Ab Lyons

Determination of lymphocyte division by flow cytometry

Techniques currently available for determining cell division are able to show one or, at best, a limited number of cell divisions. Other methods exist which can quantify overall division, but tell nothing about the division history of individual cells. Here we present a new technique in which an intracellular fluorescent label is divided equally between daughter cells upon cell division. The technique is applicable to in vitro cell division, as well as in vivo division of adoptively transferred cells, and can resolve multiple successive generations using flow cytometry. The label is fluorescein derived, allowing monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes to be used to immunophenotype the dividing cells.

Divided we stand: Tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester

Most techniques for assessing cell division can either detect limited numbers of cell divisions (bromodeoxyuridine incorporation) or only quantify overall proliferation (tritiated thymidine incorporation). In the majority of cases, viable cells of known division history cannot subsequently be obtained for functional studies. The cells of the immune system undergo marked proliferation and differentiation during the course of an immune response. The relative lack of an organized structure of the lymphohaemopoietic system, in contrast with other organ systems, makes lineage interrelationships difficult to study. Coupled with the remarkable degree of mobility engendered by recirculation, the differentiation occurring along with cell division in the immune system has not been readily accessible for investigation. The present article reviews the development of a cell division analysis procedure based on the quantitative serial halving of the membrane permeant, stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE or CFDA, SE). The technique can be used both in vitro and in vivo, allowing eight to 10 successive divisions to be resolved by flow cytometry. Furthermore, viable cells from defined generation numbers can be sorted by flow cytometry for functional analysis.

Chapter 17 Flow cytometric analysis of cell division history using dilution of Carboxyfluorescein Diacetate Succinimidyl Ester, a stably integrated fluorescent probe

Publisher Summary This chapter reviews the use of a technique based on the serial dilution of a stably binding intracellular fluorochrome, carboxyfluorescein diacetate succinimidyl ester (CFSE), which allows eight to ten sequential cell divisions to be analyzed by flow cytometry. When incubated with cells, the fluorescein-based CFSE crosses the cell membrane and attaches to free amine groups of cytoplasmic cell proteins. After enzymatic removal of carboxyl groups by endogenous intracellular esterases, CFSE acquires identical spectral characteristics to fluorescein, with optimal excitation by 488 nm argon laser light, emitting strongly at 519 nm, and as such is compatible with almost all single and multiple laser flow cytometers. On cell division, CFSE is distributed equally between progeny, allowing the division history of a cell population to be determined. This technique can be used to investigate the behavior of cells in vitro , as well as division of transferred cells in vivo . A major advantage of the CFSE based technique is that viable cells from defined division cycles can be recovered, allowing functional characteristics to be related to differentiation stage.

The Src/ABL kinase inhibitor dasatinib (BMS-354825) inhibits function of normal human T-lymphocytes in vitro

Dasatinib (BMS-354825) is a Src/ABL tyrosine kinase inhibitor currently approved for the treatment of chronic myeloid leukemia. Dasatinib has increased potency against ABL compared to the current therapy imatinib, and is effective in many cases where disease is resistant to imatinib. Dasatinib also inhibits many Src-family tyrosine kinases. We have demonstrated in this study that dasatinib is able to block the function of normal human T-lymphocytes in vitro at clinically relevant concentrations. T-cell functions including proliferation, activation and cytokine production were all uniformly inhibited in the presence of dasatinib. We also demonstrated inhibition of TCR signalling through Src-family kinase LCK, and predicted that inhibition of LCK and other kinases involved in T-cell signalling by dasatinib is responsible for the suppression of T-cell function. These findings raise the concern about potential T-cell inhibition in patients taking dasatinib, and suggest a possible application for the treatment of T-cell mediated immune disorders.

Inhibition of c-fms by Imatinib: Expanding the Spectrum of Treatment

Imatinib is a selective protein tyrosine kinase inhibitor currently used in the treatment of chronic myeloid leukaemia (CML). It specifically suppresses the growth of bcr-abl expressing CML progenitor cells by blocking the ATP-binding site of the kinase domain of bcr-abl. Imatinib also inhibits the c-abl, platelet derived growth factor receptor (PDGFR), abl-related gene and stem cell factor receptor, c-kit, protein tyrosine kinases. It is through inhibition of c-kit that imatinib is also used clinically in the treatment of gastrointestinal stromal tumours. We have recently demonstrated that imatinib also specifically targets the macrophage colony stimulating factor receptor, c-fms, at therapeutic concentrations. Although this finding has important implications with regard to potential side effects in patients currently receiving imatinib therapy, these results suggest that imatinib may also be useful in the treatment of diseases where c-fms is implicated. This includes breast and ovarian cancer and inflammatory conditions such as rheumatoid arthritis. We also speculate that imatinib may be used in diseases where bone destruction occurs due to excessive osteoclast activity, such as in the haematologic malignancy, multiple myeloma.

Resistance to c-KIT kinase inhibitors conferred by V654A mutation

Certain mutations within c-KIT cause constitutive activation of the receptor and have been associated with several human malignancies. These include gastrointestinal stromal tumors (GIST), mastocytosis, acute myelogenous leukemia, and germ cell tumors. The kinase inhibitor imatinib potently inhibits c-KIT and is approved for treatment of GIST. However, secondary point mutations can develop within the kinase domain to confer resistance to imatinib and cause drug-resistant relapse. A common mutation, which results in a V654A substitution, has been documented in imatinib-resistant GIST patients. We expressed c-KIT cDNA constructs encoding the V654A substitution alone and in combination with a typical activating exon 11 mutation characteristic of GIST, V560G, in factor-dependent FDC-P1 cells. The V654A substitution alone resulted in enhanced proliferation in c-KIT ligand (stem cell factor) but not factor independence. Cells expressing the double mutant were, like those expressing single V560G mutant c-KIT, factor independent. Analysis of cellular proliferation in the presence of imatinib showed that the V654A substitution alone conferred resistance. The difference in sensitivity was especially pronounced for cells expressing single mutant V560G c-KIT compared with double mutant V560G/V654A c-KIT. The findings were supported by studies of c-KIT phosphorylation. Analysis of the crystal structure of imatinib in complex with the kinase domain of c-KIT predicts that the V654A substitution directly affects the binding of imatinib to the receptor. Alternative c-KIT inhibitors, nilotinib (AMN107) and PKC412, were also less active on V560G/V654A c-KIT than on the V560G single mutant; however, nilotinib, like imatinib, potently inhibited the V560G mutant. PKC412 strongly inhibited imatinib-resistant D816V c-KIT. [Mol Cancer Ther 2007;6(3):1159–66]

CC Chemokine Ligand 20 and Its Cognate Receptor CCR6 in Mucosal T Cell Immunology and Inflammatory Bowel Disease: Odd Couple or Axis of Evil?

Chemokines and their cognate receptors have been identified as major factors initiating and governing cell movement and interaction. These ligands and their receptors are expressed on a wide variety of cells and act during steady-state migration as well as inflammatory recruitment. CCR6 is a non-promiscuous chemokine receptor that has only one known chemokine ligand, CCL20, and is present on B and T cells as well as dendritic cells (DCs). Two CD4+ T cell populations with opposing functions present in the intestines and the mesenteric lymph nodes express CCR6: the pro-inflammatory TH17 and regulatory Treg cells. CCL20 is also present in the intestine and is strongly up-regulated after an inflammatory stimulus. Interestingly, this ligand is also expressed by TH17 cells, which opens up the possibility of autocrine/paracrine signaling and, consequently, a self-perpetuating cycle of recruitment, thereby promoting inflammation. Recently, CCR6 has been implicated in inflammatory bowel disease (IBD) by genome wide association studies which showed an association between SNPs in the genomic region of the CCR6 gene and the inflammation. Furthermore, recent research targeting the biological function of CCR6 indicates a significant role for this chemokine receptor in the development of chronic IBD. It is therefore possible that IBD is facilitated by a disordered regulation of TH17 and Treg cells due to a disruption in the CCL20-CCR6 axis and consequently disturbed mucosal homeostasis. This review will summarize the literature on CCL20-CCR6 in mucosal immunology and will analyze the role this receptor-ligand axis has in chronic IBD.

Specific binding of human interleukin-3 and granulocyte-macrophage colony-stimulating factor to human basophils

The human T cell-derived cytokines interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were examined for their ability to bind to human basophils. Basophils were obtained from the peripheral blood of a patient with chronic myeloid leukemia undergoing basophilic differentiation after purification on a density gradient of metrizamide. Binding studies with 125I-labeled IL-3 and 125I-labeled GM-CSF demonstrated that basophils express a single class of high-affinity receptors for each of these molecules. Saturation binding curves with 125I-labeled IL-3 revealed that IL-3 bound specifically to basophils, and analysis according to the method of Scatchard revealed that basophils express 800 to 900 receptors per cell with an apparent dissociation constant of 2.6 x 10(-11) mol/L. Saturation-binding curves with 125I-labeled GM-CSF revealed that basophils express 100 to 200 receptors per cell with an apparent dissociation constant of 4 x 10(-11) mol/L. The demonstration of high-affinity receptors for IL-3 and GM-CSF on human basophils suggests a role for these cytokines in the regulation of basophil function.

Human myeloid differentiation antigens identified by monoclonal antibodies to the myelomonocytic leukemia cell line RC-2A.

&NA; Murine monoclonal antibodies to human myeloid cell surface differentiation antigens were prepared using the myelomonocytic leukemia cell line RC‐2A as immunogen. Using a highly sensitive colorimetric assay, antibodies were selected as myeloid‐associated based on their binding to RC‐2A cells, but not to cells of the autologous EBV‐transformed* B cell line Cess‐B. Antibodies to five distinct cell surface antigens were extensively characterized for their binding to normal and leukemic hemopoietic cells, and to tissue sections. Three antibodies may identify antigens previously described in the International Leucocyte Typing Workshops (CD14, CD11b and CD31). The other two antigens appear to be expressed at low levels on the surface of RC‐2A cells, and do not correspond to existing CD groups. One of these is also present on monocytes and neutrophils. Both were present on myeloid progenitor cells, as judged by depletion experiments with antibody and complement, although neither bound appreciably to myeloid leukemic cells as judged by indirect immunofluorescence. The other three antibodies bound preferentially to leukemic specimens displaying monocytic differentiation. Four of the antibodies could be demonstrated to bind to cells in frozen sections of tonsil and small intestine and all gave distinct patterns of reactivity. In particular, these antibodies differed markedly in their binding to endothelium, follicular dendritic cells and various types of tissue macrophages. These antibodies may be useful in the study of the differentiation of myeloid cells and in studies of immunologically mediated disease such as allograft rejection.

The immunomodulatory small molecule imiquimod induces apoptosis in devil facial tumour cell lines

The survival of the Tasmanian devil (Sarcophilus harrisii) is threatened by devil facial tumour disease (DFTD). This transmissible cancer is usually fatal, and no successful treatments have been developed. In human studies, the small immunomodulatory molecule imiquimod is a successful immunotherapy, activating anti-tumour immunity via stimulation of toll-like receptor-7 (TLR7) signaling pathways. In addition, imiquimod is a potent inducer of apoptosis in human tumour cell lines via TLR7 independent mechanisms. Here we investigate the potential of imiquimod as a DFTD therapy through analysis of treated DFTD cell lines and Tasmanian devil fibroblasts. WST-8 proliferation assays and annexin V apoptosis assays were performed to monitor apoptosis, and changes to the expression of pro- and anti-apoptotic genes were analysed using qRT-PCR. Our results show that DFTD cell lines, but not Tasmanian devil fibroblasts, are sensitive to imiquimod-induced apoptosis in a time and concentration dependent manner. Induction of apoptosis was accompanied by down-regulation of the anti-apoptotic BCL2 and BCLXL genes, and up-regulation of the pro-apoptotic BIM gene. Continuous imiquimod treatment was required for these effects to occur. These results demonstrate that imiquimod can deregulate DFTD cell growth and survival in direct and targeted manner. In vivo, this may increase DFTD vulnerability to imiquimod-induced TLR7-mediated immune responses. Our findings have improved the current knowledge of imiquimod action in tumour cells for application to both DFTD and human cancer therapy.