Inducing the Anti-Leukemia Effects of CD8+ T Cytotoxic Lymphocytes Derived from Umbilical Cord Cell.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4406-4406
Author(s):  
Huo Tan ◽  
Xin Liu
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Cell Line ◽  
Umbilical Cord ◽  
K562 Cell ◽  
U937 Cell ◽  
Ex Vivo ◽  
Cytotoxic Lymphocytes ◽  
Gm Csf ◽  
Cell Groups

Abstract Objective: The previous studies of our research group have proved that specific cytotoxic T lymphocytes(CTLs) can be generated from umbilical cord cells. The purpose of this study is to further demonstrate whether CD8+CTLs can be generated from cord blood (CB) and kill leukemia cells specifically ex vivo. And whether the leukemia cytotoxicity of CD8+CTLs and CD8−CTLs are different. Method: First we induce adherent cells collecting from CB samples to generate dendritic cells (DCs) by culturing with GM-CSF, IL-4 and load lysate antigen of U937 cell to immature DCs. On day 6th, TNF-αand PGE2 was added to accelerate the maturation of DCs. After co-culturing DCs with the CTLs from the same CB, then we used MidiMACS to isolate CD8+ and CD8− T cell. The cytotoxicity of the CD8+ CTLs and CD8−−CTLs to U937 cell line was evaluated with Methyl thiazolyl tetrazolium(MTT) assay. Result: The purity of CD8+CTL was(95.73±1.50)%. Among the CD8−CTLs, the percentage of CD4+CTLs was (65.01±9.29)% and CD8+T lymphocytes was (4.9±4.46)%. When E:T ratios were 40:1, 20:1 and 10:1, the mean cytotoxic rate (MCR)of CD8+ CTLs to U937 cell were (66.36±12.43)%,(35.38±9.64)% and(19.04±6.15)% respectively; the MCR of CD8−CTLs to U937 cell were (34.47±8.19)%,(21.85±7.06)% and(12.26±4.87)% and the MCR of T cell from CB to U937 cell were(15.79±4.64)%,(9.6±3.71)% and (5.69±3.14)%. The MCR of CD8+ CTLs to U937 cell were significant higher than those of CD8−CTLs and T cell groups at the same E:T ratios(P<0.05). When E:T ratios was 40:1,the MCR of CD8+CTLs and CD8−CTLs to K562 cell line were (36.77±10.24)% and (26.95±3.06)% respectively, the cytotoxicity effect of CD8+ and CD8−CTLs to K562 cell are no difference (P>0.05). Conclusions: The cytotoxicity of CD8+CTLs against U937 cell lines is more potent than that of CD8−CTLs, and this effect is specific.

A Step towards Reconstitution or Maintenance of Remission before or after Stem Cell Transplantation in AML/MDS by Ex Vivo Generation of Leukaemia-Derived DC (DCleu) and DC-Activated T-Cells: Role of the Quality and Quantity of DC/DCleu and Anti-Leukemia-Directed T-Cells To Predict the Course and Success of Immunotherapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3246-3246
Author(s):  
Helga M. Schmetzer ◽  
Anja Liepert ◽  
Christine Grabrucker ◽  
Andreas Kremser ◽  
Julia Loibl ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Leukemic Cells ◽  
Cytotoxic Lymphocytes ◽  
Activated T Cells ◽  
Gm Csf ◽  
Donor T Cells

Abstract The presentation of leukemic antigens can be improved in AML and MDS by in vitro conversion of leukemic cells in leukemia-derived DC (DCleu), thereby forming a platform for the generation of leukemia-specific cytotoxic lymphocytes (CTL). In preliminary analyses with 140 AML and 60 MDS-cases we could already define optimal serum-free culture conditions to generate DC/DCleu.(Kufner 2005 I–III). Now we want to predict or correlate the clinical response to a DC/CTL-based immunotherapy by detailed analyses of the ex vivo generated/activated DC/DCleu and T-cells: 1)By a combination of 3 different DC-generating methods (‘MCM-mimic’, Lee 2003; ‘Ca-Ionophore’, Houtenbos 2003; ‘Picibanil’, Sato 2003) we can generate DC/DCleu in every case of AML/MDS, independently from FAB-type or karyotype. DC/DCleu are quantified according to their surface DC/blast-marker profiles. On average 42–45%/39–66% DC in AML/MDS could be generated with 48–54%/39–51% mature (CD83+) and 31–34%/23–31% migratory (CCR7+) DC. 45–65% of DC were ‘DCleu’; on average 47% of blasts are convertible to DCleu.. 2) In AML-patients who had presented with a relapse after SCT we could correlate a better ex vivo convertibility of blasts to DCleu with the patients’ in vivo response to a GM-CSF/Donor-lymphocyte Infusion (DLI)-therapy of their relapse after SCT (33% vs 7% to DCleu convertible blasts in ‘non-responders’). 3) A ‘Mixed lymphocyte culture’ (MLC) of autologous AML-patients’ or allogeneic donor-T-cells showed an on average higher proliferation and stimulation of DC-primed compared to MNC-primed T-cells: Upregulation of CD80/CD86-CD28;CD40-CD154;CD137L-CD137; moreover DC-priming yielded higher proportions of CD4+ cells, CD3+CD45RO+ memory cells CCR4+ T-cells (+59%, +52%, +91%) compared to MNC-primed T-cells (+35%, +13%, +44%) and a higher leukaemia-cytolytic activity (average 62%) compared to MNC-stimulated CTL (average 26%). 4) A detailed analysis of data showed great individual variations depending on the quality and composition of DC and T-cells: a) non-DC-primed autologous or allogeneic T-cells an lead to an increase of naive blasts after 3h incubation with blasts b) in cases with an ineffective DC-mediated ex vivo lysis of naïve blasts lower proportions of mature DC (29% vs 63%), DCleu (41% vs 68%) or a reduced T-cell proliferation or even loss of CD4/CD8/memory T-cells were seen. In summary our data show 1. that DC/DCleu can be generated in every single AML/MDS-case. 2. Grade of ex-vivo generability of DC/DCleu correlates with the in vivo response to a GM-CSF/DLI-relapse therapy. 3. Composition and quality of DC and autologous or donor T-cells after DC-priming provides informations about the activability and quality of CTLs in individual patients. We conclude, that ex vivo analysis of the DC/anti-leukemic T-cell-activability is necessary to develop and select promising anti-leukemia-directed T-cells for the immunotherapy of AML and MDS.

scholarly journals CAL-1 as Cellular Model System to Study CCR7-Guided Human Dendritic Cell Migration

2021 ◽  
Vol 12 ◽  
Author(s):  
Edith Uetz-von Allmen ◽  
Guerric P. B. Samson ◽  
Vladimir Purvanov ◽  
Takahiro Maeda ◽  
Daniel F. Legler
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Neoplastic Cell ◽  
Model System ◽  
Model Systems ◽  
Cellular Model ◽  
Gm Csf ◽  
Endogenous Expression ◽  
Dendritic Cell Migration ◽  
Spatio Temporal

Dendritic cells (DCs) are potent and versatile professional antigen-presenting cells and central for the induction of adaptive immunity. The ability to migrate and transport peripherally acquired antigens to draining lymph nodes for subsequent cognate T cell priming is a key feature of DCs. Consequently, DC-based immunotherapies are used to elicit tumor-antigen specific T cell responses in cancer patients. Understanding chemokine-guided DC migration is critical to explore DCs as cellular vaccines for immunotherapeutic approaches. Currently, research is hampered by the lack of appropriate human cellular model systems to effectively study spatio-temporal signaling and CCR7-driven migration of human DCs. Here, we report that the previously established human neoplastic cell line CAL-1 expresses the human DC surface antigens CD11c and HLA-DR together with co-stimulatory molecules. Importantly, if exposed for three days to GM-CSF, CAL-1 cells induce the endogenous expression of the chemokine receptor CCR7 upon encountering the clinically approved TLR7/8 agonist Resiquimod R848 and readily migrate along chemokine gradients. Further, we demonstrate that CAL-1 cells can be genetically modified to express fluorescent (GFP)-tagged reporter proteins to study and visualize signaling or can be gene-edited using CRISPR/Cas9. Hence, we herein present the human CAL-1 cell line as versatile and valuable cellular model system to effectively study human DC migration and signaling.

scholarly journals Limiting-dilution analysis of the effects of colony-stimulating factors, phytohemagglutinin, and hydrocortisone on hematopoietic progenitor cell growth

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1611-1618
Author(s):  
Y Takaue ◽  
CL Reading ◽  
AJ Roome ◽  
KA Dicke ◽  
S Tindle ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Cell Growth ◽  
Cell Line ◽  
Conditioned Medium ◽  
Hematopoietic Progenitor ◽  
Colony Stimulating Factors ◽  
Gm Csf ◽  
Percoll Gradients ◽  
Limiting Dilution

The effects of colony-stimulating factors (CSFs), phytohemagglutinin (PHA), and hydrocortisone on the growth of human bone marrow hematopoietic progenitor cells (granulocyte-macrophage; GM) were analyzed in a limiting-dilution assay (LDA). Both low-density bone marrow cells separated by discontinuous Percoll gradients and a T cell- depleted and progenitor-enriched cell fraction obtained by the combination of counterflow elutriation centrifugation and Percoll gradients were examined in LDA. GCT (monocytoid cell line-conditioned medium containing GM-CSF), human placenta-conditioned medium, bladder carcinoma cell line 5637-conditioned medium (containing GM- and G-CSF), and recombinant CSF (G-CSF) directly induced proliferation of progenitors with single-hit kinetics. In some instances, however, PHA- stimulated lymphocyte-conditioned medium (containing G- and GM-CSF) showed deviation from single-hit kinetics, which demonstrated the presence of factor(s) suppressive to progenitor growth. In a T cell- depleted, progenitor-enriched fraction, PHA alone was found to suppress progenitor growth at a level as low as 100 ng/mL. The addition of hydrocortisone (10(-6) mol/L) increased the progenitor frequency but suppressed progenitor growth at 10(-4) mol/L. LDA appears to be a valuable method for exploring mechanisms of factors regulating hematopoietic cell growth.

FRI0010 GM-CSFR PATHWAY IS IMPLICATED IN PATHOGENIC INFLAMMATORY MECHANISMS IN GIANT CELL ARTERITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 576.1-576
Author(s):  
M. C. Cid ◽  
S. Muralidharan ◽  
M. Corbera-Bellalta ◽  
G. Espigol-Frigole ◽  
J. Marco Hernandez ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mrna Expression ◽  
Giant Cell Arteritis ◽  
Giant Cell ◽  
Signaling Pathway ◽  
Ex Vivo ◽  
Cell Marker ◽  
Gm Csf ◽  
Inflammatory Macrophages

Background:Giant Cell Arteritis (GCA) is characterized by inflammation of large and medium arteries. Classic symptoms include headaches, malaise and, in severe cases, blindness and aortic aneurysms. Corticosteroids (CS) are the first line of treatment. Relapsing disease patients undergo multiple courses of CS therapy increasing their CS exposure and toxicity. A significant unmet need for disease-modifying CS-sparing therapy remains in GCA as the efficacy of current treatment options, including tocilizumab have limitations.We have previously reported elevated expression of granulocyte-macrophage colony stimulating factor (GM-CSF) pathway transcriptomic signature in GCA vessels. GM-CSF may contribute to underlying disease mechanisms by regulating inflammatory macrophages, dendritic cells (DCs) and T helper (TH1/TH17) cells which are involved in GCA pathogenesis. GM-CSF produced by T cells1can promote polarization of inflammatory macrophages2and recruitment and differentiation of monocytes into inflammatory DCs2that can in turn recruit T cells and stimulate TH1/TH17 differentiation creating a feedback loop. GM-CSF may also exert direct effects on angiogenesis3and vessel wall remodeling4.Objectives:To demonstrate the contributing role of GM-CSF pathway to inflammation in GCA arteries.Methods:Immunostaining was used to examine expression of GM-CSF and GM-CSF-Rα proteins in temporal artery biopsies (TABs) from GCA and controls (patients with suspected but not confirmed GCA and a negative TAB). Costaining with cell markers such as CD31, CD3, and CD68 allowed visualization of cells expressing GM-CSF and GM-CSF-Rα. Expression of GM-CSF pathway molecules such as phospho-JAK2 and PU.1 proteins was detected by immunohistochemical staining of GCA and control TABs.Ex vivocultured GCA arteries treated (10 each) with mavrilimumab (anti-GM-CSF-Rα) or placebo for 5 days were assayed for gene expression by qPCR, and culture supernatants were analyzed by ELISA.Results:Endothelial cells and macrophages were the main cell types expressing GM-CSF and GM-CSF-Rα. Increased expression of phospho-JAK2 (activated signaling molecule) and nuclear-localized PU.1 (transcription factor) in GCA TABs compared to controls indicated the presence of active GM-CSF signaling pathway in GCA.Inhibition of PU.1 mRNA expression inex vivocultures of GCA arteries treated with mavrilimumab indicated blockade of GM-CSFR signaling pathway. Mavrilimumab induced decrease in mRNA expression of key cell type markers including DC and macrophage activation markers CD83 and HLA-DRA, monocyte markers CD14 and CD16, T cell marker CD3ε, and B cell marker CD20 in these GCA artery cultures. Expression of inflammatory TH1/TH17 factors IFNγ (mRNA), TNFα, CXCL10 (IFNγ-stimulated chemokine) and IL-6 (mRNA and protein) was also inhibited by mavrilimumab in GCA artery cultures.Conclusion:Increased GM-CSF, GM-CSF-Rα, and downstream pathway-associated protein levels in GCA biopsies were consistent with previously-observed increased transcriptome signature. Expression of genes associated with inflammatory cells was suppressed by mavrilimumab in cultured GCA arteries. These data implicate the GM-CSF pathway in GCA pathophysiology and increase confidence in rationale for targeting the GM-CSF pathway in GCA.References:[1]GM-CSF and T-cell responses: what we do and don’t know. Shiet al., Cell Res 2006[2]GM-CSF-Dependent Inflammatory Pathways. Hamilton, Front Immunol 2019[3]GM-CSF increases tumor growth and angiogenesis. Zhenget al., Tumour Biol 2017[4]GM-CSF deficiency affects vascular elastin production and integrity of elastic lamellae. Weissen-Plenzet al., J Vasc Res 2008Disclosure of Interests:Maria C. Cid Grant/research support from: Kiniksa Pharmaceuticals, Consultant of: Janssen, Abbvie, Roche, GSK, Speakers bureau: Vifor, Sujatha Muralidharan Shareholder of: Kiniksa, Employee of: Kiniksa, Marc Corbera-Bellalta: None declared, Georgina Espigol-Frigole Consultant of: Roche and Janssen, Javier Marco Hernandez: None declared, Amanda Denuc: None declared, Roberto Rios-Garces: None declared, Nekane Terrades-Garcia: None declared, John F. Paolini Shareholder of: Kiniksa, Employee of: Kiniksa, Annalisa D’Andrea Shareholder of: Kiniksa, Employee of: Kiniksa

scholarly journals EA-1, a novel adhesion molecule involved in the homing of progenitor T lymphocytes to the thymus.

1991 ◽  
Vol 114 (5) ◽  
pp. 1069-1078 ◽  
Author(s):  
B A Imhof ◽  
P Ruiz ◽  
B Hesse ◽  
R Palacios ◽  
D Dunon
Keyword(s):  
Endothelial Cell ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Line ◽  
Adhesion Molecule ◽  
Newborn Mice ◽  
Embryonic Mouse ◽  
Endothelial Cell Line ◽  
Mouse Tissues

The mouse progenitor T lymphocyte (pro-T) cell line FTF1 binds in vitro to thymus blood vessels, the thymic capsule, and liver from newborn mice. A mAb, EA-1, raised against an embryonic mouse endothelial cell line, blocked adhesion. The antibody also interfered with pro-T cell adhesion to a thymus-derived mouse endothelial cell line; it had no effect on the adhesion of mature T lymphocytes and myeloid cells. The antigen recognized by EA-1 is located on the vascular endothelium of various mouse tissues and absent on pro-T cells. EA-1 antibody precipitates molecules with apparent molecular weights of 110,000, 140,000, 160,000, and 200,000. Immunoclearing and binding-inhibition studies with antibodies against known adhesion molecules suggest that the EA-1 antigen is a novel adhesion molecule involved in colonization of the embryonic thymus by T cell progenitors.

Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB

2002 ◽  
Vol 20 (2) ◽  
pp. 143-148 ◽  
Author(s):  
Marcela V. Maus ◽  
Anna K. Thomas ◽  
Debra G.B. Leonard ◽  
David Allman ◽  
Kathakali Addya ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
T Cell Receptor ◽  
Cytotoxic T Lymphocytes ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Ex Vivo Expansion

scholarly journals Ex Vivo Expansion of Human Umbilical Cord Blood-Derived T-Lymphocytes with Homologous Cord Blood Plasma

2005 ◽  
Vol 205 (2) ◽  
pp. 115-122 ◽  
Author(s):  
Yong-Man Kim ◽  
Min-Hyung Jung ◽  
Ha-Young Song ◽  
Hyun Ok Yang ◽  
Sung-Tae Lee ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Blood Plasma ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord

Ex Vivo Expansion of Umbilical Cord Blood T Cells for Adoptive Immunotherapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 35-35
Author(s):  
Melissa A. Mazur ◽  
Young Ah Lee ◽  
Kurtzberg Joanne ◽  
Szabolcs Paul
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Adoptive Immunotherapy ◽  
Ex Vivo ◽  
Cell Activity ◽  
Ex Vivo Expansion ◽  
Hla Dr

Abstract Background: Viral infections cause significant morbidity & mortality in patients undergoing unrelated allogeneic BMT transplantation before immune reconstitution is completed. It poses a greater risk for recipients of unrelated umbilical cord blood (uUCB) transplants as there is no established antiviral immunity in naïve UCB lymphocytes available for adoptive transfer. UCB T cells also lack Th1/Tc1 cytokines, Granzymes & Perforin which are prerequisites to control viral pathogens. Another major limitation of uUCBT is the lack of donor cells available for post-transplant donor leukocyte infusions (DLI) to boost immunity or induce GVL. However, a fraction of the uCB graft could be available for T cell expansion. In this study we evaluated the feasibility of ex vivo expansion of UCB T cells. We postulated that following expansion naïve T cells may mature & acquire a phenotype compatible with effector function as assessed by expression of essential cytokines & de novo expression of members of the granzyme-perforin pathway. Methods: Thawed UBC research samples with a leukocyte content &lt;5% of an average UCB graft are processed. T cells are enriched with “EasySep” (StemCell Tech) to deplete CD14, CD16, CD19, CD56, & glycophorin A + cells. 5–7.5*105 T cells/ml are incubated with “CD3/28 T cell Expander” artificial APC beads (Dynal) in X Vivo-15 (BioWhittacker) + 200u/ml IL2 & 10% human serum in gas permeable bags. The initial purity of the T cells is 77–92%. The starting absolute T cell numbers ranged from 0.75 to 2*106 cells. Media & cytokines are added every other day to maintain a concentration of &lt;2*106 cells/ml. Results: At the end of 14 days UCB T Cells expanded 67 fold +/− 36, n=6. There are significant alterations in phenotype over the 2 weeks (Table 1) with up to 40% of T cells in cell cycle. Compared to the starting resting UCB T cells the majority of expanded cells have acquired the phenotype of activated (HLA-DR+, CD25+ T cells) memory cells, at the expense of naive/recent thymic immigrants (CD45RA+/CD62+). There is an inverted CD4/CD8 ratio due to the higher expansion rate of CD8 T cells (p=0.0035) while there is no difference in apoptosis (p=0.57). However, they all retain expression of CD28 (96% ±8%) along with CD27. Although some T cells have acquired the capacity to secrete granzymes A and B these are still almost a log below normal adult peripheral blood (PB) values & perforin has not been detected. Similarly, while post expansion significantly more T cells secrete cytokines upon PMA + ionomycin stimulation (Table 1) they are below levels of adult PB. Conclusions: From our preliminary results we can demonstrate effective expansion & partial maturation of UCB T Cells. For example, if one starts with 2*106 total T cells & expands them 67 fold this could provide for DLI ~5*106 T cells/kg for an average pediatric patient (25kg). We are further optimizing & characterizing this model for T cell activity & repertoire. In sum, ex vivo expansion with CD3/CD28 co-stimulation may provide clinically relevant numbers T cells available for adoptive immunotherapy that have also undergone partial maturation. Characterization of Expanded T Cells as % of all Lymphocytes Variable Median SD CD3+ 99.8 0.1 CD4+ 35 11 CD4+/CD8+ 2.3 2.8 CD45RA+/RO− 13 11 CD45RO+/RA− 55 22 CD25+ 42 21 CD45RA+/CD62+ 38 20 CD45RA+/CD27+/CD8+ 52 15 CD45RA−/CD27+/CD8+ 46 15 KI67/CD8+ 42 9 Ki67/CD4+ 32 7 HLA DR+ 40 13 Granzyme A/CD8+ 54 18 Granzyme B/CD8+ 2 2 Perforin/CD8+ 0 0

Sign in / Sign up

Export Citation Format

Share Document