Intracellular cytokine expression in T cells from patients with chronic lymphocytic leukemia

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Original research article/Praca oryginalna

Intracellular cytokine expression in T cells from patients with chronic lymphocytic leukemia

Ekspresja cytokin wewn ątrzkomórkowych w limfocytach T u chorych na przewlek łą bia łaczkę limfocytow ą

Monika Pieczykolan

^1,

*, Agnieszka Bojarska-Junak

¹

, Iwona Hus

²

, Justyna Wo ś

¹

, Sylwia Chocholska

²

, Karolina Olszewska-Bo żek

¹

, Waldemar Tomczak

²

, Pawe ł Czubak

³

, Jacek Roli ński

¹

1DepartmentofClinicalImmunology,MedicalUniversityHead,Prof.drhab.n.med.JacekRoliński,Lublin,Poland

2DepartmentofHaematooncologyandBoneMarrowTransplantation,MedicalUniversityofLublin,Poland

3DepartmentofGynaecologyandGynaecologicalEndocrinology,MedicalUniversityofLublin,Poland

Introduction

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia of adults in the Western World. It is characterizedby aheterogenousclinicalcourse withsurvi- val ranging from months to several years [1, 2]. In many casespatientsdonothaveanysymptomsforatleastafew

yearsandthediseaseisdiagnosedduringperiodicalroutine laboratory tests. Generally, two schemes of the disease course are adopted: the ﬁrst is slow and infrequently requires treatment, while the other is fast-growing, with poor prognosis and short survival [3]. The available treat- ments can often lead tothe remission of the disease, but mostpatientshaverelapses,whichimpliesthattheCLLstill remainsanincurabledisease.

article info

Articlehistory:

Received:23.05.2013 Accepted:16.06.2013 Availableonline:18.07.2013

Keywords:

CLL

T-cell

Cytokine

Słowakluczowe:

PBL

LimfocytyT

Cytokiny

abstract

FunctionaldisordersofTlymphocytes playanessentialroleinabnormalimmuneres- ponse in patients with chronic lymphocytic leukemia. The aim of this study was to assesstheprofileofcytokinesexpressedbyTcellsderivedfrompatientswithCLL.We have demonstratedthattheintracellular levels ofIL-2,IL-4,IFN-g,TNF,IL-6andIL-10 weresignificantlyhigherinTcellsofCLLpatientsthaninhealthydonors.Moreover,the percentagesofCD4+/CD3+/TNF+,CD4+/CD3+/IFN-g+,andCD4+/CD3+/IL-2+cellsweresig- nificantlyhigherinZAP-70-positivepatientscomparedwithZAP-70-negativeones.Like- wise,significantlyhigherpercentagesofCD4+/CD3+/TNF+,CD4+/CD3+/IFN-g+cellswere observed in CD38-positive than in CD38-negative cases. What is more, there was a significantdifference inmedian percentageof CD3+/CD4+cellsexpressingTNF,IL-4, IFN-g, IL-2 or IL-6 between patients carrying the 11q22.3 deletion and/or the 17p13.1 deletionandpatientswithoutthesegeneticaberrations.Ourresultsconfirmthefunctio- naldisordersofTcellsinCLLandtheirinfluenceontheclinicalcourseofthedisease.

*Correspondingauthorat:ul.Chodźki4a,20-093Lublin,Poland.Tel.:+48817564854;fax:+48817564840.

E-mailaddress:mpieczykolan@vp.pl(M.Pieczykolan).

ContentslistsavailableatSciVerseScienceDirect

Acta Haematologica Polonica

journalhomepage:www.elsevier.com/locate/achaem

http://dx.doi.org/10.1016/j.achaem.2013.07.002

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Chroniclymphocyticleukemiaischaracterizedbyexces- sive proliferation and accumulation of the long-lived mor- phologically mature but immunologically incompetent malignant-altered B lymphocytes, in blood, bone marrow andperipherallymphoidorgans[1].Theclinicaldiagnosisof CLLrequiresanassessmentofmonoclonalB-cellpopulation inperipheralbloodwithaminimumthresholdofmorethan 5000cells/ml[4].Atypicalphenotypecharacteristicofleuke- miccellsisaco-expressionofBlymphocyteantigens(CD19, CD20,CD23)andTcellmarkerCD5[5–7].

Inchroniclymphocyticleukemiathereare manyimmu- nological disorders of both the humoral and the cellular immune response that are considered to be involved in diseasepathogenesis.

Numerousobservationsindicatethatthelong-livedinvivo CLLcellsquicklyundergospontaneousapoptosiswhenthey areculturedinvitro[8].Thissuggeststhatinvivo,factorslike cytokinessigniﬁcantlyaffecttheactivation,proliferationand extended survival [9–11]. Therefore, in this study we have focusedon theassessmentof cytokines secreted byT cells withagrowth-promotingpotential,including:tumornecrosis factor(TNF)[12–14],interleukin2(IL-2)[15],interleukin6(IL- 6) [16–18],interferon IFN-gand interleukin 4(IL-4)and play aroleinthe interactionsbetweentumor cellsandimmune effectorcells[19].Moreover,weanalyzedintracellularexpres- sion of IL-10 which was found to be associated with CLL progression [20] and IL-12 was considered asan important elementofanti-tumorresponseaswell[21].

Patients

Fifty-ﬁve newly diagnosed and previously untreated, con- secutive,patientswithCLLwereenrolledforthisstudy.The diagnosisof CLLwasmadeonthebasis ofNCI-WGcriteria [22]. There were21 women and 34 men,with the median age of 66 years (range 40–83). At the time of diagnosis, patientswerestagedaccordingtotheRaistagingsystemas follows:stage0–21cases,stage1–15cases,stage2–12cases, stage3–4cases and stage4–3case.Cytogeneticanalysisat thetimeof testingwasavailablefor36 outof the55study patients.Thepatientswereassignedtotwogroupsaccord- ingtothisanalysis.Theﬁrstgroupconsisted of11patients who had 11q22.3 deletion and/or 17p13.1 deletion. The second groupconsisted of the remaining 25 patientswith- outtheseunfavorablecytogeneticabnormalities.

Controlgroupconsistedof10age-matchedhealthyvolun- teers(HV).TheLocalEthicalCommittee approvedtheproto- colofthestudyandallthepatientssignedinformedconsent.

Monoclonalantibodiesandotherreagents

The following ﬂuorochrome-conjugated monoclonal antibo- dies (MoAbs) were used for the detection of intracellular cytokines:IL-12PE,IL-2PE,IL-4PE,TNFPE,IFN-gPE,IL-10PE andIL-6PE(BectonDickinson,USA).For theidentiﬁcationof cellsurfaceantigensthefollowingantibodieswereused:anti- CD3PerCP,anti-CD4FITC,(BD,USA).TheIgG1FITCPEPerCP –conjugatemousemAbsfromBectonDickinsonwasusedas a negative control. Phorbol 12-mysistate 13-acetate (PMA), ionomycinandbrefeldinA(BFA)werepurchasedfromSigma.

CellpreparationandinvitrostimulationofTcells

All the peripheralblood sampleswere collected intohepar- inized tubes and immediately processed. Mononuclear cells were isolatedbythedensity gradientcentrifugationonGra- disolL(AquaMedica,Poland).Interphasecellswereremoved and washed twice in phosphate-buffered saline (PBS). Cells from every sample were cultured in a complete culture medium (RPMI 1640 supplemented with fetal calf serum 100IUmL ¹penicillin,100mgmL ¹streptomycin)for4h.The cells were stimulated with a mixture containing phorbol myristateacetate(PMA,25ng/ml),ionomycin(1mg/ml).Cyto- kine secretion was blocked with brefeldin (10mg/ml). The incubatorsweresetat378Cina5%CO2environment.More- over, this procedure was also performed on non-activated lymphocytesusingonlybrefeldinAinordertoassessthelevel ofresidualcytokinesynthesisfrominvitroactivation.

Surfaceandintracellularstaining

Forsurfacestaining,culturedPBMCs werewashed twicein PBSandthenincubatedwithappropriateMoAbsspecificfor CD3and CD4for 30minat 48C.After surfacestaining,the cells were washed, subsequently fixed and permeabilized withBD Cytofix/Cytoperm Fixation/Permeabilizationkit (BD Biosciences). Afterwards, the cells wereincubated with an appropriate amount of MoAbs for intracellular cytokine staining (20min at 48C in the dark). The cells were then washedtwicewithPBS,andanalyzedbyflowcytometry.

Flowcytometricanalysis

The samples were analyzed by ﬂow cytometry using aBectonDickinsonFACSCaliburinstrument.Fivedatapara- meterswereassessed:linearforward andsidescatter(FSC, SSC), FL-1(FITC), FL-2(PE) and FL-3 (PerCP). An acquisition gatewasestablishedontheirforwardandsidelightscatter properties (R1 region), which included mononuclear cells and excluded dead cells and debris (Fig. 1). The R1 gated events wereanalyzed for CD3 positive cells (R2). Theﬁnal dotplots,usingR1andR2regions,representthepercentage ofCD3+/CD4+cellsexpressingcytoplasmiccytokines.

For each analysis, 10,000 events were acquired and analyzed using CellQuest Pro software. Isotype-matched antibodieswereusedtoverifythestainingspeciﬁcityandas a guide for setting the markers to delineate positive and negativepopulations.

FlowcytometricanalysisofCD38andZAP-70expressionin CLLcells

CLL cellswerestainedfor CD38antigenandZAP-70protein expression (asdescribed previously [23]). Thecut-off point forCD38orZAP-70positivityinleukemiccellswas20%.

Statisticalanalysis

The Mann–Whitney U test was applied for statisticalcom- parisonoftheresultsbetweenCLLpatientsandHV,aswell as betweenCLL patients indifferentstages of the disease.

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Thedatawereanalyzedusing STATISTICA6.0 softwarefor Windows. Differences were considered statistically signiﬁ- cantwithap-value0.05.

Results

Wemeasuredtheintracellular cytokinesexpressionofIL-2, IL-4, IFN-g,TNF, IL-6, IL-10and IL-12inT cells of patients

with CLL and healthy control group. Representative FACS proﬁles showing intracellular staining of spontaneous and in vitroactivatedcellsare showedinFig. 2. InCLLpatients aswell asinhealthycontrol,thepercentage ofCD4+/CD3+

cellswithintracellularcytokineexpressioninnon-activation assays wasfrequently lowerthan1%,comparable withthe levelofautoﬂuorescence.

ThemeanpercentagesofCD3+CD4+cellpositiveforIL-2, IL-4, IFN-g,TNF, IL-6and IL-10weresigniﬁcantly higherin Fig.1–ThedotplotshowsrepresentativedatefromoneCLLpatient,illustratingtheanalysismethodforidentificationof CD4+Tcellsexpressingappropriatelycytokinesfollowingthree-colorstaining.(a)Thedotplotshowstheforwardscatter/

sidescatter(FSC/SSC)distributionandthegate(regionR1)usedtoselectlymphocytesforanalysis.(b)TheR1gateevents werethenanalyzedforCD3positivecells(CD3+)weregated(regionR2).ThedotplotshowstheSSCvs.CD3PerCP distribution.(c)ThefinaldotplotCD4FITCvs.IFNPE,wasestablishedbycombinedgatingofeventsusingR1andR2.The numberintheupperrightquadrantonthedotplotrepresentsthepercentageofCD3+/CD4+cellsexpressingcytoplasmicIFN Ryc.1–Przykładowyobrazzcytometruprzepływowego,przedstawiającymetodęanalizykomórekTCD4+wykazującychekspresję cytokin.(a)Obrazprzedstawiającyrozkładkomórekwzależnośćodwielkościiziarnistości(FSC/SSC),regionR1zawieralimfocyty.

(b)ZregionuR1wyodrębnionokomórkiCD3-pozytywneioznaczonojejakoR2.ObrazprzedstawiazależnośćSSCodCD3PerCP.(c) Końcowyobraz,przedstawiającyprocentkomórekCD3+/CD4+wykazującychekspresjęIFN-g,zawierakomórkizrejonówR1iR2.

Komórkiwgórnymprawymrogu,reprezentująodseteklimfocytówCD3+/CD4+zcytoplazmatycznąekspresjąIFN-g

Fig.2–IntracellularIL-2expressioninCD3+/CD4+cellsfromonerepresentativeCLLpatent.ExpressionofintracellularIL-2 withoutstimulation(a),andwithPMAandionomycinstimulation(b)

Ryc.2–PrzykładowyobrazprzedstawiającyodsetekkomórekCD3+/CD4+zwewnątrzkomórkowąekspresjąIL-2.EkspresjaIL-2 przed(a)ipostymulacjiPMAijonomycyną(b)

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CLL patients than in healthy controls (p<0.01) (Table I).

However,no signiﬁcantdifferenceswereobserved between these groups in the percentage of CD3+CD4+ cells expres- singIL-12.

The low-risk group (Rai stage 0) and the intermediate- risk group (Rai stages I and II) CLL patients expressed statistically signiﬁcant higher levels of IL-4, IFN, TNF, IL-6 andIL-10than healthyindividuals(p<0.05).Moreover,CLL patients in the Rai stages 0-II showed a higher mean expressionofIL-10andIL-6whencomparedtothegroupof patients in more advanced stages of the disease (III–IV) (Table II). However,these differenceswere notstatistically signiﬁcant.

Inourstudy,thepercentagesofCD4+/CD3+/TNF+,CD4+/

CD3+/IFN-g+, and CD4+/CD3+/IL-2+ cells were signiﬁcantly higher in ZAP-70-positive patients compared with ZAP-70- negative patients (Table III). Likewise, signiﬁcantly higher percentages of CD4+/CD3+/TNF+, CD4+/CD3+/IFN-g+ cells were observed in CD38-positive than in CD38-negative patients(TableIII).

Cytogeneticanalysisat thetimeoftestingwasavailable for 46out ofthe 55studypatients.Therewas asigniﬁcant

differenceinmedianpercentageof CD3+/CD4+cellsexpres- sing TNF,IL-4, IFN-g,IL-2or IL-6betweenpatientscarrying the 11q22.3 deletion and/or the 17p13.1 deletion and patientswithoutthesegeneticaberrations (Table IV).There were no signiﬁcant differences in CD3⁺/CD4⁺/IL-10⁺ and CD3⁺/CD4⁺/IL-12⁺cellpercentagesbetweenpatientswithdel (11q22.3) or/and del(17p13.1) and patients without these unfavorablegenetic aberrations(TableIV).Additionally,the percentage of T lymphocytes expressingall analyzed cyto- kineswashigherinthepatientwithdel(17p13.1)thaninthe patients with del(11q22.3)(Table V).However, these differ- enceswerenotstatisticallysigniﬁcant.

Discussion

For manyyears, the main function inanticancer response wasattributedtocytotoxicTlymphocytes(CD8+,Tc).Nowa- daysitisknownthatThelperlymphocytes(CD4+,Th)play an essential role in the regulation of cell mediated anti- tumor responses while their respective populations may inhibit or promote tumor growth through the cytokine secretion proﬁle. In this study, we have undertaken the assessment of the functional T helper cell function in patients with chroniclymphocytic leukemia, by evaluating theircytokinesecretion.

We have measured the intracellular expression of selected cytokines in CLL cells, demonstrating that the levels of IL-2, IL-4, IFN-g, TNF, IL-6 and IL-10 after in vitro TableI–MedianpercentageofCD3⁺CD4⁺cellswith

intracellularTNF,IFN-g,IL-4,IL-6,IL-2,IL-12andIL-10 expressioninperipheralbloodfromCLLpatientsand healthycontrol

TabelaI–ŚredniodseteklimfocytówCD3⁺CD4⁺zwe- wnątrzkomórkowąekspresjąTNF,IFN-g,IL-4,IL-6,IL-2, IL-12iIL-10wkrwiobwodowejpacjentówzPBLizdrowych dawców

Variable CLL Healthycontrol p-Value

CD3⁺CD4⁺TNF⁺ 15.17 5.46 0.003

CD3⁺CD4⁺IFN-g⁺ 6.70 4.24 0.002

CD3⁺CD4⁺IL-4⁺ 1.46 0.68 0.001

CD3⁺CD4⁺IL-10⁺ 1.06 0.88 0.009

CD3⁺CD4⁺IL-6⁺ 0.86 0.47 0.001

CD3⁺CD4⁺IL-2⁺ 13.85 8.41 0.036

CD3⁺CD4⁺IL-12⁺ 1.30 2.24 0.167

Thep-valuewas calculatedusingtheUMann–Whitneytest (p- valueof<0.05wasconsideredstatisticallysigniﬁcant).

Wartośćpobliczono,stosująctest UManna–Whitneya(p<0.05 uznanozaistotnestatystycznie).

TableII–MedianpercentageofCD3⁺CD4⁺cellswith intracellularTNF,IFN-g,IL-4,IL-6,IL-2,IL-12andIL-10 expressioninperipheralbloodfromCLLpatientsin differentstagesofdisease

TabelaII–ŚredniodseteklimfocytówCD3⁺CD4⁺zwe- wnątrzkomórkowąekspresjąTNF,IFN-g,IL-4,IL-6,IL-2, IL-12iIL-10wkrwiobwodowejpacjentówzPBLwróżnych stadiachzaawansowaniachoroby

Variable 0 I–II III–IV

CD3⁺CD4⁺TNF⁺ 15.78 11.55 21.10

CD3⁺CD4⁺IFN-g⁺ 6.70 6.54 9.28

CD3⁺CD4⁺IL-4⁺ 1.48 1.43 3.16

CD3⁺CD4⁺IL-10⁺ 1.07 1.04 1.00

CD3⁺CD4⁺IL-6⁺ 0.86 0.87 0.45

CD3⁺CD4⁺IL-2⁺ 12.63 13.88 23.99

CD3⁺CD4⁺IL-12⁺ 1.47 1.02 NE

TableIII–MedianpercentageofCD3⁺CD4⁺cellswith intracellularTNF,IFN-g,IL-4,IL-6,IL-2,IL-12andIL-10 expressioninperipheralbloodfromCLLpatientswith differentZAP-70andCD38antigenexpressions

TabelaIII–ŚredniodsetekkomórekCD3⁺CD4⁺zwewnątrz- komórkowąekspresjąTNF,IFN-g,IL-4,IL-6,IL-2,IL-12iIL- 10wkrwiobwodowejpacjentówzPBLwykazującychróżną ekspresjąantygenówZAP-70iCD38

Variable ZAP-70-

n=32

ZAP-70+

n=23

p-Value

CD3⁺CD4⁺TNF⁺ 43.14 51.55 0.01

CD3⁺CD4⁺IFN-g⁺ 16.33 22.28 0.02

CD3⁺CD4⁺IL-4⁺ 3.28 3.11 0.14

CD3⁺CD4⁺IL-10⁺ 1.55 1.57 0.49

CD3⁺CD4⁺IL-6⁺ 1.80 1.01 0.61

CD3⁺CD4⁺IL-2⁺ 17.01 26.08 0.01

CD3⁺CD4⁺IL-12⁺ 1.4 1.76 0.167

CD38- n=30

CD38+

n=25

p-Value

CD3⁺CD4⁺TNF⁺ 40.05 49.68 0.014

CD3⁺CD4⁺IFN-g⁺ 16.00 19.40 0.035

CD3⁺CD4⁺IL-4⁺ 3.45 2.78 0.16

CD3⁺CD4⁺IL-10⁺ 1.55 1.58 0.27

CD3⁺CD4⁺IL-6⁺ 17.39 20.59 0.052

CD3⁺CD4⁺IL-2⁺ 1.08 1.02 0.43

CD3⁺CD4⁺IL-12⁺ 1.39 1.94 0.067

Thep-valuewascalculated usingtheUMann–Whitneytest(p- valueof<0.05wasconsideredstatisticallysigniﬁcant).

Wartośćpobliczono,stosując testUManna–Whitneya (p<0.05 uznanozaistotnestatystycznie).

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stimulationweresigniﬁcantlyhigherinCLLthaninnormal cells. Our results suggest that the production of studied cytokinesbyTcellsmayplay arole inthemaintenanceof leukemiccellssurvivalandproliferation.

WehavealsofoundsignificantlyhigherintracellularIL-2 expressioninCD3+/CD4+T cellsfrom CLLpatientsthan in healthyindividuals,whichconfirmsearlierresultsofRooss- manns et al. who showed a significantly higher level of spontaneousproductionofIL-2inpatientswithleukemiaas compared to the control group [24]. In contrast, other investigators such asGallego et al. detecteda significantly lowerpercentageof CD4+cellsexpressingIL-2after activa- tionwithPMAandionomycininCLLpatientsascompared tothecontrolgroup[25].Theauthorsconfirmtheresultsby theELISAtest.Furthermore,incontrasttotheresultsofthe presented study, they found no differencesin the level of

intracellular cytokines such as TNF and IFN between the study and the control group. These discrepancies might result from the differentmethods used for the analysis of cytokines expression as well as the heterogeneity of the disease.

In ourstudywehave foundno correlationbetweenthe stage of thedisease and thelevel of cytokines secretedby Tcells.DifferentresultswerepresentedbyHulkkonenetal.

[26], who showed variations in the secretion of TNF and IL-6, depending on the stage of the disease. The level of secreted cytokines after stimulation with PMA was signiﬁ- cantly lower in the more advanced stages of the disease, accordingtoBinet'sclassiﬁcation.

Currently thereis a general consensus that the expression ofZAP-70 protein and CD38antigen byleukemic cells belongstotheimportant prognosticmarkers inCLL.Inour study,thehigherpercentageofTcellsexpressingTNF,IFN- gandIL-2intheZAP-70+andCD38+incomparisontoZAP- 70– and CD38- group indicates that the production of cytokinesissignificantlyconnected withtheclinicalcourse ofdisease;themoreaggressiveZAP-70+orCD38+CLLcases are characterizedby a highercapability for the production of cytokines responsible for disease pathogenesis. What is more, the intracellular cytokine expressions were significantly higherinpatientswithhigh-riskcytogeneticaberra- tions such asdeletionof 17por11q. Apartfrom providing insightsintothepathogenesis,genomicaberrationsidentify subgroups of patients with distinct clinical pictures: lym- phadenopathy (11q deletion) or resistance to therapy (17p deletion). Deletions at 11q and particularly 17p are associated with rapid disease progression or inferior survival [27].Genomicaberrations(i.e.,11qand17pdeletion)helpto define biological and clinical subgroups. In our study the intracellular cytokine expressions were higher in patients withdel(11q22.3) or/anddel(17p13.1)thanin patientswithout these unfavorablegenetic aberrations.Deletionsof 17p and 11qhave been associated withunfavorable prognosis.

Nevertheless, the deletion of 17p was described as the strongest independent predictor for aggressive behavior, resistance to chemotherapy and early death [27]. In our studyweobservedahigherintracellularcytokineexpression in patients with 17p deletion than in patients with 11q deletion. However, this difference was not statistically signiﬁcant.Inourstudythecytogeneticanalysisatthetime of testingwasavailablefor 36outof the55studypatients.

It shouldbenoted thatthe group of patientswith11q22.3 deletion or 17p13.1 deletion had only 6 and 5 patients, respectively. We can suppose that future analysis is requiredinmorenumerousgroupsofpatients.

CLLpatientsshowmuchlowerpercentagesofcirculating T-lymphocytes than it is observed in normal peripheral blood samples [28]. Although the relative percentage of T cells in the peripheral blood is decreased, the absolute number isfrequentlyincreased. Ourdata suggestthat itis verylikelythatmalignantcells,whosenumberisextremely high in the blood of patients with leukemia, regulate the productionofcytokinesbyTlymphocytes.

In summary, the possible large impairment of T lymphocyte function in the laterstages, ascompared to theearlystagesofthedisease,mayaffectcytokinesecretion TableIV–MedianpercentageofCD3⁺CD4⁺cellswith

intracellularTNF,IFN-g,IL-4,IL-6,IL-2,IL-12andIL-10 expressioninperipheralbloodfromCLLpatientssub- dividedaccordingtocytogeneticanalysis

TabelaIV–ŚredniodseteklimfocytówCD3⁺CD4⁺zwe- wnątrzkomórkowąekspresjąTNF,IFN-g,IL-4,IL-6,IL-2, IL-12iIL-10wkrwiobwodowejpacjentówzPBL,podzielo- nychwedługanalizycytogenetycznej

Variable Patientswith del(11q22.3)

or/and del(17p13.1)

n=11

Patientswithout thesegenetic

aberrations n=25

p-Value

CD3⁺CD4⁺TNF⁺ 56.38 44.16 0.01

CD3⁺CD4⁺IFN-g⁺ 31.12 11.10 0.0004

CD3⁺CD4⁺IL-4⁺ 5.71 2.52 0.001

CD3⁺CD4⁺IL-10⁺ 1.50 1.52 0.60

CD3⁺CD4⁺IL-6⁺ 2.61 0.92 0.002

CD3⁺CD4⁺IL-2⁺ 29.61 17.17 0.03

CD3⁺CD4⁺IL-12⁺ 0.25 1.79 0.43

Thep-valuewas calculatedusingtheUMann–Whitneytest(p- valueof<0.05wasconsideredstatisticallysigniﬁcant).

Wartośćpobliczono,stosując testUManna-Whitneya(p<0.05 uznanozaistotnestatystycznie).

TableV–MedianpercentageofCD3⁺CD4⁺cellswith intracellularTNF,IFN-g,IL-4,IL-6,IL-2,IL-12andIL-10 expressioninperipheralbloodfromCLLpatientswithdel (11q22.3)anddel(17p13.1)

TabelaV–ŚredniodseteklimfocytówCD3⁺CD4⁺zwe- wnątrzkomórkowąekspresjąTNF,IFN-g,IL-4,IL-6,IL-2, IL-12iIL-10wkrwiobwodowejpacjentówzPBLzdel (11q22.3)idel(17p13.1)

Variable Patientswith

del(11q22.3) n=6

Patientswith del(17p13.1)

n=5

CD3⁺CD4⁺TNF⁺ 54.43 56.60

CD3⁺CD4⁺IFN-g⁺ 24.34 37.91

CD3⁺CD4⁺IL-4⁺ 5.79 5.46

CD3⁺CD4⁺IL-10⁺ 1.39 1.68

CD3⁺CD4⁺IL-6⁺ 2.11 4.11

CD3⁺CD4⁺IL-2⁺ 28.00 29.92

CD3⁺CD4⁺IL-12⁺ 0.25 1.78

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proﬁle which is involved in the clinical course of chronic lymphocyticleukemia.

Authors' contributions/Wkład autorów

Accordingtoorder.

Conﬂict of interest/Konﬂikt interesu

Nonedeclared.

Financial support/Finansowanie

Thisworkwassupportedbyresearchgrants:NN402351438, N N402 439139, N N404 682440, N N402 683140 and from State Funds for Scientiﬁc Research of National Science Centre(NCN).

The paper was developed using the equipment pur- chased within the Project: ‘‘The equipment of innovative laboratories doing research of new medicines used in the therapy of civilization and neoplastic diseases’’ within OperationalProgram Development of Eastern Poland2007– 2013,PriorityAxisIModernEconomy,OperationsI.3Innova- tionPromotion.

Ethics/Etyka

Thework described inthis article has been carriedout in accordance with The Code of Ethics of the World Medical Association(Declaration of Helsinki)for experimentsinvol- ving humans; EU Directive 2010/63/EU for animal experi- ments;UniformRequirementsformanuscriptssubmittedto Biomedicaljournals.

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