Interterleukin-6 (IL-6) Produced by Monocyte Activation Reduces Dendritic Cell (DC) Differentiation in Patients with Multiple Myeloma (MM) and Malignant Lymphoma (ML): Role of CNTO 328, an Anti-IL-6 Monoclonal Antibody (Mab) and Possible Clinical Applications.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2662-2662
Author(s):  
Jean-Francois Rossi ◽  
Anne-Marie Conge ◽  
Catherine Barjot ◽  
Mohamed H. Zaki ◽  
Marian T. Nakada ◽  
...  
Keyword(s):  
Large Scale ◽  
Ex Vivo ◽  
Immune Therapy ◽  
Human Albumin ◽  
Autologous Tumor ◽  
Vaccination Programs ◽  
Gm Csf ◽  
Free Process ◽  
Tnf Α ◽  
Dc Maturation

Abstract We developed a serum-free process in a closed system using culture cassettes and bags for large-scale and clinical-grade DC vaccination, accepted by the “Afssaps-French drug Agency” (Tarte K. et al. Leukemia2000; 14:2152 & patent). Intermediate mature DCs are generated from mononucleated cells obtained by mobilized leukapheresis, followed by Mo selection using adherence in specific cassettes (CLINIcell, Mabiol). Non-adherent cells are removed and Mo are cultured for 5 days (D) in X-VIVO15 medium (Cambrex) with 2% of human albumin, 100ng/ml of GM-CSF (Leukine, Berlex) and 25 ng/ml of IL-4 (CellGenix-Cellgen). At D5, immature DCs are harvested, pulsed with autologous tumor lysate (or peptides) for 4 h in X-VIVO15 medium + GM-CSF (100ng/ml) and maturation factors (TNF-α: 20ng/ml, CellGenix-CellGen, and PGE2: 100ng/ml; Prostine, Pharmacia). Maturation of DCs was allowed to proceed for 20 h with TNF-α and PGE2. Mo-conditioned media, or IL-6 as well as IL-1 are used for enhancing ex vivo DC maturation by different groups in spite of the fact that IL-6 has been described as a blocker of DC differentiation from CD34+ cells particularly in MM. We demonstrated that in our process, IL-6 is produced by activated Mo during their selection (mean= 378pg/mL, range 37–1219). The amount of the IL-6 released in the medium correlated with the % of CD14+ cells obtained at D5 (CD14<2.8%: mean IL-6=73.1 pg/mL; CD14>22.6%: mean IL-6=682.9 pg/mL), indicating that the intrinsic production of IL-6 is one major parameter of variability of the cellular product. By adding IL-6 from D1 to D5, the percentage of CD14+ cells at D5 was enhanced by a mean of 23-fold in samples from patients with MM (n=7) and 17-fold in ML (n=7). The modifications of other DCs markers including CD1a, CD 84 and CCR7 were modest. By using CNTO 328, an anti-IL-6 MAb (Centocor Inc) at 1–10μg/mL, we totally blocked the activity of added IL-6 and samples with high IL-6 intrinsic production, with a reduction of CD14+ cells at D5. In contrast, neither IL-6 nor CNTO 328 had any activity on terminal DC maturation after D5. IL-6 and CNTO328 are tested on DC functions. This means that in B-cell malignancies and other solid tumors with high levels of circulating IL-6: 1) anti-IL-6 treatment such as CNTO 328 may be associated with active immune therapy, including vaccinations; 2) mature and intermediate mature DCs are the only cells to be administered in vaccination programs because of a de-differentiation effect of immature DCs due to IL-6; 3) anti-IL-6 MAbs, particularly CNTO 328 could be added for ex vivo DC differentiation, instead of IL-6. mean % (range) of CD14+ cells at Day5 samples MM ML Control 2.9 (0.1–7.1) 12.2 (0–44.8) IL-6 (100ng/mL) 20 (6–35) 34.2 (0–71.4) IL-6+CNTO328 1μg/mL 2.8 (0.5–7.5) 15.7 (0–45.6) IL-6+CNTO328 10μg/mL 0.4 (0–0.8) 6.8 (0–20.3) CNTO328 1μg/mL 0.4 (0.1–0.7) 6.5 (0–19.5) CNTO328 10μg/mL 0.2 (0–0.4) 5.3 (0–15.3)

scholarly journals Defining the inflammatory signature of human lung explant tissue in the presence and absence of glucocorticoid

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 460 ◽  
Author(s):  
Tracy L Rimington ◽  
Emily Hodge ◽  
Charlotte K Billington ◽  
Sangita Bhaker ◽  
Binaya K C ◽  
...  
Keyword(s):  
Respiratory Diseases ◽  
Human Lung ◽  
Inflammatory Responses ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
Chronic Obstructive ◽  
Interleukin 1 Beta ◽  
Gm Csf ◽  
Tnf Α ◽  
Anti Inflammatory

Background: Airway inflammation is a feature of many respiratory diseases and there is a need for newer, more effective anti-inflammatory compounds. The aim of this study was to develop an ex vivo human lung explant model which can be used to help study the mechanisms underlying inflammatory responses and which can provide a tool to aid drug discovery for inflammatory respiratory diseases such as asthma and COPD. Method: Parenchymal lung tissue from 6 individual donors was dissected and cultured with two pro-inflammatory stimuli, lipopolysaccharide (LPS) (1 µg/ml) and interleukin-1 beta (IL-1β) (10 ng/ml) in the presence or absence of dexamethasone (1 µM).  Inflammatory responses were assessed using Luminex analysis of tissue culture supernatants to measure levels of 21 chemokines, growth factors and cytokines. Results: A robust and reproducible inflammatory signal was detected across all donors for 12 of the analytes measured following LPS stimulation with a modest fold increase (<2-fold) in levels of CCL22, IL-4, and IL-2; increases of 2-4-fold in levels of CXCL8, VEGF and IL-6 and increases >4-fold in CCL3, CCL4, GM-CSF, IL-10, TNF-α and IL-1β.  The inflammatory signal induced by IL-1β stimulation was less than that observed with LPS but resulted in elevated levels of 7 analytes (CXCL8, CCL3, CCL4, GM-CSF, IL-6, IL-10 and TNF-α).  The inflammatory responses induced by both stimulations was supressed by dexamethasone for the majority of analytes. Conclusions: These data provide proof of concept that this ex vivo human lung explant model is responsive to inflammatory signals and could be used to investigate the anti-inflammatory effects of existing and novel compounds.  In addition this model could be used to help define the mechanisms and pathways involved in development of inflammatory airway disease. Abbreviations: COPD: Chronic Obstructive Pulmonary Disease; ICS: inhaled corticosteroids; LPS: lipopolysaccharide; IL-1β: interleukin-1 beta; PSF: penicillin, streptomycin and fungizone

A novel cancer vaccine platform utilizing autologous tumor lysate loaded into capped yeast cell wall particles.

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 169-169
Author(s):  
Kaitlin M. Peace ◽  
Timothy J Vreeland ◽  
Diane F Hale ◽  
Doreen O Jackson ◽  
Julia M Greene ◽  
...  
Keyword(s):  
Cell Wall ◽  
Yeast Cell ◽  
Ex Vivo ◽  
Yeast Cell Wall ◽  
Autologous Tumor ◽  
Tumor Lysate ◽  
Vaccine Platform ◽  
Gm Csf ◽  
Murine Melanoma

169 Background: Our current tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine employs yeast cell wall particles (YCWP) to deliver antigen to DC ex vivo and is being tested in a randomized phase IIb trial. This strategy may be improved by injecting TL-loaded YCWP as an intradermal vaccine, with in vivo DC uptake. Silicate capping should allow YCWP to retain TL longer and stimulate DC uptake. Here, we present preclinical data on the tumor lysate, particle only (TLPO) vaccine concept vs. TLPLDC. Methods: To test the TLPO concept, YCWP were loaded with fluorescence (flr) labeled albumin and capped (cp) vs. uncapped (ucp) particles were compared for flr leak. Next, YCWP were added to cultured macrophages to evaluate DC uptake. Cells were then lysed, centrifuged, and flr in the cytoplasm vs. organelles measured. 3 C57B mice were then injected with 100ml NaCl, 106empty cp YCWP, or 1mcg GM-CSF. 5 hours (hr) post-injection, 100ml NaCl was injected into the same site, withdrawn, and examined via microscope to count monocytes. TLPO was compared to TLPLDC in a B16 murine melanoma survival model. Finally, 4 grey horses with equine melanoma were treated with autologous TLPO, injected biweekly for 4 vaccinations. Target lesions were assessed over 6 months. Results: Compared to ucp YCWP, cp had decreased flr leak at 1 (15.8% vs. 24.7%) and 2 hr (6.7% vs. 16.6%), increased uptake by DC (2hr flr readings 11065 vs. 3928) and higher delivery to DC cytoplasm (68.9% vs. 48.8%). Empty cp YCWP showed increased recruitment of monocytes (276/hpf) vs. GM-CSF (55/hpf) or NaCl alone (18/hpf). In the B16 murine melanoma model, the median survival time in days was 21 for controls (n=5), 42 for TLPLDC (n=10), and 56 for TLPO (n=20). At 6 months, the equine model revealed 1 complete and 3 partial responses (50%, 68%, and 45% tumor reduction). Conclusions: Silicate capping of the YCWP effectively limits leakage of contents, improves uptake by DC and delivery to the cytoplasm without the need for GM-CSF. In early animal studies, TLPO appears to have equivalent efficacy to TLPLDC, but eliminates the need for in vitro DC loading. We plan to perform a bridging study comparing TLPO to TLPLDC after enrollment is complete in our phase IIb TLPLDC melanoma trial.

scholarly journals An Inducible Leukemia-Associated Transcription Factor Facilitates Large-Scale Ex Vivo Generation of Functional Human Macrophages

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2805-2805
Author(s):  
Roland Windisch ◽  
Sarah Soliman ◽  
Adrian Hoffmann ◽  
Linping Chen-Wichmann ◽  
Sebastian Lutz ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Board Of Directors ◽  
Blood Cells ◽  
Large Scale ◽  
Ex Vivo ◽  
Advisory Committees ◽  
Gm Csf ◽  
Human Cd34 ◽  
Protein Switch

Abstract Long-term ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells (HSPCs) proves to be unfeasible as cellular differentiation occurs when HSPCs are detached from their supporting bone marrow stem cell niche. This issue renders it difficult to make use of the proliferation capacity of HSPCs to subsequently produce functional blood cells in relevant numbers, e.g. for cell therapy approaches. To circumvent this challenge, leukemia-associated chimeric transcription factors, including MLL fusion proteins, can be exploited for their pronounced ability to propel cell proliferation while preserving cell immaturity. By designing the protein's activity controllable, the immature state can be abolished at an arbitrary point in time enabling terminal differentiation. In this study, we employed the fusion gene mixed lineage leukemia/eleven nineteen leukemia (MLL-ENL) for engineering an inducible protein switch. For this purpose, we fused the coding sequence of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the transcription factor MLL-ENL and subsequently expressed the protein switch (DD-MLL-ENL) in human CD34+ HSPCs derived from adult healthy donors. In the presence of the specific ligand Shield1, DD-mediated protein degradation is prevented leading to massive and long-term expansion of HSPC-derived late monocytic precursors in the presence of IL-3, IL-6, SCF, FLT3-L, TPO and GM-CSF. The cells do not exhibit additional driver mutations, feature a normal karyotype and telomere length, and sustain immaturity that is strictly dependent on Shield1 supplementation every other day even after two years of ex vivo culture. Upon Shield1 deprivation, the cells completely lost self-renewal and colony-forming properties and spontaneously differentiated. By changing the cytokines to GM-CSF in combination with IFN-γ and LPS we differentiated the progenitor cells into macrophages (MΦ) (Fig. 1 A, B). Immunophenotypic characterization revealed upregulation of the monocyte/macrophage-associated surface markers CD14, CD80, CD86, CD163 and MHC class I and II, concordant with monocytic morphology as judged by cytospin preparations. Analysis of the transcription of selected inflammatory genes, including IL-6 and IL-10, revealed overlapping M1 and M2 macrophage characteristics. Furthermore, mRNA expression profiles using nCounter Systems technology covering a total of 770 myeloid innate immunity-related genes proves the cells' identity as differentiated phagocytes shown by upregulation of gene clusters involved in Fc receptor signaling, TLR signaling, antigen presentation and T cell activation. In functional assays, we demonstrated the ability of the obtained cells to migrate towards the chemokine CCL2 in a 3D chemotaxis assay, attach to VCAM-1 under flow and shear stress and produce reactive oxygen species. Regarding the cells' phagocytic capability, we could verify the uptake of bacterial particles as well as apoptotic cells in efferocytosis assays. Finally, we demonstrated IgG Fc region recognition and binding by the expressed Fcγ receptors enabling phagocytosis of lymphoblastic tumor cells, including Daudi, Raji and patient-derived MCL cells in an antibody-dependent manner using rituximab (RTX), daratumumab (Dara) and trastuzumab (Trast) as a negative control (Fig. 1C). Overall, we could demonstrate the conversion of a harmful leukemic transcription factor into a useful molecular tool for large-scale ex vivo production of functional blood cells. Such engineered controllable protein switches might have the potential to be employed as molecular tools to produce functional immune cells for cell-based immunotherapeutic approaches. Figure 1 Figure 1. Disclosures Redondo Monte: Minaris Regenerative Medicine: Current Employment. Beier: Alexion: Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Other: Travel reembursement. Weigert: Janssen: Speakers Bureau; Epizyme: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding. Greif: AstraZeneca: Honoraria.

scholarly journals Ex Vivo Cytokine Release, Determined by a Multiplex Cytokine Assay, in Response to Coccidioidal Antigen Stimulation of Whole Blood among Subjects with Recently Diagnosed Primary Pulmonary Coccidioidomycosis

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Neil M. Ampel ◽  
Ian Robey ◽  
Chinh T. Nguyen ◽  
Brentin Roller ◽  
Jessica August ◽  
...  
Keyword(s):  
Antifungal Therapy ◽  
Whole Blood ◽  
Ex Vivo ◽  
Valley Fever ◽  
Blood Samples ◽  
Antigen Stimulation ◽  
Gm Csf ◽  
Tnf Α ◽  
Inflammatory Proteins ◽  
Ifn Γ

ABSTRACT The elements of the cellular immune response in human coccidioidomycosis remain undefined. We examined the ex vivo release of an array of inflammatory proteins in response to incubation with a coccidioidal antigen preparation to ascertain which of these might be associated with diagnosis and outcome. Patients with a recent diagnosis of primary pulmonary coccidioidomycosis and a control group of healthy subjects were studied. Blood samples were incubated for 18 h with T27K, a soluble coccidioidal preparation containing multiple glycosylated antigens, and the supernatant was assayed for inflammatory proteins using the multiplex Luminex system. The presentation and course of illness were compared to the levels of the inflammatory proteins. Among the 31 subjects studied, the median time from diagnosis to assay was 15 days. Of the 30 inflammatory proteins measured, the levels of only 7 proteins, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 receptor alpha (IL-1RA), interleukin-1β (IL-1β), interferon gamma (IFN-γ), IL-2, IL-13, and tumor necrosis factor alpha (TNF-α), were more than 10-fold above the levels seen without antigen stimulation. The levels of IFN-γ and IL-2 were significantly elevated in those subjects not receiving triazole antifungal therapy compared to those who were receiving triazole antifungal therapy. While the levels of IL-1RA were nonspecifically elevated, elevated levels of IL-13 were seen only in those with active pulmonary coccidioidomycosis. Only six cytokines were specifically increased in subjects with recently diagnosed primary pulmonary coccidioidomycosis. While IFN-γ, IL-2, and TNF-α have been previously noted, the finding of elevated levels of the innate cytokines GM-CSF and IL-1β could suggest that these, as well as IL-13, are early and specific markers for pulmonary coccidioidomycosis. IMPORTANCE Coccidioidomycosis, commonly known as Valley fever, is a common pneumonia in the southwestern United States. In this paper, we examined the release of 30 inflammatory proteins in whole-blood samples obtained from persons with coccidioidal pneumonia after the blood samples were incubated with a preparation made from the causative fungus, Coccidioides . We found that six of these proteins, all cytokines, were specifically released in high concentrations in these patients. Three of the cytokines were seen very early in disease, and an assay for all six might serve as a marker for the early diagnosis of Valley fever.

Role of Human NK Cells in Dentric-Cell Based Vaccine.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1550-1550
Author(s):  
Gerard M.J. Bos ◽  
Janine CHMJ Van Elssen ◽  
Joris Vanderlocht ◽  
Brigitte LMG Senden-Gijsbers ◽  
Wilfred LMG Germeraad
Keyword(s):  
Nk Cells ◽  
Human Study ◽  
Dependent Manner ◽  
Gm Csf ◽  
Tnf Α ◽  
Toll Like Receptor 2 ◽  
Ifn Γ ◽  
Th1 Polarization ◽  
Dc Maturation

Abstract Figure Figure Besides their prominent role in the destruction of altered self-cells, natural killer (NK) cells have been shown to potentiate T cell responses by interacting with dendritic cells (DC). In mouse models as well as in a recent human study it has been demonstrated that DC might activate NK cells. In the context of dendritic cell-based vaccines – i.e. optimising the optimal maturation cocktail - it remains to be determined if and how NK-DC interactions depend on differential DC maturation and what factors influence the NK activation.. By comparing differential DC differentiation (IL-4/GM-CSF and IL-13/GMCSF) and maturation cocktails (IFN-γ/FMKp and PGE2/TNF-α), we show that the ability of human DCs to attract NK cells is imprinted during DC maturation. Only FMKP/IFN-γ (stimulation Toll like receptor 2 and 4) maturated DCs have the capacity to actively recruit NK cells in vitro and our data indicate that CCR5 is the dominant chemokine receptor in this recruitment (Figure 1). Furthermore, in contrast to PGE2/TNF-α matured DC, FMKP/IFN-γ maturated DCs activate NK cells to produce IFN-γ in a IL-12/IL18 dependent manner, of which we show it contributes to strong TH1 polarization. In addition upon contact with these DCs NK cells upregulate their lymph node homing receptors, possibly inducing secondary migration to the lymph nodes. In conclusion, besides the identification of a superior DC maturation cocktail which contributes to NK-DC interactions, we identified a novel recruitment mechanism for peripheral human NK cells which may contribute to secondary, central DC-NK interactions and strong TH1 polarization.

scholarly journals Ex vivo expansion of enriched peripheral blood CD34+ progenitor cells by stem cell factor, interleukin-1 beta (IL-1 beta), IL-6, IL-3, interferon-gamma, and erythropoietin

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2579-2584 ◽  
Author(s):  
W Brugger ◽  
W Mocklin ◽  
S Heimfeld ◽  
RJ Berenson ◽  
R Mertelsmann ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Interferon Gamma ◽  
Large Scale ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
High Dose Chemotherapy ◽  
High Dose ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Factor Combination

Abstract To provide sufficient numbers of peripheral blood progenitor cells (PBPCs) for repetitive use after high-dose chemotherapy, we investigated the ability of hematopoietic growth factor combinations to expand the number of clonogenic PBPCs ex vivo. Chemotherapy plus granulocyte colony-stimulating factor (G-CSF) mobilized CD34+ cells from 18 patients with metastatic solid tumors or refractory lymphomas were cultured for up to 28 days in a liquid culture system. The effects of interleukin-1 beta (IL-1), IL-3, IL-6, granulocyte-macrophage-CSF (GM-CSF), G-CSF, macrophage-CSF (M-CSF), stem cell factor (SCF), erythropoietin (EPO), leukemia inhibitory factor (LIF), and interferon- gamma, as well as 36 combinations of these factors were tested. A combination of five hematopoietic growth factors, including SCF, EPO, IL-1, IL-3, and IL-6, was identified as the optimal combination of growth factors for both the expansion of total nucleated cells as well as the expansion of clonogenic progenitor cells. Proliferation peaked at days 12 to 14, with a median 190-fold increase (range, 46- to 930- fold) of total clonogenic progenitor cells. Expanded progenitor cells generated myeloid (colony-forming unit-granulocyte-macrophage), erythroid (burst-forming unit-erythroid), as well as multilineage (colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte) colony-forming units. The number of multilineage colonies increased 250- fold (range, 33- to 589-fold) as compared with pre-expansion values. Moreover, the absolute number of early hematopoietic progenitor cells (CD34+/HLA-DR-; CD34+/CD38-), as well as the number of 4-HC-resistant progenitors within expanded cells increased significantly. Interferon- gamma was shown to synergize with the 5-factor combination, whereas the addition of GM-CSF significantly decreased the number of total clonogenic progenitor cells. Large-scale expansion of PB CD34+ cells (starting cell number, 1.5 x 10(6) CD34+ cells) in autologous plasma supplemented with the same 5-factor combination resulted in an equivalent expansion of progenitor cells as compared with the microculture system. In summary, our data indicate that chemotherapy plus G-CSF-mobilized PBPCs from cancer patients can be effectively expanded ex vivo. Moreover, our data suggest the feasibility of large- scale expansion of PBPCs, starting from small numbers of PB CD34+ cells. The number of cells expanded ex vivo might be sufficient for repetitive use after high-dose chemotherapy and might be candidate cells for therapeutic gene transfer.

scholarly journals Investigating the Early Events after Skin-Barrier Disruption Using Microdialysis—A Human Ex Vivo Skin Model

Dermato ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 47-58
Author(s):  
Katrine Baumann ◽  
Niels Peter Hell Knudsen ◽  
Anne-Sofie Østergaard Gadsbøll ◽  
Anders Woetmann ◽  
Per Stahl Skov
Keyword(s):  
Interstitial Fluid ◽  
Ex Vivo ◽  
Skin Barrier ◽  
Microdialysis Sampling ◽  
Sample Analysis ◽  
Skin Model ◽  
Barrier Disruption ◽  
Gm Csf ◽  
Tnf Α ◽  
Subsequent Sample

Skin-barrier restoration following abrasive trauma is facilitated by mediator release from skin-resident cells, a process that has been investigated primarily in mice or simplified human systems with previous studies focusing on a limited number of biomarkers. Here, we demonstrate how early events caused by skin-barrier disruption can be studied in a human ex vivo skin model. Ten relevant biomarkers were recovered from the interstitial fluid by skin microdialysis with subsequent sample analysis using a multiplex platform. As a control, the biomarker profiles obtained from microdialysis sampling were compared to profiles of skin biopsy homogenates. We found that nine (GM-CSF, CXCL1/GROα, CXCL8/IL-8 CXCL10/IP-10, IL-1α, IL-6, MIF, TNF-α, and VEGF) of the 10 biomarkers were significantly upregulated in response to abrasive trauma. Only dialysate levels of CCL27/CTACK were unaffected by skin abrasion. Biomarker levels in the homogenates corresponded to dialysate levels for CCL27/CTACK, CXCL1/GROα, CXCL8/IL-8, and IL-6. However, IL-1α showed an inverse trend in response to trauma, and biopsy levels of MIF were unchanged. GM-CSF, CXCL10/IP-10, TNF-α, and VEGF were not detected in the biopsy homogenates. Our results suggest that the human ex vivo skin model is a reliable approach to study early events after disruption of the skin barrier.

scholarly journals Ex vivo expansion of enriched peripheral blood CD34+ progenitor cells by stem cell factor, interleukin-1 beta (IL-1 beta), IL-6, IL-3, interferon-gamma, and erythropoietin

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2579-2584 ◽  
Author(s):  
W Brugger ◽  
W Mocklin ◽  
S Heimfeld ◽  
RJ Berenson ◽  
R Mertelsmann ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Interferon Gamma ◽  
Large Scale ◽  
Ex Vivo ◽  
Interleukin 1 ◽  
High Dose Chemotherapy ◽  
High Dose ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Factor Combination

To provide sufficient numbers of peripheral blood progenitor cells (PBPCs) for repetitive use after high-dose chemotherapy, we investigated the ability of hematopoietic growth factor combinations to expand the number of clonogenic PBPCs ex vivo. Chemotherapy plus granulocyte colony-stimulating factor (G-CSF) mobilized CD34+ cells from 18 patients with metastatic solid tumors or refractory lymphomas were cultured for up to 28 days in a liquid culture system. The effects of interleukin-1 beta (IL-1), IL-3, IL-6, granulocyte-macrophage-CSF (GM-CSF), G-CSF, macrophage-CSF (M-CSF), stem cell factor (SCF), erythropoietin (EPO), leukemia inhibitory factor (LIF), and interferon- gamma, as well as 36 combinations of these factors were tested. A combination of five hematopoietic growth factors, including SCF, EPO, IL-1, IL-3, and IL-6, was identified as the optimal combination of growth factors for both the expansion of total nucleated cells as well as the expansion of clonogenic progenitor cells. Proliferation peaked at days 12 to 14, with a median 190-fold increase (range, 46- to 930- fold) of total clonogenic progenitor cells. Expanded progenitor cells generated myeloid (colony-forming unit-granulocyte-macrophage), erythroid (burst-forming unit-erythroid), as well as multilineage (colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte) colony-forming units. The number of multilineage colonies increased 250- fold (range, 33- to 589-fold) as compared with pre-expansion values. Moreover, the absolute number of early hematopoietic progenitor cells (CD34+/HLA-DR-; CD34+/CD38-), as well as the number of 4-HC-resistant progenitors within expanded cells increased significantly. Interferon- gamma was shown to synergize with the 5-factor combination, whereas the addition of GM-CSF significantly decreased the number of total clonogenic progenitor cells. Large-scale expansion of PB CD34+ cells (starting cell number, 1.5 x 10(6) CD34+ cells) in autologous plasma supplemented with the same 5-factor combination resulted in an equivalent expansion of progenitor cells as compared with the microculture system. In summary, our data indicate that chemotherapy plus G-CSF-mobilized PBPCs from cancer patients can be effectively expanded ex vivo. Moreover, our data suggest the feasibility of large- scale expansion of PBPCs, starting from small numbers of PB CD34+ cells. The number of cells expanded ex vivo might be sufficient for repetitive use after high-dose chemotherapy and might be candidate cells for therapeutic gene transfer.

Deficient Th-1 Responses from TNFα-Matured Dendritic Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3839-3839
Author(s):  
William K. Decker ◽  
Sufang Li ◽  
Dongxia Xing ◽  
Simon N. Robinson ◽  
Hong Yang ◽  
...  
Keyword(s):  
Tumor Antigens ◽  
Myelogenous Leukemia ◽  
Active Immunotherapy ◽  
Normal Donor ◽  
Becton Dickinson ◽  
Cd8 Cells ◽  
Gm Csf ◽  
Tnf Α ◽  
Th 1 ◽  
Dc Maturation

Abstract INTRODUCTION: The ultimate success of dendritic cell (DC) vaccination for the active immunotherapy of neoplasia involves a wide variety of variables including the method of DC generation, loading, and maturation; the DC dose and route of administration. A maturation cocktail comprising IL-1b, TNF-a, IL-6, and PGE2 (ITIP) is commonly employed clinically, as is the use of the single inflammatory cytokine TNF-a. Here we compared these two methods with respect to DC maturation, phenotype, and function. METHODS: CD14+ monocytes were isolated from normal donor G-CSF-mobilized peripheral blood progenitor cells using the mini-MACS system (Miltenyi Biotec), and cultured for 6 days in AIM-V medium containing GM-CSF and IL-4 (Cellgenix). Immature DCs were loaded with both acute myelogenous leukemia (AML) tumor lysate and mRNA as previously described1 and then matured with the ITIP cocktail, TNF-α alone, or a combination of TNF-α and CD40 agonism (Becton-Dickinson). Mature DCs were analyzed phenotypically and then cultured with autologous T-cells. RESULTS: Data generated from three different normal donors indicated that DCs matured with TNF-α were deficient in important costimulatory surface molecules CD80, CD83, and CD40 as well as in IL-12 production (p=0.001 in representative experiment). Analysis of T-cell functionality by IFN-γ ELISpot also demonstrated greatly decreased Th-1 functionality among lymphocytes stimulated by TNFα-matured DCs in comparison to ITIP-matured DCs (p=0.004 in representative experiment). Additionally, lymphocytes stimulated by TNFα-matured DCs were not able to proliferate significantly whereas ITIP-matured DCs mediated robust lymphocyte expansions consisting of up to 70% CD8+ cells. The addition of a CD40 agonist antibody to TNF-α during the DC maturation process could partially, but not completely overcome these observed functional deficiencies. CONCLUSIONS: The data suggest that DCs loaded with primary tumor antigens and then matured with TNF-a might be deficient in the induction of type 1 T-helper responses. The absence of critical costimulatory markers and IL-12 secretion suggest that DCs matured with TNF-α might be suboptimal for the priming of naïve CD8 responses. These findings might help to improve the clinical efficacy of future DC vaccination studies.

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