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Demonstration of MPLW515K, but not JAK2V617F, in in vitro expanded CD4+ T lymphocytes

Leukemia volume 21, pages 2206–2207 (2007)Cite this article

JAK2V617F is a recurrent mutation in the classic BCR-ABL-negative myeloproliferative disorders (MPDs); allelic frequency is estimated at 95% for polycythemia vera (PV) and 50% each for essential thrombocythemia (ET) and primary myelofibrosis (PMF).1 More recently, other JAK2 mutations (exon 12 mutations) have been described in JAK2V617F-negative PV and MPL mutations (MPLW515L/K) in PMF and ET. JAK2V617F occurs in hematopoietic stem cells and myeloid-lineage cells, and some studies have suggested lymphocyte involvement as well: in natural killer,2 as well as in T and B lymphocytes.3 However, T-cell involvement was not recognized in many of the initial JAK2V617F studies,4, 5 including those that used sensitive allele-specific (AS)-PCR assays.6 Obviously, a negative study in this regard does not exclude the possibility that T-cell involvement is either limited to only a subset of JAK2V617F-positive cases, or is accompanied by an allele burden that is below the limit of detection for a particular mutation screening assay, especially if PCR sequencing is used alone. At the same time, however, the use of ultrasensitive assays could produce false-positive results in the absence of a myeloid cell contamination-free T-cell enrichment process.

To clarify the issue of T-lymphocyte involvement, we prospectively studied four PMF patients harboring MPLW515 mutations (two each with MPLW515L and MPLW515K), and two PV patients harboring homozygous JAK2V617F. The current study was approved by our institutional review board, and all patients provided verbal and written informed consent for study participation. In each case, peripheral blood mononuclear cells (PBMC) were cultured in vitro to yield CD4+ T lymphocytes. PBMC or granulocytes were isolated by double Ficoll separation as described previously.5 T-lymphocyte expansion was performed using the Dynabeads® CD3/CD28 T cell expander kit (Dynal, Oslo, Norway), as per protocol. Briefly, previously frozen PBMC were thawed and plated in X-vivo medium (Lonza, Walkersville, MD, USA) with 5% fetal calf serum (FCS) at 2 × 106 cells/ml. Dynabeads were added (150 μl) and cells incubated at 37°C. On day 3, human recombinant interleukin (rIL)-2 (100 U/ml) was added. On days 5 and 7, the cells were washed in a Big Easy EasySep® magnet (Stem Cell Technologies, Vancouver, CA, USA), and re-suspended in culture medium with IL-2 at 0.5 × 106 cells/ml. Cells were harvested on day 9 for flow cytometric analysis of purity as follows: an aliquot of expanded T lymphocytes was resuspended in 1 × phosphate-buffered saline + 3% FCS, and stained with CD4-FITC or isotype control antibodies in the dark at room temperature for 15 min. T-lymphocyte purity was analyzed using a FACS Calibur flow cytometry with CellQuest Pro Software (BD Biosciences, San Jose, CA, USA).

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  1. Division of Hematology, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA

    A Pardanani, T L Lasho, C Finke, S N Markovic & A Tefferi

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  1. A Pardanani
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  2. T L Lasho
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Correspondence to A Pardanani.

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Pardanani, A., Lasho, T., Finke, C. et al. Demonstration of MPLW515K, but not JAK2V617F, in in vitro expanded CD4+ T lymphocytes. Leukemia 21, 2206–2207 (2007). https://doi.org/10.1038/sj.leu.2404749

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