doi:10.1182/blood-2013-09-5248272014 123: 594-595   

 Roland JacobsDebanjana Chatterjee, Dejene Milkessa Tufa, Heike Baehre, Ralf Hass, Reinhold Ernst Schmidt and mesenchymal stem cellsNatural killer cells acquire CD73 expression upon exposure to

http://bloodjournal.hematologylibrary.org/content/123/4/594.full.htmlUpdated information and services can be found at:

(5140 articles)Immunobiology   �Articles on similar topics can be found in the following Blood collections

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

Copyright 2011 by The American Society of Hematology; all rights reserved.Washington DC 20036.by the American Society of Hematology, 2021 L St, NW, Suite 900, Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly    

only.For personal use at UNIVERSITY OF NEWCASTLE on January 28, 2014. bloodjournal.hematologylibrary.orgFrom

only.For personal use at UNIVERSITY OF NEWCASTLE on January 28, 2014. bloodjournal.hematologylibrary.orgFrom

Correspondence

To the editor:

Natural killer cells acquire CD73 expression upon exposure to mesenchymal stem cells

We read with great interest the article by Saze et al,1 which waspublished recently in Blood as a plenary paper. The authors il-lustrated that human peripheral blood B cells express ectonucleo-tidases CD39 and CD73. Thus, B cells are capable of hydrolyzingadenosine triphosphate to adenosine 59-monophosphate (59-AMP)via CD39 and subsequently to adenosine (ADO) by means of CD73enzymatic activity.

In our laboratory, we study the interaction between human naturalkiller (NK) cells and mesenchymal stem cells (MSCs). Due to theirimmunosuppressive properties and inherent immunopriviledged na-ture, MSCs can be used as biological tolerance inducers in inflam-matory and autoimmune diseases.2,3 In this work, we coculturedfluorescence-activated cell sorter (FACS)-sorted allogeneic NK cells

with human umbilical cord (UC)-derived MSCs obtained from con-senting donors. We found that freshly isolated NK cells expressCD39, and the level of CD39 expression remains unaltered oncoculture with MSCs (Figure 1A). The percentage of CD73-expressingNK cells in peripheral blood is very low (;1%). However, oncoculture with MSCs, the percentage of CD731 NK cells increasessignificantly (Figure 1B). Thus, a subpopulation of NK cells acquiresthe ability to convert AMP into ADO on exposure to MSCs.

ADO has been shown to suppress inflammation by regulating var-ious immune cells.4,5 Saze et al1 also showed that 2-chloroadenosine(CADO), a stable ADO analog, could suppress B-cell functions likecytokine production, suggesting an autocrine regulatory role for ADOin B-cell physiology. To determine whether CADO also affects

Figure 1. CD39 andCD73 expression onNKcells and the effect of ADOanalog onNKcell functions.Blood sampleswere obtained from healthy volunteers, and peripheral

blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation. CD561CD32 NK cells were sorted out from PBMCs using FACS and cultured

overnight in the presence/absence ofMSCs. (A) CD39 and (B) CD73 expressionwas analyzed on theNK cells using flow cytometry. (C-D) NK cellswere stimulatedwith 1000 IU/mL

interleukin-2 (IL-2) in the presence/absence of 15mMCADO and incubated overnight. Brefeldin A was added to the cultures after 1 hour of incubation. The cells were then washed,

fixed, and stained intracellularly for (C) tumor necrosis factor (TNF)-a or (D) interferon (IFN)-g. (E) NK cells were cultured with/without 15 mMCADO overnight, washed, and further

incubatedwithK562cells for 4 hours. CD107a surface expressionwasanalyzedon theNKcells asa readout forNKcell degranulative capacity. (F-G)NKcells culturedwith orwithout

MSCs were cultivated in PBS with 59AMP as a substrate for 30 minutes at 37°C. Cell-free supernatants were collected and analyzed using chromatography with tandem mass

spectrometry for the levels of (F) residual 59-AMP (as a readout of substrate utilization) or (G) ADO (as a read out of product accumulation). Paired 2-tailed Student t tests were

performed using GRAPHPAD PRISM V5.00 software. Levels of significance are shown as P values (*P , .05, **P, .01). Bar graphs represent mean6 standard error of mean.

594 BLOOD, 23 JANUARY 2014 x VOLUME 123, NUMBER 4

only.For personal use at UNIVERSITY OF NEWCASTLE on January 28, 2014. bloodjournal.hematologylibrary.orgFrom

NK cell function, we cultured NK cells overnight with 1000 IU/mL ofIL-2 in the presence/absence of 15 mM CADO. We observed thatCADO significantly suppresses IL-2 induced tumor necrosisfactor-a and interferon-g production by NK cells (Figure 1C-D).We also studied the effect of overnight CADO treatment on un-stimulated NK cells. When these CADO pretreated NK cells wereexposed to K562 leukemic cells, the former exhibited significantlyreduced degranulation capacity compared with NK cells culturedwithout CADO (Figure 1E).

Finally, using mass spectroscopy, we could demonstrate thatup-regulation of ectonucleotidase CD73 on NK cells is also ac-companied by increased enzyme activity (Protocol as describedin supplemental Material 1). First, we could document increasedutilization of the CD73 substrate 59-AMP by NK cells that wereprecultured with MSCs (Figure 1F). Second, we could directlyestablish that NK cells precultivated with MSCs were able to producesignificantly higher amounts of ADO compared with NK cellscultured alone (Figure 1G).

MSCs have the ability to home to sites of inflammation invivo.6,7 NK cells interacting with these MSCs are likely to acquireCD73 expression. These CD731 NK cells have the potential toregulate NK cell activation in an autocrine or paracrine manner.This might have widespread implications for immunomodulationin the inflammatory microenvironment.

Debanjana Chatterjee

Department of Clinical Immunology and Rheumatology,

Hannover Medical School,

Hannover, Germany

Dejene Milkessa Tufa

Department of Clinical Immunology and Rheumatology,

Hannover Medical School,

Hannover, Germany

Heike Baehre

Institute of Pharmacology and Research Core Unit Metabolomics,

Hannover Medical School,

Hannover, Germany

Ralf Hass

Laboratory of Biochemistry and Tumor Biology,

Clinic of Obstetrics and Gynecology, Hannover Medical School,

Hannover, Germany

Reinhold Ernst Schmidt

Department of Clinical Immunology and Rheumatology,

Hannover Medical School,

Hannover, Germany

Roland Jacobs

Department of Clinical Immunology and Rheumatology,

Hannover Medical School,

Hannover, Germany

The online version of this article contains a data supplement.

Acknowledgments: This work was supported by the Deutsche

Forschungsgemeinschaft (DFG; German Research Foundation),

Sonderforschungsbereich, grant 738/A5, REBIRTH (Regenative Biology to

Reconstructive Therapy) by the Cluster of Excellence from the DFG, and

Niedersachsische Krebsgesellschaft.

Contribution:D.C., D.M.T., H.B., R.H., and R.J. performed experiments; D.C.,

H.B., and R.J. analyzed the results andmade the figures; D.C., R.E.S., and R.J.

designed the research; and D.C. wrote the letter.

Conflict-of-interest disclosure: The authors declare no competing financial

interests.

Correspondence: Roland Jacobs, Department of Clinical Immunology and

Rheumatology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625

Hannover, Germany; e-mail: jacobs.roland@mh-hannover.de.

References

1. Saze Z, Schuler PJ, Hong C-S, Cheng D, Jackson EK, Whiteside TL. Adenosineproduction by human B cells and B cell-mediated suppression of activatedT cells. Blood. 2013;122(1):9-18.

2. Voswinkel J, Francois S, Simon J-M, et al. Use of mesenchymal stem cells(MSC) in chronic inflammatory fistulizing and fibrotic diseases: a comprehensivereview. Clin Rev Allergy Immunol. 2013;45(2):180-192.

3. MacDonald GI, Augello A, De Bari C. Role of mesenchymal stem cells inreestablishing immunologic tolerance in autoimmune rheumatic diseases.Arthritis Rheum. 2011;63(9):2547-2557.

4. Hasko G, Linden J, Cronstein B, Pacher P. Adenosine receptors: therapeutic aspectsfor inflammatory and immune diseases. Nat Rev Drug Discov. 2008;7(9):759-770.

5. Cronstein BN. Adenosine, an endogenous anti-inflammatory agent. J ApplPhysiol (1985). 1994;76(1):5-13.

6. Karp JM, Leng Teo GS. Mesenchymal stem cell homing: the devil is in thedetails. Cell Stem Cell. 2009;4(3):206-216.

7. Deak E, Seifried E, Henschler R. Homing pathways of mesenchymal stromal cells(MSCs) and their role in clinical applications. Int Rev Immunol. 2010;29(5):514-529.

© 2014 by The American Society of Hematology

To the editor:

The FIP1L1-PDGFRA fusion gene and the KIT D816V mutation are coexisting in a small subsetof myeloid/lymphoid neoplasms with eosinophilia

Spindle-shaped tryptase1/CD251/CD1171/CD21/2mast cells (MCs)are a hallmark of the bone marrow (BM) in myeloid/lymphoidneoplasms harboring a FIP1L1-PDGFRA (FP) fusion gene.1,2 Theseneoplasms that may present as chronic eosinophilic leukemia (CEL),acute myeloid leukemia/blast phase, myeloid sarcoma, or lympho-blastic lymphoma/leukemia rarely fulfill the criteria of the WorldHealth Organization category “systemic mastocytosis with associatedhaematological non-mast cell disorders” (SM-AHNMD).1-5 Among123 SM-AHNMDpatients, only 12 carried an FP fusion gene.5 KITD816V analysis performed on 8 of these FP-positive patients andon 2 cases included in another study were negative.5,6 Thus, therelationship between MCs in FP-positive CEL and KIT D816V-positive SM remains elusive.

We microdissected tryptase1 MCs from formalin-fixed, EDTA-decalcified, paraffin-embedded BM trephine biopsies of 19 FP-positive CEL patients using a PALM MicroBeam (Carl ZeissMicroscopy, Oberkochen, Germany). DNA was extracted from 100to 250 pooled MCs per biopsy (QIAamp DNA Mini Kit; Qiagen,Hilden, Germany). KIT D816V mutation was evaluated by analyz-ing KIT exon 17 nested polymerase chain reaction products byLightCycler-based high-resolution melting and conventional directsequencing.6 Genomic DNA from the KIT D816V-positive MCleukemia cell line HMC-1 and NB4 cells harboring wild-type KITserved as controls. The polymerase chain reaction reactions and finalresults were evaluated in a blinded fashion. Low levels of KITD816V-mutated DNA ranging from,10% to 20% were detected in

BLOOD, 23 JANUARY 2014 x VOLUME 123, NUMBER 4 CORRESPONDENCE 595

only.For personal use at UNIVERSITY OF NEWCASTLE on January 28, 2014. bloodjournal.hematologylibrary.orgFrom