Accepted Manuscript

CRISPR knock out CTLA-4 enhances the anti-tumor activity of

cytotoxic T lymphocytes

Long Shi, Tongyu Meng, Zhilong Zhao, Jinsheng Han, Wei

Zhang, Fei Gao, Jianhui Cai

PII: S0378-1119(17)30719-9
DOI: doi: 10.1016/j.gene.2017.09.010
Reference: GENE 42159
To appear in: Gene
Received date: 17 May 2017
Revised date: 12 August 2017
Accepted date: 5 September 2017

Please cite this article as: Long Shi, Tongyu Meng, Zhilong Zhao, Jinsheng Han, Wei
Zhang, Fei Gao, Jianhui Cai , CRISPR knock out CTLA-4 enhances the anti-tumor activity
of cytotoxic T lymphocytes, Gene (2017), doi: 10.1016/j.gene.2017.09.010

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 ACCEPTED MANUSCRIPT

CRISPR knock out CTLA-4 enhances the anti-tumor activity of cytotoxic T lymphocytes

Long Shi1, Tongyu Meng4, Zhilong Zhao5, Jinsheng Han6, Wei Zhang7, Fei Gao3, Jianhui Cai 2,3

1. Department of Oncology, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei,

China 050000

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2. Department of Surgery, Hebei Medical University, Shijiazhuang, Hebei, China 050000

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3. Department of Surgery＆Oncology, Hebei General Hospital, Shijiazhuang, Hebei, China 050000

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4.Department of Gynecologic Oncology, The first hospital of SJZ, Shijiazhuang, Hebei, China 050000

5. Department of Surgery, the third affiliated hospital of Jinzhou medical university, Jinzhou, Liaoning,
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China 121000
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6. Department of Surgery, Cangzhou hospital of Traditional Chinese Medicine and Western Medicine
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integrated Hebei, Cangzhou, Hebei, China 601000

7. Department of Surgery, HanDan Central Hospital, Handan, Hebei, China 056001

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Correspondence to Jianhui Cai; Tel: 86-311-85988977; Fax: 86-311-85988902.

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Email: jianhuicai2002@163.com
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Abstract

T cell-mediated anti-tumor immunity plays a pivotal role in cancer immune surveillance. Cytotoxic T-

lymphocyte-associated protein 4 (CTLA-4) is a protein receptor mainly expressed in activated T cells and

regulatory T cells. CTLA-4 competes with CD28 for ligand binding and generates inhibitory signals to

attenuate T cell activation. The blockade of CTLA-4 mediated immune inhibitory checkpoint has been

associated with enhanced anti-tumor immunity. In this study, we use CRISPR-Cas9 system to knock out
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(KO) CTLA-4 from cytotoxic T lymphocytes (CTLs) and evaluate its effect on the anti-tumor activity of

the CTLs. CTLA-4 KO CTLs robustly enhanced tumor cell death by 40% compared to the control and

facilitated apoptosis and caspase activities in tumor cells. The knockout of CTLA-4 also increased TNF-α

and IFN-γ secretion of the CTLs by approximately 2-fold. The effectiveness of CTLA-4 KO in enhancing

anti-tumor activity of the CTLs was verified in vivo using mouse xenograft model. The xenografted mice

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treated with CTLA-4 KO CTLs demonstrated repressed tumor growth and prolonged survival compared

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to the control group. Our data suggest that CRISPR targeting CTLA-4 immune checkpoint could

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significantly improve the anti-tumor activity of CTLs.

CRISPR-Cas9 - Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein
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CTLA-4 - Cytotoxic T-lymphocyte-associated protein 4
KO – Knock out
CTL - cytotoxic T lymphocytes
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TNF-α - Tumor necrosis factor-α
IFN-γ - interferon gamma
DC - dendritic cells
CIK - Cytokine-induced killer cells
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APC - antigen-presenting cell

CAR - Chimeric antigen receptor
TRAC - T-cell receptor α constant locus
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PBMC - peripheral blood mononuclear cells

GM-CSF - Granulocyte-macrophage colony-stimulating factor
IL-4 - interleukin 4
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IL-1β - Interleukin 1 beta

SDS-PAGE - sodium dodecyl sulfate polyacrylamide gel electrophoresis
ELISA - enzyme-linked immunosorbent assay
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SCID - Severe Combined Immunodeficiency

PARP - Poly (ADP-ribose) polymerase
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Keywords: CRISPR, CTLA-4, cytotoxic T lymphocytes, T cell-mediated antitumor immunity,

cancer immunotherapy

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1. Introduction

Human adaptive immunity is finely tuned by the balance between stimulatory and inhibitory signals[1].

Two stimulatory signals are required for naive T cells activation [2-5]. The interaction of T-cell receptor

(TCR) with the antigenic peptide presented by the antigen-presenting cell (APC) provides the antigen-

specific signal[6]. The interaction of T cell surface molecule CD28 with the B7 molecules (CD80 and

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CD86) on APC generates co-stimulatory signal[7-16]. Activation of the TCR in the presence of co-

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stimulatory signals leads to T cell clonal expansion and initiation of effector functions. Cytotoxic T-

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lymphocyte-associated protein 4 (CTLA-4), also known as cluster of differentiation 152 (CD152), is a

protein receptor primarily expressed in activated T cells and regulatory T cells [17]. CTLA-4 is

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homologous to the T cell co-stimulatory protein CD28 and competes with CD28 in binding the B7

ligands[18]. Unlike CD28 that delivers a co-stimulatory signal for T cells activation, CTLA-4 transmits
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an inhibitory signal to T cells through interacting with B7 ligands[17, 19]. CTLA-4 down-regulates
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immune response through inhibiting IL-2 production and cell cycle progression[19, 20].
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The co-stimulatory and co-inhibitory signals mediated by CD28 and CTLA-4 deliberately modulate the T

cell-mediated immunity. The blockade of CTLA-4 with monoclonal antibodies led to enhanced the anti-
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tumor immune response in a range of preclinical investigations[21-31]. These preclinical findings suggest

the activation of cytotoxic T-lymphocytes (CTLs) trough targeting CTLA-4 is a feasible strategy in
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cancer immunotherapy.
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The emerging CRISPR gene editing technology has taken cancer immunotherapy to the next level.

CRISPR has been recently used to precisely deliver Chimeric antigen receptor (CAR) to the T-cell

receptor α constant (TRAC) locus to improve the endurance and potency of CAR T cells. In this study,

we edited CTLA-4 out of CTLs with the CRISPR-Cas9 system to investigate its effect on T cell

activation and cytotoxicity. This study will provide the foundation for the future application of CRISPR

technology in CTLA-4 targeting.

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2. Materials and methods

2.1 Cell culture and reagents

Peripheral blood from healthy donors was obtained were with the written consent and approved from

Hebei Blood Center (Hebei, China). Human PBMCs (peripheral blood mononuclear cells) isolated from

peripheral blood were cultured in RPMI-1640 complete medium (Gibco BRL, USA) supplemented with

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10% fetal bovine serum (FBS, Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin (Life

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Technologies, Rockville, MD, USA). Human colonic adenocarcinoma cell line HCT116 cell line [32] was

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purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in RPMI-

1640 medium containing 10% FBS and antibiotics in a humidified 37 °C incubator with 5% CO2.

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2.2 DCs induction and maturation
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PBMCs were isolated from whole blood samples by Ficoll-Paque density gradient centrifugation (GE

Healthcare, Chicago, IL, USA). Mononuclear cells from the interphase were collected, washed with
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complete medium three times, and then cultured in the complete medium at 37 oC with 5% CO2. After 2h
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incubation, unattached cells and medium were removed. Attached cells were washed and then cultured in

RPMI complete medium supplemented with 100ng/ml human recombinant GM-CSF (PeproTech, Rocky
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Hill, NJ, USA) and 100ng/ml human recombinant IL-4 (PeproTech) for 6 days. On day 6, irradiated

HCT116 cell lysates were added into cultured DCs. After 24h co-culture, 25 ng/ml IL-1β (PeproTech)
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and 100 ng/ml TNF-α (Millipore Sigma, St. Louis, MO, USA) were added into the culture. Mature DCs
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were tested and collected on day 8.

2.3 Generation of Cytokine-induced killer cells (CIK) and CTLs

After 2h incubation in the complete medium at 37 °C with 5% CO2, unattached PBMCs were used to

generate CIKs according to the protocol by Schmidt-Wolf et al [33]. Briefly, unattached cells were

cultured in RPMI-1640 complete medium supplemented with 1000U/ml IFN-γ (Millipore Sigma),

1000U/ml IL-2 (Millipore Sigma), 5µg/ml CD3 monoclonal antibody (Millipore Sigma). The medium
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was half changed every 2-3 days with new medium contain 1000U/ml IL-2. CIK cells were mature and

ready to use after two weeks. CIKs were co-cultured with the mature DCs at the ratio of DC: T = 1:20 for

4 days to induce antigen-specific CTLs. CTLs were expanded in RPMI-1640 complete medium

supplemented with 50U/ml recombinant human IL-2.

2.4 Detection of immunophenotyping by flow cytometry

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The FC500 flow cytometer (Beckman Coulter, USA) was used to characterize the immune phenotypes of

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DCs, CIKs, and CTLs. DCs were evaluated by CD80/CD86/CD83/CD11c; CIKs were evaluated by

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CD3+/CD56+; CTLs were evaluated by CD3+/CD8+. All the antibodies were purchased from BioLegend

(San Diego, CA, USA). The flow cytometry data were analyzed with FlowJo v.10.2 (TreeStar, Ashland,

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OR, USA).
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2.5 CRISPR/Cas9 Lentivirus production

The non-targeting single guide RNA control and three single guide RNA sequences targeting human
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CTLA-4 gene were designed. Control: ACGGAGGCTAAGCGTCGCAA; CTLA-4 sg1:

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CCTTGGATTTCAGCGGCACA; CTLA-4 sg2: GCACAAGGCTCAGCTGAACC; and CTLA-4 sg3:

CTCAGCTGAACCTGGCTACC. Single guide RNAs were cloned into a LentiCrispr v2 vector at BsmBI
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site following the protocol described previously[34]. Human embryonic kidney 293T cells were co-

transfected with single guide RNA containing LentiCrispr V2, packaging vector ps-PAX2 vector, and
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envelop vector pCL-VSVG. The culture medium was removed and changed to RPMI-1640 complete
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medium 24h later. The supernatant containing lentivirus was harvested 48h later and filtered through

0.45µm syringe filter.

2.6 Generation of CTLA-4 knockout cell lines

CTLs were transduced with lentivirus expressing control or CTLA-4 sgRNAs. After 48h incubation,

growth medium was changed to selection medium containing 1µg/ml puromycin. Once growing normally
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in selection medium, CTLs were then expanded in RPMI-1640 complete medium supplemented with

50U/ml recombinant human IL-2. The knockdown of CTLA-4 was confirmed using western blot analysis.

2.7 Western blot analysis

Cells were pelleted and lysed in lysis buffer (150 mM NaCl, 1% Triton X-100, 0.5% Na deoxycholate,

0.1% SDS, 50mM Tris-HCL, pH 7.4 with protease and phosphor-protease inhibitors). After incubation

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for 15min on ice, cell lysates were centrifuged at 15,000 rpm for 15 min and then the supernatant was

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collected. Protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA,

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USA). Equal amounts of total cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane

and then immunoblotted with antibodies against CTLA-4 or GAPDH followed by horseradish

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peroxidase-conjugated secondary antibodies. Blots were then visualized by enhanced chemiluminescence

(Bio-Rad Laboratories).
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2.8 Cell viability assay
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HCT116 cell viability was measured using WST-1 assay. Briefly, HCT116 cells were seeded in triplicate
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wells in 96-well-plates at the density of 5 x 103/well. After attachment, the control or CTLA-4 KO CTLs

were added to the plates at the density of 5 x 104/well. CTLs were co-cultured with HCT116 cells for
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another 18h at 37°C. HCT116 cell viability was assessed by WST-1 assay as described previously [35].

Briefly, 10 µl of WST-1 reagent was added into each well of the cell culture and incubated at 37 °C for 1
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h. Absorbance at wavelength 450 nm was read by standard microplate reader, and data were recorded and
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normalized by T cell culture alone.

2.9 Caspase enzymatic activity assay

Caspase assay buffer and caspase substrates were purchased from Enzo Life Science, Inc. (Farmingdale,

NY, USA). Caspase-3 substrate (Ac-DMQD-AMC), caspase-8 substrate(Ac-LETD-AFC) and caspase-9

substrate (Ac-LEHD-AFC) were used to test the activity of caspase-3, caspase-8 and caspase-9

respectively following the manufacture’s instruction.

2.10 cytokine induction assay

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1.5 x 104 HCT116 cells (target cells) were seeded in U-bottom 96-well-plate overnight at 37°C. On the

second day, 1.5 x 105 control CTLs or CTLA-4 KO CTLs (effector cells) were added to the culture. After

another 24 h, cell-free supernatants was collected by centrifuging at 1,000 g for 10 min and assayed for

cytokine release. Human IFN-gamma ELISA kit (Thermo Fisher Scientific Inc., Waltham, MA, USA)

and TNF-alpha ELISA Kit (Abcam Inc., Cambridge, MA, USA) were used to test IFN gamma, TNF

alpha, and IL-2 release following manufacture’s protocol.

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2.11 Xenograft mice model

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All the animal studies were approved by Hebei Medical University Animal Care and Use Committee.

HCT116 cells were washed and suspended in phosphate-buffered saline at the concentration of 5 × 107/ml.

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100 µl of HCT116 cells were subcutaneously injected into the right flank of CB-17 SCID mice (6 weeks

old) that received irradiation (200 rad) on day 0. Mice were randomly separated into two groups (10 mice
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each group). On day 7 and day 14, 1 x 107 control CTLs or CTLA-4 KO CTLs were injected
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intravenously into the mice. Tumors were measured twice a week by a digital caliper. Tumor volume was

calculated using the formula v = ab2 x 0.5, where a is the length and b is the width of the tumor. Mice
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were sacrificed when mice died, or tumor size reached 1000 mm3. Overall survival was examined by
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Kaplan-Meier method.

2.12 Statistical analysis

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All experiments were independently repeated three times. The results were presented as the mean 
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standard deviation. Two-tailed Student’s t test was used to determine statistical significance. The

GraphPad prism 5 software was used to evaluate the significance of the difference between groups. P

value less than 0.05 was considered as statistically significant.

3. Result

3.1 Immunophenotypes of DCs, CIKs and CTLs

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Flow cytometry was used to monitor the immunophenotypes of the ex vivo cultivated cells. The high-

level expression of CD11c (97.53 ± 1.08%; Figure 1A), co-stimulatory molecules CD80 (83.12 ± 1.15%;

Figure 1B), CD86 (93.49 ± 3.52%; Figure 1C) and the maturation marker CD83(83.24 ± 0.74%, Figure

1D) at day 8 demonstrated the maturation of DCs. The proportion of CD3+/CD56+ CIK cells reached

21.73 ± 2.04% (Figure 1E) on Day 10. The proportion of CD3+/CD8+ CTLs reached 87.86 ± 1.73%

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(Figure 1F) at day 13, indicating the successful activation and expansion of CTLs by mature DCs.

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3.2 CRISPR knockout of CTLA-4 increased the anti-tumor activity of the CTLs

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The CTLs were transduced with lentivirus expressing guide RNA against CTLA-4 sequence to generate

CTLA-4 KO CTLs. The lentivirus expressing non-targeting guide RNA was used to generate parallel

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control CTLs with intact CTLA-4. The efficiency of CTLA-4 knockout in the CTLs was tested 10 days

after lentivirus transduction. An equal amount of cell lysates was subjected to SDS-PAGE and then
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transferred to a PVDF membrane for western blot analysis. Compared to the control, the CTLA-4
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expression was significantly reduced in the CTLs transduced with lentivirus expressing CTLA-4 guide

RNA, indicating the successful knockout of CTLA-4 from most of the CTLs by the CRISPR-Cas9 system
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(Figure 2A).
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We next examined if the knockout of CTLA-4 could improve the anti-tumor activity of the CTLs. We

first assayed the cytotoxicity of CTLA-4 KO CTLs on HCT116 colon cancer cell line. After being co-
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cultured with CTLs (control or CTLA-4 KO) for 24 h, HCT116 cells was assessed for cell viability using
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WST-1 assay. Results showed that the HCT116 cell viability in CTLA-4 KO co-culture decreased to

58.26% ± 4.55% of that in the control treatment (p < 0.005, Figure 2B), clearly demonstrating that

CTLA-4 KO enhanced the cytotoxicity of CTLs.

3.3 CTLA-4 KO CTLs enhance apoptosis and caspase activities in tumor cells

The apoptosis of HCT116 cells were analyzed by Annexin V/PI staining followed by flow cytometry

analysis after being co-cultured with CTLs for 12 h. 40.3% of HCT116 cells entered late apoptotic stage
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in the CTLA-4 KO co-culture, whereas only 20.3% of HCT116 cells entered this stage in the control co-

culture (Fig 3A). Accordingly, the percentage of early apoptotic HCT116 cells in the CTLA-4 KO and

control co-culture are 22.7% and 18.3% respectively. These data clearly show that CTLA-4 KO CTLs

induced a higher level of apoptosis in the co-cultured tumor cells than did the control CTLs.

Caspases play essential roles in programmed cell death. We found the HCT116 cells in the CTLA-4 KO

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co-culture have more cleaved form of caspase-3, caspase-7, caspase-8, caspase-9 and PARP than the

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control (Fig 3B), showing that CTLA-4 KO CTLs lead to an elevated caspase activation in tumor cells.

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We then incubated HCT116 cell lysates with different caspase substrates to test their caspase activities.

Results confirmed that the caspase-3, caspase-8, and caspase-9 activities of the HCT116 cells in CTLA-4

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KO co-culture are respectively 10.35 ± 1.53, 9.18 ± 0.67 and 7.92 ± 0.25 times higher than that of the

control.
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3.4 CTLA-4 KO CTLs produce increased cytokines
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CTLs release cytokines such as Tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), which is

associated with the activation of host immune response to kill viral infected cells and tumor cells. Since
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we found that CTLA-4 KO enhanced the anti-tumor activity of the CTLs, we then examined if CTLA-4
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KO could enhance the cytokines secretion of CTLs. ELISA assays showed that CTLA-4 KO CTLs

secreted 2.38 ± 0.45 and 2.17 ± 0.26 times more TNF-α and IFN-γ respectively than did the control CTLs
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(Figure 4A and 4B). This result clearly shows that CTLA-4 KO could increase the cytokines production

of CTLs.
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3.5 CTLA-4 KO CTLs decrease the growth of HCT116 cells xenografted in mice

The efficacy of CTLA-4 KO CTLs in repressing tumor growth was validated in vivo using human tumor

xenograft model. Two groups of CB-17 SCID mice were subcutaneously inoculated with 5 x 10 6

HCT116 cells following 200 rad irradiation. The mice were then treated with either control CTLs

(control group) or CTLA-4 KO CTLs for four different times to evaluate the repression of CTLs on tumor

growth. The tumor became detectable after 2 weeks and the tumor growth in the CTLA-4 KO CTLs
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treated mice was dramatically repressed compared to that in the control group (Figure 5A, p < 0.01). All

the mice in the control group died from progressive tumors by 62 days. In contrast, only 10% of CTLA-4

KO CTLs treated mice died at the same time (Figure 5B, p < 0.0001). Thus, CTLA-4 KO in CTLs

effectively inhibited human MM cell growth in vivo and significantly improved overall survival of the

xenografted mice.

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4. Discussion

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T-cell mediated anti-tumor responses are the major component of cancer immune surveillance that

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controls tumor development and progression [36]. Tumor cells can escape immune surveillance in the

presence of tumor-associated antigen-specific T cells [37]. The mechanisms utilized by cancer cells to

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repress T-cell function including the production of soluble immunosuppressive factors (e.g. TGF-β, IL-10)

and the recruitment of suppressor immune cells. The cellular molecules involved in the inhibition of T
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cell mediated anti-tumor response have been intensively investigated and a group of inhibitory molecules
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have been identified. Among these molecules, PD-1 and CTLA-4 are two most studied targets for the

development of cancer therapy[17, 38, 39].

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CTLA-4 plays an essential role in modulating the T-cell mediated anti-tumor response [40]. The blockade
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of CTLA-4 mediated co-inhibitory signaling with antibody has been shown to enhance the immune

response against cancer cells [41]. The monoclonal CTLA-4 antibody ipilimumab has been approved for
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melanoma treatment and others are in development [42-44]. In addition to therapeutic antibody

development, the application of CRISPR gene-editing technology in T cell genome engineering also
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holds great promise in cancer immunotherapy. The advantage of CRISPR is the sequence specific

interventions in cells. Recent studies have demonstrated the potential of CRISPR technology in

improving the efficacy of CAR T cells.

In this study, we used CRISPR technology to disrupt the CTLA-4 mediated co-inhibitory signaling and

tested its effect on CTLs’ anti-tumor activity. Compared to the control, the CTLA-4 KO CTLs could

further reduce tumor cell viability by about 40%. The mechanism study showed the enhanced anti-tumor
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effect is associated with the increased apoptosis and augmented caspase activities in tumor cells. Since

secretion of cytokines (primarily TNF-α and IFN-γ) is one of the mechanisms for CTLs to kill tumor cells,

we next measured if knockout of CTLA-4 in CTLs increases TNF-α and IFN-γ secretion. The production

of TNF-α and IFN-γ in CTLA-4 KO CTLs increased about 2-fold compared to that in the control CTLs.

In vivo animal study shows that CTLA-4 KO CTLs are active in the xenograft mouse model. The CTLA-

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4 KO CTLs treatment inhibited tumor growth more efficiently than did the control and prolonged the

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overall survival of the host.

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In conclusion, our data showed that CRISPR knockout CTLA-4 enhanced the T cell mediated anti-tumor

immunity, suggesting the potential application of CRISPR technology in immune checkpoints targeting.

Conflicts of interest
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The authors declare that there is no conflict of interest regarding the publication of this paper.
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Figure 1. Flow cytometry analysis of the immunophenotypes of the ex vivo cultivated cells. A-D, the DC
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cell marker CD80, CD83, CD11c and CD83 were analyzed. E, The CD3+ cells were further gated for
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CD56 to show the percentage of CIK cells. F, The CTL cells were analyzed by CD3 and CD8 antibody.
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Figure 2. CRISPR knockout of CTLA-4 in CTLs. A. western blot analysis showing knockout of CTLA-4

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in CTLs. GAPDH is used as loading control. B. The cell viability of HCT116 cells co-cultured with the

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control or CTLA-4 KO CTLs were compared and normalized to the control. Data is presented as mean 

standard deviation, experiment was independently repeated for 3 times.

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Figure 3. CTLA-4 disruption in CTLs increased apoptosis and caspase activities in tumor cells. A.

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Annexin V/PI staining shows that HCT116 cells co-cultured with CTLA-4 KO CTLs have higher level of

early stage and late stage apoptotic cells. Annexin V+/PI- means early apoptotic, Annexin V+/PI+ means

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late apoptotic. B. Compared to the control, HCT116 cells co-cultured with CTLA-4 KO CTLs
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demonstrated higher level of the cleaved form (CF) of PARP, caspase-3, caspase-7, caspase-8 and

caspase-9. C. Compared to the control, HCT116 cells co-cultured with CTLA-4 KO CTLs demonstrated
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higher level of caspase enzymatic activity.

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Figure 4. CTLA04 KO increased the cytokine secretion of CTLs. A. The TNF-α secretion in the control

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and CTLA-4 KO CTLs were tested and normalized to control CTLs. B. The IFN-γ secretion in the control

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and CTLA-4 KO CTLs were tested and normalized to control CTLs.

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Figure 5. CTLA-4 KO CTLs repress tumor growth more efficiently than the control CTLs in xenografted

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mice. A. The tumor growth in human HCT116 bearing mice treated with either control or CTLA-4 KO

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CTLs. Data were presented as mean  standard deviation. B. Overall survival was examined by Kaplan-

Meier method (n = 10, p < 0.0001).

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Highlights
 CTLA-4 knockout increases the anti-tumor activity of the CTLs
 CTLA-4 knockout CTLs enhance apoptosis and caspase activities in tumor cells
 CTLA-4 knockout increases the secretion of cytokines in CTLs

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