Phosphospecific flow cytometry for pharmacodynamic drug monitoring: Analysis of the JAK-STAT signaling pathway

Department of Internal Medicine, Erasmus MC, University medical Hospital Rotterdam, The Netherlands.
Clinica chimica acta; international journal of clinical chemistry (Impact Factor: 2.76). 01/2012; 413(17-18):1398-405. DOI: 10.1016/j.cca.2011.12.023
Source: PubMed

ABSTRACT Cytokines of the IL-2 receptor family act via activation of the JAK-STAT (janus tyrosine kinase-signal transducer and activator of transcription) signaling pathway. These cytokines are pivotal for the development and function of lymphocyte subsets involved in the immune response after organ transplantation including T, B and natural killer cells. The new small drug molecule and JAK1/3 inhibitor, tofacitinib, is currently being tested in phase II and III clinical trials for rheumatoid arthritis, psoriasis and in organ transplantation. This agent specifically targets the JAK-STAT signaling pathway. Here we discuss phosphospecific flow cytometry as a novel tool to monitor the JAK-STAT signaling pathway in kidney transplant patients and speculate that through the use of this pharmacodynamic tool the efficacy of immunosuppressive drugs can be assessed enabling optimization of the immunosuppressive therapy for individual transplant patients.

1 Follower
  • [Show abstract] [Hide abstract]
    ABSTRACT: Advanced single cell analysis technologies (e.g. Mass Cytometry) that help in multiplexing cellular measurements in limited volumes of primary cells are critical in bridging the discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multi-parametric single-cell analysis. Mass cytometers (also known as CyTOF or Cytometry by Time-of-Flight) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Standard flow cytometry is limited in the number of parameters that can be measured due to the overlap in signal when detecting the fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, requiring minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this perspective we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss emerging and potential applications of CyTOF technology in the pharmaceutical industry that include quantitative and qualititative deep profiling of immune cells and their use application in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies based on phenotype and/or function. We end with a discussion on the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering the drug's pharmacokinetics and pharmacodynamics relationship.
    Drug metabolism and disposition: the biological fate of chemicals 10/2014; 43(2). DOI:10.1124/dmd.114.060798 · 3.33 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Psoriasis is a chronic inflammatory disease of the skin. The causes of psoriasis are unknown, although family and twin studies have shown genetic factors to play a key role in its development. The many genes associated with psoriasis and the immune response include TNF α , IL23, and IL12. Advances in knowledge of the pathogenesis of psoriasis have enabled the development of new drugs that target cytokines (e.g., etanercept, adalimumab, and infliximab, which target TNF α , and ustekinumab, which targets the p40 subunit of IL23 and IL12). These drugs have improved the safety and efficacy of treatment in comparison with previous therapies. However, not all patients respond equally to treatment, possibly owing to interindividual genetic variability. In this review, we describe the genes associated with psoriasis and the immune response, the biological drugs used to treat chronic severe plaque psoriasis, new drugs in phase II and III trials, and current knowledge on the implications of pharmacogenomics in predicting response to these treatments.
    08/2013; 2013:613086. DOI:10.1155/2013/613086
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: T-cell depletion therapy is associated with diminished interleukin (IL)-7/IL-15-dependent homeostatic proliferation resulting in incomplete T-cell repopulation. Furthermore, it is associated with impaired T-cell functions. We hypothesized that this is the result of impaired cytokine responsiveness of T cells, through affected signal transducer and activator of transcription (STAT)5 phosphorylation and upregulation of coinhibitory molecules. Patients were treated with T cell-depleting rabbit antithymocyte globulin (rATG) (6 mg/kg, n = 17) or nondepleting, anti-CD25 antibody (basiliximab, 2 × 40 mg, n = 25) induction therapy, in combination with tacrolimus, mycophenolate mofetil, and steroids. Before and the first year after transplantation, IL-7 and IL-2 induced STAT5 phosphorylation, and the expression of the coinhibitory molecules programmed cell death protein 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), cluster of differentiation (CD) 160, and CD244 was measured by flow cytometry. The first year after rATG, CD4+, and CD8+ T cells were affected in their IL-7-dependent phosphorylation of STAT5 (pSTAT5) which was most outspoken in the CD8+ memory population. The capacity of CD4+ and CD8+ T cells to pSTAT5 in response to IL-2 decreased after both rATG and basiliximab therapy. After kidney transplantation, the percentage of TIM-3+, PD-1+, and CD160+CD4+ T cells and the percentage of CD160+ and CD244+CD8+ T cells increased, with no differences in expression between rATG- and basiliximab-treated patients. The decrease in pSTAT5 capacity CD8+ T cells and the increase in coinhibitory molecules were correlated. We show that memory T cells in kidney transplant patients, in particular after rATG treatment, have decreased cytokine responsiveness by impaired phosphorylation of STAT5 and have increased expression of coinhibitory molecules, processes which were correlated in CD8+ T cells.
    Transplantation 03/2015; DOI:10.1097/TP.0000000000000674 · 3.78 Impact Factor


Available from
Oct 20, 2014