TUMOR CELLS FORM INVADOPODIA THAT IS INITIATED BY MACULAR EXPRESSION OF FORMIN-BINDING PROTEIN(FBP) 17.

INTRODUCTION AND OBJECTIVES: To induce a potent cytotoxic T lymphocyte (CTL) response in dendritic cell (DC)-based immunotherapy against bladder cancer, various tumor antigens need to be loaded onto DCs. The aim of this study was to establish a method of immunotherapy for bladder cancer using bladder cancer-specific CTLs generated in vitro by DCs. METHODS: Monocyte-derived DCs from bladder cancer patients were induced to mature in a standard cytokine cocktail (in IL-1b, TNF-a, IL-6, and PGE2: standard DCs, sDCs) or in an a-type 1-polarized DC (aDC1) cocktail (in IL-1b, TNF-a, IFN-b, IFN-g, and polyinosinic:polycytidylic acid) and then loaded with the UVB-irradiated bladder cancer cell line T24. Antigen-loaded aDC1s were evaluated by morphological and functional assays, and the bladder cancer-specific CTL response was analyzed by cytotoxic assay. RESULTS: The aDC1s significantly increased the expression of several molecules related to DC maturation, regardless of whether the aDC1s were loaded with tumor antigens or not, compared to sDCs. The aDC1sshowedahigherproductionof interleukin-12bothduringmaturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumor antigens as compared to standard DCs (sDCs). Bladder cancer-specific CTLs against autologous bladder cancer cells were successfully induced by aDC1s loaded with dying T24 cells. CONCLUSIONS: Autologous aDC1s loaded with an allogeneic bladder cancer cell line can generate greater bladder cancer-specific CTL responses and may provide a novel source of DC-based vaccines that can be used for the development of immunotherapy in patients with bladder cancer.

MP28-01 GENERATION OF POTENT CYTOTOXIC T LYMPHOCYTES AGAINST BLADDER CANCER CELLS BY DENDRITIC CELLS LOADED WITH DYING T24 BLADDER CANCER CELLS

INTRODUCTION AND OBJECTIVES: To induce a potent cytotoxic T lymphocyte (CTL) response in dendritic cell (DC)-based immunotherapy against bladder cancer, various tumor antigens need to be loaded onto DCs. The aim of this study was to establish a method of immunotherapy for bladder cancer using bladder cancer-specific CTLs generated in vitro by DCs. METHODS: Monocyte-derived DCs from bladder cancer patients were induced to mature in a standard cytokine cocktail (in IL-1b, TNF-a, IL-6, and PGE2: standard DCs, sDCs) or in an a-type 1-polarized DC (aDC1) cocktail (in IL-1b, TNF-a, IFN-b, IFN-g, and polyinosinic:polycytidylic acid) and then loaded with the UVB-irradiated bladder cancer cell line T24. Antigen-loaded aDC1s were evaluated by morphological and functional assays, and the bladder cancer-specific CTL response was analyzed by cytotoxic assay. RESULTS: The aDC1s significantly increased the expression of several molecules related to DC maturation, regardless of whether the aDC1s were loaded with tumor antigens or not, compared to sDCs. The aDC1sshowedahigherproductionof interleukin-12bothduringmaturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumor antigens as compared to standard DCs (sDCs). Bladder cancer-specific CTLs against autologous bladder cancer cells were successfully induced by aDC1s loaded with dying T24 cells. CONCLUSIONS: Autologous aDC1s loaded with an allogeneic bladder cancer cell line can generate greater bladder cancer-specific CTL responses and may provide a novel source of DC-based vaccines that can be used for the development of immunotherapy in patients with bladder cancer.

Generation of Potent Cytotoxic T Lymphocytes Against Castration‐Resistant Prostate Cancer Cells by Dendritic Cells Loaded With Dying Allogeneic Prostate Cancer Cells

To induce a potent cytotoxic T lymphocyte (CTL) response in dendritic cell (DC)-based immunotherapy against prostate cancer, various tumour antigens should be loaded onto DCs. The aim of this study was to establish a method of immunotherapy for castration-resistant prostate cancer (CRPC) using prostate cancer-specific CTLs generated in vitro by DCs. Monocyte-derived DCs from patients with CRPC were induced to mature using a standard cytokine cocktail (in IL-1β, TNF-α, IL-6 and PGE(2) : standard DCs, sDCs) or using an α-type 1-polarized DC (αDC1) cocktail (in IL-1β, TNF-α, IFN-α, IFN-γ and polyinosinic:polycytidylic acid) and loaded with the UVB-irradiated CRPC cell line PC-3. Antigen-loaded DCs were evaluated by morphological and functional assays. The αDC1s significantly increased the expression of several molecules related to DC maturation, regardless of whether the αDC1s were loaded with tumour antigens or not, compared to sDCs. The αDC1s showed a higher production of interleukin-12 both during maturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumour antigens, as compared to standard DCs (sDCs). Prostate cancer-specific CTLs against autologous CRPC cells were successfully induced by αDC1s loaded with dying PC-3 cells. Autologous αDC1s loaded with an allogeneic CRPC cell line can generate greater CRPC-specific CTL responses as compared to sDCs and may provide a novel source of DC-based vaccines that can be used for the development of immunotherapy in patients with CRPC.

Generation of Red Blood Cells From Human Embryonic Stem Cells (ES) with Increased Efficacy

Abstract 4334It is estimated that for every unit of donated blood, two units are required in North America. The current rate of blood donation is stagnant while the need increases by 6–8% annually. In order to overcome this difficulty, we have developed an improved method to generate red blood cells from human embryonic stem cells (H9) with increased efficacy. In addition to xeno-free conditions and standard cytokine cocktail used for hematopoietic differentiation of human embryonic stem cells (Carrier et al. J Transl Med. 2009; Vol 7: 27), we have introduced a new method of improved growth and differentiation of human ES cells with hypoxia-induced mesenchymal stem cells, obtained from allogeneic adult bone marrow donors. This technique increased efficacy of red blood cell production by 5–25 fold.We have developed a bioscaffold–> microsphere-based culture system with highly porous surface allowing culturing of a very large number of embryonic stem cells per one culture condition. This culture system avoids shear forces and damage to the cells, and facilitates removal and recycling of the microspheres. The in vitro obtained human ES-derived red blood cells are enucleated and do not produce tumors (efficacy of enucleation is 65–95%). The laser-based system is utilized to eliminate nucleated cells from the culture.The problem with hES-derived red blood cells is that they are produced in small numbers and process is very costly. We are developing a 3-phase bioreactor with computerized programming, which will increase every step of the differentiation process and allow recycling of feeder cells and cytokines. In this system we will utilize iron-loaded microspheres coated with hypoxia-processed mesenchymal stem cells as a main culture unit. The in vitro generated human ES-derived red blood cells upscaled in a bioreactor will be used for the off-shelf production of red blood cells for clinical use. Disclosures:Srivastava:Giostar: Employment, Equity Ownership. Azad:Dnamicroarray, Inc.: Employment, Equity Ownership. Carrier:Giostar: Consultancy; Samaritan Pharmaceuticals: Consultancy; Entest Biomedical: Consultancy, Equity Ownership; America Stem Cells: Consultancy, Equity Ownership; Millenium: Speakers Bureau; NovaRx: Employment, Equity Ownership.

Bromelain Treatment Leads to Maturation of Monocyte‐derived Dendritic Cells but Cannot Replace PGE2 in a Cocktail of IL‐1β, IL‐6, TNF‐α and PGE2

Immunotherapy using dendritic cells (DC) has shown promising results. However, the use of an appropriate DC population is critical for the outcome of this treatment, and the search for an optimal DC subset is still ongoing. The DC used in immunotherapy today are usually matured with a cytokine cocktail consisting of TNF-α, IL-1β, IL-6 and PGE(2). These cells have deficits in their cytokine production, particularly IL-12p70, mainly because of the presence of PGE(2). Bromelain is a pineapple stem extract containing a mixture of proteases that has been used clinically in adjuvant cancer treatment. In this study, we analysed the effect of bromelain on human monocyte-derived DC. We added bromelain to the cytokine cocktail and modified cytokine cocktails with either no PGE(2) or reduced amounts of PGE(2), respectively. Combining bromelain with the cytokine cocktails containing PGE(2) resulted in an increased surface expression of CD83, CD80 and CD86. The chemokine receptor CCR7 was also considerably upregulated in these DC populations compared with DC treated with the cytokine cocktail alone. Removal or reduction of PGE(2) from the cytokine cocktail did not increase the IL-12p70 secretion from stimulated DC, and addition of bromelain to the different cytokine cocktails resulted in only a minor increase in IL-12p70 production. Moreover, combining bromelain with the cytokine cocktails did not improve the T cell stimulatory capacity of the generated DC populations. In conclusion, bromelain treatment of monocyte-derived DC does not improve the functional quality compared with the standard cytokine cocktail.

Low Dose IGFBP1, IGFBP2 and ANGPTL3 Coordinately Stimulate Ex Vivo Expansion of Human Umbilical Cord Blood (UCB) Hematopoietic Stem Cells (HSCs).

AbstractAbstract 1546Ex vivo expansion of umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may overcome the obstacle of low cell dose for UCB transplantation in adults. Insulin like growth factors (IGFs), IGF binding proteins (IGFBPs) and angiopoietin like proteins (ANGPTLs) can further enhance the ex vivo expansion of HSCs when used with a standard cytokine cocktail of stem cell factor (SCF), thrombopoietin (TPO) and FLT3-ligand (FL). Current doses of IGFBPs and ANGPTLs are in the range of 100∼500ng/ml, but these concentrations may not be optimal and high concentrations could be costly for clinical use. In order to determine the optimal dosage of IGFs, IGFBPs and ANGPTLs, 4×105cells/mL of cryopreserved clinical UCB was inoculated in serum-free Stemspan® medium supplied with standard basal cytokine combination of 100ng/ml SCF, 50ng/ml FL and 100ng/ml TPO on an MSCs stromal layer and with individually varied doses of IGFBP1, IGFBP2, IGF2 and ANGPTL3 in the range of 0∼200ng/ml. In order to determine optimal cytokine combination, complete permutation was carried out after establishing the optimal dosage of each cytokine. On day 7, the same amount of Stemspan® medium with the indicated cytokine combination was replenished to the culture system. Cord blood cells were harvested after 12 days ex vivo culture and assayed for total cell count, cell surface phenotype (viability determined by CD45/AnnV/7AAD staining, primitive progenitor determined by CD45/CD34/CD38/CD90 staining) and functional studies (Colony-forming unit-granulocyte and macrophage (CFU-GM) was determined by methylcellulose colony culture). Paradoxically, the highest expansion of CD34+CD38-CD90+ primitive progenitor was at a low dose of 20ng/ml for IGFBP1, IGFBP2, IGF2 and ANGPTL3 when concentrations of 0, 20, 50, 100 and 200 ng/ml were studied (Fig. 1A). Based on this results the cytokine dosage range was narrowed down to 0∼50ng/ml and experiments (Fig. 1B) showed that the optimal cytokines dosages were 20ng/ml of IGFBP2 and ANGPTL3, 15ng/ml IGFBP1 and 10ng/ml IGF2, which could stimulate 13.0±1.1 fold, 13.3±2.4 fold, 11.0±0.8 fold and 14.3±2.1 fold expansion of CD34+CD38-CD90+ primitive progenitor compared to 6.8±0.2 fold with standard cytokine control (p =0.01). Studying multiple permutations, combination “ABD” comprising 15ng/ml IGFBP1, 20ng/ml IGFBP2 and 20ng/ml ANGPTL3 had the highest expansion of CD34+CD38-CD90+ primitive progenitor (27.7±2.2 fold compared to 8.5±1.1 fold with standard cytokines, p =0.01), was found to be superior to all other combinations, including combinations “A”, “B”, “BC”, “BD”, “CD” and “ABCD”, which could stimulate over 2 fold expansion of primitive progenitor compare to control (Figure 1C). Interestingly, despite expansion of primitive CD34+CD38-CD90+ cells, there was no further enhancement of the expansion of total cells and general progenitors compare to control (data not shown), suggesting that the cyokine cocktail enhanced only the earliest progenitors. In conclusion, IGFBP1, IGFBP2, IGF2 and ANGPTL3 can stimulate the expansion of CD34+CD38-CD90+ primitive progenitor at low dosage, and the optimal combination comprises IGFBP1, IGFBP2 and ANGPTL3. Further in vivo experimentation is in progress to verify the effect of our optimized cytokine combination culture system on ex vivo expansion of cryopreserved unselected clinical UCB HSCs. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Improved activation of specific T-lymphocyte responses to the human telomerase reverse transcriptase (hTERT) in patients with advanced non-small cell lung cancer (NSCLC).

e13114 Background: hTERT is expressed in 93% to 100% of NSCLC, making the enzyme a target for immunotherapy. Present vaccination studies show a survival advantage in patients with a hTERT-specific immune response (Kosmatopoulos et al., 2007), but the number of immune responders is still limited. In the current study we tried to improve the activation of hTERT- specific T-cell responses in NSCLC-patients of different HLA-types who simultaneously received platinum-based standard first-line chemotherapy. Methods: After depletion of regulatory T-cells the lymphocytes were stimulated in vitro with mature peptide loaded dendritic cells (DC). The DC were either matured with the standard cytokine cocktail consisting of GM-CSF, IL-4, IL-6, IL-1β, TNF-α, and PGE2 or with a TLR7/8- in combination with a CD40-ligand. The activation of hTERT-specific T-cells was measured by flow cytometric analysis of intracellular IFN-γ. Results: In 2 patients the hTERT-specific T-cell response was more intensively activated by cytokine matured DC than by TLR7/8- and CD40-L matured DC. Using this standard approach 0.1-0.6% IFNg+ CD8+ T-cells cells were activated. In contrast in 3 other patients hTERT-specific T cells could be more effectively activated by TLR7/8- and CD40-L matured DC. We could activate between 0.1% and 2.1% IFN+CD8+ T-cells. Similarly in one healthy donor the hTERT-specific T-cell response was more intensively activated by cytokine matured DC showing up to 0.5% IFNg+ CD8+ T- cells. However, in another healthy donor hTERT- specific T-cell responses could be more effectively activated by TLR7/8- and CD40-L with up to 0.7 % IFNg+CD8+ cells. In 2 other patients we could not activate any hTERT-specific IFNg+ T-cell response. Conclusions: These data indicate that by using different DC maturation protocols hTERT-specific T-cells can be activated in the majority of patients. Even concomitant to standard chemotherapy clinical vaccination protocols may be feasible. However, DC maturation protocols may need to be individualized. The very low percentage of hTERT-specific T-cells in some patients implicate the need for even more potent immune adjuvants. No significant financial relationships to disclose.

ERK/p38 MAP‐kinases and PI3K are involved in the differential regulation of B7‐H1 expression in DC subsets

Regulatory molecules of the B7-H-family expressed by DC are important for immune homeostasis, but their regulation is largely unknown. When investigating the pathways regulating B7-H1 expression in monocyte-derived DC (MoDC), freshly isolated myeloid DC (mDC) and plasmacytoid DC, respectively, we showed that in MoDC and mDC B7-H1 expression was upregulated by a standard cytokine cocktail, poly I:C or LPS. MoDC utilize ERK and PI3K pathways to upregulate B7-H1 in response to cytokines, whereas p38 kinase was predominantly utilized in response to poly I:C. In mDC, ERK and p38 pathways are involved in B7-H1 regulation, and similar to MoDC, mainly p38 signaling was required for poly I:C-induced expression of B7-H1. Plasmacytoid DC responded only to CpG with upregulation of B7-H1 and in addition to p38 also utilized the PI3K and ERK pathways to regulate B7-H1 expression. As a functional consequence of B7-H1 expression on DC, we demonstrate downmodulation of IFN-gamma production in T cells. Thus, the differential regulation of B7-H1 on DC subsets may suppress immune responses variably, depending on the target DC population. Further analysis of the regulatory mechanisms may facilitate the development of new immunosuppressive therapies, utilizing the regulation of B7-H1 expression on DC.

Effect of Ex Vivo Culture of CD34 + Bone Marrow Cells on Immune Reconstitution of XSCID Dogs Following Allogeneic Bone Marrow Transplantation

Successful genetic treatment of most primary immunodeficiencies or hematological disorders will require the transduction of pluripotent, self-renewing hematopoietic stem cells (HSC) rather than their progeny to achieve enduring production of genetically corrected cells and durable immune reconstitution. Current ex vivo transduction protocols require manipulation of HSC by culture in cytokines for various lengths of time depending upon the retroviral vector that may force HSC to enter pathways of proliferation, and possibly differentiation, which could limit their engraftment potential, pluripotentiality and long-term repopulating capacity. We have compared the ability of normal CD34(+) cells cultured in a standard cytokine cocktail for 18hours or 4.5 days to reconstitute XSCID dogs following bone marrow transplantation in the absence of any pretransplant conditioning with that of freshly isolated CD34(+) cells. CD34(+) cells cultured under standard gamma-retroviral transduction conditions (4.5 days) showed decreased engraftment potential and ability to sustain long-term thymopoiesis. In contrast, XSCID dogs transplanted with CD34(+) cells cultured for 18hours showed a robust T cell immune reconstitution similar to dogs transplanted with freshly isolated CD34(+) cells, however, the ability to sustain long-term thymopoiesis was impaired. These results emphasize the need to determine ex vivo culture conditions that maintain both the engraftment potential and "stem cell" potential of the cultured cells.

Mesenchymal Stromal Cell Co-Culture Enhances Insulin-Like Growth Factor Binding Protein 2 - Augmented Ex Vivo Cultures of Human Cord Blood in a Viability Supporting, Contact-Dependent Process Involving the Rescue of Cells from Early Apoptosis

The use of cord blood (CB) for adult transplantation is limited by the number of hematopoietic stem (HSC) and progenitor cells (HPC) residing in the CB. Successful ex vivo expansion of HSC and HPC may overcome this limitation. An ex vivo expansion system (Zhang et. al., 2008, Blood) based on the additional of Angiopoietin-like 5 and/or Insulin-like Growth Factor Binding Protein 2 (IGFBP2) to a standard cytokine cocktail of stem cell factor, thrombopoietin and FLT3-ligand has recently been reported to expand CB HSC by ~20-fold in a SCID-repopulating cell (SRC) assay. In this study, we further investigated if co-culture of the CB cells with mesenchymal stromal cells (MSC) would enhance the ex vivo expansion of CB in IGFBP2-augmented cultures. Using thawed unselected CB cells, IGFBP2-augmented cultures were able to expand CD45(+)/CD34(+) cells 49-fold and retain in vitro colony-forming units granulocyte-macrophage (CFU-GM) colony size with a concomitant 15-fold increase in number. Under the same conditions, co-culture with MSC resulted in a further 2.9-fold enhancement of CD45(+)/CD34(+) cell expansion (144-fold expansion) and a 2.2-fold enhancement of CFU-GM expansion (33- fold expansion). The numbers reflect true expansion of cell numbers as no prior CD34/ CD133 selection was employed. We further studied the mechanism of MSC enhancement of HSC and HPC expansion. Rather than an increase in cellular proliferation rate, the MSC enhancement effect was associated with 2- to 5-fold increase in viability of a population of lymphocyte-like cells [FS(low) SS(low) CD45(+)] in CB, as determined by annexin-V/7AAD staining. Moreover, direct contact with MSC was necessary for optimal expansion and viability support as shown by use of conditioned media and transwell assays. Thawed mononucleated CB cells exhibited a high level of positivity for Annexin-V, which translated to subsequent 7AAD positivity during ex vivo expansion. MSC co-culture resulted in reversion of Annexin-V positivity within 3–6 hours, and reached a maximum on day 3 (80–90% viability with MSC versus 40–60% without, as defined by double negative staining for Annexin-V and 7AAD). Concomitantly, the loss of mitochondrial membrane potential as determined by JC-1 was also reduced by MSC co-culture. There was a reduction of Caspase 9 activity (12.7% with MSC versus 23.8% without) at day 3 and a reduction in Caspase 3 and 7 activity (5.5% with MSC versus 15.5% without) at day 7 with MSC co-culture. In further experiments, an increase of lysosomal activity in CB, as determined by acridine orange staining, was found to be reduced in MSC co-culture. The use of a non-viable MSC layer did not support CB viability and subsequent expansion. We hypothesized that cytosolic transfer of nutrient or other survival factors from MSC to CB could be a potential mechanism responsible for the rescue of thawed CB from loss of viability and subsequent enhancement in CB ex vivo expansion. Using genetically modified stromal cells expressing with the fluorescent protein containing mitochondria targeting sequence (DsRed-mito) and time-lapse (>10 hours) confocal imaging, we observed potential cytosolic transfer activities between the stromal cells. In conclusion, IGFBP2-augmented cultures result in successful CB expansion, which is further enhanced by MSC co-culture in a viability supporting, contact-dependent process that could involve reversal of cells from early apoptosis possibly mediated through cytosolic transfer of nutrient or cellular material. Further in vivo experimentation is in progress to verify the effect of our combined MSC-IGFBP2 culture system on HSC expansion.

Generation of functionally mature dendritic cells from elutriated monocytes using polyinosinic : polycytidylic acid and soluble CD40 ligand for clinical application

Despite the increasing use of dendritic cell (DC) vaccination in clinical trials, optimal conditions for the generation of functionally mature DCs remain to be established. The current standard DC maturation protocol for clinical trials has been used as an inflammatory cytokine cocktail [tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and prostaglandin E(2)], but this cocktail induced insufficient maturation of DCs derived from elutriated monocytes when cultured in X-VIVO 15. The aim of this study was to define effective combinations of stimulators for generating functionally mature DCs from elutriated monocytes under current good manufacturing practice conditions. We compared the functional capacity of DCs in response to all possible pairwise combinations of four different classes of stimuli: TNF-alpha, peptidoglycan, polyinosinic : polycytidylic acid [poly(I:C)] and soluble CD40 ligand (CD40L). Maturation status of DCs stimulated with combination of four stimuli was similar to that of the cytokine cocktail as assessed by the cell surface phenotype. However, only the combination of poly(I:C) + CD40L induced complete functional activation of the whole DC population, assessing IL-12p70 production, allostimulatory activity, migratory response to CCL19 and T helper 1-polarizing capacity. Thus, the protocol based on the combination of poly(I:C) and CD40L is more effective for the induction of clinical-grade DCs from elutriated monocytes than the standard cytokine cocktail.

Generation of alpha-Type-1 Polarized Dendritic Cells as a Potent Immunogen in Patients with Chronic Lymphocytic Leukemia.

We tested the feasibility of generating alpha-type-1 polarized DC (αDC1) from the blood of chronic lymphocytic leukemia (CLL) patients and the in vitro effectiveness of autologous tumor-loaded αDC1s in inducing CTLs against CLL. CD14+ cells were isolated from the peripheral blood of CLL patients and were cultured in rhu GM-CSF and IL-4. On day 6, DC maturation was induced using either a standard cytokine cocktail (in IL-1β, TNFα, IL-6, and PGE2) or αDC1-polarizing cocktail (in IL-1β, TNFα, IFNα, IFNγ, and poly-I:C). In either case, the maturing DCs were loaded with gamma-irradiated autologous CLL cells. αDC1s from CLL patients expressed substantially higher levels of several costimulatory molecules (CD83, CD86, CD80, CD11c and CD40) than standard DCs (sDCs) while CCR7 showed lower expression. Gamma-irradiated CLL cells were more efficienly taken up by DCs compared to UVC-irradiated CLL cells, but the ability of tumor antigen uptake was similar between αDC1 and sDCs. The capacity of IL-12p70 secretion in DCs (baseline and after stimulation with CD40L-transfected J558 cells) was significantly elevated in αDC1s (10–60 times) compared to sDCs. αDC1s also induced significantly higher numbers of functional CD8+ T cells against CLL cells, as determined by IFN-γ ELISPOT, using anti-MHC class I blocking mAb (W6/32) to verify the specificity of CTL responses. Our data demonstrate that αDC1s are a potent alternative to standard DCs as inducers of T cells for adoptive immunotherapy in patients with CLL and a clinical trial will be initiated.

Parvovirus H-1-Induced Tumor Cell Death Enhances Human Immune ResponseIn Vitrovia Increased Phagocytosis, Maturation, and Cross-Presentation by Dendritic Cells

Oncotropic and oncolytic viruses have attracted high attention as antitumor agents because they preferentially kill cancer cells in vitro and reduce the incidence of spontaneous, induced, or implanted animal tumors. Some autonomous parvoviruses (H-1, minute virus of mice) and derived recombinant vectors are currently under preclinical evaluation. Still not fully understood, their antitumor properties involve more than just tumor cell killing. Because wild-type parvovirus-mediated tumor cell lysates (TCLs) may trigger antigen-presenting cells (APCs) to augment the host immune repertoire, we analyzed phagocytosis, maturation, and crosspresentation of H-1-induced TCLs by human dendritic cells (DCs). We first established H-1-mediated oncolysis in two HLA-A2(+) and A2(-) variant melanoma cell clones. Monocyte-derived immature DCs phagocytosed H- 1-infected TCLs as well as ultraviolet-induced apoptotic TCLs and better than freeze-thaw-induced necrotic TCLs. Immature DCs incubated with H-1-induced TCLs acquired specific maturation markers comparable to a standard cytokine cocktail. Furthermore, A2(+) DCs pulsed with H-1-infected A2(-) TCLs cross-presented melanoma antigens to specific cytotoxic T lymphocytes (CTLs) and released proinflammatory cytokines. This shows for the first time that tumor cell killing by a wild-type oncolytic virus directly stimulates human APCs and CTLs. Because H-1-infected tumors enhance the immune repertoire, the clinical perspectives of parvoviral vectors are even more promising.