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ABSTRACT: The technique described in this unit uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE) to track proliferating cells. Covalently bound CFSE is divided equally between daughter cells, allowing discrimination of successive rounds of cell division. The technique is applicable to in vitro cell division, as well as to in vivo division of adoptively transferred cells and can resolve eight or more successive generations. CFSE is long lived, permitting analysis for several months after cell transfer, and has the same spectral characteristics as fluorescein, so monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes may be used to immunophenotype the dividing cells. In addition, information is given on a second-generation dye, Cell Trace Violet (CTV), excited by 405-nm blue laser light. CTV is chemically related to CFSE, but allows the 488-nm line of the Argon laser to be used for other probes. Curr. Protoc. Cytom. 64:9.11.1-9.11.12. © 2013 by John Wiley & Sons, Inc.
Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.] 04/2013; Chapter 9:Unit9.11.
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ABSTRACT: Development of small molecule tyrosine kinase inhibitors for the treatment of chronic myeloid leukemia has been astonishingly successful; however, their off-target effects have generated both challenges and opportunities for extending their clinical application. Dasatinib and imatinib are two of the most commonly used tyrosine kinase inhibitors and both have been shown to impact T-cell function. Due to this activity, their use as potential immune suppressants has been proposed. In this report, we investigated drug interactions with cyclosporine A in suppressing T-cell proliferation. Dasatinib and imatinib were titrated against varying concentrations of cyclosporine in the cultures and T-cell proliferation assessed by 5-6-carboxyfluorescein diacetate, succinimidyl ester dye dilution. These proliferation data were then used to determine the combination index to evaluate additive, synergistic, or antagonistic interactions between the drugs. This analysis uncovered a number of different drug interactions affecting T-cell proliferation. Cyclosporine had an additive or synergistic effect on T-cell proliferation when combined with dasatinib and imatinib for 3 of the 4 methods of stimulating T-cell proliferation. However, when T cells were stimulated with anti-CD3 and anti-CD28 antibodies, this interaction was found to be strongly antagonistic at low dasatinib concentrations. In contrast, this strong antagonism was not observed when imatinib was used in combination with cyclosporine A. This study suggests drug interactions affecting T cells may need to be carefully taken into account when using tyrosine kinase inhibitors. Furthermore, the technique to evaluate drug interactions is novel, and applicable to study any interaction affecting proliferation.
Experimental hematology 04/2012; 40(8):612-21.e6. · 3.11 Impact Factor
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ABSTRACT: Oncogene-specific downregulation mediated by RNA interference (RNAi) is a promising avenue for cancer therapy. In addition to specific gene silencing, in vivo RNAi treatment with short interfering RNAs (siRNAs) can initiate immune activation through innate immune receptors including Toll-like receptors, (TLRs) 7 and 8. Two recent studies have shown that activation of innate immunity by addition of tri-phosphate motifs to oncogene-specific siRNAs, or by co-treatment with CpG oligos, can potentiate siRNA antitumor effects. To date, there are no reports on applying such approach against human papillomavirus (HPV)-driven cancers. Here, we characterized the antitumor effects of non-modified siRNAs that can target a specific oncogene and/or recruit the innate immune system against HPV-driven tumors. Following the characterization of silencing efficacy and TLR7 immunostimulatory potential of 15 siRNAs targeting the HPV type 16 E6/E7 oncogenes, we identified a bifunctional siRNA sequence that displayed both potent gene silencing and active immunostimulation effect. In vivo systemic administration of this siRNA resulted in reduced growth of established TC-1 tumors in C57BL/6 mice. Ablation of TLR7 recruitment via 2'O-methyl modification of the oligo backbone reduced these antitumor effects. Further, a highly immunostimulatory, but non-HPV targeting siRNA was also able to exert antitumoral effects although for less prolonged time compared with the bifunctional siRNA. Collectively, our work demonstrates for the first time that siRNA-induced immunostimulation can have antitumoral effects against HPV-driven tumors in vivo, even independent of gene silencing efficacy.
Immunology and Cell Biology 03/2011; 90(2):187-96. · 3.66 Impact Factor
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Journal of Cellular and Molecular Medicine 04/2009; 13(3):599-601. · 4.13 Impact Factor
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ABSTRACT: Dasatinib (BMS-354825) is a small molecule Src/Abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Members of the Src family of kinases are involved in the induction of innate and adaptive immunity. The purpose of this study was to evaluate the inhibitory action of dasatinib on antigen-specific CD8(+) and CD4(+) T-cell function, as well as natural killer (NK) cell cytotoxicity.
To assess dasatinib-mediated inhibition of antigen-specific T-cell proliferation, transgenic CD4(+) and CD8(+) T cells specific for ovalbumin were utilized. Endogenous CD4(+) and CD8(+) T-cell responses were determined following immunization of dasatinib-treated or control mice with a nonreplicating recombinant virus. Clearance of the RMA-S cells, a major histocompatibility complex (MHC) class I-deficient thymoma sensitive to NK-cell lysis, was analyzed in mice undergoing dasatinib treatment.
Dasatinib inhibited antigen-specific proliferation of murine CD4(+) and CD8(+) transgenic T cells in vitro and in vivo. Endogenous antigen-specific helper T-cell recall responses and induction of T-cell-mediated cytotoxicity following immunization with a nonreplicating recombinant virus were also inhibited. So to was the ability of NK cells to eliminate MHC class I-deficient cells in vivo.
These findings suggest that dasatinib has the potential to modulate the host immune response at clinical doses and highlights scope for off target applications, e.g., therapeutic immunosuppression in the context of autoimmune pathogenesis and allogeneic tissue transplantation.
Experimental hematology 01/2009; 37(2):256-65. · 3.11 Impact Factor
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Blood 05/2008; 111(8):4415-6. · 9.90 Impact Factor
