Fig S1. Wild type and TCL1 B-1 cells and TCL1-192 cells express BCRs reactive to PtC The

percentage of cells with the ability to bind PtC in (A) B-1 cells (B) CLL cells and (C) B2 cells

collected from WT and TCL1 mice at different ages. Data are representative results from 3 animals

per group. (D) 1x106 TCL1-192 cells taken from the spleen were pre-incubated with 1µg anti-IgM

pAbs. SmIgM internalization was evaluated. Cells incubated with isotype control antibody (gray

line) served as controls. Control cells (black line) and anti-IgM pAbs exposed cells (red line) were

then incubated with PtC liposomes. Data represent three experiments. Scatter graphs indicate mean ± SD. (E) 1x106 of PtC+ and PtC- splenic CLL cells from 4th transfer TCL1-192 mice were

injected into 3 SCID mice (gating strategy shown in left). 5 weeks later, spleens were analyzed for

percentage cells that recognized PtC (P<0.001, unpaired t test by Prism). (F) The average MFI of

smIgM and PtC binding were then calculated in PtC+ and PtC- cells harvested from each recipient.

(* P<0.05, ** P<0.01, unpaired t test by Prism).

Figure S2. Trafficking dynamics and kinetics of the disease (A-C) 5x106 TCL1-192 cells were

injected into 37 SCID mice. Three mice were sacrificed at weeks 2, 3, and 4, and 25 mice were

sacrificed at week 6. The presence of CLL cells in lymph node (LN), spleen (SP), blood (PB),

peritoneal cavity (PC), bone marrow (BM) and liver at indicated time points analyzed by flow

cytometry and H&E staining were shown in (A) and (B). In vivo CLL cell proliferation in different

locations was evaluated in 6 animals. BrdU+ cells shown in (C) were gated based on the negative

control cells collected from mice without BrdU injection. Mean values and SD are indicated with

filled circles and error bars (***P<0.001, unpaired T test using GraphPad Prism software). (D-E)

25x106 TCL1-192 cells were iv or ip injected into SCID mice. Three animals from each group were

sacrificed 1 or 5 weeks after injection. The proliferation and distribution of CLL cells injected in

different locales were studied. Flow cytometry results shown in (D) are representative data of 3

mice from each group at each time point. BrdU incorporation analysis shown in (E) was from all

samples except one that was not analyzed (n.a.) due to minimal numbers of CLL cells. Data shown

are means ± SD. * P<0.05, ** P<0.01 by student T test analyzed with GraphPad Prism software.

A B

Spleen

Peritoneum

Peripheral blood

� B220

�C

D5

WK2 WK3 WK6

WK2

WK3

WK4

WK6

Spleen Liver

WK6

Bone

Marrow

Lymph

Node

C

LN SP

PB PC

WK6

���� BrdU

BM

23% 18%

1% 1% 4%

D E

0%

20%

40%

% B

rdU

+ C

LL

ce

lls

SP PC SP PC

WK1 WK5

i.p. injectioni.v. injection

n.a.

*

**

***

SP PC PB

WK5

WK1

i.v.

i.p.

i.v.

i.p.

� B220

�C

D5

43.7% 73%

10.1% 82.3%

92.5% 92.8%

88.7% 86.1%

16%

3.1%

83.1%

85.3%

******

SP LN PC PB BM0

5

10

15

20

25

30

35%

Brd

Uin

copora

t io

nin

24

hrs

Figure S3. Genes changed in PtC+ versus PtC- cells (A) A heatmap of 40 known genes of the

53 genes (56 probes) significantly changed in splenic PtC+ versus PtC- cells from 2 TCL1-192 mice

is shown, along with the expression of these genes in PtC+ cells sorted from B-1 cells in wild type

(WT) mice, young (Yg) original TCL1 mice, and peritoneal PtC+ and PtC- cells collected from TCL1-

192 mice. (B) A heatmap of 147 up-regulated (left) and 172 down-regulated (right) known genes

identified in peritoneal PtC+ versus PtC- cells from 2 TCL1-192 mice. Expression of these genes in

PtC+ cells sorted from B-1 cells in wild type (WT) mice, young (Yg) original TCL1 mice, and splenic

PtC+ and PtC- cells collected from TCL1-192 mice is also shown for comparison. (C) Molecules

involved in BCR signaling pathway that were identified as differently expressed in PtC+ versus PtC-

cells were verified by SYBR-Green real time PCR in PtC+ versus PtC- cells from a total of 3 mice

(top). Values obtained from PtC- cells obtained from peritoneal cavity (PC) were set as 1 for their comparison to splenic (SP) and PC PtC+ cells. Data shown in bar graph are means ± SD. Bottom

shown are values in the gene-expression profile of genes verified by real time PCR.

WT

B1

Yg

TC

L1

SP

PtC

-

SP

PtC

-

SP

PtC

+

SP

PtC

+

PC

PtC

-

PC

PtC

-

PC

PtC

+

PC

PtC

+A

B

WT

B1 PtC+

CLL

PtC-

Yg

TCL1 1 2

SP

CLL

PtC+

1 2

SP

CLL

PtC-

1 2

PC

CLL

PtC+

1 2

PC WT

B1 PtC+

CLL

PtC-

Yg

TCL1 1 2

SP

CLL

PtC+

1 2

SP

CLL

PtC-

1 2

PC

CLL

PtC+

1 2

PC

C

NF

AC

1

RE

LA

SH

IP1

(IN

PP

5D

)

SH

IP2

(IN

PP

L1

)

PIK

3C

D

PIK

3A

P1

RT

-PC

R R

ela

tive

Exp

ressio

nA

rra

y A

na

lysis

Da

ta

2

1.5

1

0.5

0

2

1.5

1

0.5

0

A.

B.

Figure S4. Changes of genes involved in BCR, PI3Kinase and NFkB pathways in peritoneal

PtC+ versus PtC- cells (A) Genes involved in BCR, PI3Kinas, ERK/MAPKinase and NFκB

pathways identified in microarray analysis. (B) Networks of CD40-regulated genes in PtC+ and

PtC- cells from spleens and peritoneal cavity: Ingenuity Pathways Analysis (IPA) software was

used to analyze identified genes (n=68) found differently expressed in both splenic (Left) and

peritoneal (Right) PtC+ and PtC- cells. Genes labeled in red and green were those identified as

up- and down-regulated and other genes were those related to the regulated genes on the basis of the network analysis.

Spleen PtC+ vs PtC-: CD40 signaling Peritoneum PtC+ vs PtC-: CD40 signaling

Increased

Decreased

Supplemental Table S1. Results of cDNA sequencing on VH and Vκκκκ transcripts

Tissue PtC binding Heavy chain #/total sequenced clones Light chain #/total sequenced clones

Donor TCL1-192 Spleen Unsort VH11-2/D1-1/J1 14/24

VH12-3/D3-2/J1 6/24

VH2-9-1/D2-13/J4 1/24

VH1-82/D2-1/J2 1/24

VH1-80/D2-11/J1 1/24

VH1-55/D1-1/J4 1/24

1st transfer (n=2) Spleen PtC+ VH11-2/D1-1/J1 34/34 VK14-111/JK4 22/22

PtC- VH11-2/D1-1/J1 22/23 VK14-111/JK4 18/18

VH1-55/D1-1/J1 1/23

Peritoneum PtC+ VH11-2/D1-1/J1 34/34 VK14-111/JK4 22/22

PtC-

VH11-2/D1-1/J136/36

VK14-111/JK433/33

2nd transfer (n=3) Spleen PtC+ VH11-2/D1-1/J1 21/21 VK14-111/JK4 35/35

PtC- VH11-2/D1-1/J1 29/30 VK14-111/JK4 35/35

VH1-64/D3-1/J4 1/30

Peritoneum PtC+ VH11-2/D1-1/J1 19/19 VK14-111/JK4 34/34

PtC- VH11-2/D1-1/J1 22/22 VK14-111/JK4 37/37

3rd transfer (n=3) Spleen PtC+ VH11-2/D1-1/J1 10/10 VK14-111/JK4 10/10

PtC- VH11-2/D1-1/J1 9/9 VK14-111/JK4 9/9

Peritoneum PtC+ VH11-2/D1-1/J1 10/10 VK14-111/JK4 10/10

PtC- VH11-2/D1-1/J1 9/9 VK14-111/JK4 11/11

PC PC

Supplemental Table S2. Genes changed in splenic PtC+ vs PtC- cells. Total

56 probes changed in splenic PtC+ versus PtC- cells (set as 1). The difference

between peritoneal PtC+ versus PtC- (set as 1) in these genes is also shown in

the table.

Supplemental Table S3. Genes changed in peritoneal PtC+ vs PtC- cells. Total 492 probes

changed in peritoneal PtC+ versus PtC- cells. The difference between splenic PtC+ versus PtC-

in these genes is also shown in the table.

Supplemental Table S4A. 68 genes changed discordantly in spleen and peritoneum

PtC+ vs PtC- cells

Supplemental Table S4B. 33 genes changed concordantly in spleen and peritoneum PtC+

vs PtC- cells

Supplemental Table S5. 272 genes changed >2-fold in splenic versus peritoneal PtC+ cells.

Table S6. 28 genes changed in both human CLL LN versus PB and TCL1-192

SP versus PC

1 BCAR3 Up in TCL1-192 SP vs PC and human CLL LN vs PB

2 NUSAP1 Up in TCL1-192 SP vs PC and human CLL LN vs PB

3 KIF23 Up in TCL1-192 SP vs PC and human CLL LN vs PB

4 AICDA Up in TCL1-192 SP vs PC and human CLL LN vs PB

5 CCND2 Up in TCL1-192 SP vs PC and human CLL LN vs PB

6 DTNB Up in TCL1-192 SP vs PC and human CLL LN vs PB

7 CDCA3 Up in TCL1-192 SP vs PC and human CLL LN vs PB

8 RGS1 Up in TCL1-192 SP vs PC and human CLL LN vs PB

9 GADD45B Up in TCL1-192 SP vs PC and human CLL LN vs PB

10 CD69 Up in TCL1-192 SP vs PC and human CLL LN vs PB

11 ABCG1 Up in TCL1-192 SP vs PC and human CLL LN vs PB

12 KLF6 Up in TCL1-192 SP vs PC and human CLL LN vs PB

13 SOCS3 Up in TCL1-192 SP vs PC and human CLL LN vs PB

14 CCNL1 Up in TCL1-192 SP vs PC and human CLL LN vs PB

15 KLF4 Up in TCL1-192 SP vs PC and human CLL LN vs PB

16 CTLA4 Up in TCL1-192 SP vs PC and human CLL LN vs PB

17 SNF1LK Up in TCL1-192 SP vs PC and human CLL LN vs PB

18 MKI67 Up in TCL1-192 SP vs PC and human CLL LN vs PB

19 DUSP2 Up in TCL1-192 SP vs PC and human CLL LN vs PB

20 JUN Up in TCL1-192 SP vs PC and human CLL LN vs PB

21 JUNB Up in TCL1-192 SP vs PC and human CLL LN vs PB

22 DUSP1 Up in TCL1-192 SP vs PC and human CLL LN vs PB

23 EGR3 Up in TCL1-192 SP vs PC and human CLL LN vs PB

24 LYZ Down in TCL1-192 SP vs PC and human CLL LN vs PB

25 H3F3B Up in TCL1-192 SP vs PC but Down in human CLL LN vs PB

26 STK17B Down in TCL1-192 SP vs PC but Up in human CLL LN vs PB

27 SLC2A3 Down in TCL1-192 SP vs PC but Up in human CLL LN vs PB

28 CDCA7 Down in TCL1-192 SP vs PC but Up in human CLL LN vs PB

hCLL

LN vs PB

(n=128)

hCLL

BM vs PB

(n=23) (n=3)

hCLL

LN vs PB

(n=128)

TCL1-192

SP vs PC

(n=285)

Common (n=28)