TACI expression and signaling in chronic lymphocytic leukemia

Research ArticleTACI Expression and Signaling in ChronicLymphocytic Leukemia

Antigoni Mamara1 Anastasios E Germenis1 Maria Kompoti1 Maria Palassopoulou2

Eudokia Mandala3 Anastasia Banti4 Nikolaos Giannakoulas2 and Matthaios Speletas1

1Department of Immunology amp Histocompatibility Faculty of Medicine School of Health Sciences University of ThessalyBiopolis 3 41500 Larissa Greece2Department of Hematology Faculty of Medicine School of Health Sciences University of Thessaly Biopolis 3 41500 Larissa Greece3Fourth Department of Internal Medicine Ippokration Hospital Aristotle University of Thessaloniki Konstantinoupoleos 4954642 Thessaloniki Greece4Department of Hematology PapageorgiouGeneral Hospital Periferiaki OdosThessalonikis Nea Efkarpia 56429Thessaloniki Greece

Correspondence should be addressed to Matthaios Speletas maspelmeduthgr

Received 20 November 2014 Accepted 15 March 2015

Academic Editor Jacek Tabarkiewicz

Copyright copy 2015 Antigoni Mamara et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

TACI is a membrane receptor of BAFF and APRIL contributing to the differentiation and survival of normal B cells Althoughmalignant B cells are also subjected on TACI signaling there is a remarkable intradisease and interindividual variability of TACIexpression in B-cell malignancies The aim of our study was to explore the possible role of TACI signaling in the biology ofchronic lymphocytic leukemia (CLL) including its phenotypic and clinical characteristics and prognosis Ninety-four patientsand 19 healthy controls were studied CLL patients exhibited variable TACI expression with the majority of cases displaying lowto undetectable TACI along with low to undetectable BAFF and increased APRIL serum levels compared to healthy controls CLLcells with high TACI expression displayed a better survival capacity in vitro when cultured with BAFF andor APRIL MoreoverTACI expression was positively correlated with the presence of monoclonal gammopathy and inversely with CD11c expressionTherefore our study provides further evidence for the contribution of BAFFAPRIL signaling to CLL biology suggesting also thatTACI detection might be useful in the selection of patients for novel targeting therapeutic approaches

1 Introduction

B-cell chronic lymphocytic leukemia (CLL) is the mostcommon hematologic malignancy in theWestern world Thedisease is characterized by proliferation and accumulation ofCD5+ CD19+ CD20low and surface immunoglobulin (Ig)lowexpressing B cells with balanced homeostasis in patientswith stable lymphocyte count and imbalanced homeostasisin patients with increasing lymphocyte count [1 2] CLL isan incurable and heterogeneous disease clinically dividedinto two major entities based on the presence or absence ofsomatic mutations in the variable region of the immunoglob-ulin heavy-chain (IGHV) gene [3 4] There is no uniquegenetic defect identified in CLL although over 80 of thepatients present with cytogenetic lesions [5 6]

Signals delivered by direct cell contact or soluble factorswhich may or may not occur concurrently with B-cell-receptor engagement propagate the growth of CLL cellsThe disruption of this cross talk is an attractive novelstrategy for treating the affected patients [7 8] Recentstudies have demonstrated that tumor necrosis factor (TNF)superfamily members may contribute to the enhanced sur-vival potential of malignant lymphocytes In particularneoplastic B cells express variable protein levels of B-cell-activating factor (BAFF) receptor (BAFFR encoded byTNFRSF13C) transmembrane activator and calcium mod-ulator and cyclophilin ligand interactor (TACI encodedby TNFRSF13B) and B-cell maturation antigen (BCMAencoded by TNFRSF17) When these receptors are bound toBAFF (also known as BLyS TALL-1 zTNF4 and THANK)

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2015 Article ID 478753 12 pageshttpdxdoiorg1011552015478753

2 Journal of Immunology Research

andor A proliferation-inducing ligand (APRIL) whichare produced by antigen presenting cells (APCs) stromalendothelial cells nurse-like andor malignant cells they canevade apoptosis and promote tumor cell survival in vitro [9ndash14] BAFF and APRIL bind TACI and BCMA while BAFFbut not APRIL binds BAFFR [15 16] Normally BAFF andAPRIL are required for the development of most but notall normal mature B-cell populations found in the periphery[17 18]

BAFF and APRIL in CLL cells induce activation of thenuclear factor-kappaB1 (NF-120581B1) namely the classical NF-120581Β pathway through BCMA and TACI However BAFFbut not APRIL also induces the activation of NF-120581B2p100namely the alternative NF-120581Β pathway apparently throughbinding to BAFFR [12 13] At the same time the blocking ofthe classical NF-120581Β pathway blocked the capacity of BAFFto support neoplastic B-cell survival without any effect onthe survival of normal B cells in which the activation ofalternative NF-120581Β pathway prevails [13] Interestingly CLLcells may variably express TACI and very low to undetectableBCMA [9 11 13 19] but all cells express BAFFR with a ratherlower intensity compared to normal B cells [9 11 13 14 2021] These findings suggest that BAFFAPRIL signaling inneoplastic CLL cells might be important

Considering that CLL is characterized by a variableclinical and laboratory presentation and prognosis and thatthe majority of previous studies have enrolled a few CLLpatients (ranged from 10ndash25) the contribution of TACIexpression in the disease phenotype remains as yet obscureThe clarification of its role might be of outstanding inter-est since novel anti-BAFFAPRIL treatments have recentlybeen approved for the management of patients with B-cell-mediated autoimmune diseases [22 23] suggesting that thetargeting of TACI might be also a therapeutic candidate inB-cell malignancies [24ndash26] Therefore the aim of this studywas to investigate the contribution of TACI signaling to thebiological clinical laboratory and prognostic characteristicsof CLL

2 Material and Methods

21 Subjects Ninety-four CLL patients (malefemale 5341mean age 686 range 39ndash87) diagnosed by standard criteria[2] were enrolled in the study Among them 54 wereanalyzed at diagnosis 21 had not received any treatment inthe past and the rest 19 patients were analyzed at relapseFor the majority (90 out of 94) of patients flow cytometricand molecular analyses were performed in peripheral blood(PB) in 4 patients in bone marrow (BM) and in 3 patientssimultaneously in both PB and BM Moreover in 19 healthyindividuals (malefemale 712 mean age 628 range 29ndash84) a detailed analysis of TACI expression on B-cell sub-populations and measurement of BAFF and APRIL serumlevels as described below was also performed No patientor healthy individual was receiving any chemotherapy andorimmunosuppressive treatment over the last sixmonths beforeenrollment

Medical records of all CLL patients were reviewed anddata regarding the frequency and the location of infections

the presence of autoantibodies and autoimmune manifesta-tions the presence of hypogammaglobulinemia or mono-clonal M component and treatment modalities (for patientsat relapse) were recorded Data from conventional andormolecular cytogenetic analyses and ZAP-70 expression werenot available for all patients at enrollment and for this reasonthey were not included in the statistical analysis

The study was conducted in accordance with the prin-ciples of the Helsinki declaration and was approved by theInstitutional Review Board All patients and healthy controlagreed by written informed consent and the proceduresfollowed were in accordance with institutional guidelines

22 Immunophenotyping Studies Immunophenotyping wasperformed by flow cytometry on Coulter FC-500 instrument(Epics XL-MCL 4 color analysis Beckman-CoulterBCHialeah FL USA) using a multistaining protocol andcommercially available reagents Mouse anti-humanimmunoglobulin G (IgG) monoclonal antibodies were usedto detect in patients molecules that reacted with CD3 (cloneUCHT1) CD19 (clone J4119) CD45 (clone J33) CD5 (cloneBL1a) CD20 (clone B9ER) CD23 (clone 9P25) CD38 (cloneT16) CD43 (clone DFT1) CD10 (clone ALB1) CD11c (cloneBU15) CD79b (clone CB 3-1) and FMC7 (clone FMC7)All the above antibodies were purchased by BC and wereconjugated with the appropriate fluorochrome (fluoresceinisothiocyanate FITC phycoerythrin PE PE-cyanine5PE-Cy5) The clonality of B cells was assessed using rabbitanti-human polyclonal antibodies (anti-kappa light chainsFITC anti-lambda light chains PE) purchased by Dako(High Wycombe UK) The percentage of fluorescent cellsand the mean fluorescence intensity (MFI) were determinedin each case corrected for background fluorescence usingFITC PE and PE-Cy5-labelled control IgG antibodies

In healthy individuals peripheral blood samples wereanalyzed in order to indicate the different white blood cellsubpopulations Appropriate mouse anti-human IgG mono-clonal antibodies were used to detect molecules reacting withCD3 (clone UCHT1) CD4 (clone 13B82) CD8 (clone B911)CD19 (clone J4119) and CD45 (clone J33) all purchasedby BC and conjugated with the appropriate fluorochrome(FITC PE PE-Cy5 and ECD) The different cell populationswere gated using forward and side light scatter characteristicsas well as using the expression of specific markers Thepercentage of fluorescent cells and the MFI were determinedas above

The expression of TACI on CLL cells was determinedon 1 times 105 cells after erythrocyte lysis (Versalyse BC) andwashing with 1x phosphate buffered saline (PBS) After-wards the cells were incubated with 3 normal rat blockingserum (eBioscience San Diego USA) at 4∘C for 20minand subsequently were labeled with anti-TACI (clone 1A1PE conjugated Abcam Cambridge UK) anti-CD19 (FITCconjugated BC) and anti-CD45 (PE-Cy-5 conjugated BC)monoclonal antibodies for 15min at room temperature Thepercentage of TACI-positive B cells and their MFI werecorrected for background fluorescence using isotype controlIgG2a-PE antibody (eBioscience) Ten CLL patients exhibit-ing either high or very low to undetectable TACI by flow

Journal of Immunology Research 3

cytometry were also analyzed for IgD and CD27 expressionusing a polyclonal anti-IgD antibody (clone F0189 FITCconjugated DAKO) and a monoclonal anti-CD27 antibody(clone 1A4CD27 PE-Cy-5 conjugated BC)The expression ofTACI on B-cell subpopulations of 19 healthy individuals wasdetermined by using the monoclonal antibodies anti-CD19(ECD conjugated) anti-TACI (PE conjugated) anti-IgD(FITC conjugated) and anti-CD27 (PE-Cy-5 conjugated)

Taking into account the high total number of patientsand controls whole blood andor bonemarrow samples wereanalyzed in batches of 2ndash10 samples per day in order tominimize the day-to-day variation especially for the MFImeasurement

23 TNFRSF13BTACI mRNA Expression PB or BM sam-ples from 19 patients with CLL exhibiting a proportion ofneoplastic cells more than 80 as well as CD19+ B cellsfrom 2 healthy individuals isolated by magnetic sortingusing the Easy Sep Kit on a RobosepTM instrument (STEM-CELL Technologies Inc Vancouver Canada) were subjectedto total RNA extraction The latter was performed usingTRI (Ambion Life Technologies Thessaloniki Greece)according to manufacturerrsquos instructions ComplementaryDNA (cDNA) was reversed transcribed from 1mg of RNAusing a random 6-mer oligonucleotide primer (50 pmol120583L)(F Hoffmann-La Roche Basel Switzerland) and M-MLVreverse transcriptase (Invitrogen Life Technologies Thes-saloniki Greece) according to manufacturerrsquos instructionsThe mRNA levels of TNFRSF13BTACI were determined inquantitative real-time reverse-transcriptase PCR (qRT-PCR)reaction using Platinum-SYBR-Green PCR Supermix (Invit-rogen Life Technologies) in the automated thermocyclerRotorGene 6000 (Corbett Life Science Sydney Australia)The beta-2-microglobulin (B2M) gene was used as endoge-nous control for sample normalization (reference gene) An120 aliquot of the cDNA reaction product was used in dupli-cate qRT-PCR reactions and all measurements were aver-aged The primers for the amplification of TNFRSF13BTACIwere designed with the aid of Oligo 60 software (NBIPlymouth MN USA) and along with the thermocyclingPCR conditions are presented in Table 1 The efficiency ofeach qRT-PCR reaction ranged between 09 and 105 In orderto verify the specificity of the PCR products melting curveanalysis was performed from 65∘C to 95∘C with 01∘C persec intervals and stepwise fluorescence acquisition Relativequantification and calculation of the range of confidencewereperformed using the comparativeΔΔCT method as described[27] The relative expression of each gene is presented as amultiple of the respective gene expression in isolated B cellsof a healthy individual

24 Mutational Status of IGHV Gene RearrangementsGenomic DNA was extracted from PB or BM using theQIAamp DNA Blood Mini Kit (Qiagen Crawley UK)according to the manufacturerrsquos instructions The IGHVmutational status was assessed by a 2-step seminestedPCR reaction as previously described [28] with somemodifications (Table 1) All reactions were performedin a PCR-engine apparatus PTC-200 MJ-Research

(Watertown-Massachusetts) and the PCR productswere analyzed in 3 TBE agarose gels stained withethidium bromide and visualized under UV light Afterthe identification of the a clonal IGHV rearrangement theamplified PCR products were purified with QIAquick gelextraction kit (Qiagen) and direct sequenced using an ABIPrism 310 Genetic Analyzer (Applied Biosystems FosterCity CA) and a Big Dye Terminator DNA sequencingkit (Applied Biosystems) The sequences were comparedwith the sequence of the germline variable (V) regionas presented in the National Center for BiotechnologyInformation website (httpwwwncbinlmnihgovigblast)using the Basic Local Alignment Search Tool (IgBLAST)Thepresence of IGHV somatic hypermutation (SHM) is definedas greater or equal to 2 different from the germline V genesequence (mutated M-CLL) whereas less than 2 differenceis considered evidence of no SHM (unmutated U-CLL)

25 Functional Assays and Study of Apoptosis PB mononu-clear cells from six patients with CLL exhibiting high (gt403 patients) or very low to absent (lt10 3 patients) TACIexpression were isolated by density gradient centrifuga-tion and diluted with Iscoversquos Modified Dulbeccorsquos Medium(IMDM Life Technologies) supplemented with 10 fetalbovine serum (FBS) at a concentration of 8 times 105 cellsmLAfterwards aliquots of 05mL of cells were plated on a96-well plate and stimulated by either 1 120583gmL of recombi-nant BAFF (RampD Systems Mineapolis USA) or 200 ngmLAPRIL (RampD Systems) or combinations of the above dilutedinto 01mL IMDM Another aliquot of 05mL of cells supple-mented with 01mL IMDMwithout any stimulus was used asinternal control Samples were then incubated at 37∘C in thepresence of 10 CO

2for 24 h Subsequently apoptosis was

measured by flow cytometry using an Annexin V-FITC7-AAD (7-AAD) kit (BC) according to the manufacturerrsquosinstructions

26 Quantification of Soluble BAFF and APRIL SolubleBAFF and APRIL levels were determined in the serumof 56 patients with CLL and 19 healthy individuals usingthe BAFF Soluble (human hypersensitive) ELISA Kit (Adi-pogen Liestal Switzerland) and APRIL ELISA kit (IBLMinneapolis USA) respectively according tomanufacturersrsquoinstructions

27 Measurement of Immunoglobulin Levels For the deter-mination of the IgG IgM and IgA levels we used com-mercially available immunonephelometric assays (Immulite-2000 Siemens Medical Solutions Llanberis Gwynedd UK)with N antiserum to human IgG (product code OSAS) Nantiserum to human IgA (OSAT09) N antiserum to humanIgM (OSAS09) and the N protein standard SL (OQIM13)according to the manufacturerrsquos instructions

28 Statistical Analysis Categorical variables were analyzedwith Fisherrsquos exact test Normality of continuous variableswas assessed with Kolmogorov-Smirnov test Normally dis-tributed data were analyzed with Studentrsquos 119905-test Skewed data


Table1Prim

ersa

ndPC

Rcond

ition

sfor

thea

mplificatio

nof

thea

nalyzedgenes

Gene

Prim

ers

Sequ

ence

PCRcond

ition

s

TNFR

SF13B

Forw

ard

Reverse

51015840-TCT

GCC

TGTG

TGCC

GTC

CTC-

3101584051015840-C

GGCT

TCCA

TCGCG

TGAT

-31015840

95∘

Cfor2

minfollowed

by40

cycle

s(95∘

Cfor10s

64∘

Cfor6

0s)

B2M

Forw

ard

Reverse

Com

merciallyob

tained

byQiagen

CatNoPP

H01094E

95∘

Cfor10m

infollowed

by40

cycle

s(95∘

Cfor15s

60∘

Cfor6

0s)

IGHV

A1015840

PCR

FR2A

FR1C

LGH

51015840-TGG(A

G)TCC

G(C

A)C

AG(G

C)C

(TC)(TC)

C(AG

TC)-31015840

51015840-AGGTG

CAGCT

G(C

G)(AT)G

(CG)A

GTC

(AG

T)G

G-31015840

51015840-TGAG

GAG

ACGGTG

ACC-

31015840

95∘

Cfor2

minfollowed

by5cycle

s(95∘

Cfor3

0s63∘C

for3

0s72∘Cfor3

0s)then

by25

cycle

s(95∘

Cfor3

0s

57∘

Cfor3

0s72∘Cfor3

0s)andafi

nalelong

ationat

72∘

Cfor5

min

IGHV

neste

dPC

R

FR2A

VH1

VH2

VH3

VH4

VLJH

Similarw

ithA1015840

PCR

51015840-ACT

AGTC

GAC

CTCA

GTG

AAG

GT-31015840

51015840-ACT

AGTC

GAC

GTC

CTGVG

CTGGTG

AAA(G

C)C

CACA

C-31015840

51015840-ACT

AGTG

ACGGGTC

CCTG

AGAC

TCTC

CTGTG

CAG-31015840

51015840-ACT

AGTC

GAC

CCTG

TCCC

TCAC

CTGC(

AG

)CTG

TC-31015840

51015840-G

TGAC

CAGGGT(AG

CT)C

CTTG

GCC

CCAG

-31015840

95∘

Cfor2

minfollowed

by30

cycle

s(95∘

Cfor3

0s

63∘

Cfor3

0s72∘Cfor3

0s)andafi

nalelong

ationat

72∘

Cfor5

min

B2Mbeta-2-microglob

ulinIGHV

immun

oglobu

linheavychainvaria

bler

egion


were analyzed withMann-Whitney test Survival analysis wasperformed with Kaplan-Meier procedure and survival rateswere comparedwith log-rank test Logistic regressionmodelsand Cox proportional hazards regression models were fittedas appropriate Data analysis was performed with SPSS 170(IBM Corporation NY 2008) Graphs were made on Exceland Prism 6 for Mac For all analyses alpha was set at 005(2-sided)

3 Results

31 Clinical and Laboratory Findings of CLL Patients and Con-trols The clinical and laboratory characteristics of the ana-lyzed patients are presented in Table 2 About half of patients(41 436) exhibited the most characteristic phenotype ofthe disease (CD5+CD23+FMC7minusCD20lowsIglowCD79blow B-CLL score 5) among the rest the majority (43 patients458) displayed a B-CLL score of 4 with either FMC7+or sIghigh or CD79bhigh while 10 patients (106) had B-CLL score of 3 (CD5+CD23+ and CD20high andor sIghigh

andor CD79bhigh andor FMC7+) Moreover all CLL cellsanalyzed were double positive for CD27 and sIgD expression(Figure 1) similar with previous reports in the literature[29 30] Seven patients (75) displayed monoclonal M-component in their serum (Table 2) and all were negative forthe MyD88-L265P mutation [31]

During the follow-up period (median 38 months range3ndash68) 20 patients (213) died due to CLL progressionand disease complications (Table 2) Therefore the mediansurvival of the enrolled patients was 586 months (range 3ndash158)Theolder age and the severity of the disease (RAI stagingsystem) were significant risk factors affecting the diseaseoutcome (119875 lt 0001 and 119875 = 0024 resp) Additionalrisk factors for a worse outcome were the developmentof monoclonal gammopathy (119875 = 0033) (SupplementaryFigure 1 see Supplementary Material available online athttpdxdoiorg1011552015478753) and the presence ofhypogammaglobulinemia (with total immunoglobulin levelsbelow 600mgdL 119875 = 0020) (Supplementary Figure 1)alongwith the emergence of infections (119875 = 0020) especiallythose of the respiratory track (119875 = 0013)

All CLL patients were also analyzed for IGHVmutationalstatus and CD38 expression 35 patients (376) displayedunmutated IGHV and 59 (628) were mutated (with SHMgreater or equal to 2 different from the germline V genesequence) Among the latter group (M-CLL) 4 patientsdisplayed borderline IGHV status with SHM between 97 and98 therefore using as cut-off the SHM greater or equal to3 U-CLL patients were 39 (425) and M-CLL 55 (575)The pattern of IGHV mutational status of our CLL patientswas similar with previous studies [32] and is presented indetail in Supplementary Figure 2

The most frequent IGHV genes in our cohort of patientswere IGHV1-69 (14 patients) IGHV1-2 (7) IGHV3-23 (7)IGHV3-30 (6) and IGHV3-33 (7) (total 41 patients 436)Within theU-CLL group 16CLL cells (410) used amemberof the IGHV1 family with the IGHV1-69 to be the mostcommon type (accounted for 308 of all U-CLL patients and

750 of U-CLL patients using IGHV1 genes) Within theM-CLL group only 14 CLL cells (255) used a member ofthe IGHV1 family whereas 33 of malignant cells (601) useda member of the IGHV3 family

Although U-CLL patients (with a SHM cut-off of 2)exhibited a rather worse outcome compared to M-CLL onesthe difference was not reached to be significant (119875 = 0191)however when the SHM cut-off was considered as 3 theIGHV mutational status was significantly associated withprognosis (119875 = 0022) (Supplementary Figure 2)

Moreover the proportion of leukemic cells expressingCD38 above isotype control level ranged from 00 to 822with a median of 18 and a mean of 114 Subsequentlydifferent cut-offs were considered to define the CD38 posi-tivity thus 27 patients (287) displayed CD38 expressionabove 10 21 (223) above 20 and 12 (128) above 30However in our cohort of patients CD38 expression was notfound to affect the disease outcome (119875 gt 005 in all cases)

Finally an overview of PB immunophenotyping of 19healthy individuals with leukocyte and lymphocyte subpop-ulations is presented in Supplementary Table 1

32 TACI Expression The immunophenotyping on B-cellsubpopulations of healthy individuals indicated that TACIis expressed only on switched and nonswitched memoryB cells (CD27+IgDminus and CD27+IgD+ resp) (Figure 1) inaccordancewith previous reports [33] On the other hand theproportion of CLL patients expressing TACI above isotypecontrol level ranged from 01 to 578 (median 74interquartile range [IQR] 21ndash208) Interestingly the greatmajority of CLL patients (69 750) presented with very lowTACI expression (lt20) and only 14 patients (149) dis-played TACI gt30 Further molecular analyses revealed thatthe mRNA TNFRSF13B expression was equivalent to proteinmembrane expression (Supplementary Figure 3) indicatingthat the lowTACI expression observed in themajority of CLLcells is due to a dysregulated TNFRSF13B gene expressionConsidering that all CLL cells were CD27+ and the majorityof them also IgD+ (Figure 1) TACI expression was notcorrelated with CD27 and IgD

To further characterize the effect of TACI on CLL cellswe investigated whether BAFF or APRIL could enhance thesurvival of CLL cells in vitroWe demonstrated that CLL cellswith highTACI expression (gt40) displayed a better survivalcapacity compared to CLL cells with low to undetectableTACI expression when cultured in the presence of BAFFandor APRIL (Figure 2) These results indicate that TACIsignaling protects CLL cells from apoptosis in vitro

In addition we investigated whether TACI expressionon malignant cells is associated with CLL clinical andorlaboratory phenotype We identified that TACI expressionwas significantly associated with CD11c expression and thepresence of monoclonal M-component In particular weobserved that TACI expression gt20 and a high CD11c(gt70) were mutually exclusive events (119875 = 0008) whilepatients with monoclonal gammopathy have higher TACIexpression onmalignant B cells compared to patients withoutmonoclonal M-component (mean plusmn SD 262 plusmn 173 versus126 plusmn 147 119875 = 0020 confirmed also by ROC curve


Table 2 Overview of clinical and laboratory characteristics of the patients of the study

Total At diagnosis During follow-upNo treatment Relapse1

No () 94 (1000) 54 (574) 21 (223) 19 (203)Gender (MF) 5341 3420 1011 910Age (y mean plusmn SD) 686 plusmn 100 687 plusmn 105 657 plusmn 99 713 plusmn 78

At enrollmentWBC (times109L) (median IQR) 220 (138ndash342) 190 (142ndash314) 210 (194ndash317) 345 (142ndash615)B cells (times109L) (median IQR) 160 (57ndash271) 99 (53ndash202) 118 (52ndash200) 236 (54ndash399)Rai staging system

Stage O (119899 ) 42 447 28 519 8 380 6 316Stage I (119899 ) 22 234 9 166 10 477 3 158Stage II (119899 ) 11 117 7 130 2 95 2 105Stage III (119899 ) 10 106 8 148 0 0 2 105Stage IV (119899 ) 9 96 2 37 1 48 6 316

ImmunophenotypingCD5 (median IQR) 975 (868ndash994) 956 (833ndash989) 986 (971ndash997) 993 (824ndash997)CD23 (median IQR) 911 (818ndash965) 921(844ndash970) 938 (841ndash975) 846 (815ndash910)CD20 (median IQR) 920 (838ndash975) 930 (844ndash983) 925 (853ndash979) 910 (754ndash959)CD79b (median IQR) 924 (827ndash988) 927 (808ndash982) 964 (840ndash999) 885 (818ndash988)CD43 (median IQR) 933 (846ndash971) 928 (814ndash970) 942 (868ndash976) 947 (806ndash971)FMC7 (median IQR) 427 (289ndash594) 445 (307ndash612) 442 (308ndash616) 358 (150ndash589)CD38 (median IQR) 18 (06ndash155) 14 (05ndash86) 17 (07ndash191) 120 (02ndash271)CD11c (median IQR) 346 (174ndash520) 347 (174ndash507) 371 (201ndash621) 286 (119ndash551)CD10 (median IQR) 100 (48ndash177) 87 (47ndash164) 110 (53ndash193) 113 (38ndash187)Kappa (119899 ) 61 650 36 666 15 714 10 526Lambda (119899 ) 33 350 18 333 6 286 9 474

HypogammaglobulinemiaTotal Igs lt 600mgdL 15 160 4 74 3 143 8 429Total Igs lt 300mgdL 3 32 0 0 0 0 3 158

Monoclonal M-component2 7 74 2 37 2 95 3 158At enrollment and during follow-up

Autoimmuneautoinflammatory manifestations 18 191 10 185 1 48 7 368Coombs direct (119899 )3 7 74 4 74 0 0 3 158Other (119899 )4 11 117 6 113 1 48 4 210

Infections (119899 )5 19 202 8 148 1 48 10 526Respiratory (119899 ) 10 106 3 55 1 48 6 316Urinary (119899 ) 2 21 0 0 0 0 2 105Herpes viruses (119899 )6 5 53 2 37 0 0 3 158Other (119899 )7 5 53 3 55 0 0 2 105

Death (119899 ) 20 213 11 203 2 95 7 368Survival (mo) (median IQR) 525 385ndash722 435 115ndash532 730 525ndash1155 930 590ndash1340Abbreviations F female Igs immunoglobulins IQR interquartile range M male mo months SD standard deviation WBC white blood cell count1Patients at relapse had received in the past chlorambucil-based medication (10 patients) fludarabine-based medication (3) only corticosteroids (1)cyclophosphamidevincristine-based medication (2) or multiple consecutive treatments (3) Anti-CD20 (rituximab) treatment had been administered in 6patients in combination with other medications 2IgG (5 patients) IgM (2 patients)3a patient at diagnosis and another at relapse developed also autoimmune hemolytic anemia4Including rheumatic polymyalgia (one patient) Crohn disease (one patient) thrombopenic purpura (one patient) hyperthyreodism (antithyroglobulinanti-TG antibodies one patient) while 7 patients displayed autoantibodies without clinical signs of autoimmunity (antinuclearANA andor anti-neutrophilcytoplasmicANCA andor anti-smooth muscleASMA andor anti-extractable nuclearENA andor antiphospholipidAPA antibodies)53 patients at relapse displayed infections at multiple locations 6herpes simplex (a patient at diagnosis) and varicella zoster virus (4 patients) 7including sepsis(2 patients) encephalitis (1) salmonellosis (1) and tuberculosis (1)


41

CD19 ECD

Healthy individual641

TACI PE

1 2

0 97

38 51

1 10

CD27

PC5

IgD FITCIgD FITC

359

100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

CD27

PC5

100

101

102

103

100 101 102 103

TACIminus cellsCD19+ TACI+ cellsCD19+

(a)

841

CD19 ECD TACI PE

CD27

PC5

IgD FITC

12 81

1 6

100 101 102 103

100

101

102

103

100 101 102 103100 101 102 103

526 CLL_1

(b)

Figure 1 Continued


IgD FITC

782

CD19 ECD

48

TACI PE

43 44

100 101 102 103

100 101 102 103

100 101 102 103

CD27

PC5

100

101

102

103

5 8

CLL_2

(c)Figure 1 TACI along with CD27 and IgD expression on B cells established by flow cytometry in a healthy individual and 2 patients withCLL TACI is expressed only on normal switched and nonswitched memory B cells (CD27+IgDminus and CD27+IgD+ resp) (a) CLL cells wereCD27+ and the majority of them also IgD+ with high (b) and low (c) TACI expression

analysis AUC = 0765 95CI 0626ndash0964) Further logisticregression analysis after adjustment for patient gender andage revealed that TACI expression on CLL cells gt10 wassignificantly associated with a 69-fold probability of thepresence of monoclonal M-component (OR 0866 95 CI1211ndash38925119875 = 0030) Finally no significant associations ofTACI expression with autoimmune manifestations (clinicalandor laboratory) the presence of hypogammaglobulinemia(and its severity) the emergence of infections and the overallsurvival were found (119875 gt 005 in all cases)

At the end we investigated whether TACI expressionis correlated with the well-established prognostic factors ofCLL namely CD38 expression and IGHV mutational status[3 4 34] but no significant associationswere found (119875 gt 005in all cases)

33 BAFF and APRIL Expression and Their Correlationswith Disease Phenotype We demonstrated that CLL patientsdisplayed significantly lower levels of serum BAFF comparedto healthy controls (median IQR 0 pgmL 0ndash207 versus283 pgmL 216ndash481 119875 le 0001) (Figure 3) while themajority of patients (30 out of 57 analyzed 526) exhibitedundetectable levels using a hypersensitive BAFF detectionkit Interestingly 5 patients displayed very high levels ofBAFF in their serum (Figure 3) and 3 of them also displayedsevere autoimmune manifestations (autoimmune hemolytic

anemia lupus like symptoms with high levels of antinuclearand antiphospholipid antibodies and vasculitis with high lev-els of antinuclear and anti-neutrophil cytoplasmic antibodiesresp) No correlation of BAFF levels with other clinical andlaboratory characteristics of CLL patients were found (119875 gt005 in all cases)

On the other hand CLL patients displayed significantlyincreased levels of serum APRIL compared to healthy con-trols (median IQR 51 ngmL 37ndash78 versus 35 ngmL 25ndash47 119875 = 0003) (Figure 3) Interestingly serum APRIL levelswere positively correlated with the number of peripheralblood B cells and the percentage of bone marrow infiltration(119903 = 0305 119875 = 0023 and 119903 = 0312 119875 = 0019 resp)and inversely with IgA and IgG serum levels (119903 = minus0390119875 = 0004 and 119903 = minus0284 119875 = 0037 resp) and theexpression of FMC7 (119903 = minus0303 119875 = 0023)

Finally no significant correlations of BAFF and APRILserum levels with TACI expression on CLL cells wereobserved

4 Discussion

Our study provides clear evidence that CLL cells are char-acterized by variable TACI expression (with the majorityof cases displaying low expression) which was positivelycorrelated with the presence of monoclonal gammopathy


51

7-A

AD

PC7

Control (without BAFF andor APRIL)

0 464 32

0 639 55

0 644 50

TACI PE Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

0 464 32

0 639 55

0 644 50

100

101

102

103

100 101 102 103100 101 102 103

100

101

102

103

100 101 102 103

100

101

102

103

100 101 102 103

APRIL (200ngmL)BAFF (1120583gmL)

(a)

66578

TACI PE

059 35

060 34

0 856 36

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

6059 35

6060 34

0 856 36

100 101 102 103100 101 102 103 100 101 102 103 100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

(b)

1000900800700600500400300200100

00

1000900800700600500400300200100

00

1000900800700600500400300200100

00Control APRILBAFF Control APRILBAFF Control APRILBAFF

Necrotic cells (annexin-V+7-AAD+)Apoptotic cells (annexin-V+7-AADminus)

TACI lt 10TACI gt 40



(annexin-Vminus7-AADminus)Viable cells

(c)

Figure 2 TACI expression protects CLL cells from apoptosis in vitro CLL cells with very low (a) or high (b) TACI expression were culturedwith IMDMmediumwithout any stimulus (internal control) or stimulated by either BAFF orAPRIL for 24 h Cells were harvested and stainedwith Annexin V (119909-axis) and 7-AAD (119910-axis) to discriminate between viable (double negative) apoptotic (Annexin V-FITC positive 7-AADnegative) and necrotic (double positive) cells Results are summarized in the bar graphs in the lower panel of the figure (c) and are obtainedfrom three independent experiments in 6 CLL patients with low (3 patients mean 32 range 17ndash51) and high TACI expression (3patients mean 497 range 401ndash578)

and inversely with CD11c expression However no prognosticsignificance of TACI expression in CLL was found althoughTACI signaling seems to protect CLL cells from apoptosisin vitro Finally we identified that CLL patients displayedlow to undetectable BAFF and increased APRIL serum levelscompared to healthy individuals similarly with previousreports [35ndash38]

To the best of our knowledge this is the largest studyanalyzing the role of TACI expression in CLL It is notable

that the initial studies reported a rather high TACI expressionin the majority of CLL patients however the number ofthe enrolled patients was low ranged from only 9 to 23[9 11 13] In a subsequent larger study Bojarska-Junak et alanalyzed 62 CLL patients and demonstrated that themajorityof them displayed a low to undetectable TACI expression[19] similarly with our results Moreover they suggested arather prognostic significance of TACI expression in CLLsince they reported a correlation of TACI with CD38 and


19 healthy individuals 57 CLL patients

800

600200

100

0

BAFF

seru

m le

vels

P lt 0001

(a)


70

60

50

40

30

20

10

0

APR

IL se

rum

leve

ls

P = 0003

(b)

Figure 3 (a) BAFF levels in CLL patients and healthy controls (b) APRIL serum levels in CLL patients and controls Lines indicate medianvalues The significant 119875 values refer to Mann-Whitney 119880 test

ZAP-70 expression [19] However such correlations werenot confirmed in our study and TACI expression was notassociated with the most important marker of prognosis inCLL namely the IGHV mutational status At the end ourstudy also confirms the findings of Kern et al that TACIexpression protects CLL cells from apoptosis in vitro [11]

Previous studies have reported that gene expressionsignatures of CLL cells were similar to those observed formemory B cells and marginal zone (MZ) or MZ-like Bcells irrespective of IGHV mutational status suggesting acommon mechanism of transformation andor cell of origin[39 40] In this context we actually demonstrated that CLLcells express CD27+ irrespective of their mutational statusHowever CLL cells display a variable expression of TACI thisis an unexpected finding since normal CD27+memory B cellsare characterized by constitutive TACI expression [33] Afterthat the notion that the origin of CLL cell is a memory B cellis rather disputable

The abovementioned argument (that CLL cell is not amemory B cell) is also supported by the recent results ofSeifert et al suggesting that CLL cells derived from CD5+ B-cell populations already found in young healthy adults andcharacterized by deregulated molecules and pathways com-paredwith normalCD5+ or conventional B cells Consideringalso the major disease subsets these researchers suggest thatU-CLL derives from unmutated mature CD5+ B cells whileM-CLL derives from a distinct previously unrecognizedCD5+CD27+ postgerminal center and antigen-exposedB-cellsubset [41] Along these lines it can be assumed that CD27and TACI represent a part of deregulated proteins duringtransformation similarly with other known transcriptionfactors and proteins that are necessary for the survival anddevelopment of healthy B cells

Despite the fact that our findings can support the above-mentioned model in regard to the origin of CLL cell thepossibility that this cell is derived from a virgin B-cellcannot be excluded In such a case the phenotypic andmolecular characteristics we detected might result eitherfrom activation of mutational machinery (possibly againstauto-antigens) or frommodifications (transcriptional andor

posttranscriptional) of the expressed molecules The dereg-ulatory events of emerging subclones could therefore affecttheir survival capacity (as on TACI expression) and their finalpredominance

Over the recent years it became increasingly clear thatexternal signals from the leukemia microenvironment makepivotal contributions to CLL pathophysiology and progres-sion [42 43] This microenvironment includes both BAFFand APRIL which can rescue highly purified leukemic CLLcells from spontaneous apoptosis in vitro and thus maycontribute to their prolonged survival in vivo [44]Thereforethe targeting of BAFFAPRIL receptors could represent anideal treatment approach In this context considering thatonly a minority of CLL patients displays a high TACI expres-sion and previous studies have shown that BCMA is ratherabsent on CLL cells [9 13] the novel anti-APRIL therapeuticapproaches should be tested only after the demonstration ofTACI expression on CLL cells

5 Conclusions

Our study provides further evidence on the role of TACIexpression in CLL In particular we demonstrated that thereis a remarkable interindividual variability of TACI expressionin CLL although the majority of patients display low toundetectable TACI Moreover CLL cells expressing TACIdisplay a better survival capacity in vitro suggesting thatTACI detection might be useful in the selection of patientsfor novel targeting therapeutic approaches

Conflict of Interests

The authors have no conflict of interests in connection withthe submitted paper

Acknowledgments

This study has been cofinanced by the European Union(European Social Fund-ESF) and Greek national fundsthrough the Operational Program ldquoEducation and Lifelong


Learningrdquo of the National Strategic Reference Framework-(NSRF-) Research Funding Program ldquoHeracleitus II Invest-ing in knowledge society through the European Social FundrdquoThe authors would like to thank Dr F Kalala Dr F BardakaDr Z Kartasis Dr C Kartsios Dr S Lafioniatis and DrN Papadoulis for collecting patientsrsquo and healthy individualsrsquosamples

References

[1] N Chiorazzi ldquoCell proliferation and death forgotten featuresof chronic lymphocytic leukemia B cellsrdquo Best Practice andResearch Clinical Haematology vol 20 no 3 pp 399ndash413 2007

[2] N E Kay T J Hamblin D F Jelinek et al ldquoChronic lympho-cytic leukemiardquo HematologyAmerican Society of HematologyEducation Program vol 2002 pp 193ndash213 2002

[3] T J Hamblin Z Davis A Gardiner D G Oscier and F KStevenson ldquoUnmutated Ig V

119867genes are associated with a more

aggressive form of chronic lymphocytic leukemiardquo Blood vol94 no 6 pp 1848ndash1854 1999

[4] R N Damle T Wasil F Fais et al ldquoIg V gene mutation statusand CD38 expression as novel prognostic indicators in chroniclymphocytic leukemiardquo Blood vol 94 no 6 pp 1840ndash18471999

[5] H Dohner S Stilgenbauer A Benner et al ldquoGenomic aberra-tions and survival in chronic lymphocytic leukemiardquo The NewEngland Journal of Medicine vol 343 no 26 I pp 1910ndash19162000

[6] C Moreno and E Montserrat ldquoGenetic lesions in chroniclymphocytic leukemia whatrsquos ready for prime time userdquoHaematologica vol 95 no 1 pp 12ndash15 2010

[7] M T Scupoli and G Pizzolo ldquoSignaling pathways activated bythe B-cell receptor in chronic lymphocytic leukemiardquo ExpertReview of Hematology vol 5 no 3 pp 341ndash348 2012

[8] P Oppezzo and G Dighiero ldquoRole of the B-cell receptor andthemicroenvironment in chronic lymphocytic leukemiardquoBloodCancer Journal vol 3 article e149 2013

[9] A J Novak R J Bram N E Kay and D F Jelinek ldquoAberrantexpression of B-lymphocyte stimulator by B chronic lympho-cytic leukemia cells a mechanism for survivalrdquo Blood vol 100no 8 pp 2973ndash2979 2002

[10] B He A Chadburn E Jou E J Schattner D M Knowlesand A Cerutti ldquoLymphoma B cells evade apoptosis through theTNF family members BAFFBLyS and APRILrdquo The Journal ofImmunology vol 172 no 5 pp 3268ndash3279 2004

[11] C Kern J-F Cornuel C Billard et al ldquoInvolvement of BAFFand APRIL in the resistance to apoptosis of B-CLL through anautocrine pathwayrdquo Blood vol 103 no 2 pp 679ndash688 2004

[12] M Nishio T Endo N Tsukada et al ldquoNurselike cells expressBAFF and APRIL which can promote survival of chroniclymphocytic leukemia cells via a paracrine pathway distinctfrom that of SDF-1120572rdquo Blood vol 106 no 3 pp 1012ndash1020 2005

[13] T Endo M Nishio T Enzler et al ldquoBAFF and APRILsupport chronic lymphocytic leukemia B-cell survival throughactivation of the canonical NF-kappaB pathwayrdquo Blood vol 109no 2 pp 703ndash710 2007

[14] M Cols C M Barra B He et al ldquoStromal endothelial cellsestablish a bidirectional crosstalk with chronic lymphocyticleukemia cells through the TNF-related factors BAFF APRILand CD40Lrdquo The Journal of Immunology vol 188 no 12 pp6071ndash6083 2012

[15] G Yu T Boone J Delaney et al ldquoAPRIL and TALL-1 andreceptors BCMA and TACI system for regulating humoralimmunityrdquoNature Immunology vol 1 no 3 pp 252ndash256 2000

[16] L G Ng A P R Sutherland R Newton et al ldquoB cell-activatingfactor belonging to the TNF family (BAFF)-R is the principalBAFF receptor facilitating BAFF costimulation of circulating Tand B cellsrdquoThe Journal of Immunology vol 173 no 2 pp 807ndash817 2004

[17] F Mackay P Schneider P Rennert and J Browning ldquoBAFFand APRIL a tutorial on B cell survivalrdquo Annual Review ofImmunology vol 21 pp 231ndash264 2003

[18] K Pieper B Grimbacher and H Eibel ldquoB-cell biology anddevelopmentrdquo The Journal of Allergy and Clinical Immunologyvol 131 no 4 pp 959ndash971 2013

[19] A Bojarska-Junak I Hus M Sieklucka et al ldquoThe role of TACIexpression in chronic lymphocytic leukemiardquoCentral-EuropeanJournal of Immunology vol 36 no 1 pp 46ndash50 2011

[20] J Briones J M Timmerman D M Hilbert and R Levy ldquoBLySand BLyS receptor expression in non-Hodgkinrsquos lymphomardquoExperimental Hematology vol 30 no 2 pp 135ndash141 2002

[21] S A Mihalcik R C Tschumper and D F Jelinek ldquoTranscrip-tional and post-transcriptional mechanisms of BAFF-receptordysregulation in human B lineage malignanciesrdquo Cell Cycle vol9 no 24 pp 4884ndash4892 2010

[22] S S Kim K A Kirou and D Erkan ldquoBelimumab in systemiclupus erythematosus an update for cliniciansrdquo TherapeuticAdvances in Chronic Disease vol 3 no 1 pp 11ndash23 2012

[23] K Fairfax I R Mackay and F Mackay ldquoBAFFBLyS inhibitorsa new prospect for treatment of systemic lupus erythematosusrdquoIUBMB Life vol 64 no 7 pp 595ndash602 2012

[24] S M Ansell T E Witzig D J Inwards et al ldquoPhase I clinicalstudy of atacicept in patients with relapsed and refractory B-cellnon-Hodgkinrsquos lymphomardquoClinical Cancer Research vol 14 no4 pp 1105ndash1110 2008

[25] D M Kofler B B Gawlik T Elter A Gianella-Borradori C-M Wendtner and M Hallek ldquoPhase 1b trial of atacicept arecombinant protein binding BLyS and APRIL in patients withchronic lymphocytic leukemiardquo Leukemia vol 26 no 4 pp841ndash844 2012

[26] M Guadagnoli F C Kimberley U Phan et al ldquoDevelopmentand characterization of APRIL antagonistic monoclonal anti-bodies for treatment of B-cell lymphomasrdquo Blood vol 117 no25 pp 6856ndash6865 2011

[27] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minusΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[28] H Hojo Y Sasaki N Nakamura and M Abe ldquoAbsenceof somatic hypermutation of immunoglobulin heavy chainvariable region genes in precursor B-lymphoblastic lymphomaa study of four cases in childhood and adolescencerdquo AmericanJournal of Clinical Pathology vol 116 no 5 pp 673ndash682 2001

[29] E A Ranheim M J Cantwell and T J Kipps ldquoExpression ofCD27 and its ligand CD70 on chronic lymphocytic leukemiaB cellsrdquo Blood vol 85 no 12 pp 3556ndash3565 1995

[30] R N Damle F Ghiotto A Valetto et al ldquoB-cell chronic lym-phocytic leukemia cells express a surface membrane phenotypeof activated antigen-experienced B lymphocytesrdquo Blood vol99 no 11 pp 4087ndash4093 2002

[31] N Argentou G Vassilopoulos M Ioannou A E Germenisand M Speletas ldquoRapid detection of MYD88-L265P muta-tion by PCR-RFLP in B-cell lymphoproliferative disordersrdquoLeukemia vol 28 no 2 pp 447ndash449 2014


[32] P Ghia K Stamatopoulos C Belessi et al ldquoGeographicpatterns and pathogenetic implications of IGHV gene usagein chronic lymphocytic leukemia the lesson of the IGHV3-21generdquo Blood vol 105 no 4 pp 1678ndash1685 2005

[33] J R Darce B K Arendt X Wu and D F Jelinek ldquoRegulatedexpression of BAFF-binding receptors during human B celldifferentiationrdquo Journal of Immunology vol 179 no 11 pp7276ndash7286 2007

[34] F Malavasi S Deaglio R Damle G Cutrona M Ferrariniand N Chiorazzi ldquoCD38 and chronic lymphocytic leukemiaa decade laterrdquo Blood vol 118 no 13 pp 3470ndash3478 2011

[35] M Kreuzaler M Rauch U Salzer et al ldquoSoluble BAFF levelsinversely correlate with peripheral B cell numbers and theexpression of BAFF receptorsrdquoThe Journal of Immunology vol188 no 1 pp 497ndash503 2012

[36] L Planelles S Castillo-Gutierrez J P Medema A Morales-Luque H Merle-Beral and M Hahne ldquoAPRIL but not BLySserum levels are increased in chronic lymphocytic leukemiaprognostic relevance ofAPRIL for survivalrdquoHaematologica vol92 no 9 pp 1284ndash1285 2007

[37] A Bojarska-Junak I Hus S Chocholska et al ldquoBAFF andAPRIL expression in B-cell chronic lymphocytic leukemiacorrelation with biological and clinical featuresrdquo LeukemiaResearch vol 33 no 10 pp 1319ndash1327 2009

[38] S Haiat C Billard C Quiney F Ajchenbaum-Cymbalista andJ-P Kolb ldquoRole of BAFF and APRIL in human B-cell chroniclymphocytic leukaemiardquo Immunology vol 118 no 3 pp 281ndash292 2006

[39] U Klein Y Tu G A Stolovitzky et al ldquoGene expressionprofiling of B cell chronic lymphocytic leukemia reveals ahomogeneous phenotype related to memory B cellsrdquo TheJournal of Experimental Medicine vol 194 no 11 pp 1625ndash16382001

[40] A Rosenwald A A Alizadeh G Widhopf et al ldquoRelationof gene expression phenotype to immunoglobulin mutationgenotype in B cell chronic lymphocytic leukemiardquo The Journalof Experimental Medicine vol 194 no 11 pp 1639ndash1647 2001

[41] M Seifert L Sellmann J Bloehdorn et al ldquoCellular origin andpathophysiology of chronic lymphocytic leukemiardquoThe Journalof Experimental Medicine vol 209 no 12 pp 2183ndash2198 2012

[42] J A Burger ldquoNurture versus nature the microenvironmentin chronic lymphocytic leukemiardquo HematologyThe EducationProgram of the American Society of Hematology vol 2011 pp96ndash103 2011

[43] MTheodorou M Speletas A Mamara et al ldquoIdentification ofa STAT5 target gene Dpf3 provides novel insights in chroniclymphocytic leukemiardquo PLoS ONE vol 8 no 10 Article IDe76155 2013

[44] H Ghamlouch H Ouled-Haddou G Damaj B Royer BGubler and J-PMarolleau ldquoA combination of cytokines rescueshighly purified leukemic CLL B-cells from spontaneous apop-tosis in vitrordquo PLoS ONE vol 8 no 3 Article ID e60370 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


andor A proliferation-inducing ligand (APRIL) whichare produced by antigen presenting cells (APCs) stromalendothelial cells nurse-like andor malignant cells they canevade apoptosis and promote tumor cell survival in vitro [9ndash14] BAFF and APRIL bind TACI and BCMA while BAFFbut not APRIL binds BAFFR [15 16] Normally BAFF andAPRIL are required for the development of most but notall normal mature B-cell populations found in the periphery[17 18]

BAFF and APRIL in CLL cells induce activation of thenuclear factor-kappaB1 (NF-120581B1) namely the classical NF-120581Β pathway through BCMA and TACI However BAFFbut not APRIL also induces the activation of NF-120581B2p100namely the alternative NF-120581Β pathway apparently throughbinding to BAFFR [12 13] At the same time the blocking ofthe classical NF-120581Β pathway blocked the capacity of BAFFto support neoplastic B-cell survival without any effect onthe survival of normal B cells in which the activation ofalternative NF-120581Β pathway prevails [13] Interestingly CLLcells may variably express TACI and very low to undetectableBCMA [9 11 13 19] but all cells express BAFFR with a ratherlower intensity compared to normal B cells [9 11 13 14 2021] These findings suggest that BAFFAPRIL signaling inneoplastic CLL cells might be important

Considering that CLL is characterized by a variableclinical and laboratory presentation and prognosis and thatthe majority of previous studies have enrolled a few CLLpatients (ranged from 10ndash25) the contribution of TACIexpression in the disease phenotype remains as yet obscureThe clarification of its role might be of outstanding inter-est since novel anti-BAFFAPRIL treatments have recentlybeen approved for the management of patients with B-cell-mediated autoimmune diseases [22 23] suggesting that thetargeting of TACI might be also a therapeutic candidate inB-cell malignancies [24ndash26] Therefore the aim of this studywas to investigate the contribution of TACI signaling to thebiological clinical laboratory and prognostic characteristicsof CLL

2 Material and Methods

21 Subjects Ninety-four CLL patients (malefemale 5341mean age 686 range 39ndash87) diagnosed by standard criteria[2] were enrolled in the study Among them 54 wereanalyzed at diagnosis 21 had not received any treatment inthe past and the rest 19 patients were analyzed at relapseFor the majority (90 out of 94) of patients flow cytometricand molecular analyses were performed in peripheral blood(PB) in 4 patients in bone marrow (BM) and in 3 patientssimultaneously in both PB and BM Moreover in 19 healthyindividuals (malefemale 712 mean age 628 range 29ndash84) a detailed analysis of TACI expression on B-cell sub-populations and measurement of BAFF and APRIL serumlevels as described below was also performed No patientor healthy individual was receiving any chemotherapy andorimmunosuppressive treatment over the last sixmonths beforeenrollment

Medical records of all CLL patients were reviewed anddata regarding the frequency and the location of infections

the presence of autoantibodies and autoimmune manifesta-tions the presence of hypogammaglobulinemia or mono-clonal M component and treatment modalities (for patientsat relapse) were recorded Data from conventional andormolecular cytogenetic analyses and ZAP-70 expression werenot available for all patients at enrollment and for this reasonthey were not included in the statistical analysis

The study was conducted in accordance with the prin-ciples of the Helsinki declaration and was approved by theInstitutional Review Board All patients and healthy controlagreed by written informed consent and the proceduresfollowed were in accordance with institutional guidelines

22 Immunophenotyping Studies Immunophenotyping wasperformed by flow cytometry on Coulter FC-500 instrument(Epics XL-MCL 4 color analysis Beckman-CoulterBCHialeah FL USA) using a multistaining protocol andcommercially available reagents Mouse anti-humanimmunoglobulin G (IgG) monoclonal antibodies were usedto detect in patients molecules that reacted with CD3 (cloneUCHT1) CD19 (clone J4119) CD45 (clone J33) CD5 (cloneBL1a) CD20 (clone B9ER) CD23 (clone 9P25) CD38 (cloneT16) CD43 (clone DFT1) CD10 (clone ALB1) CD11c (cloneBU15) CD79b (clone CB 3-1) and FMC7 (clone FMC7)All the above antibodies were purchased by BC and wereconjugated with the appropriate fluorochrome (fluoresceinisothiocyanate FITC phycoerythrin PE PE-cyanine5PE-Cy5) The clonality of B cells was assessed using rabbitanti-human polyclonal antibodies (anti-kappa light chainsFITC anti-lambda light chains PE) purchased by Dako(High Wycombe UK) The percentage of fluorescent cellsand the mean fluorescence intensity (MFI) were determinedin each case corrected for background fluorescence usingFITC PE and PE-Cy5-labelled control IgG antibodies

In healthy individuals peripheral blood samples wereanalyzed in order to indicate the different white blood cellsubpopulations Appropriate mouse anti-human IgG mono-clonal antibodies were used to detect molecules reacting withCD3 (clone UCHT1) CD4 (clone 13B82) CD8 (clone B911)CD19 (clone J4119) and CD45 (clone J33) all purchasedby BC and conjugated with the appropriate fluorochrome(FITC PE PE-Cy5 and ECD) The different cell populationswere gated using forward and side light scatter characteristicsas well as using the expression of specific markers Thepercentage of fluorescent cells and the MFI were determinedas above

The expression of TACI on CLL cells was determinedon 1 times 105 cells after erythrocyte lysis (Versalyse BC) andwashing with 1x phosphate buffered saline (PBS) After-wards the cells were incubated with 3 normal rat blockingserum (eBioscience San Diego USA) at 4∘C for 20minand subsequently were labeled with anti-TACI (clone 1A1PE conjugated Abcam Cambridge UK) anti-CD19 (FITCconjugated BC) and anti-CD45 (PE-Cy-5 conjugated BC)monoclonal antibodies for 15min at room temperature Thepercentage of TACI-positive B cells and their MFI werecorrected for background fluorescence using isotype controlIgG2a-PE antibody (eBioscience) Ten CLL patients exhibit-ing either high or very low to undetectable TACI by flow














Table1Prim

ersa

ndPC

Rcond

ition

sfor

thea

mplificatio

nof

thea

nalyzedgenes

Gene

Prim

ers

Sequ

ence

PCRcond

ition

s

TNFR

SF13B

Forw

ard

Reverse

51015840-TCT

GCC

TGTG

TGCC

GTC

CTC-

3101584051015840-C

GGCT

TCCA

TCGCG

TGAT

-31015840

95∘

Cfor2

minfollowed

by40

cycle

s(95∘

Cfor10s

64∘

Cfor6

0s)

B2M

Forw

ard

Reverse

Com

merciallyob

tained

byQiagen

CatNoPP

H01094E

95∘

Cfor10m

infollowed

by40

cycle

s(95∘

Cfor15s

60∘

Cfor6

0s)

IGHV

A1015840

PCR

FR2A

FR1C

LGH

51015840-TGG(A

G)TCC

G(C

A)C

AG(G

C)C

(TC)(TC)

C(AG

TC)-31015840

51015840-AGGTG

CAGCT

G(C

G)(AT)G

(CG)A

GTC

(AG

T)G

G-31015840

51015840-TGAG

GAG

ACGGTG

ACC-

31015840

95∘

Cfor2

minfollowed

by5cycle

s(95∘

Cfor3

0s63∘C

for3

0s72∘Cfor3

0s)then

by25

cycle

s(95∘

Cfor3

0s

57∘

Cfor3

0s72∘Cfor3

0s)andafi

nalelong

ationat

72∘

Cfor5

min

IGHV

neste

dPC

R

FR2A

VH1

VH2

VH3

VH4

VLJH

Similarw

ithA1015840

PCR

51015840-ACT

AGTC

GAC

CTCA

GTG

AAG

GT-31015840

51015840-ACT

AGTC

GAC

GTC

CTGVG

CTGGTG

AAA(G

C)C

CACA

C-31015840

51015840-ACT

AGTG

ACGGGTC

CCTG

AGAC

TCTC

CTGTG

CAG-31015840

51015840-ACT

AGTC

GAC

CCTG

TCCC

TCAC

CTGC(

AG

)CTG

TC-31015840

51015840-G

TGAC

CAGGGT(AG

CT)C

CTTG

GCC

CCAG

-31015840

95∘

Cfor2

minfollowed

by30

cycle

s(95∘

Cfor3

0s

63∘

Cfor3

0s72∘Cfor3

0s)andafi

nalelong

ationat

72∘

Cfor5

min

B2Mbeta-2-microglob

ulinIGHV

immun

oglobu

linheavychainvaria

bler

egion



3 Results


























41

CD19 ECD


TACI PE

1 2

0 97

38 51

1 10

CD27

PC5

IgD FITCIgD FITC

359

100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

CD27

PC5

100

101

102

103

100 101 102 103


(a)

841

CD19 ECD TACI PE

CD27

PC5

IgD FITC

12 81

1 6

100 101 102 103

100

101

102

103

100 101 102 103100 101 102 103

526 CLL_1

(b)

Figure 1 Continued


IgD FITC

782

CD19 ECD

48

TACI PE

43 44

100 101 102 103

100 101 102 103

100 101 102 103

CD27

PC5

100

101

102

103

5 8

CLL_2








4 Discussion



51

7-A

AD

PC7


0 464 32

0 639 55

0 644 50


7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

0 464 32

0 639 55

0 644 50

100

101

102

103

100 101 102 103100 101 102 103

100

101

102

103

100 101 102 103

100

101

102

103

100 101 102 103


(a)

66578

TACI PE

059 35

060 34

0 856 36

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

6059 35

6060 34

0 856 36

100 101 102 103100 101 102 103 100 101 102 103 100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

(b)

1000900800700600500400300200100

00

1000900800700600500400300200100

00

1000900800700600500400300200100







(c)







800

600200

100

0

BAFF

seru

m le

vels

P lt 0001

(a)


70

60

50

40

30

20

10

0

APR

IL se

rum

leve

ls

P = 0003

(b)








5 Conclusions




Acknowledgments




References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























Table1Prim

ersa

ndPC

Rcond

ition

sfor

thea

mplificatio

nof

thea

nalyzedgenes

Gene

Prim

ers

Sequ

ence

PCRcond

ition

s

TNFR

SF13B

Forw

ard

Reverse

51015840-TCT

GCC

TGTG

TGCC

GTC

CTC-

3101584051015840-C

GGCT

TCCA

TCGCG

TGAT

-31015840

95∘

Cfor2

minfollowed

by40

cycle

s(95∘

Cfor10s

64∘

Cfor6

0s)

B2M

Forw

ard

Reverse

Com

merciallyob

tained

byQiagen

CatNoPP

H01094E

95∘

Cfor10m

infollowed

by40

cycle

s(95∘

Cfor15s

60∘

Cfor6

0s)

IGHV

A1015840

PCR

FR2A

FR1C

LGH

51015840-TGG(A

G)TCC

G(C

A)C

AG(G

C)C

(TC)(TC)

C(AG

TC)-31015840

51015840-AGGTG

CAGCT

G(C

G)(AT)G

(CG)A

GTC

(AG

T)G

G-31015840

51015840-TGAG

GAG

ACGGTG

ACC-

31015840

95∘

Cfor2

minfollowed

by5cycle

s(95∘

Cfor3

0s63∘C

for3

0s72∘Cfor3

0s)then

by25

cycle

s(95∘

Cfor3

0s

57∘

Cfor3

0s72∘Cfor3

0s)andafi

nalelong

ationat

72∘

Cfor5

min

IGHV

neste

dPC

R

FR2A

VH1

VH2

VH3

VH4

VLJH

Similarw

ithA1015840

PCR

51015840-ACT

AGTC

GAC

CTCA

GTG

AAG

GT-31015840

51015840-ACT

AGTC

GAC

GTC

CTGVG

CTGGTG

AAA(G

C)C

CACA

C-31015840

51015840-ACT

AGTG

ACGGGTC

CCTG

AGAC

TCTC

CTGTG

CAG-31015840

51015840-ACT

AGTC

GAC

CCTG

TCCC

TCAC

CTGC(

AG

)CTG

TC-31015840

51015840-G

TGAC

CAGGGT(AG

CT)C

CTTG

GCC

CCAG

-31015840

95∘

Cfor2

minfollowed

by30

cycle

s(95∘

Cfor3

0s

63∘

Cfor3

0s72∘Cfor3

0s)andafi

nalelong

ationat

72∘

Cfor5

min

B2Mbeta-2-microglob

ulinIGHV

immun

oglobu

linheavychainvaria

bler

egion



3 Results


























41

CD19 ECD


TACI PE

1 2

0 97

38 51

1 10

CD27

PC5

IgD FITCIgD FITC

359

100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

CD27

PC5

100

101

102

103

100 101 102 103


(a)

841

CD19 ECD TACI PE

CD27

PC5

IgD FITC

12 81

1 6

100 101 102 103

100

101

102

103

100 101 102 103100 101 102 103

526 CLL_1

(b)

Figure 1 Continued


IgD FITC

782

CD19 ECD

48

TACI PE

43 44

100 101 102 103

100 101 102 103

100 101 102 103

CD27

PC5

100

101

102

103

5 8

CLL_2








4 Discussion



51

7-A

AD

PC7


0 464 32

0 639 55

0 644 50


7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

0 464 32

0 639 55

0 644 50

100

101

102

103

100 101 102 103100 101 102 103

100

101

102

103

100 101 102 103

100

101

102

103

100 101 102 103


(a)

66578

TACI PE

059 35

060 34

0 856 36

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

6059 35

6060 34

0 856 36

100 101 102 103100 101 102 103 100 101 102 103 100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

(b)

1000900800700600500400300200100

00

1000900800700600500400300200100

00

1000900800700600500400300200100







(c)







800

600200

100

0

BAFF

seru

m le

vels

P lt 0001

(a)


70

60

50

40

30

20

10

0

APR

IL se

rum

leve

ls

P = 0003

(b)








5 Conclusions




Acknowledgments




References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3 Results


























41

CD19 ECD


TACI PE

1 2

0 97

38 51

1 10

CD27

PC5

IgD FITCIgD FITC

359

100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

CD27

PC5

100

101

102

103

100 101 102 103


(a)

841

CD19 ECD TACI PE

CD27

PC5

IgD FITC

12 81

1 6

100 101 102 103

100

101

102

103

100 101 102 103100 101 102 103

526 CLL_1

(b)

Figure 1 Continued


IgD FITC

782

CD19 ECD

48

TACI PE

43 44

100 101 102 103

100 101 102 103

100 101 102 103

CD27

PC5

100

101

102

103

5 8

CLL_2








4 Discussion



51

7-A

AD

PC7


0 464 32

0 639 55

0 644 50


7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

0 464 32

0 639 55

0 644 50

100

101

102

103

100 101 102 103100 101 102 103

100

101

102

103

100 101 102 103

100

101

102

103

100 101 102 103


(a)

66578

TACI PE

059 35

060 34

0 856 36

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

6059 35

6060 34

0 856 36

100 101 102 103100 101 102 103 100 101 102 103 100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

(b)

1000900800700600500400300200100

00

1000900800700600500400300200100

00

1000900800700600500400300200100







(c)







800

600200

100

0

BAFF

seru

m le

vels

P lt 0001

(a)


70

60

50

40

30

20

10

0

APR

IL se

rum

leve

ls

P = 0003

(b)








5 Conclusions




Acknowledgments




References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























41

CD19 ECD


TACI PE

1 2

0 97

38 51

1 10

CD27

PC5

IgD FITCIgD FITC

359

100 101 102 103

100 101 102 103100 101 102 103

100

101

102

103

CD27

PC5

100

101

102

103

100 101 102 103


(a)

841

CD19 ECD TACI PE

CD27

PC5

IgD FITC

12 81

1 6

100 101 102 103

100

101

102

103

100 101 102 103100 101 102 103

526 CLL_1

(b)

Figure 1 Continued


IgD FITC

782

CD19 ECD

48

TACI PE

43 44

100 101 102 103

100 101 102 103

100 101 102 103

CD27

PC5

100

101

102

103

5 8

CLL_2








4 Discussion



51

7-A

AD

PC7


0 464 32

0 639 55

0 644 50


7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

0 464 32

0 639 55

0 644 50

100

101

102

103

100 101 102 103100 101 102 103

100

101

102

103

100 101 102 103

100

101

102

103

100 101 102 103


(a)

66578

TACI PE

059 35

060 34

0 856 36

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

6059 35

6060 34

0 856 36

100 101 102 103100 101 102 103 100 101 102 103 100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

(b)

1000900800700600500400300200100

00

1000900800700600500400300200100

00

1000900800700600500400300200100







(c)







800

600200

100

0

BAFF

seru

m le

vels

P lt 0001

(a)


70

60

50

40

30

20

10

0

APR

IL se

rum

leve

ls

P = 0003

(b)








5 Conclusions




Acknowledgments




References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















IgD FITC

782

CD19 ECD

48

TACI PE

43 44

100 101 102 103

100 101 102 103

100 101 102 103

CD27

PC5

100

101

102

103

5 8

CLL_2








4 Discussion



51

7-A

AD

PC7


0 464 32

0 639 55

0 644 50


7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

0 464 32

0 639 55

0 644 50

100

101

102

103

100 101 102 103100 101 102 103

100

101

102

103

100 101 102 103

100

101

102

103

100 101 102 103


(a)

66578

TACI PE

059 35

060 34

0 856 36

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

6059 35

6060 34

0 856 36

100 101 102 103100 101 102 103 100 101 102 103 100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

(b)

1000900800700600500400300200100

00

1000900800700600500400300200100

00

1000900800700600500400300200100







(c)







800

600200

100

0

BAFF

seru

m le

vels

P lt 0001

(a)


70

60

50

40

30

20

10

0

APR

IL se

rum

leve

ls

P = 0003

(b)








5 Conclusions




Acknowledgments




References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















51

7-A

AD

PC7


0 464 32

0 639 55

0 644 50


7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

0 464 32

0 639 55

0 644 50

100

101

102

103

100 101 102 103100 101 102 103

100

101

102

103

100 101 102 103

100

101

102

103

100 101 102 103


(a)

66578

TACI PE

059 35

060 34

0 856 36

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

7-A

AD

PC7

Annexin V-FITC

6059 35

6060 34

0 856 36

100 101 102 103100 101 102 103 100 101 102 103 100 101 102 103

100

101

102

103

100

101

102

103

100

101

102

103

(b)

1000900800700600500400300200100

00

1000900800700600500400300200100

00

1000900800700600500400300200100







(c)







800

600200

100

0

BAFF

seru

m le

vels

P lt 0001

(a)


70

60

50

40

30

20

10

0

APR

IL se

rum

leve

ls

P = 0003

(b)








5 Conclusions




Acknowledgments




References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















800

600200

100

0

BAFF

seru

m le

vels

P lt 0001

(a)


70

60

50

40

30

20

10

0

APR

IL se

rum

leve

ls

P = 0003

(b)








5 Conclusions




Acknowledgments




References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















References





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















TACI expression and signaling in chronic lymphocytic leukemia

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Transcript of TACI expression and signaling in chronic lymphocytic leukemia