Supplementary Methods

Fluorescence in situ hybridization (FISH). The FISH analysis was performed according to the standard method. In brief, harvested MCL cells were suspended in fixative (acetic acid and methanol, Sigma-Aldrich, A6283 and 32213, St.Louis, MO), deposited on the slides, and left to dry. The dual color gene fusion probe IgH(green)/CCND1(orange) (Abbott Molecular, 05J72-001, Abbott Park, IL), was applied in the hybridization buffer solution. The slides were cover-slipped, sealed, and left inside the HYBrite chamber at 37 C for 6 hr. After removal of the sealant and the coverslip, the slides were washed twice, blotted, dried, and counterstained with DAPI (LifeTechnologies, D1306, Grand Island, NY). The slides were then examined under fluorescent microscope, with a minimum of 200 nuclei evaluated in each specimen.

MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) enzymatic conversion assay. Cells were suspended at 2x104/well and incubated at 37°C in microtiter plates in the presence of various concentrations of ibrutinib (2 nM -10 uM) or drug vehicle alone for up to 48 hr, then incubated with MTT (Promega, G4000, Madison, WI) for 4 hr. Well contents were solubilized overnight in medium containing 10% SDS and 0.01M HCl. Absorbance at 570 nm in each well was measured using a Titertek Multiskan reader (Thermo Scientific, Waltham, MA).

Western blot analysis. For western blot analysis cells were washed, centrifuged, and lysed using a buffer supplemented with 0.5 mmol/L phenylmethylsulfonyl fluoride (Sigma-Aldrich, P7626), phosphatase inhibitor cocktails I and II (Roche, Basel, Switzerland), according to the manufacturer's specifications. For normalization of gel loading, the protein extracts were assayed by the Lowry method (Bio-Rad Dc protein assay 500-0111, Hercules, CA). Typically, 10 to 20 µg of the protein per lane were loaded. After protein transfer, the membranes were incubated with antibodies specific for p-BTK (pY180) (BD biosciences, 562753, San Jose, CA), -pERK, -Rb, -ITK (Cell Signaling, Danvers, MA), p-ITK (BD biosciences, 562753) and actin (Santa Cruz, Dallas, TX). The membranes were incubated with the appropriate secondary peroxidase-conjugated antibodies. The blots were developed using the enhanced chemiluminescence (ECL) plus Western blotting detection system (Amersham WB system, 29-0320-30, GE Healthcare, Pittsburgh, PA).

Multiparametric flow cytometry. Anti-human –mouse antibodies were purchased from multiiple vendors (listed in Table S1). Cells were isolated from in vitro culture or from animals, washed once in PBS (Gibco, Life Technologies, 14190-136), supplemented with 2% fetal bovine serum (Gemini, 100-106, West Sacramento, CA) and stained for 15 minutes at room temperature. For cell number quantitation, Countbright (Invitrogen, LifeTechnologies, C36950) beads were used according to the manufacturer’s instructions. In all analyses, the population of interest was gated based on forward vs. side scatter characteristics followed by singlet gating, and live cells were gated using Live Dead Fixable Aqua (Invitrogen, LifeTechnologies, L34957) or 7-AAD (Invitrogen, LifeTechnologies, A1310). Time gating was included for quality control. Surface expression of CAR19 was detected as previously described. (Kalos, M, Sci. Transl. Med., 2011) Flow cytometry was performed on a four-laser Fortessa-LSR II cytometer (Becton-Dickinson, San Jose, CA) and analyzed with FlowJo X 10.0.7r2 (Tree Star, Ashland OR).

Specificity fluorochrome clone vendor cat #Annexin-V APC BioLegend 640920bcl-2 FITC Bcl-2/100 eBioscience BMS1028FICCR6 BV605 G034e3 BioLegend 353420CD107a PE-Cy7 H4A3 Biolegend 328618CD127 (IL7R) BV711 A019D5 Biolegend 351328CD137 (41-BB) PE-Cy5 4B4-1 BioLegend 309808CD14 V500 M5E2 BD Horizon 561391CD152 (CTLA-4) PE-Cy7 L3D10 BioLegend 349914CD19 PE-Cy7 SJ25C1 eBioscience 25-0198-42CD19 PerCP Cy5.5 HIB19 Biolegend 302230CD19 PE HIB19 eBioscience 12-0088-42CD19 APC HIB19 eBioscience 17-0199-42CD194 (CCR4) PE 1.291H4 Biolegend 359412CD197 (CCR7) PE 3D12 eBioscience 12-1979-42CD223 (LAG3) FITC 2DS223H eBioscience 11-2239-42CD23 PE EBVCS-5 Biolegend 338508CD27 BV650 O323 Biolegend 302828CD279 (PD-1) BV711 EH12.2H7 BioLegend 329927CD28 BV605 CD28.2 BD Horizons 562976CD3 PE-Cy7 UCHT1 eBioscience 25-0038-42CD3 PE OKT3 eBioscience 12-0037-42CD3 BV711 OKT3 BioLegend 317328CD4 BV605 OKT4 Biolegend 317438CD45 BV421 HI30 Biolegend 304032CD45RA FITC L48 BD Biosciences 347723CD5 PE-Cy7 UCHT2 eBioscience 25-0059-42CD8 BV605 RPA-T8 Biolegend 301040CD8 PE-Cy7 RPA-T8 eBioscience 25-0088-42CD184 (CXCR4) PE-CY7 12G5 BioLegend 306508GM-CSF BV421 BVD2-21C11 BD Biosciences 562930IFNgamma PE 4S.B3 BioLegend 502509IL-2 PECF594 5344.111 BD Biosciences 562384Ki67 PerCP-eF710 20Raj1 eBioscience 46-5699-42TIM3 PE F38-2E2 BioLegend 345006TNF-a AF700 Mab11 BioLegend 502928murine CD45 APC Cy7 30-F11 Biolegend 103116tBET PE eBio4B10 eBioscience 12-5825-80Perforin AF488 K86-1161 BD Biosciences 560135MIP-1b PE-CY7 D21-1351 BD Biosciences 560687CD253 (TRAIL) PE N2B2 BioLegend 109306

Table S1. List of the antibodies used for multiparametric flow cytometry.

Degranulation assay. Briefly, T cells were incubated with target cells at a 1:5 ratio in T cell media. Anti-CD107a-PECY7 (Biolegend), anti-CD28 (BD Biosciences), anti-CD49d (BD Biosciences) antibodies and monensin (BD Biosciences) were added to the co-culture. After 4 hours, cells were harvested and stained for CAR expression, CD3, CD8 and Live Dead aqua staining (Invitrogen). Cells were fixed and permeabilized (Invitrogen, LifeTechnologies, GAS003, FIX & PERM Cell Fixation & Cell Permeabilization Kit) and intracellular staining was then performed to detect multiple cytokines (IFN, TNFα, IL-2, GM-CSF, MIP1β, all from eBioscience or BD).

Proliferation assay. T cells were washed and resuspended at 1x107/ml in 100 ul of PBS and stained with 100 ul of CFSE 2.5 uM (Invitrogen, LifeTechnologies, C34554) for 5 minutes at 37° C. The reaction was then quenched with cold media, and cells were washed three times. Targets were irradiated at a dose of 100 Gy. T cells were incubated at a 1:1 ratio with irradiated target cells for 120 hours, adding media at 24 hours. Cells were then harvested, stained for CD3, CAR and Live Dead aqua (Invitrogen), and Countbright beads (Invitrogen) were added prior to flow cytometric analysis for absolute quantification.

Cytotoxicity assays. Luciferase/eGFP+ NALM-6 or MCL-RL cells were used for cytotoxicity assay as previously described. (Gill S., Blood, 2014) In brief, targets were incubated at the indicated ratios with effector T cells for 24 hours. Killing was calculated by bioluminescence imaging on a Xenogen IVIS-200 Spectrum camera (Caliper LifeSciences, Inc., PerkinElmer, IVISSPE , Waltham , MA).

Cytokine measurements. Effector and target cells were co-incubated at a 1:1 ratio in T cell media for 24. Supernatant was harvested and analyzed by 30-plex Luminex array (Luminex Corp, FLEXMAP 3D, Austin, TX) according to the manufacturer’s protocol (Invitrogen, Liftechnologies, LHC6003).

Animal experiments. NOD-SCID-γ chain-/- (NSG) originally obtained from Jackson Laboratories (Bar Harbor, ME) were purchased from the Stem Cell and Xenograft Core of the University of Pennsylvania. All experiments were performed according to a protocol (#803230) approved by the Institutional Animal Care and Use Committee (IACUC). Animals were weighted and clinically monitored every day by the authors and/or the ULAR staff. The pre-established inclusion criteria for treatment were: confirmed engraftment of the tumor, good clinical conditions, and age of 6-9 weeks. All experiments were conducted with gender balanced groups of animals. Sample size was chosen to ensure adequate power (80%) to detect at least a 30% increase in response rate with a p-values of 0.05. Mice were randomized to treatment according to tumor burden, weight, age and gender in order to obtain homogeneus groups. Cells (MCL cell lines or T cells) were injected in 200 ul of PBS (Gibco, LifeTechnologies) at the indicated concentration into the tail veins of mice. Bioluminescent imaging was performed using a Xenogen IVIS-200 Spectrum camera and analyzed with LivingImage software v. 4.4 (Caliper LifeSciences, Inc., PerkinElmer). Animals were euthanized at the end of the experiment or when they met pre-specified endpoints according to the IACUC protocols. The animal technician was blinded during the experiment and assessment of outcome.

Supplementary Figures

AFigure S1

Ibrutinib concentration

MTT

Con

v. (%

)MCL-RL JEKO

C

Ibrutinib concentration

B

Figure S1. Different sensitivity to ibrutinib in hematological cell lines. A. Ibrutinib induces apoptotic cell death of the sensitive MCL cells. Dose-dependent DNA fragmentation is observed in the ibrutinib-sensitive MCL-RL (top) but not in the ibrutinib-resistant cell line Jeko-1 (bottom). Cells were incubated with increasing dose of ibrutinib (10-1000 nM) and analyzed by tunel assay after 72 hrs. Cells unexposed to terminal deoxynucleotidyl transferase (TdT) served as negative control. B. Western blot analysis of p-BTK inhibition by ibrutinib in MCL cell lines. MCL-RL and JEKO cells were incubated for 2 hours in the presence of different concentrations of ibrutinib (100 and 500 nM) or control (DMSO). Results showed significant inhibition of the phosphorylation of BTK, more pronounced in MCL-RL cell line. C. MTT assay of (non-MCL) leukemia cell lines. K562, and NALM6 cells resulted highly resistant to ibrutinib and, to a lesser degree, also MOLM14.

Figure S2

Figure S2. MCL-RL engrafted mice: tumor burden and histology. A. In vivo engraftment of MCL-RL cells in NSG mice. One million luciferase-positive MCL-RL cells were injected in NSG mice and tumor burden was followed up over time. Significant accumulation of MCL-RL cells in the liver and spleen was detectable starting day 5 with gradual dissemination over time to other organs including bone marrow and lungs (note change in scale, as depicted in the boxes above the BLI). B. Diffuse involvement of the spleen and peri-portal infiltration of liver was detected by immunohistochemistry; neoplastic B cells were PAX-5+, cyclin D1+ and SOX11+ (note the high background).

A

B

Figure S3

VL

VH

CD8αH

4-1BB

CD3-ζ

Ant

i-CD

19

scFv

(FM

C63

)

A BFigure S3. CTL019 design and production. A. CAR19 gene construct. Construct of the murine anti-CD19 chimeric –antigen receptor (CAR19). This is the same construct currently used in the CTL019 clinical trials at the University of Pennsylvania; it is a second-generation CAR that includes CD8 hinge, 4-1BB costimulatory and CD3 zeta signaling domains. B. CTL019 production schema. Normal donor CD4 and CD8 T cells (selected from PBMC using anti-CD4 and anti-CD8 microbeads (Miltenyi, San Diego, CA, USA) or PB mononuclear cells (PBMC) were obtained from the Human Immunology Core of the University of Pennsylvania. T cells were plated at 1x106/ml with a CD4:CD8 ratio of 1:1 and expanded in X-vivo 15 media (Lonza, 04-380Q), supplemented with human AB serum 5% (Gemini, 100-512), penicillin/streptomycin (Gibco, 15070063) and Glutamax (Gibco, LifeTechnologies, 35050061) using anti-CD3/CD28 Dynabeads (Invitrogen, Life Technologies, 11161D) added on the day 1 of culture and removed on day 6. T cells were transduced with lentivirus on day 2. T cells were expanded in culture for 8-15 days and harvested when the median cell volume was below 300 fl. T cells were then cryopreserved in FBS with 10% DMSO (Sigma, D2650) for future experiments. Prior to all experiments, T cells were thawed and rested overnight at 37° C.

0 2 4 6 8 10 120

200

400

600

800

BM #002

days

Cell

Volu

me

(fl)

UTDCART19

0 2 4 6 8 10 12 14 160

200

400

600

800

PB #001

daysCe

ll Vo

lum

e (fl

)

UTDCART19

CD

19C

D19

CD5

Figure S4A

B

CD5

CAR19

TNFa

IL-2

CAR19

CAR19C

D

MCL-RL JEKO0

200

400

600

800

1000

1200

T ce

ll/ul

*

MCL-RL JEKO0

10

20

30

40

50

APO

PTO

TIC

%

***

ALONE PI CD19- CL JEKO-1 MCL-RL0

20

40

60

80

%CA

R+ IL

-2 c

ells

UTDCART19

ALONE PI CD19- CL JEKO-1 MCL-RL0

10

20

30

40

50

%CA

R+

TNFa

+ ce

lls

Figure S4. CTL019 cell production from primary MCL samples. A. Presence of malignant B cells in MCL samples. Both PB#001 and BM#002 samples obtained from PB and BM respectively showed presence of malignant MCL B cells (>90% of B cells) as defined by the CD19+ and CD5+ characteristic phenotype (gated on CD19+ B cells). B. CTL019 expansion from MCL samples: T cell volume. During anti-CD3/CD28 bead expansion T cells showed increasing cell volume with a peak on day 6-8 with reduction to < 300 fl after day 10. T cells were frozen when cell volume was < 300 fl. C. CTL019 in vitro effector functions. CAR19+ T cells showed intense specific intra-cytoplasmic cytokine production, including IL-2 and TNFa when co-cultured with JEKO-1 and MCL-RL MCL cell lines but not with untransduced T cells (UTD); PMA and ionomycin stimulation (PI) was used as positive control and culture media as negative control. D. Early apoptotic death of CART-19 cells exposed to MCL-RL cells as compared to JEKO-1. CTL019 cells were co-cultured with irradiated MCL-RL or JEKO-1 cells for 24 hours; absolute T cell numbers were calculated by flow-cytometry using CountBright beads and gating on live, single CD3+ cells. CART-19 cells exposed to MCL-RL showed a significant reduction in cell number as compared to JEKO-1 exposed CART-19 (as determined by 7-AAD and Annexin V flow cytometry analysis).

Figure S5

A

TRAILPERFORIN

DM

SOIB

RU

10

nM

IBR

U 1

00

nMIB

RU

10

00 n

M

B C

CART19DMSO

CART19 IBRU 10nM

CART19 IBRU 100nM

CART19 IBRU 1000

nM

CC

R4

CXCR3

Th2

Th1

CD4+ CCR6-

Figure S5. Effect of ibrutinib in T cell function in vitro and in vivo. A. CTL019 proliferation assay in the presence of ibrutinib. CTL019 cells were co-cultured with lethally irradiated JEKO-1 cells or controls (no stimulation and PMA-Ionomycin stimulation) in the presence of increasing doses of ibrutinib (10-1000 nM, added at every change of media). At day 5 total T cell numbers were evaluated via CountBright quantification. Decrease T cell numbers were observed at the highest ibrutinib concentrations. This decrease was found to be corrleated to an increase in T cell apoptosis (as determined by 7-AAD and Annexin V flow cytometry analysis). B. Th2/Th1 T cell subtype analysis during CTL019 expansion in the presence of ibrutinib. CD4+/CD8+ CTL019 were expanded with anti-CD3/CD28 beads in the presence of increasing doses of ibrutinib (10-1000 nM). At the end of expansion (day 10) Th1 and Th2 CD4+ cells were defined using CCR6, CCR4 and CXCR3 expression; no difference in Th1/Th2 ratio was observed with increasing doses of ibrutinib. C. CTL019 intrinsic cytotoxic machinery in the presence of ibrutinib. Normal donor T cells were expanded with anti-CD3/CD28 beads in the presence of different doses of ibrutinib (10-1000 nM) or vehicle. TRAIL and perforin expression was analyzed by flow cytometry and no significant difference in mean fluorescence intensity MFI was observed. Gated on CD3+ CD8+ CAR19+.

0

2000

4000

6000

8000

TOT T CELLS JEKO DAY 5 IBRU DAY 0-1-3

CD

3+/u

l

CART-19 ALONECART-19 PICART-19 NO IBRUCART-19 IBRU 10 nMCART-19 IBRU 100 nMCART-19 IBRU 200 nMCART-19 IBRU 500 nMCART-19 IBRU 1000 nM

0

10

20

30

40

APOPTOTIC% JEKO DAY 5

APO

PTO

TIC

%

Figure S6

A

Figure S6. Peripheral blood T cell subsets in mice treated with CART19 or CART19-ibrutinib. A. No difference in tBET expression. PB T cells from mice receiving CART19 or CART19 plus ibrutinib were analyzed by flow cytometry (Gated on murine CD45-, human CD3+ and CD4+) for the Th1 transcription factor tBET one week after T cell infusion. Both groups showed similar tBET expression. B and C. No difference in T cell subset. PB T cells from mice receiving CART19 or CART19 plus ibrutinib were analyzed by flow cytometry (Gated on murine CD45-, human CD3+) for T cell subsets 2 weeks after T cell injection. NAÏVE cells were defined as CD45RA+CCR7+, CENTRAL MEMORY (CM) as CD45RA-CCR7+, EFFECTOR MEMMORY (EFF. MEM.) as CD45RA-CCR7+ and EFF. MEM. CD45RA+ as CD45RA+CCR7-. Both groups showed similar distribution of these subsets.

tBET CELLS PB CART19 2E6 WK 1

CART19 CART19+IBRUTINIB0

20

40

60

80

100tB

ET%

NAIVE CM

EFF. MEM.

TEMRANAIVE CM

EFF. MEM.

TEMRA0

20

40

60

80

100

DAY 22 T CELL SUBSETS

%C

D3+

CART19CART19+IBRUTINIB

CART19 CART19 + IBRUTINIB

CD45RA-FITC

CC

R7-

PE

B

C

Figure S7A

TIM3LAG3

CD137

CTLA4TIM

3LAG3

CD137

CTLA4TIM

3LAG3

CD137

CTLA4TIM

3LAG3

CD137

CTLA40

20

40

60

DAY 15 INHIBITORY LIGANDS

%C

D3+

UTD 2E6UTD+IBRUTINIBCART19CART19+IBRUTINIB

CXCR4 WK1

CART19 CART19+IBRUTINIB0

10

20

30

40

% C

XCR

4

p=NS

Figure S7. CXCR4 and inhibitory/costimulatory receptor expression in peripheral blood of mice treated with CART19 or CART19-ibrutinib. A. No difference in CXCR4 expression. PB T cells from mice receiving CART19 or CART19 plus ibrutinib were analyzed by flow cytometry (Gated on murine CD45-, human CD3+) for the CXCR4 one week after T cell infusion. Both groups showed similar CXCR4 expression. B No significant difference in inhibitory/costimulatory receptor expression in the PB of mice treated with CART19 with or without ibrutinb. PB T cells from mice receiving CART19 or CART19 plus ibrutinib were analyzed by flow cytometry (Gated on murine CD45-, human CD3+) for T cell subsets 2 weeks after T cell injection. Both groups showed similar distribution of these subsets. C Trend in reduced PD-1 expression in CART19 cells exposed to ibrutinib in vivo. however expression of PD-1 3 weeks after T cell infusion was lower in mice treated with untransduced T cells (UTD) or CART19 plus ibrutinib.

0

50

100

DAY 22 PD1+

%PD

-1+

UTD+IBRUTINIBCART19CART19+IBRUTINIB

UTD

0

50

100

DAY 22 PD1+

%PD

-1+

UTD+IBRUTINIBCART19CART19+IBRUTINIB

UTD

B

C

0

50

100

DAY 22 PD1+

%PD

-1+

UTD+IBRUTINIBCART19CART19+IBRUTINIB

UTD

Figure S8

Figure S8. A. Ibrutinib did not enhance the anti–tumor activity of control untransduced T cells. Tumor burden of mice engrafted with luciferase + MCL-RL was analyzed 13 days after treatment with vehicle, ibrutinib 125mg/kg/day, UTD or UTD. Ibrutinib alone or in combination with UTD let to partial tumor regression while UTD and vehicle alone did not. Graph representative of 2 experiments, each with 5 mice per group.

MCL-15 BLI

NO TREAT

IBRUTINIB

UTD+VEHICLE

UTD+IB

RUTINIB0

5.010 6

1.010 7

1.510 7

2.010 7

2.510 7

NO TREATIBRUTINIB 125 mg/Kg/day

UTD+VEHICLEUTD 2E6+IBRUTINIB 125mg/K

Mea

n BL

I (p/

sec)

****

NO TREAT IBRUTINIB UTD UTD + IBRUTINIB

****

ns

A