CD62L as a Therapeutic Target in Chronic Lymphocytic …midino-2-phenylindole for nuclear staining before being mounted using Vectashield (Vector Laboratories, Inc.). Immunoﬂuorescent

Cancer Therapy: Preclinical

CD62L as a Therapeutic Target in Chronic LymphocyticLeukemia

Melinda Burgess1,2, Devinder Gill1,2, Richa Singhania1, Catherine Cheung2, Lynne Chambers2,Brent A. Renyolds3, Louise Smith2, Peter Mollee2, Nicholas Saunders1, and Nigel AJ McMillan4

AbstractPurpose:Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the disease remains

incurable with standard therapies and relapse is inevitable. A growing body of evidence indicates that

alterations in the adhesion properties of neoplastic cells play a pivotal role in the development and

progression of CLL.

Experimental Design: The expression of 71 cell surface molecules was examined on CLL peripheral

blood mononuclear cells (PBMCs) over 3 weeks in culture. The most highly upregulated marker, CD62L,

was examined further for expression on CD5þ/CD19þ CLL cells in vitro and in lymph node and bonemarrow biopsies. The prosurvival role of CD62L was examined using a functional blocking antibody and

therapeutic potential evaluated by comparison with current chemotherapy agents.

Results: Blocking CD62L resulted in apoptosis of CLL cells but not PBMCs from healthy donors

suggesting a novel role for CD62L in CLL cell survival. The beneficial effect of coculturing CLL cells with

bonemarrow stromal cells or endothelial cells does not protect CLL cells from anti-CD62L–related toxicity.

Moreover, combining fludarabine or mafosfamide with the anti-CD62L in vitro produced an additive effect

both with and without stromal cells.

Conclusion: This is the first reported data showing that blocking the activation and homing marker,

CD62L, regulates CLL cell survival in vitro. These data also suggest that therapeutic antibodies against CD62L

may provide additional clinical benefit to patients with CLL receiving current standard chemotherapy

protocols. Clin Cancer Res; 19(20); 5675–85. �2013 AACR.

IntroductionChronic lymphocytic leukemia (CLL) is the most com-

mon leukemia in adults in Western society and is charac-terized by a progressive accumulation of monoclonalCD5þ/CD19þ B lymphocytes in the peripheral blood, bonemarrow, and lymphatic tissue. The clinical course is variablewith early stage disease having life expectancymore than 10years, but patients with more advanced disease have amedian survival of 18 months to 3 years. The current

standard therapy of fludarabine, cyclophosphamide, andrituximab (FCR therapy) has improved complete responsesand overall survival (1), but relapse is inevitable and thetherapy is associated with significant toxicities. Therefore,identifying new agents with novel mechanisms of actionthat complement current chemotherapies and abrogate theCLL drug-resistance factors will be necessary if furtherimprovements in CLL patient outcomes are to be realized.

The tumor microenvironment is essential for tumor pro-liferation, survival, angiogenesis, and metastasis in a rangeof malignancies including CLL (2). Studies using heavywater suggest that CLL consists of a pool of quiescent cellscirculating in the peripheral blood together with a reservoirof proliferating cells within the bone marrow and lymphnodes (3, 4). This suggests a proproliferative environment islocated within bonemarrow and lymph node, whereas CLLcells within blood are associated with long-term survival.CLL proliferation, survival, homing, and drug resistance (5–12) have been attributed to numerous factors such aschemokines, cytokines, and direct cell–cell interactionsmediated via adhesionmolecules in the lymphnode, spleen,and bone marrow microenvironment which suggests thesefactors may represent important therapeutic targets. Forexample, the Bruton tyrosine kinase (BTK) inhibitor, ibru-tinib, impairs B-cell receptor and chemokine-controlledretention of CLL cells in themarrow and lymph nodes. This

Authors' Affiliations: 1University of Queensland Diamantina Institute,Brisbane, Australia; 2Department of Haematology, Princess AlexandraHospital, Brisbane, Australia; 3Department of Neurosurgery, McKnightBrain Institute, University of Florida,Gainesville, Florida; and 4GriffithHealthInstitute and School of Medical Sciences, Griffith University, Southport,Queensland, Australia

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

M. Burgess, D. Gill, N. Saunders, and N.A.J. McMillan contributed equallyto this work.

Corresponding Author:Nicholas Saunders, TheUniversity of QueenslandDiamantina Institute, Translational Research Institute, Level 7, Kent Street,Brisbane, Queensland 4102, Australia. Phone: 617-3443-7091; Fax: 617-3443-6966; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-1037

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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on June 9, 2021. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst August 15, 2013; DOI: 10.1158/1078-0432.CCR-13-1037

http://clincancerres.aacrjournals.org/

deprives the CLL cells of their microenvironmental survivalsignals leading to disease regression (13). Consequently,there is growing interest in targeting the survival cues asso-ciated with the tumor microenvironment. Understandingthe molecular and biologic basis for these microenviron-ment-mediated responses will be critical to understandingCLL and developingmore potent or effective therapies (12).

Our previous work has concentrated on the developmentof a long-term culture systemwhere culturing CLL PBMCs athigh density provides a significant survival advantage to CLLcells (14). This culture system was also confirmed by therecent report of Seiffert and colleagues (15). Having estab-lished that culturing CLL PBMCs at high density gave asignificant survival advantage to CLL cells, we set aboutinvestigating what cell–cell interactions might be importantfor survival by assessing expression levels of cell surfacemarkers. Here, we report a significant upregulation of severalmarkersCD184 (CXCR4), CD26, CD40,CD58, CD103, andCD62L (L-selectin) on the survivingCD5þ/CD19þCLL cells.On the basis of CD62L’s known role in normal lymphocytemigration (16, 17), and initial reports of its prognosticsignificance in CLL (18), we hypothesized that CD62L maybe important in microenvironmental retention, homing, orsurvival of CLL cells and may offer a potential therapeutictarget. We (i) examined the expression of CD62L in CLL, (ii)investigated the effect of anti-CD62L antibody therapy onCLL cell survival in vitro, and (iii) assessed the cytotoxicity ofCD62L blockade in combination with the standard agentsused in the treatment of CLL. Results presented in this studysupport the potential of anti-CD62L as a therapeutic agent.

Materials and MethodsPatients and cell purification

Blood was collected into EDTA tubes from healthydonors or patients with CLL after informed consent accord-

ing to protocols approved by the Princess Alexandra Hos-pital (PAH, Brisbane, Australia) Human Research EthicsCommittee. Diagnosis of CLL was made according to NCIcriteria. Patients with CLL were either untreated (39 sam-ples) or a median of 24 months after their last treatment(range 12–57 months, 14 samples; Supplementary TableS1). Mononuclear cells were isolated by density-gradientcentrifugation over Histopaque-1077 (Sigma-Aldrich).

CLL culture and cell viabilityPurified PBMCs were resuspended in RPMI-1640 medi-

um (Gibco Invitrogen), supplemented with 10% heat inac-tivated fetal calf serum, 100 U/mL penicillin G, 100 mg/mLstreptomycin, and2.92mg/mLglutamine (Gibco; completemedia) at a cell concentration of 20–60� 106/mL. It shouldbe noted that these long-term cultures of PBMCs containnon-CLL cells derived from circulating white blood cellsplus an adherent population of cells which includes nurse-like cells and endothelial cells. Cell viability was conductedby Trypan blue exclusion and survival was expressed asnumber of viable cell counts relative to initial plating countsor normalized to untreated CLL PBMC survival. Cell via-bility and apoptosis was determined by flow cytometryusing Annexin V/PI staining kit (Roche) according to themanufacturers’ instructions. CD19þ CLL cells were purifiedusing MACS cell separation and CD19 Microbeads (Milte-nyi Biotec) according to the manufacturer’s protocol.

Flow cytometry screening analysis of surface antigensCLL PBMCs from 3 patients were screened for the expres-

sion of 71 cell surface antigens (outlined in SupplementaryTable S2) directly after isolation from the patient and after3 weeks of high-density culture (2 � 107/mL). Cells werewashed with PBS and analyzed on a BD LSRII flow cyt-ometer (BD Biosciences). Viable cells were gated on thebasis of forward and side scatter profile. Data were analyzedusing BD FACSDiVa software (BD Biosciences). Followinginitial FACS screen, CD62L expression was confirmed onadditional patient samples using flow cytometry. Cells wereincubated with APC-conjugated anti-CD19, FITC-conjugat-ed anti-CD5 (both from BD Biosciences), and PE-conju-gated anti-CD62L antibodies (BioLegend) and analyzed ona FACSCanto (BD Biosciences). Data were analyzed usingBD FACSDiVa software.

In vitro CD62L assaysThe effect of neutralizing CD62L was examined by cul-

turing CLL PBMCs or PBMCs isolated from healthy donorsin the presence of LEAF Purified anti-human CD62L anti-body (DREG56; 0.1 mg/mL; BioLegend), anti-humanCD62L antibody (DREG200, 0.1 mg/mL, ebioscience), orLEAF PurifiedMouse IgG1, k Isotype Control Antibody (0.1mg/mL; BioLegend) and cell viability was examined asdescribed above. For coculture experiments, human umbil-ical vein endothelial cells (HUVEC) or HS-5 cells wereseeded at a density of 2,000 cells per well in 96-well plates24 hours before the addition of CLL PBMCs with/withoutCD62L antibody.

Translational RelevanceAlthough the management of patients with chronic

lymphocytic leukemia (CLL) has improved over the pastdecade it still remains an incurable disease. Contributingto the poor outcome is a lack of knowledge of theunderlying pathobiology of CLL. High levels of CD62Lhave previously been shown to contribute to the homingand retention of lymphocytes to the bone marrow andlymph nodes. In this study, we show, for the first time,that the expression of CD62L also activates a novelprosurvival signal in CLL cells. Significantly, inhibitionof CD62L using therapeutic antibodies induces apopto-sis equivalent to that seen with current chemotherapeu-tics and antagonizes stromal-induced survival. Thus, wehave identified a novel prosurvival signal in CLL cellsthat when antagonized can induce cell death equivalentto current CLL chemotherapeutics and when combinedwith current chemotherapeutic cocktails can add signif-icantly to the cytotoxic response.

Burgess et al.

Clin Cancer Res; 19(20) October 15, 2013 Clinical Cancer Research5676




Chemotherapy combination assaysTo examine the therapeutic potential of CD62L antibody

therapy, CLL PBMCs were cultured either alone, or in thepresence of HS-5 cells, with varying combinations of thefollowing agents; CD62L antibody (DREG56; 0.1 mg/mL),rituximab (10 mg/mL, Mabthera, Roche) both on day 0and fludarabine (1 mg/mL, Fludara), and/or mafosfamide(1 mg/mL; Santa Cruz Biotechnology) on day 4.

Western blottingCultured CLL cells were lysed in radioimmunoprecipita-

tion assay buffer containing phosphatase and proteaseinhibitors and electrophoresed onto 10% SDS-polyacryl-amide gels. Membranes were developed using SuperSignalWest Pico Chemiluminescence substrate (Thermo Scientif-ic). The following antibodies were used: PARP, LC3 A/B,pAKT (Ser 437)XP, total AKT, phosphor-p44/42MAPK(Thr202/Tyr 204), total MAPK (all from Cell Signaling Tech-nology), GAPDH (Sigma-Aldrich), and B-actin (Sigma-Aldrich).

Immunofluorescence staining and confocalmicroscopyEthics Committee approval was obtained to use the

redundant portion of bone marrow trephine biopsies frompatients with untreated CLL. Sections from an uninvolvedlymphoma bone marrow, from a normal lymph node, andfrom an uninvolved node from a patient with lymphomawere used as controls. Primary antibodies include mousemonoclonal anti-human-CD62L (Clone 9H6;Novocastra),mouse monoclonal anti-human-CD20 (Clone L26; Dako).Secondary antibodies include Alexa Fluor 555 goat-anti-

mouse and Alexa Fluor 488 goat-anti-mouse antibodies(Invitrogen). Sections were counterstained with 40, 6-dia-midino-2-phenylindole for nuclear staining before beingmounted using Vectashield (Vector Laboratories, Inc.).Immunofluorescent signals were viewed through a CarlZeiss LSM 510 Meta confocal microscopy system (CarlZeiss) and analyzed using Zen imaging software

Data analysis and statisticsResults are shown as mean � SEM of at least 3 experi-

ments each conducted in triplicate. For statistical compar-ison, the Student unpaired or paired t test was used, two-tailed with 95% confidence intervals. Pearson correlationtests were used for examining the relationship of clinicalparameters with response to CD62L treatment. Response toCD62L antibody treatment was normalized to viability inuntreated cultures before correlation analysis. Findingswere considered significant if P � 0.05ResultsCD62L expression is upregulated on CLL cells in long-term culture

Immunophenotyping was carried out on 3 patients withCLL using 71 antibodies, comparing the percentage pos-itive cells at day 0 with those after 3 weeks in culture. Thisanalysis was conducted on suspension cells only and didnot include adherent cell populations and nurse like cells.CD5þ/CD19þ cells constituted more than 80% of the totalcells in culture at day 0 and this was maintained at week 3,indicating persistence of the CLL population (Fig. 1Aand B). Significant changes were observed for 25 surfaceantigens, with increases observed in the expression of

Figure 1. Cell surface markersshowing differential expression.CLL PBMCs were cultured at highdensity for 3 weeks and expressionof 71 cell surface molecules wasexamined and compared withbaseline levels using flowcytometry. Paired t testsdetermined which cell surfacemarkers were differentiallyexpressed, either an increase (A) ora decrease in expression observedover time in culture (B). Data aredisplayed as the mean � SEM(n ¼ 3; �P < 0.05; ��P < 0.01;��P < 0.001).

CD62L in CLL Survival

www.aacrjournals.org Clin Cancer Res; 19(20) October 15, 2013 5677




CD26, CD40, CD58, CD62L, and CD103 (Fig. 1A) anddecreased expression found in the other markers includingCD11c, CD32, CD49f, CD62P, CD80, CD106, CD140a,CD141, CD184, CD206, and CD273 (Fig. 1B). The com-plete data set is presented in Supplementary Fig. S1. Someof the observed differences in cell surface markers may bedue to changes in the non-CLL cell population duringculture. For example, CD206 is expressed on immaturedendritic cells and macrophages, cells that are expected tobe lost in culture. However, the main power of our screen-ing approach is that we were able to identify those mole-cules for which expression is highly upregulated during theculture of CLL cells such as CD62L (Fig. 1A). The veracityof the screen was also validated by the identification ofmolecules previously identified in CLL biology includingCD58 (LFA3), a cell adhesion molecule whose increasedexpression has been associated with advanced disease state(19) and CD184 (CXCR4), the receptor for SDF-1 whichhas been shown to protect cells from apoptosis in vitro(20). Several of the other molecules which exhibit signif-icant changes in expression are related to cell adhesionincluding a downregulation of CD18 (integrin b-2) whichis the beta subunit of 4 structures, of which 2 are alsodifferentially downregulated in our screen, CD11a andCD11c. Overall, the most significantly upregulated markerwas CD62L.

To directly confirm that the increased levels of CD62Lwere expressed on CLL cells, we undertook double-labelingof CLL PBMCs with CD19 and CD62L antibodies andmonitored their levels over 7 days in culture. It was foundthat the number of CD19þ/CD62Lþ cells was below 1%when isolated from the patient, but rapidly climbed, withan increase to 36.8% within 24 hours and this level con-tinued to increase to above 80% by day 7. A representativeresult is shown in Fig. 2A. Examination of nine furtherprimary cultures showed that there was a significantincrease in CD62L levels in each case, with the averageCD19þ/CD62Lþ populationmoving from12� 6% to 81�5% (P < 0.0001; Fig. 2B). As CD62L has previously beenshown to be expressed on a range of other lymphocytesincluding T cells (21), we undertook flow cytometry anal-ysis of both B (CD5þ/CD19þ) and T cells (CD5þ/CD19�)within the same PBMC culture to determine which cellswere upregulating the CD62L. Our data show that it was infact the CLL cells that became positive for CD62L over timerather than T cells within the culture (Fig. 2C). The upre-gulation of CD62L was maximal by day 5 and was main-tained in CD5þ/CD19þ cells for up to 21 days (Fig. 2C). Itwas also critical to show that the specificity of this increasedexpression was an inherent property of CLL cells and not acharacteristic of normal B cells. Therefore, PBMCs from ahealthy 60 year old female volunteer were cultured for 7

Figure 2. IncreasingCD62L expression onCLL cells in vitro. A, CLLPBMCswere cultured at high density and expression of CD62L onCD19-positive cells wasexamined using flow cytometry over 7 days in culture. Several patient samples were used and this data is representative of one patient. B, an additional 9patients were examined for expression of CD62L- on CD19-positive cells after 7 days in culture. C, flow cytometry analysis of CD62L expression onCD19þ/CD5þ CLL cells and CD5þ T cells throughout 14 days in culture. Data are representative of one patient. D, PBMCs from healthy donors wereexamined for expression of CD62L before and after 7 days in culture using flow cytometry. A total of 3 donors were examined with 1 donor represented.E, induction ofCD62L expressionwas also examined onCD19-purifiedCLLPBMCspre andpost in vitro culture. Data is displayed as themean�SEM. F, flowcytometry analysis of CD62L expression on annexin V-negative cells. Data are representative of one patient.

Burgess et al.





days at the same seeding density asCLL PBMCs and the levelof CD62L expressionmeasured by flow cytometry. The levelof CD19þ/CD62Lþ cells increasedminimally from 2.8% to5.8% over 7 days which indicates increased CD62L expres-sion is a feature of CLL cells rather than normal B cells (Fig.2D). The induction of CD62L was also examined in CD19purified CLL cells to determine whether accessory cellswithin the PBMC culture were essential for the increasedexpression of CD62L. This is shown in Fig. 2Ewhere there isno significant difference in CD62L expression between CLLcells in PBMC culture and CD19 purified cells from thesame patients. The expression of CD62L was also examinedon viable cells using Annexin V staining after 7 days inculture to confirm that this upregulation was related to CLLcell survival. Only cells which were low in Annexin Vpositivity showed expression of CD62L (Fig. 2F). Finally,the induction of CD62L expression was not due to theexpansion/proliferation of the CLL fraction as BrdUrdlabeling studies indicated that the proliferation index couldnot account for the large expansion inCD62L-positive cells.Thus, the increased expression of CD62L is due to a time-dependent induction of expression of CD62L on CD5þ/CD19þ CLL cells.

CD62L expression is upregulated in CLL cells in lymphnode and bone marrowAs CD62L is involved in homing and migration of lym-

phocytes to lymphoid organs, we examined the expressionof CD62L in bone marrow and lymph node biopsies fromCLL patients using immunofluorescent staining. Wedetected CD62L expression on a large proportion of CD20positive B-cells in lymph node sections from patients withCLL (Fig. 3A). Sections fromanormal lymphnode and froma noninvolved lymph node from a patient with lymphoma

served as controls, with a very small number of CD20-positive cells positive for CD62L in the normal lymph nodecontrol and no CD62L expression detected in the lympho-ma control. The CD62L expression in the bonemarrowwasevenmore strikingwith a high proportion of CD20-positivecells coexpressing CD62L (Fig. 3B). Conversely, a fewCD20positive cells were present in the lymphoma control bonemarrow and there was no CD62L expression evident. Toexamine whether there was any association with expressionof CD62L in the lymph node and bone marrow compart-ments comparedwith the periphery, CD62L expressionwasexamined on CLL PBMCs from matched patients used inabove immunofluorescence studies (Fig. 3C). CD62Lexpression in CLL PBMCs varied from 0.9% to 43.5%between the 3 patients used, but this positivity did notdirectly reflect the CD62L expression observed in the lymphnode and bone marrow. For example, CLL patient 16 hadhigh expression of CD62L in the bonemarrow but very lowCD62L expression inCLLPBMCs (0.9%). Thiswas also seenfor CLL patient 17, with high expression of CD62L onCD20-positive cells in the bone marrow, but only 17.3%CD62L positivity in CLL PBMCs. These results indicate thepresence of CD62L in proliferation and survival nichesinvolved in CLL.

Blocking CD62L promotes apoptosis of CLL cellsGiven the highly induced expression of CD62L in sur-

viving CLL cells, we examined the effect of a neutralizingCD62L antibody. Dose response analysis (0–2 mg/mL) wasinitially conducted to determine the optimal dose of anti-CD62L antibody that induced cell death.Maximal responsewas achieved at 0.1 mg/mL (data not shown). This trendwasobserved regardless of patient white cell counts. Initially, 7CLL patients’ PBMCs were cultured as described previously

Lymph nodeA B C

Bone marrow(Red=CD20, Green=CD62L, Blue=DAPI) (Red=CD20, Green=CD62L, Blue=DAPI)

CLL Pt16Positive Abs

CLL Pt16Isotype Abs


CLL Pt18Isotype Abs


CLL Pt16Isotype Abs


CLL Pt16

0.9%

17.3%

43.5%

CLL Pt17

CLL Pt18

CD19

CD

62L

CLL Pt17Isotype Abs

NormalLymph NodePositive Abs

NormalLymph NodeIsotype Abs

LymphomaControl

Positive Abs

LymphomaControl

Isotype Abs

LymphomaControl

Positive Abs

LymphomaControl

Isotype Abs

Figure 3. CD62L is detected in CD20þ cells in CLL lymph nodes and bonemarrow trephines. Representative sections were presented for 2 CLL lymph nodes,an uninvolved lymphoma control, and a normal lymph node control trephine (A). Representative sectionswere presented for aCLL trephine and an uninvolvedlymphoma control (B). Confocal microscopy was conducted for double-staining of CD62L (green) and CD20 (red). Images were presented for sectionsincubated with the positive antibodies (CD62L and CD20) and with the isotype control antibodies (the neg Ab control). C, flow cytometry analysis of PBMCsfrom time-matched patients used in (A) and (B) were examined for CD19 and CD5 and CD62L positivity.






inmedia containing either anti-CD62L (DREG-56 clone) oran isotype control antibody,withPBMCs fromage-matchedhealthy individuals acting as controls. The neutralization ofCD62L resulted in a significant loss of CLL cell survival(11.27% � 1.638) compared with untreated CLL PBMCs(49.12� 3.554; P < 0.0001; Fig. 4A). CLL cells treated withan isotype control antibody had the same survival profile asuntreated CLL PBMCs with 42.87% and 49.12% survival

respectively (P ¼ 0.1649). Importantly, PBMCs from nor-mal healthy controls were unaffected by treatment withanti-CD62L antibody reinforcing that the response isrestricted to CLL cells. Further testing of 36 CLL patientsamples confirmed the role of CD62L blockade in CLL cellsurvival with all patients showing a reduction in survivingcells (Fig. 4B)with amean reductionof 69%(P¼0.001). Todetermine the mechanism underlying the reduced cell

Figure 4. Blocking CD62L results ina specific reduction of CLL cells. A,CLL PBMCs or PBMCs isolatedfrom healthy donors were culturedin the presence of anti-humanCD62L antibody (DREG56) or anisotype control antibody, both at aconcentration of 0.1 mg/mL, andcell survival examined after 7 days.Data are displayed as the mean �SEM and is representative of 7 CLLpatients and 4 healthy donors,each conducted in triplicate;��, P < 0.001. B, As for (A). butexamined in an additional 37patients. Data are displayed as themean � SEM; a paired t test wasconducted with P � 0.0001. C,apoptosis was examined byAnnexin V/PI staining on cellscultured as described above andexamined using flow cytometry.Percentages represent lateapoptotic cells; data arerepresentative of 1 patient. D, flowcytometry analysis of CLL PBMCcultures treated with CD62Lantibody were examined for CD19and CD5 positivity after 7 days inculture. The percentage isrepresentative of CLL cells anddata is representative of onepatient. E, antibody response wasvalidated using a different CD62Lantibody clone (DREG200,0.1 mg/mL) and cell survivalexamined after 7 days. Data isdisplayed as the mean � SEM andis representative of 3 CLL patients,each conducted in triplicate;��,P

survival in response to CD62L blocking, we measuredapoptosis using flow cytometry. It was observed that cellswere undergoing apoptosis as a result of the neutralizationof CD62L (Fig. 4C) with an increase in late apoptotic cellsfrom 5.2% to 71.1% when treated with the CD62L anti-body compared with untreated PBMCs and similarly whencompared with the isotype control. Significantly, theCD62L antibody treatment was causing a specific reduc-tion of CD5þ/CD19þ cells with a 49% reduction in theproportion of CLL cells in the PBMC culture when com-pared with untreated PBMCs (Fig. 4D). The specificity oftargeting CD62L was confirmed by using a different neu-tralizing antibody, DREG-200, which, like DREG-56 tar-gets the lectin binding domain of CD62L. Using the sameculture and antibody treatment conditions, DREG-200significantly reduced the survival of CLL cells to 55.93%,although not as dramatically as observed for DREG-56with a reduction of survival to 45.33% (P < 0.01 for bothDREG56 and DREG200; Fig. 4E). In addition, annexin V/PI staining confirms that both antibodies initiate increasedapoptosis of CLL cells and corroborates the cell survivaldata (Fig. 4F). Together these data indicate that treatmentwith a CD62L antibody results in apoptosis in a CLL cell-specific manner.To further dissect the mechanism of CD62L antibody

mediated cytotoxicity, we examined the induction of com-mon survival pathway markers such as PARP cleavage,MAPK (total and phosphorylated), and Akt (total andphosphorylated). However, there was no differenceobserved in expression levels, or activation state betweenuntreated CLL PBMCs and CD62L antibody-treated cells(Fig. 5A andC). In addition, we observed no changes for theautophagymarker, light chain 3 (LC3 A/B) expression, (Fig.5B) following treatment. These data indicate that the apo-ptosis-inducing actions of anti CD62L are independent ofobjective changes in the activity of the AKT,MAPK, PARP, orautophagy pathways.

Associationof response toCD62Lblockade and clinicalparameters

Having established the specific effect of CD62L anti-body treatment on CLL PBMC cultures, we examinedwhether any associations were apparent between anti-CD62L induced CLL death and clinical parameters. Themost significant association was a positive correlationbetween CD62L antibody response and CD5þ/CD19þ

percentage (P ¼ 0.0142) and lymphocyte count (P ¼0.0533) at time of sample collection and a negativecorrelation for haemoglobin (P ¼ 0.0348; SupplementaryFig. S2A–S2C). Together, these associations indicate arelationship between low leukemic burden and moreeffective in vitro antibody response. However, CLL PBMCsisolated from patients with unmutated immunoglobulinheavy-chain variable-region (IgVH) gene mutations had asuperior response to the CD62L antibody comparedwith those who were mutated (Supplementary Fig.S2F). In addition, no significant associations wereobserved between CD62L response and CD38 expression(Supplementary Fig. S2D), Zap70 expression (Supple-mentary Fig. S2E), or Rai stage (Supplementary Fig.S2G). Other clinical parameters which showed no corre-lation with effectiveness of CD62L antibody treatmentinclude lymphocyte doubling time, time till first treat-ment, age, and platelet count, albumin, hypogammaglo-bulinaemia, LDH, and b-2 microglobulin levels (Supple-mentary Fig. S3).

Effect of CD62L blockade is not abrogated byco-culture with supporting microenvironmental cells

CLL cell survival has previously been shown to beenhanced by coculture of CLL cells with both bone marrowstromal cells (HS-5) and HUVEC by recapitulating theprosurvival signals that may exist in the leukemic micro-environment in vivo (22, 23). As CD62L is overexpressed insurviving CLL cells in vitro and CD62L-specific antibodies

Figure 5. CD62L related cytotoxicity is not dependent on Akt, MAPK or autophagy pathways. CLL PBMCs were cultured with or without CD62L antibody andprotein expression of PARP (A), LC3A/B (B), and total and phosphorylated Akt andMAPK (C) examined by immunolot. Representative blots from 1 patient areshown. Analysis was conducted for a total of 3 patients.






are able to induce apoptosis we examined the possibilitythat the cytotoxic response to CD62L antibody could beantagonized by a prosurvival interaction of CD62Lexpressed onCLL cells with stromal cells. HS-5 cells providea survival advantage over CLL PBMC culture alone with9.5% increase in overall cell survival, but do not protectagainst anti-CD62L antibody treatment with 37.6% and38.9% reduction in survival for CLL PBMCs culture andHS5-5 coculture respectively (P < 0.0001; Fig. 6A). Similarresults were observed for HUVEC coculture experiments(Fig. 6A). These results indicate that anti-CD62L therapymay be effective in vivo where prosurvival microenviron-mental interactionsmay contribute to the oncogenic expan-sion of CLL cells.

Therapeutic potential of CD62L antibody therapyAs CD62L seems to modulate the survival response of

CLL cells in vitro, we extended this to determine whetherCD62L blockade may also modulate sensitivity to conven-tional chemotherapeutics used in the treatment of CLL.Experiments were conducted combining maximal dosesof CD62L antibody (0.1 mg/mL) added on day 0 andmaximal cytotoxic doses of fludarabine (1 mg/mL) addedon day 4 post CD62L antibody treatment. Cell survival wasthen examined at 7 days. These experiments showed thatfludarabine and CD62L antibody, were similar in theircytotoxic potency with a 55.5% and 43.65% reduction insurvival, respectively (Fig. 6B). However, a combination ofCD62L antibody plus fludarabine gave significantly greater

Figure 6. Therapeutic potential of CD62L antibody therapy. A, CLL PBMCs were cultured for 7 days either alone or cocultured with HS-5 or HUVEC cells for 1week with anti-human CD62L antibody. Overall cell survival was examined by Trypan blue exclusion and data is displayed as the mean � SEM (n ¼ 7for HS5 and n¼ 3 for HUVEC; �, P < 0.05 ��, P < 0.001). CLL PBMCs were cultured alone or with HS5 cells and treated with CD62L antibody on day 0 eitheralone or in combination with fludarabine (B), mafosfamide (C), or rituximab (D), and cell survival examined on day 7. Data are displayed as mean � SEM(n¼ 8; ��, P < 0.01; ��, P < 0.001). E, CLL PBMCs were cultured with either CD62L antibody, fludarabine, or mafosfamide alone or in combinations as shownand survival examined. Data are displayed asmean�SEM (n¼ 8; ��,P < 0.01; ��,P < 0.001). F, CLLPBMCswere cultured using the therapy combinations asshown and cell survival examined after 7 days. G, CD19 and CD5 positive cells were examined after 7 days following treatment using flow cytometry.

Burgess et al.





cytotoxic responses compared with each drug given alonewith a 70.53% reduction in cell survival (Fig. 6B). Thisresponse was not altered when fludarabine was addedbefore the addition of the CD62L antibody (data notshown). This trend was also observed when CLL PBMCswere cultured with HS-5 cells (Fig. 6B). Similarly, whenCD62L antibody responses were compared with mafosfa-mide (1mg/mL), a cyclophosphamide analog used in in vitroexperiments, similar potencies were observed when used assingle agents, but an additive effect when used in combi-nation (Fig. 6C). These responses were maintained whencultured with HS-5 cells. However, the cytotoxic responseswith CD62L antibody treatment and rituximab (10 mg/mL)showed no significant difference when used either as singleagents or in combinations (Fig. 6D). However, when cul-tured with HS-5 cells, there was a significant differencebetween rituximab treatment alone and the combinationwith CD62L antibody (Fig. 6D). Given the additive effectobserved for CD62L with fludarabine or mafosfamide, weexamined the consequence of combining the three agents.There was a significant increase in cytotoxic responses whenCD62L was combined with both fludarabine and mafosfa-mide compared with either each agent alone, or any twoagents combined (Fig. 6E). Next, we examined whether theaddition of CD62L antibody treatment was comparablewith current chemoimmunotherapy strategies that use acombination of fludarabine, cyclophosphamide, and ritux-imab therapy (FCR). Therewas no significant increase in theresponse betweenfludarabine,mafosfamide, and rituximabcombination and fludarabine, mafosfamide, and CD62Lantibody combinations with survival reduction of 74.4%and 81.36%, respectively (P¼ 0.140, Fig. 6F). There was nosignificant advantage of combining all four agents (85.56%reduction in cell survival). Flow cytometric analysis of thesurviving CLL cells following treatment with CD62L anti-body, fludarabine, mafosfamide, or rituximab shows thatCD62L blockade is the most efficient in specifically target-ing CLL cells with a reduction from 80.2% to 62.7% after 7days (Fig. 6G). Conversely, fludarabine (85.2%), mafosfa-mide (88.0%), and rituximab (86.5%) showed no specificreduction in CLL percentages. These data indicate thatcombining the cytotoxic activity of current CLL therapieswith the antisurvival activity of CD62L inhibition mayenhance cytotoxic responses of CLL cells.

DiscussionCLL is a B-cell malignancy characterized by aberrant

control of apoptosis. Recently, we and others have shownthat death, in vitro, could be reduced by culture of CLL cellsat highdensity and/or in thepresenceof accessory andnurselike cells (14, 15). These data and other accumulatingevidence indicate that CLL cell resistance to apoptosis isattributable to microenvironmental factors mediated viacell–cell interactions and/or dysregulation of proapoptoticor prosurvival cytokines, and their cognate receptors andsignaling pathways. In this study,we show, for the first time,that (i) survival of CLL cells, in vitro, is accompanied byprofound increases in the expression of CD62L, (ii) pro-

survival signals from stromal cells are CD62L-dependant asinhibition of CD62L, by therapeutic antibodies, reducesCLL cell survival, (iii) that CD62L-induced survival path-ways are independent of those pathways mediatingfludarabine ormafosfamide-induced cytotoxicity as CD62Lantibody treatment potentiates the cytotoxic effects of flu-darabine, mafosfamide beyond those observed using amaximal dose of fludarabine or mafosfamide alone and,(iv) CD62L antibody treatment, in vitro, can overcome theprosurvival signals provided by stromal cells. This latterpoint is of particular relevance as we show that CD62Lexpression is profoundly overexpressed on malignant B-cells located within lymph nodes and the bone marrowparticularly in the proliferation centres (pseudofollicles).The malignant B-cells residing in the bone marrow andlymph node compartments are particularly resistant toapoptosis due to the prosurvival signals within these niches.Thus, CD62L is a novel prosurvival effector that may rep-resent an attractive therapeutic target in CLL.

CD62L (L-selectin, LAM-1, LECAM1, SELL) is a memberof the selectin family of adhesionmolecules and is known toplay an important role in the trafficking/homing of lym-phocytes to the lymph node. For example, CD62L bindsseveral ligands involved in homing and migration such as,CD34, GlyCAM-1, and MadCAM-1 (21). Moreover, highCD62L andCXCR4 expression on lymphocytes is needed tomigrate to lymphnodes andbonemarrow (24).Once in thelymph nodes and bone marrow environment lymphocytesmigrate into the proliferating centres under the influence ofchemokines such as CXCL12 (25). Conversely, lower levelsof CD62L expression on CLL cells has been associated withan impaired capacity to migrate under endothelial cells invitro (26). Furthermore, lower CD62L and CXCR4 expres-sion is necessary for CLL cells to exit these microenviron-mental niches (27). Consistent with these observations onthe role of CD62L in lymphocyte homing it has also beenshown that BCR activation results in downregulation ofCD62L and CXCR4 (27). Thus, the dysregulation of CD62Lexpression on CLL cells is likely to contribute to the path-ological disturbance in total CLL numbers and the accu-mulation of CLL cells in the peripheral blood and lymphnodes.

The results of this study show that in vitro addition of anti-CD62L to primary CLL results in apoptotic loss of CD5þ/CD19þ cell comparable with fludarabine-induced cytotox-icity. More importantly PBMCs from normal healthycontrols were unaffected by treatment with anti-CD62Lantibody reinforcing that the response is restricted to CLLcells. This effect of CD62L blockade is not abrogated by thepresence of stromal cell line HS-5 suggesting that anti-CD62L therapy may be effective in vivo where prosurvivalmicroenvironmental interactions may contribute to theoncogenic expansion of CLL cells. There was a significantincrease in cytotoxic responses when CD62Lwas combinedwith both fludarabine and mafosfamide compared witheither each agent alone or any two agents combined. Thesefindings indicate that different pathways in survival/deathpathways in response to CD62L antibody or fludarabine






andmafosfamide exist. These data also highlight the poten-tial therapeutic value of combining anti-CD62L therapieswith current chemotherapy agents in patients with CLL.

The mechanism by which CD62L antibody producescytotoxicity CLL cells is currently unknown. As we havepreviously shown that CLL cell survival, in vitro, is modu-lated by secreted factor(s) as well as cell–cell interactions, itis likely that themechanisms involved are complex andmayinvolve multiple independent signaling events or indepen-dent signaling events that converge on a common prosur-vival pathway. Significantly, we found no evidence forconstitutive activation or induction of common survivalpathways such as AKT or MAPK signaling pathways in CLLcells taken from patients or following culture in vitro.Furthermore, addition of a neutralizing CD62L antibodyfailed to alter AKT orMAPK activity. These data indicate thatCD62L-mediated survival is independent of alterations inAKT and MAPK signaling. Given that cytotoxicity inducedby CD62L-neutralizing antibody took several days to occur,we considered the possibility that it may be driven byautophagy. However, death induced by CD62L antibodywas not associated with changes in the expression of theknown autophagy marker, LC3A/B. Thus, CD62L signalingwas independent of AKT and MAPK pathways and waslinked to the suppression of apoptosis. Our data suggestthat there may be other hitherto unknown roles for CD62Lin CLL.

Finally, the present study identified a number of cellsurface markers associated with primary CLL cell survival.While these markers were not examined in detail theirexpression served to validate our approach, as many ofthese cell surface markers have previously been reported toplay roles in CLL cell survival. For example, we identifiedincreased levels of expression of CD184 (CXCR4), CD26,CD40, CD58, CD62L, and CD103. CD60L and CXCR4 arekey molecules in CLL cell trafficking. We observed thatCD40 was increased in surviving CLL cells. Interactionbetween B cells and Th cells in lymph node proliferatingcentres is mediated via CD40 crosslinking with CD40L toactivate protein kinases such as Lyn and Syk and suchinteractions have been observed in CLL patient lymphnodes (28). The fact that CD26 was increased in survivingCLL cells is indicative of B-cell activation (29, 30), which isconsistent with the upregulation of CD58, another markerof costimulation. It has been shown that incubation of CLLcells with human CD40L-expressing T cells results in

increased CD54, CD58, CD80, and CD86 (31). Furtherstudy of these molecules may provide insight into thepathways that drive the dysregulation of B-cell survival/apoptosis in CLL.

In conclusion, our work establishes an important role ofCD62L indifferent prosurvival stimuli provided toCLL cellsby themicroenvironment. Our data contribute to the estab-lishment of CD62L inhibition as a rational therapeuticprinciple in CLL. Anti-CD62L antibody has marked in vitroactivity against human CLL cells at concentrations that arereadily attainable in the clinic. In addition to the cytoxicityof CD62L in CLL, our study adds the important prospect ofpreventing survival of drug-resistant CLL cells in protectiveniches by direct disruption of stromal signals by inhibitionof CD62L.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Burgess, D.S. Gill, N.A. Saunders, N.A.McMillanDevelopment of methodology: M. Burgess, D.S. Gill, C. Cheung, B.A.Reynolds, P. Mollee, N.A. McMillanAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.):D.S. Gill, R. Singhania, C. Cheung, L. Chambers,L. Smith, P. MolleeAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): M. Burgess, D.S. Gill, C. Cheung, P.Mollee, N.A. Saunders, N.A. McMillanWriting, review, and/or revisionof themanuscript:M. Burgess, D.S. Gill,B.A. Reynolds, P. Mollee, N.A. Saunders, N.A. McMillanAdministrative, technical, or material support (i.e., reporting or orga-nizingdata, constructingdatabases):M.Burgess, D.S. Gill, B.A. Reynolds,L. Smith, N.A. SaundersStudy supervision: D.S. Gill, N.A. Saunders, N.A. McMillan

AcknowledgmentsUQDI gratefully acknowledges all those patients who donated tissue for

this project.

Grant SupportThis work was funded by grants from the Australian Leukaemia Research

Foundation (to D.S. Gill and N.A. McMillan), Uniquest (to N.A. McMillan),and the Princess Alexandra Hospital Cancer Collaborative Group (to D.S.Gill, P. Mollee, N.A. McMillan). N.A. Saunders is supported by a CancerCouncil Queensland Senior Research Fellowship.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 22, 2013; revised July 14, 2013; accepted July 31, 2013;published OnlineFirst August 15, 2013.

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2013;19:5675-5685. Published OnlineFirst August 15, 2013.Clin Cancer Res Melinda Burgess, Devinder Gill, Richa Singhania, et al. CD62L as a Therapeutic Target in Chronic Lymphocytic Leukemia

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