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The role of APRIL (TNFSF13) in carcinogenesis

Lascano, V.

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Citation for published version (APA):Lascano, V. (2014). The role of APRIL (TNFSF13) in carcinogenesis.

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CHAPTER

Chronic Lymphocytic Leukemia

Disease Progression Is Accelerated

By APRIL / TACI Interaction in The

TCL1 Transgenic Mouse Model

Valeria Lascano1*, Marco Guadagnoli1*, Jan G

Schot2, Dieuwertje M Luijks2, Jeroen EJ Guikema3,

Katherine Cameron1, Michael Hahne 4, Steven Pals

3,7,Erik Slinger2, Thomas J Kipps5, Marinus HJ van

Oers2,7, Eric Eldering6,7, Jan Paul Medema1 #,

Arnon P Kater2,7#§

* these authors share first authorship

# these authors share senior authorship

Blood. 2013 Dec 5;122(24):3960-3

2

Chapter 2

36

CLL progression Is accelerated by APRIL / TACI interaction

37

Abstract

Although in vitro studies pointed to the TNF family member APRIL in mediating survival of

chronic lymphocytic leukemia (CLL), clear evidence for a role of APRIL in leukemogenesis

and progression in CLL is lacking. We observed that APRIL significantly prolonged in vitro

survival of CD5+B220dull leukemic cells derived from the murine Eμ-‐TCL1-‐Tg (TCL1-‐Tg) model

for CLL. APRIL-‐TCL1 double-‐Tg mice showed a significant earlier onset of leukemia,

disruption of splenic architecture and survival of double-‐Tg mice was significantly reduced.

Interestingly, clonal evolution of CD5+B220dull cells, as judged by BCR clonality, seemed not

to be accelerated by APRIL and both mouse strains were oligoclonal at 4 months.

Furthermore, although APRIL binds different receptors, APRIL-‐mediated leukemic cell

survival depended on TACI ligation. These findings indicate an important role for APRIL in

CLL and indicate that the APRIL-‐TACI interaction might be a selective novel therapeutic

target for human CLL.

Chapter 2

38

Introduction

The microenvironment is thought to protect chronic lymphocytic leukemia (CLL) cells from

cytotoxic drugs serving as a niche from which relapse can occur. Most evidence for the

presence of such signals in CLL thus far has been obtained from correlative studies and

artificial in vitro model systems (reviewed by1).

Direct comparison of gene and protein expression patterns of CLL cells residing in the LN

versus circulating CLL cells pointed to TNF receptor family mediated activation of the

nuclear factor-‐kappa B (NF-‐κB) transcription factor within the LN microenvironment2,3. CLL

cells indeed express various TNF receptor family members including BCMA, TACI, and BR3,

which are receptors for APRIL and BAFF4. Interaction of BAFF and APRIL with their cognate

receptors promote CLL cell survival in vitro either in an autocrine5 or in a paracrine fashion,

for instance by APRIL and BAFF expression on monocyte-‐derived nurse like cells (NLC)6. We

showed that, in contrast to BAFF, APRIL serum levels were increased in CLL patients and

correlated with a worse prognosis7. Further knowledge on the role of BAFF and APRIL in CLL

biology becomes increasingly important as development of monoclonal antibodies against

these ligands and/or their cognate receptors allows for specific targeting of these

ligand/receptor complexes8.

We recently found that TCL1-‐Tg mice crossed with mice over-‐expressing BAFF had

accelerated rates of leukemogenesis because of reduced spontaneous B-‐cell apoptosis

relative to TCL1-‐Tg mice9. In the current study we address the role of APRIL in CLL

leukemogenesis in the TCL1-‐Tg mouse model.

Study Design

Mice

Heterozygous human APRIL-‐Tg mice10 and TCL1-‐Tg mice11 were used to generate APRILxTCL-‐

1 double-‐Tg mice (from now on called double-‐Tg) in our local conventional animal facility

under virus-‐free conditions. Both TCL1-‐Tg and APRIL-‐Tg mice had been backcrossed more

than 10 generations onto the C57Bl/6 background. Survival data were obtained in a cohort

of 32 mice with 9 mice per group for TCL1-‐Tg and double-‐Tg. APRIL-‐Tg (n=8) and WT mice

(n=6) were used as control. All animal experiments were approved by the animal ethical

committee of the Academic Medical Center, University of Amsterdam (DSK102031).


39

In vitro stimulation of TCL1-‐Tg derived leukemic cells.

TCL1-‐Tg derived CD5+CD19+B220dull cells were isolated from spleens of >11 month-‐old mice

with an overt leukemia phenotype characterized by marked lymphocytosis and

splenomegaly. All mice presented with >70% CD5+B220dull cells among the CD19+/CD3-‐

splenic population (B cells). Stimulation experiments are described in detail in the

Supplementary Methods section.

Results and Discussion

Responsiveness of TCL1-‐Tg derived CD5+ splenocytes to APRIL was first examined in vitro.

Both APRIL and BAFF stimulation resulted in decreased spontaneous cell death over time

(Supplemental Figure 1A). The APRIL-‐induced survival was specific as addition of hA.01A

(inhibitory anti-‐APRIL) completely prevented its effect (Supplemental Figure 1B).

To study the effect of APRIL on development of leukemia, TCL1-‐Tg mice were crossed with

APRIL-‐Tg mice. The four offspring genotypes (WT, APRIL-‐Tg, TCL1-‐Tg and double-‐Tg) were

monitored for leukemia development in peripheral blood over time. Whereas TCL1-‐Tg mice

developed a clear leukemic CD5+B220dull population at around the age of eighth months,

double-‐Tg animals developed this population at the fourth months of life (Figure 1A). A

more rapid expansion of this population in double-‐Tg mice was evident in both relative and

absolute cell numbers (Figure 1B) and was independent of gender.

Splenic sections showed a grossly distorted architecture in double-‐Tg mice at the age of 4

months (Figure 1C) with a loss of demarcation between B and T cell areas. TCL1-‐Tg mice also

developed distorted splenic architecture but at much later time points (Supplemental Figure

2A,B).

Faster leukemia development in double-‐Tg mice correlated with a shorter life-‐span (Figure

1D). While the mean life-‐span of TCL1-‐Tg mice was 393 days, the average life-‐span of

double-‐Tg mice was 292 days. Since BAFF and APRIL are believed to induce partly

overlapping responses in CLL cells, we studied whether BAFF levels are compensatorily

down regulated in the double-‐Tg mice. As shown by ELISA, BAFF levels were unaffected by

increased levels of APRIL (Supplemental Figure 3).

We next compared the development of clonality at four and eight months of age. FACsorted

CD5-‐ B cells showed a Gaussian distribution of BCR CDR3-‐lengths, indicating the presence of

a polyclonal B cell population, at both time points. In contrast, at four months of age

Chapter 2

40

CD5+B220dull cells of both TCL1 and double-‐Tg mice showed a more restricted

representation of BCR VDJ-‐lengths and were therefore oligoclonal (Figure 1E). At eight

months of age, both TCL1 and double-‐Tg derived CD5+ B220dull cells showed one dominant

BCR rearrangement suggesting monoclonal expansion during progressive disease (Figure 1E).

To characterize the signaling pathway that mediates the stimulation effect of APRIL, we first

measured expression patterns of APRIL/BAFF receptors on CD5+ B splenocytes derived from

aged TCL1-‐Tg mice. A strong expression of TACI and BR-‐3 expression was observed while

expression of BCMA was consistently low (Figure 2A). Interestingly, comparison of TACI and

BCMA expression levels between leukemic and normal B cells of TCL1-‐Tg mice revealed a

remarkably increased TACI expression in the CD5+ malignant B cell fraction, while the

expression of BCMA was only marginally affected (Figure 2B).

Dependency of APRIL-‐induced survival signaling in the CLL-‐like cells on BCMA or TACI was

studied using a recently developed receptor-‐selective APRIL variant characterized by very

low affinity for TACI, but unchanged affinity for BCMA12. Consistent with this affinity profile,

APRIL-‐R206E, even though it was expressed at the same level as APRIL-‐WT (Supplemental

Figure 4), failed to kill Jurkat cells expressing a TACI:Fas fusion protein, while BCMA:Fas

expressing Jurkat cells were as effectively killed as with APRIL-‐WT (Supplemental Figure 5).

We subsequently incubated TCL1-‐Tg-‐derived CLL cells with conditioned media containing

either APRIL-‐WT, APRIL-‐R206E or MOCK, and observed that APRIL lacking the capacity to

bind TACI did not confer the survival benefits of APRIL-‐WT (Figure 2 C,D). This indicates that

the stimulatory effect of APRIL is mediated via TACI and not BCMA.


41

Figure 1. APRIL accelerates leukemic onset in TCL1-‐Tg mice. A) Detection of peripheral blood leukemic B cells in 4, 6 and 8 month-‐old mice belonging to the 4 different genotypes. CD5+/B220-‐ CLL cells were discriminated on CD19+ gated B cells. B) Bar graphs representing % CD5+CD19+ cells (top) or CD5+CD19+ absolute numbers (bottom). C) Spleens from double-‐Tg report a grossly disturbed architecture with indistinguishable B and T-‐cell areas, a hallmark of an advanced disease stage. Representative consecutive spleen sections of 4 month-‐old mice with indicated genotypes (n=3 per genotype) stained for H&E, CD3 and B220. 40X magnification. D) Kaplan-‐Meier survival curve for the 4 different genotypes (WT n=6; APRIL-‐Tg n=8; TCL1-‐Tg n=9; double-‐Tg n=9). Mean life-‐span (days) is indicated on the TCL1-‐Tg and double-‐Tg survival curves. Log-‐rank test p-‐value 0.0011 as significance level comparing TCL1-‐tg to double-‐Tg. E) TCL1-‐Tg and double-‐Tg leukemic cells are characterized by the same B cell receptor clonality peak patterns. RNA from sorted normal B (B220high/CD5-‐) and leukemic B cells (B220dull/CD5+) was used to assess B cell receptor clonality by analysis of CDR3-‐family VDJ genes. Spectra of individual 4 months and 8

WT APRIL-Tg TCL1-Tg double-Tg 4

mon

ths

6 m

onth

s8

mon

ths

B220CD5

A.

Figure 1. APRIL accelerates leukemic onset in TCL1-Tg mice

C.

100 200 300 400 5000

20

40

60

80

100

TCL1-Tgdouble-Tg

WT APRIL-Tg

days

% o

vera

ll sur

vival

292 393

P=0.0011

0.554 2.55 6.42 32.2

5.11 2.51 18.7 51.9

2.12 3.41 48.4 63.6

B.

20

40

60

80

WTAPRIL-Tg

TCL1-Tg double-Tg

% C

D5+ CD

19+

cells

4 6 80

5

10

15

months

x106

CD5+ CD

19+

cells

/ml

ns

***

***

ns

ns

*

***

ns

*

******

ns***

ns

H&E

CD3

TCL1-Tg double-Tg

B220

CD5+

B ce

llsCD

5-B

cells

4 months 8 months

Lenght of VHDJH junction

Fluor

esce

nce

4 months 8 months

D.

E.double-TgTCL1-Tg

Chapter 2

42

month-‐old mice showing an oligoclonal arising leukemic population in both genotypes.sterisks denote p values < 0.05 (*), < 0.01 (**) and < 0.005 (***).

Taken together, these data show that ectopic APRIL expression accelerates the onset of

TCL1-‐driven leukemia formation mainly through TACI activation, shortening the mean life-‐

span by around 100 days.

Interestingly, we found CD5+ B cells in TCL1-‐Tg mice to have oligoclonal BCR gene

rearrangements already at four months of age. Our non-‐quantitative data on a limited

number of mice suggests that BCR clonal evolution develops at the same pace in TCL1-‐Tg

and double-‐Tg mice (at least from 4 months of age onwards). This suggests that the actual

outgrowth of leukemic cells and therefore the leukemic load is affected by ectopic APRIL

levels. Leukemic cells in the single TCL1-‐Tg mice do eventually develop lethal leukemia

pointing to the possibility that leukemia cells create and shape a supportive

microenvironment (as has been reviewed by Facteau and Kipps13). Alternatively, one could

speculate that clonal selection of specific BCRs is an independent event and that full

transformation of the TCL-‐1 leukemic cells requires a stochastic second hit. In the APRIL-‐Tg

mice this hit is more likely to occur as the leukemic cell load is higher, explaining why these

mice require less time to full transformation. Either way, our data point to the fact that

APRIL itself is not driving clonal selection, but rather affects leukemic cell survival.

Relevant with regards to therapeutic intervention is our finding that APRIL-‐induced viability

mainly relies on TACI, as CLL cells could not be stimulated with an APRIL form that

specifically binds BCMA12. Dissection of APRIL receptor deployment is of importance since

TACI and BCMA play roles at different stages of B cell development4. Selectively targeting

the interaction between APRIL and TACI will likely inhibit APRIL-‐induced survival signaling in

CLL, but may leave APRIL-‐dependent BCMA signaling in normal B cells intact.


43

Figure 2. APRIL-‐mediated effect depends on TACI and not BCMA A) Splenic CD19+CD5+ cells were isolated from TCL1-‐Tg mice with an overt leukemic phenotype (>11 month-‐old), stained with anti-‐BCMA, -‐TACI and -‐BR-‐3 fluorescent-‐conjugated antibodies and analyzed by FACS. Spleens contained more than 70% leukemic cells (CD5+B220dull) among the B cell gate (CD3-‐, CD19+, left column). Unfilled bold line represents the specific staining, the filled curve represents the isotype control. Data shown from a representative mouse (n=6) B) TACI and BCMA expression represented as MSFI (Mean Specific Fluorescence Intensity) (n=3). C) 100.000 splenic CLL cells from 8-‐12 month TCL1-‐Tg mice, that were > 70% CD5+CD19+B220dull, were stimulated using either APRIL-‐WT, mock, or a mutant of APRIL that binds to BCMA and not TACI (APRIL-‐R206E). At the indicated time points, cell viability was measured. D) Absolute number of DioC6 positive cells following 4 days of stimulation with indicated conditioned media. Asterisks denote p values < 0.05 (*), < 0.01 (**) and < 0.005 (***).

Figure 2. APRIL-mediated effect depends on TACI and not BCMA

A. TACIBCMA BR-3

B220

CD5

CD3-/CD19+

96.2

B.

BCMA TACI0

10

20

30

40

50 CD5- B cellsCD5+ B cells

MSF

I

1 3 6 910

30

40

50

60

20MOCK

APRIL-WTAPRIL-R206E

days

% li

ve c

ells

C.***

ns

APRIL-WT

MOCK

APRIL-R206E

20

30

40

50

60 **

Tota

l num

ber D

ioC 6+

cel

ls(1

.102 )

D.

Chapter 2

44

Acknowledgements

This research was performed within the framework of project T3-‐504 of the Dutch Top

Institute Pharma and Dutch Cancer Society projects (UvA2007-‐3750 and UvA2009-‐4440).

APK is funded by a personal Dutch Cancer Society Clinical Fellowship grant.

Contribution: V.L, M.G, J.G.S, D.L, E.S, K.C. and J.E.J.G performed experiments; V.L., M.G.,

J.G.S. analyzed results and made the figures; M.H, M.vO., E.E., T.J. K., J.P.M. and A.K.

designed and directed the research. S.P. is the pathologist of reference who supervised the

histology. V.L., G.M, J.P.M and A.K. wrote the manuscript.


45

Reference list

1. Burger JA, Ghia P, Rosenwald A, Caligaris-‐Cappio F. The microenvironment in mature B-‐cell

malignancies: a target for new treatment strategies. Blood 2009;114:3367-‐3375.

2. Smit LA, Hallaert DY, Spijker R et al. Differential Noxa/Mcl-‐1 balance in peripheral versus lymph node chronic lymphocytic leukemia cells correlates with survival capacity. Blood 2007;109:1660-‐1668.

3. Herishanu Y, Perez-‐Galan P, Liu D et al. The lymph node microenvironment promotes B-‐cell receptor signaling, NF-‐{kappa}B activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 2011;117:563-‐574.

4. Bossen C, Schneider P. BAFF, APRIL and their receptors: Structure, function and signaling. Seminars in Immunology 2006;18:263-‐275.

5. Kern C, Cornuel JF, Billard C et al. Involvement of BAFF and APRIL in the resistance to apoptosis of B-‐CLL through an autocrine pathway. Blood 2004;103:679-‐688.

6. Nishio M, Endo T, Tsukada N et al. Nurselike cells express BAFF and APRIL, which can promote survival of chronic lymphocytic leukemia cells via a paracrine pathway distinct from that of SDF-‐1alpha. Blood 2005;106:1012-‐1020.

7. Planelles L, Castillo-‐Gutierrez S, Medema JP et al. APRIL but not BLyS serum levels are increased in chronic lymphocytic leukemia: prognostic relevance of APRIL for survival. Haematologica 2007;92:1284-‐1285.

8. Guadagnoli M, Kimberley FC, Phan U et al. Development and characterization of APRIL antagonistic monoclonal antibodies for treatment of B-‐cell lymphomas. Blood 2011;117:6856-‐6865.

9. Enzler T, Kater AP, Zhang W et al. Chronic lymphocytic leukemia of Emu-‐TCL1 transgenic mice undergoes rapid cell turnover that can be offset by extrinsic CD257 to accelerate disease progression. Blood 2009;114:4469-‐4476.

10. Planelles L, Carvalho-‐Pinto CE, Hardenberg G et al. APRIL promotes B-‐1 cell-‐associated neoplasm. Cancer Cell 2004;6:399-‐408.

11. Bichi R, Shinton SA, Martin ES et al. Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression. Proc.Natl.Acad.Sci.U.S.A 2002;99:6955-‐6960.

12. Kimberley FC, van der Sloot AM, Guadagnoli M et al. The design and characterization of receptor-‐selective APRIL variants. J.Biol.Chem. 2012;287:37434-‐37446.

13. Fecteau JF, Kipps TJ. Structure and function of the hematopoietic cancer niche: focus on chronic lymphocytic leukemia. Front Biosci.(Schol.Ed) 2012;4:61-‐73.

Chapter 2

46

Supplemental Materials Contents

Supplemental Methods

Flow Cytometry

Surface markers were stained with anti-‐CD19 (FITC), anti-‐CD5 (PerCP-‐Cy5.5), anti-‐

B220/CD45R (APC), anti-‐CD3 (PE-‐Cy7), anti-‐IgM (FITC), anti-‐CD43b (PE), CD23 (PE-‐Cy7), anti-‐

CD138 (PE), anti-‐CD268/BAFF-‐R (APC) and anti-‐CD267/TACI (PE) which were purchased from

eBioscience (Vienna, Austria). Anti-‐BCMA (FITC) was purchased from R&D systems

(Abingdon, UK). For measuring apoptosis, cells were stained with 40 nM 3,3ʹ′

dihexyloxacarbocyanine iodide (DiOC6; Invitrogen, Cat. No. D-‐273) and propidium iodide (PI;

Sigma, Cat. No. P4864) for 30 min at 37°C as previously described1. All antibodies were used

according to manufacturer’s specifications. Flow cytometry was performed on a FACSCalibur

or FACSCanto II (BD) and data analyzed with FlowJo software (TreeStar Inc).

In vitro stimulation of TCL1-‐Tg derived leukemic cells.

For in vitro stimulation, 2x105 cells were plated in a U shaped 96 well plate in 100 μl of

RPMI-‐1640 complemented with 8% FCS, Glutamine and Penicilin/Streptavidin and β-‐

mercaptoethanol 50 μM. 100 μl of APRIL-‐WT, MOCK (empty vector) or APRIL-‐R206E (a

BCMA-‐specific APRIL variant2) conditioned media was added to the plated cells and

incubated at 37 oC. Conditioned media was prepared by transfection of 293T cells with

plasmid DNA coding for the different variants of APRIL, followed by collection of

supernatant following 4 days of culture. The amount of APRIL was evaluated by ELISA.

(Supplemental Fig 3). Activity was checked by measuring cell death in 2 modified Jurkat cell

lines that express either extracellular TACI -‐ intracellular FAS receptor or extracellular BCMA

-‐ intracellular FAS receptor (Suppl. Fig 6) as has recently been described2. For BAFF

stimulation, 200 ng/ml of purified recombinant human BAFF (a kind gift from Dr G. Zhang,

National Jewish Medical and Research Center, Denver, CO) was added to MOCK medium.

Histopathology and Immuno-‐histochemistry

Spleens fixed in 10 % buffered formalin for 48 hr and embedded in paraffin. Sections (4 μm

thick) were cut, deparaffinized and stained with hematoxylin and eosin (H&E) according to

standard protocols and analyzed. H&E and immunohistochemistry were performed on

consecutive sections. For the immunostaining, the sections were heated for 15 min at 60°C,


47

followed by xilene and rehydration steps through a graded ethanol series and PBS, the

Antigen retrieval was performed with citrate buffer pH=6 for 15 min at 98°C. Endogenous

peroxidase was blocked with 1% H2O2 in PBS for 15 min. In order to block non-‐specific

staining we used Ultra-‐V block (TA-‐125-‐UB, Immunologic, Duiven, The Netherlands). We

stained for CD3 (clone SP7, dilution 1:1000, Neomarkers, Fremont, CA, USA); B220

(MCA1258 GT/RAT9-‐6B2 dilution 1:7000, Serotec, Puchheim, Germany); CD5 (53-‐7,3,

dilution 1:1000, BD Biosciences). CD3 antibody was detected with Brightvision anti-‐rabbit

HRP (DPVR110AP, Immunologic). B220 antibody was followed by rabbit-‐anti-‐rat (6130-‐01,

1:3000 diluted in 10% normal mouse serum, Southernbiotec, Uden, The Netherlands) for 30

min at RT. Followed by Brightvision anti-‐rabbit HRP. CD5 antibody was followed by rabbit-‐

anti-‐FITC Ab (4510-‐7804, 1:1000, Bioconnect, Huissen, The Netherlands) 30 min at RT and

then detected with Brightvision anti-‐rabbit HRP.

Clonality analysis and spectratyping of B cell populations

CD5+ and CD5-‐ B cell populations were obtained after FACsorting splenocytes from TCL1-‐Tg

and TCL1xAPRIL double-‐Tg based on CD5 and B220 expression. Total RNA was extracted

using the RNeasy isolation kit from QIAGEN. VH-‐DJH gene rearrangements from B cell

populations were amplified using PCR primers specific for the J558 VH region gene together

with a primer specific for the Cμ constant region gene. Using a FAM-‐conjugated Cμ constant

region or a JH gene-‐specific primer in a run-‐off reaction, PCR products were labeled and

subsequently analyzed on a capillary sequencer (ABI3100; Applied Biosystems, Leusden, NL)

by fragment-‐length analysis. Sequences of primers were published earlier 3.

BAFF ELISA

We used a commercial kit to measure mouse BAFF levels, BAFF Quantikine ELISA KIT cat.

num. MBLYS0 R&D systems. We used 20 μl of mice serum and we followed the standard

protocol described by the user’s manual.

ELISA to quantify APRIL levels in 293T supernatants

96-‐wells flat bottom are coated with anti-‐Flag(M2) Sigma at 1 µg/m in coating buffer. Left

overnight at 4 ºC. Blocked with PBS/BSA (BSA 1%) for 1 hour at 37 ºC. the different

preconditioned media are diluted in serial dilutions ½ in PBS/1% BSA and incubated for 2h at

37 ºC. To detect APRIL we used Aprily-‐5-‐bio (ALX-‐804-‐801, Alexis) at 1 μg/ml in PBS/BSA,

Chapter 2

48

incubated for 1h at 37 ºC. And detected using Strept-‐Avidin-‐HRP (Jackson-‐Immuno) to

1μg/ml in PBS/BSA

Statistics

Data are expressed as mean +/-‐ SEM. Statistical analyses were performed using SPSS version

19. Statistical analysis was performed using a 2-‐tailed unpaired Student t-‐test. Comparison

of more than three samples was performed by non-‐parametric one-‐way ANOVA followed by

Tuckey’s multiple comparison correction. Overall survival was tested using Kaplan-‐Meier

analysis with a log-‐rank test. Differences were considered significant when p values < 0.05

(*), < 0.01 (**) and < 0.005 (***).

Supplemental figures

Supplemental Figure 1. CD5+ leukemic B cells derived from TCL1-‐Tg mice respond to APRIL and BAFF in vitro. Leukemic cells are clearly identified by FACS as B220dull and CD5int in 8-‐12 month old TCL1-‐Tg mice (> 70% CD5+CD19+B220dull) A) Effect of APRIL and BAFF on leukemic cell viability over time. Error bars represent SEM (n=6) B) The anti-‐APRIL antagonistic antibody hA.01A fully prevents APRIL-‐mediated leukemic cell stimulation.

1" 3" 6"days"

%"live"cells"

A.#

days"

%"live"cells"

B.#

CONTROL APRIL APRIL""+"hA.01A"0"

20"

40"

60"

**"**"

1" 3" 6" 9"

CONTROL"APRIL BAFF

0"

20"

40"

60"**" **"

*"

Supplemental#Figure#1.##CD5+#leukemic#B#cells#derived#from#TCL1@Tg#mice#respond##to#APRIL#and#BAFF#in#vitro#


49

Supplemental Figure 2. Splenic sections of TCL1-‐Tg mice show mild enlargement of the marginal zones at four months of age and distorted splenic architecture at eight months of age. A) Splenic section of a TCL1-‐Tg mouse (4 months old) presenting an enlarged marginal zone. Pictures show highly compact cell areas which lack B220 expression, indicating an abnormal expansion of the marginal zone cells. 10X magnification. B) Representative consecutive spleen sections of TCL1-‐Tg mice stained for H&E, CD3 and B220. Spleens from TCL1-‐Tg report increased disruption in splenic architecture with indistinguishable B and T-‐cell areas, at 8 month of age. 4X magnification.

Supplemental*Figure*2.*Splenic*sec3ons*of*TCL1:Tg*mice*show*mild*enlargement*of*the*marginal*zones*at*four*months*of*age*and*distorted*splenic*architecture*at*eight*months*of*age.**

H&E$

WT$ TCL1*Tg$

B220

$H&

E$CD

3$B2

20$

8$months$4$months$

A.*

B.*

Chapter 2

50

Supplemental Figure 3. hAPRIL in the double-‐Tg does not affect mouse BAFF (mBAFF) levels. BAFF ELISA on serum of mice. Serum levels of BAFF are comparable in wild type, TCL1-‐Tg and double-‐Tg mice (n=3). Serum levels of BAFF-‐Tg mice are shown as control.

Supplemental Figure 4. Quantification of APRIL levels in the different conditioned media used in TCL1-‐Tg in vitro stimulation. ELISA using anti-‐Flag coated plates to compare the amount of the different APRIL mutants added onto the TCL1-‐Tg cells in vitro. The detection of APRIL is with APRILY-‐5 Ab. Different bars represent limiting dilutions 1 in 2 of each conditioned media.

Supplemental*Figure*3.*hAPRIL*in*the*double:Tg*does*not*affect*mBAFF*levels*

0"

2"

4"

6"

8"

10"

mBA

FF"ng/ml""

ns"

Supplemental*Figure*4.*Quan2fica2on*of*the*different*condi2oned*media*used*in*TCL1?Tg*in#vitro*s2mula2on*

0.0#

0.1#

0.2#

0.3#

0.4#

OD#49

0#nm

#


51

Supplemental Figure 5. APRIL-‐R206E variant is able to signal via BCMA but not TACI. Binding activity of the different APRIL variants were tested on BCMA:Fas or TACI:Fas Jurkats cells. The ligand binding is directly associated with induced cell death measure by % of DNA fragmentation.

Reference List

1. Enzler T, Kater AP, Zhang W et al. Chronic lymphocytic leukemia of Emu-‐TCL1 transgenic mice undergoes rapid cell turnover that can be offset by extrinsic CD257 to accelerate disease progression. Blood 2009;114:4469-‐4476.

2. Kimberley FC, van der Sloot AM, Guadagnoli M et al. The design and characterization of receptor-‐selective APRIL variants. J.Biol.Chem. 2012;287:37434-‐37446.

3. Duy C, Yu JJ, Nahar R et al. BCL6 is critical for the development of a diverse primary B cell repertoire. J.Exp.Med. 2010;207:1209-‐1221.

Supplemental*Figure*5.*APRIL6R206E*variant*is*able*to*signal*via*BCMA*but*not*TACI*

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