scholarly journals Exposure of Cord Blood to Mycobacterium bovis BCG Induces an Innate Response but Not a T-Cell Cytokine Response

2008 ◽  
Vol 15 (11) ◽  
pp. 1666-1673 ◽  
Author(s):  
M. L. V. Watkins ◽  
P. L. Semple ◽  
B. Abel ◽  
W. A. Hanekom ◽  
G. Kaplan ◽  
...  
Keyword(s):  
Cord Blood ◽  
Mycobacterium Bovis ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cytokine Response ◽  
Healthy Human ◽  
Blood Samples ◽  
Ifn Γ

ABSTRACT Despite routine vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) soon after birth, tuberculosis in babies and adults remains epidemic in South Africa. The immune responses of the naïve newborn child and how they are affected by vaccination with BCG are as yet not fully understood. Immunity during pregnancy and in healthy human newborns may be skewed toward type 2 cytokine production; however, it is type 1 cytokines that are required for protection against M. tuberculosis infection. To better understand neonatal cytokine responses prior to and following exposure to mycobacteria, we have collected cord blood and peripheral blood samples and evaluated the cytokine response following ex vivo incubation with BCG. Gamma interferon (IFN-γ), interleukin 10 (IL-10), IL-12, and low levels of IL-13 and IL-5 but no IL-4 were secreted into the culture supernatant of cord blood mononuclear cells. Intracellular staining showed that IL-10 and IL-12 were produced by monocytes and that IFN-γ was produced by natural killer (NK) cells but not by CD4+ or CD8+ T cells. In contrast, in the peripheral blood samples collected from babies 13 weeks post-BCG vaccination, IFN-γ was detected within CD4+ and CD8+ cells. Taken together, the data suggest a central role for Th1 cytokines in naïve as well as BCG-vaccinated neonates in the protective immune response to tuberculosis. NK cell-derived IFN-γ produced in naïve neonates likely plays a key protective role via monocyte activation and the priming of a subsequent adaptive Th1 response.

scholarly journals Combined Stimulation with Interleukin-18 and Interleukin-12 Potently Induces Interleukin-8 Production by Natural Killer Cells

2017 ◽  
Vol 9 (5) ◽  
pp. 511-525 ◽  
Author(s):  
Sophie M. Poznanski ◽  
Amanda J. Lee ◽  
Tina Nham ◽  
Evan Lusty ◽  
Margaret J. Larché ◽  
...  
Keyword(s):  
Immune Response ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Critical Role ◽  
Interleukin 12 ◽  
Tnf Α ◽  
Ifn Γ

The combination of interleukin (IL)-18 and IL-12 (IL-18+IL-12) potently stimulates natural killer (NK) cells, triggering an innate immune response to infections and cancers. Strategies exploiting the effects of IL-18+IL-12 have shown promise for cancer immunotherapy. However, studies have primarily characterized the NK cell response to IL-18+IL-12 in terms of interferon (IFN)-γ production, with little focus on other cytokines produced. IL-8 plays a critical role in activating and recruiting immune cells, but it also has tumor-promoting functions. IL-8 is classically produced by regulatory NK cells; however, cytotoxic NK cells do not typically produce IL-8. In this study, we uncover that stimulation with IL-18+IL-12 induces high levels of IL-8 production by ex vivo expanded and freshly isolated NK cells and NK cells in peripheral blood mononuclear cells. We further report that tumor necrosis factor (TNF)-α, produced by NK cells following IL-18+IL-12 stimulation, regulates IL-8 production. The IL-8 produced is in turn required for maximal IFN-γ and TNF-α production. These findings may have important implications for the immune response to infections and cancer immunotherapies. This study broadens our understanding of NK cell function and IL-18+IL-12 synergy by uncovering an unprecedented ability of IL-18+IL-12-activated peripheral blood NK cells to produce elevated levels of IL-8 and identifying the requirement for intermediates induced by IL-18+IL-12 for maximal cytokine production following stimulation.

scholarly journals A Murine Model for Human Cord Blood Transplantation: Near-Term Fetal and Neonatal Peripheral Blood Cells Can Achieve Long-Term Bone Marrow Engraftment in Sublethally Irradiated Adult Recipients

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 1089-1099 ◽  
Author(s):  
Andromachi Scaradavou ◽  
Luis Isola ◽  
Pablo Rubinstein ◽  
Yelena Galperin ◽  
Vesna Najfeld ◽  
...  
Keyword(s):  
Cord Blood ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Cord Blood Transplantation ◽  
Blood Transplantation ◽  
Near Term ◽  
Ex Vivo Culture

Abstract The purposes of the research reported here were first to explore a murine model for human placental and umbilical cord blood transplantation and second to evaluate the engraftment ability of ex vivo cultured hematopoietic cells. Murine near-term fetal and neonatal peripheral blood (FNPB) cells, genetically marked with the human multiple drug resistance transgene (MDR1) were used for syngeneic transplants into sublethally irradiated adult mice. Donor cells were transplanted either fresh and untreated, or after ex vivo culture in the presence of the hematopoietic growth factors recombinant murine stem cell factor, recombinant human interleukin-3 (rHu IL-3), and rHu IL-6, in a liquid culture system. To evaluate, count, and characterize FNPB progenitor cell-derived colonies, neonatal mouse mononuclear cells were cultured directly in methylcellulose with growth factors. To assess their ex vivo expansion ability, FNPB mononuclear cells were first cultured in liquid medium for 3 to 8 days and then transferred to semisolid assay plates. Evaluation of the cell counts after liquid culture showed a 1.4- to 11.6-fold increase, and the numbers of colonies observed in methylcellulose were similar to those produced by fresh FNPB cells. Donor-type engraftment was demonstrated by polymerase chain reaction (PCR) amplification of the human MDR1 transgene in the peripheral blood of all surviving animals (5 of 7 recipients of the fresh, and 3 of 8 recipients of the ex vivo–cultured cells) 2 to 4 months after transplantation. The proportion of donor leukocytes in the peripheral blood of the recipients (chimerism) was evaluated using fluorescence in situ hybridization (FISH) analysis 4 to 6 months after transplantation and ranged from 2% to 26%. In addition, bone marrow cultures were obtained from two recipient animals: one had received fresh-untreated cells and was evaluated 8 months after transplant, the other had received ex vivo cultured cells and was tested 14 months after grafting. The derived hematopoietic colonies were tested by PCR and the transgene was detected, conclusively proving long-term engraftment of donor cells. These results indicate that FNPB transplants can be successfully performed in sublethally irradiated mice with and without ex vivo culture. Long-term donor-type engraftment with sustained chimerism has been demonstrated. Thus, murine neonatal blood grafts can be used as an animal model for cord blood transplantation for gene therapy studies where complete myeloablation is not desirable and partial replacement of defective marrow may be sufficient. Furthermore, the possibility of numerically expanding hematopoietic progenitor cells contained in neonatal blood without affecting their engraftment ability could facilitate use of cord blood grafts in adult recipients.

Dysregulation of Interleukin (IL)-18 Gene Expression and Protein Production in Basal and Activated Cord Blood (CB) vs. Peripheral Blood (PB) Mononuclear Cells and Differential Effects of IL-18 in CB vs. PB T and NK Cell Immunity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2660-2660
Author(s):  
Prakash Satwani ◽  
Janet Ayello ◽  
Lynn Simpson ◽  
Carmella van de Ven ◽  
Mitchell S. Cairo
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Mrna Expression ◽  
Half Life ◽  
Protein Production ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Ifn Γ

Abstract IL-12 and IL-15 are critically important cytokines that regulate the activation of T-cell lymphocyte and NK cell subsets. We have previously demonstrated reduced IL-12 & IL-15 gene and protein expression between activated CB vs. PB mononuclear cells (MNC) (Lee/Cairo, Blood; 1996 & Qian/Cairo, Blood; 1997). IL-18 has been shown to enhance T-cell cytolytic activity & provide antitumor immunity (Osaki et al, J Immunol 1998). In the present study, we compared IL-18 gene expression, protein production and IL-18 mRNA transcript half-life in CB vs. APB MNC and the effects of IL-18 on IFN-γ protein production from CB vs. APB MNC and ex-vivo expansion and activation of fresh CB MNC upon stimulation with IL-12 + IL-2+anti-CD3 ±IL-18. MNC were isolated from CB and APB by Ficoll gradient centrifugation and cultured in RPMI for 2 hours before the addition of staphylococcus aureus enterotoxin (SEB). 24 hours after SEB, the supernatant was measured for IL-18 by ELISA. Furthermore, basal and activated MNC IL-18 mRNA expression was measured by qRT-PCR in APB & CB. APB & CB IL-18 mRNA Actinomycin D half-life studies were performed as previously described (Lee/Cairo, Blood, 1996). IFN-γ production from IL-18 ± IL-12 activated CB vs. APB MNC was measured by ELISA at 48 hours. For ex-vivo expansion, non-adherent CB MNC were cultured for 48 hours with either AIM-V media or AIM-V + IL-2 (5ng/mL) + IL-12 (10ng/mL) + anti-CD3 (50ng/mL) ± escalating doses of IL-18 (1, 10, or 100ng/mL). We demonstrated that the constitutive levels of IL-18 secreted by APB MNC were significantly higher than CB MNC (16.09±6.09 vs. <10 pg/ml, not detectable, p<0.05). SEB dramatically increased IL-18 levels in APB > CB MNC (20hrs) (628.41±34.08 vs. 358.23±9.82 pg/ml, p<0.05). IL-18 mRNA and protein levels were significantly lower in CB compared to APB MNC (2hrs) (0.329 ± 0.131 vs.1.482 ± 0.505, p<0.05). IL-18 mRNA half life was significantly shorter in CB vs. APB MNC (3.281±0.222 vs. 4.967±0.411 min, p<0.05). IL-18 independently or synergistically with IL-12 induced IFN-γ production from both APB and CB MNC (313.67±83.18 vs. 50.00±6.66 pg/ml, p<0.05). Lymphocyte subset expansion of CD8+/25+, CD4+/25+ and CD16+/56+ was significantly increased with IL-12 +IL-2+anti-CD3 and IL-18(10ng/ml) compared to media alone (CD8+/25+: 31±4.9 vs. 0.25±0.8%, p<0.001; CD4+/25+: 61±9 vs. 2.6±0.7%, p<0.001; CD3−/CD16+/56+: 58±11.2 vs. 16.5±5.5%, p<0.001, respectively). Furthermore, there was also significant increase in CD16+/56+ subset cultured in IL-2, IL-12, anti-CD3 vs. IL-2, IL-12, anti-CD3 and IL-18 at 1, 10 and 100ng/ml (16.07±0.87 vs.39.38±9.92 vs.58.27±11.17 vs.66.71±6.25%, p<0.001). There was also significant increase in NK cytotoxicity with IL-2, IL-12, anti-CD3 vs. IL-2, IL-12, anti-CD3 and IL-18 (10ng/ml) (43.1±3.2 vs. 91.5±2.42; p<0.01). These results suggest a significant decrease in IL-18 mRNA expression and protein production in activated CB vs. APB MNC, which was in part secondary to increased degradation of CB IL-18 mRNA. IL-18 in combination with IL-12, IL-2 and anti-CD3 showed enhanced CB NK expansion and cytotoxicity. These results may have important implications in immune reconstitution and graft versus tumor activity following UCBT.

scholarly journals Modulation of Mycobacterium bovis-Specific Responses of Bovine Peripheral Blood Mononuclear Cells by 1,25-Dihydroxyvitamin D3

2001 ◽  
Vol 8 (6) ◽  
pp. 1204-1212 ◽  
Author(s):  
W. R. Waters ◽  
B. J. Nonnecke ◽  
T. E. Rahner ◽  
M. V. Palmer ◽  
D. L. Whipple ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cell Subset ◽  
No Production ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

ABSTRACT Historically, administration of vitamin D has been considered beneficial in the treatment of tuberculosis. The interaction of this vitamin {i.e., 1,25-dihdroxyvitamin D3[1,25(OH)2D3]} with the antitubercular immune response, however, is not clear. In the present study, in vitro recall responses of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis were used to study the immune-modulatory effects of 1,25(OH)2D3 on M. bovis-specific responses in vitro. Addition of 1 or 10 nM 1,25(OH)2D3 inhibited M. bovis-specific proliferative responses of PBMC from M. bovis-infected cattle, affecting predominately the CD4+ cell subset. In addition, 1,25(OH)2D3 inhibited M. bovis-specific gamma interferon (IFN-γ) production yet enhanced M. bovis-specific nitric oxide (NO) production. Lymphocyte apoptosis, measured by flow cytometry using annexin-V staining, was diminished by addition of 1,25(OH)2D3 to PBMC cultures. These findings support the current hypothesis that 1,25(OH)2D3enhances mycobacterial killing by increasing NO production, a potent antimicrobial mechanism of activated macrophages, and suggest that 1,25(OH)2D3 limits host damage by decreasingM. bovis-induced IFN-γ production.

Mobilization of Human Hemopoietic Progenitor Stem Cell Via Modulating CXCR4/SDF-1 Using Galactofucan Sulfate.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1971-1971
Author(s):  
Mohammad R. Irhimeh ◽  
Ray M. Lowenthal ◽  
J. Helen Fitton
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Autologous Transplantation ◽  
White Blood Cells ◽  
Healthy Human ◽  
Cytokine Analysis ◽  
Ifn Γ

Abstract Mobilization of peripheral blood progenitor cells for hematopoietic rescue following autologous transplantation is usually achieved with the chemokine G-CSF. Engraftment potential is increased when higher levels of the receptor CXCR4 are noted on the hematopoietic progenitor stem cells (HPCs). It was previously reported that intravenous treatment with fucoidan or dextran sulfate, which are sulfated high molecular weight compounds, increased numbers of circulating mature white blood cells and HPCs in mice and nonhuman primates. This treatment also led to an increase in the pro-inflammatory cytokines IFN-γ and IL-12 levels in mice. In vitro treatment of bone marrow mononuclear cells with IFN-γ can up-regulate the expression of CXCR4 on granulocyte precursors and monocytes. We obtained ethics approval and informed consent to study the mobilization effect of orally ingested GFSTM (Galactofucan Sulfate), a seaweed-derived fucoidan, in healthy human volunteers in a single blinded placebo controlled phase I/II clinical study. Flow cytometry was used to monitor CXCR4 receptor on CD34+ stem cells. When moderate quantities (3 g/day) of seaweed containing 10% GFSTM were ingested, a slight increase in the total number of HPCs (CD34+) in the peripheral blood (PB) was observed, from 1.38 to 1.69 cells/μL (p=0.22, n=6). In addition, there was a small increase in the percentage of HPCs that expressed CXCR4 surface receptor, from 0.59 to 1.47 cells/μL, which is equivalent to 43% to 63% (p=0.19, n=6). Moreover, when 3 g/day of 75% GFSTM was ingested, a greater increase in the total number of HPCs (CD34+) in PB was observed, from 1.65 to 1.84 cells/μL (p=0.04, n=23). Furthermore, the percentage of HPCs that expressed CXCR4 increased from 0.746–1.652 cells/μL, which is equivalent to 45% to 90% (p=0.0002, n=23). Cytokine analysis, which was performed using ELISA to test for SDF-1 and IFN-γ, showed a significant increase in the plasma level of these cytokines. SDF-1 level was elevated from 1979 to 2068 pg/mL (p=0.051, n=10) and the level of IFN-γ from 9.04 to 9.90 pg/mL (p=0.007, n=10). These results suggest that GFSTM may modulate CXCR4 or disturb the SDF-1 gradient between bone marrow and PB. IFN-γ might play a role in the up-regulation of the expression of CXCR4 on CD34+ cells. To the authors’ knowledge, this is the first report of mobilization of HPCs by disruption of CXCR4/SDF-1 interaction using oral fucoidan.

Patients’ Peripheral Blood as a Possible Source of Donor-Derived NK Cells for Ex Vivo Expansion and Genetic Modification for Post-Transplant Cellular Immunotherapy In Cord Blood Recipients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2094-2094
Author(s):  
Chihaya Imai ◽  
Sakiko Yoshida ◽  
Takayuki Takachi ◽  
Masaru Imamura ◽  
Ryosuke Hosokai ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Genetic Modification ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
Cellular Immunotherapy ◽  
Healthy Individuals ◽  
Expansion Range

Abstract Abstract 2094 Haploidentical natural killer (NK) cells can induce and consolidate remission in patients with high-risk acute myeloid leukemia (AML) (Rubnitz et al. J Clin Onc 24: 371, 2010). Recently, significantly reduced relapse rates were observed in AML patients who received killer immunoglobulin-like receptor ligand-mismatched cord blood, suggesting effective alloreactivity of cord blood-derived NK cells (Willemze et al. Leukemia 23: 492, 2009). Cord blood transplantation (CBT) is an effective alternative source for allogeneic hematopoietic cell transplantation in both children and adults. However, its therapeutic efficacy for malignant diseases is limited by the lack of available donor effector cells, such as cytotoxic T lymphocytes, lymphokine-activated killer cells, NK-like T cells and NK cells, for treatment of hematological relapse and posttransplant lymphoproliferative disorder and/or for scheduled posttransplant cellular immunotherapy against refractory diseases. We previously reported a method that induces NK cells to proliferate and reliably allows their genetic modification in healthy individuals and leukemia patients in remission receiving maintenance chemotherapy (Imai et al. Blood 106: 376, 2005). To explore the possibility of using patients’ peripheral blood as a source for posttransplant NK cell therapy, we used our method to expand donor-derived NK cells from peripheral blood of CBT recipients early after engraftment. We also examined whether NK cells can be rendered cytotoxic against original leukemia blasts by transferring an antigen-specific artificial immunoreceptor gene. This study was approved by an institutional ethical committee. Patients received CBT for consolidation of hematological malignancy (n=7), neuroblastoma (n=1) or resolution of refractory EBV-associated hemophagocytic syndrome (n=1) with myeloablative (n=7) or reduced intensity conditioning (RIC) regimens (n=2). The patients were enrolled in the study after engraftment and peripheral blood was obtained after appropriate written consent was obtained. A chimerism study using short tandem repeat assays showed complete donor chimerism in all patients except one who received RIC-CBT. The peripheral blood was obtained at a median of 92 days post-CBT (range: 46–303 days) and subjected to ex vivo activation and expansion using a previously described protocol with slight modifications. Briefly, peripheral blood was coincubated with modified K562 cells expressing membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL) in the presence of low-dose IL-2 (10 U/mL). Most patients were on maintenance immunosuppressive therapy with calcineurin inhibitors with (n=3) or without (n=6) systemic corticosteroids. After 7 days of culture, a median 11.0-fold expansion (range: 5.3–28.9-fold) was observed in all but one patient who had been administered chemotherapy with Mylotarg for relapsed AML a few days before the blood sampling. The expansion rate in the first week was less efficient in CBT recipients than in healthy individuals (>20-fold), probably because of the immunosuppressants administered. However, an additional 2-week culture in the presence of high-dose IL-2 (1000 U/mL) yielded a median 206-fold expansion (range: 101–1381-fold in 21 days). The expanded NK cells exhibited upregulation of activating receptors including NKG2D, NCRp30 and NCRp44, and vigorous cytotoxicity against K562 cells (86.8–97.7% at an E/T ratio of 1:1). The NK cells were susceptible to retroviral genetic modification with the MSCV-IRES-GFP vector (median GFP-positive cells, 52.7%, n=10). Finally, peripheral NK cells from patients with acute lymphoblastic leukemia were expanded and transduced with the chimeric immunoreceptor gene anti-CD19-BB-ζ. The donor-derived NK cells expressed large amounts of anti-CD19 chimeric receptors on their surface and killed original leukemia blasts that were highly resistant to NK cell lysis (e.g. anti-CD19 vs. non-signaling receptor: 69% vs. 0% at an E/T ratio of 1:1). These results suggest that, in CBT recipients, ex vivo expansion and genetic modification of donor-derived NK cells from the patients’ peripheral blood is feasible. Because peripheral blood can be easily and repeatedly obtained, the method described here will allow multiple scheduled infusions. This preliminary study may lead to a novel strategy for posttransplant donor-NK cell therapy in CBT recipients. Disclosures: No relevant conflicts of interest to declare.

Ex vivo activation and expansion of γδ T cells for immunotherapy of glioblastoma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 2553-2553
Author(s):  
C. Suarez-Cuervo ◽  
N. L. Bryant ◽  
R. D. Lopez ◽  
G. Y. Gillespie ◽  
L. S. Lamb
Keyword(s):  
T Cells ◽  
Human Serum ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Primary Cultures ◽  
Γδ T Cells ◽  
Laboratory Method ◽  
Ifn Γ

2553 Background: Activated γδ T cells will efficiently kill established glioblastoma (GBM) cell lines in vitro. However, their potential for cell therapy has been limited by their relatively small numbers in the peripheral blood and their sensitivity to activation induced cell death (AICD) during ex vivo expansion. Our laboratory developed a method whereby γδ T cells, when stimulated by IFN-γ, IL- 12, and anti-CD2, acquire resistance to AICD and can be expanded in culture with anti-CD3 and IL-2. The γδ T cells expanded by this method are highly cytotoxic to GBM. We have now sought to adapt the laboratory method for human use. Methods: The laboratory method and selection procedure were modified to employ pharmaceutical-grade reagents. Peripheral blood mononuclear cells (1.0 × 106/ml) were stimulated overnight with media containing cGMP grade human serum, IFN-γ, IL-12, and anti-CD2. Media containing human serum, IL-2 and anti-CD3 was then added (1 v/v) and refreshed 1 v/v 3× weekly. The cultures were harvested after two weeks followed by depletion of CD4+ and CD8+ T cells, thereby enriching γδ T cells. Cytotoxicity of expanded of γδ T cells was evaluated against GBM primary tumor cultures, established GBM cell lines, and cultured astrocytes using a commercial flow cytometric method. Results: After two weeks in culture, γδ T cells expanded up to 1000 fold (usually 200–400 fold), consistent with results from our previous research-scale method. Positive selection yielded a γδ T cell product with >80% purity and >70% recovery. The cell products exhibited incremental cytotoxicity against several primary GBM cultures and established GBM cell lines. Astrocytoma cells were not killed. Conclusions: Apoptosis-resistant γδ T cells can be expanded and selected using clinically approvable reagents and in numbers sufficient for immunotherapy of malignant brain tumors. Initial data show that expanded γδ T cells retain cytotoxicity against the GBM primary cultures and spare normal astrocytes. No significant financial relationships to disclose.

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