Molecular Immunology 158 (2023) 68–78

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Molecular Immunology
journal homepage: www.elsevier.com/locate/molimm

Sirtuin 6 is a negative regulator of the anti-tumor function of natural killer

cells in murine inflammatory colorectal cancer
Fei Xiao , Bo Hu , Zhilong Si , Huanbin Yang , Jun Xie *
The Division of Gastrointestinal Surgery, Wuhan Fourth Hospital (Tongji Medical College Affiliated Wuhan Puai Hospital), 473 Hanzheng Street, Qiaokou District,
Wuhan, Hubei Province 430033, China

A R T I C L E I N F O A B S T R A C T

Keywords: The immune system plays a crucial role in controlling colorectal cancer (CRC) development. Natural killer (NK)
Natural killer cells cells are tumoricidal but undergo exhaustion in CRC patients. The current research aims to understand the role of
Colorectal cancer sirtuin 6 (SIRT6) in CRC-associated NK cell exhaustion in a murine inflammatory colorectal cancer model. To this
Sirtuin 6
end, inflammatory CRC was induced by treating mice with azoxymethane plus dextran sulfate sodium. The
T cell immunoreceptor with Ig and ITIM
domains
expression of SIRT6 in NK cells in murine mesenteric lymph nodes (mLNs) and the CRC tissue was characterized
Lymphocyte-activation protein 3 by Immunoblotting. SIRT6 knockdown was achieved by lentiviral transduction of murine splenic NK cells, fol­
lowed by evaluation of NK cell proliferation and the expression of cytotoxic mediators using flow cytometry. NK
cell cytotoxicity was measured by cytotoxicity assays. Adoptive transfer of murine NK cells was applied to
analyze the effect of SIRT6 knockdown in vivo. We found that SIRT6 was up-regulated in infiltrating NK cells in
the murine CRC tissue, especially NK cells with an exhausted phenotype and impaired cytotoxicity. SIRT6
knockdown significantly boosted murine splenic NK cell functionality, as evidenced by accelerated proliferation,
increased production of cytotoxic mediators, and higher tumoricidal activity both in vitro and in vivo. Further­
more, the adoptive transfer of SIRT6-knockdown NK cells into CRC-bearing mice effectively suppressed CRC
progression. Therefore, SIRT6 up-regulation is essential for murine NK cell exhaustion in CRC because it impedes
the tumoricidal activity of murine NK cells. Artificial SIRT6 down-regulation could boost the function of infil­
trating NK cells to oppress CRC progression in mice.

1. Introduction
Colorectal cancer (CRC) is the world’s third most commonly diag­
nosed and second most fatal cancer (Hossain et al., 2022). The immune
system plays a significant role in the surveillance and control of CRC
development (Mezheyeuski et al., 2021; Kather and Halama, 2019).
Immune cells participating in CRC immunity include natural killer (NK)
cells, CD4+ T cells, CD8+ cytotoxic T cells, B cells, macrophages, and
dendritic cells (Kather and Halama, 2019; Guo et al., 2020). Notably, NK
cells are potent tumoricidal cells. Upon encountering malignant cells, an
array of surface molecules on NK cells elicit activating or inhibitory
signals to modulate NK cells to release cytotoxic mediators such as
perforin and granzymes (Wu et al., 2020). NK cells, either circulating or
intratumoral, are reported as a favorable prognostic marker in CRC
(Tang et al., 2020; Coca et al., 1997; Donadon et al., 2017). In a rat CRC
model, follicle-like NK cell aggregates were found to be localized in the
tunica mucosa and tunica submucosa of colorectal tumors, suggesting
their role in fighting CRC development (Bahr et al., 2021). NK cell-based
immunotherapy has been regarded as a promising option for CRC
treatment. However, CRC-associated NK cell exhaustion impairs NK cell
tumoricidal capacity especially in the local CRC tissue to incur immune
escape of CRC (Sorrentino et al., 2021; Rocca et al., 2016, 2013). NK
cells exhibited multiple phenotypic changes and a profound
down-regulation of NK cell-activating receptors in CRC patients (Rocca
et al., 2013). Therefore, NK cell exhaustion or dysfunction may severely
reduce the efficiency of NK cell-based immunotherapy against CRC.
Unfortunately, the factors resulting in CRC-associated NK cell exhaus­
tion, either intrinsic or extrinsic, have not been completely understood.

Abbreviations: AOM, azoxymethane; CRC, colorectal cancer; DSS, dextran sodium sulfate; GFP, green fluorescence protein; LAG3, lymphocyte-activation protein
3; mLN, mesenteric lymph node; NK cells, natural killer cells; PD-1, programmed cell death 1; SIRT6, sirtuin 6; TIGIT, T cell immunoreceptor with Ig and ITIM
domains; TIM3, T-cell immunoglobulin and mucin domain 3.
* Corresponding author.
E-mail address: xyxiej@hotmail.com (J. Xie).

https://doi.org/10.1016/j.molimm.2023.04.011
Received 3 March 2023; Received in revised form 20 April 2023; Accepted 30 April 2023
Available online 3 May 2023
0161-5890/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
F. Xiao et al.
Sirtuin 6 (SIRT6) belongs to the Sirtuin family of NAD-dependent
enzymes that modulate cellular stress response, genome stability,
aging, and metabolism (Korotkov et al., 2021). Localized to the nucleus,
the ADP-ribosyl transferase and histone deacetylase activities of SIRT6
make it essential for DNA repair, maintenance of telomeric chromatin,
inflammation, lipid and glucose metabolism (Chang et al., 2020). SIRT6
deacetylates histone H3 lysine 9 (H3K9), H3K18, and H3K56 to induce
transcriptional repression (Pillai and Gupta, 2021). SIRT6 is also
implicated in transcriptional repression independently of its deacetylase
activity (Etchegaray et al., 2019; Ravi et al., 2019). Recent studies imply
that SIRT6 could be a master regulator of immune function. High SIRT6
expression is observed in immune organs including the spleen, lymph
node, and bone marrow, as well as immune cells including white blood
cells (Pillai and Gupta, 2021). Indeed, SIRT6 has been implicated in the
regulation of neutrophil and macrophage activities (Lee et al., 2013; Koo
et al., 2019). Nonetheless, little is known about the role of SIRT6 in NK
cell functionality.
In this research using a murine CRC model, we characterized the
expression and function of SIRT6 in NK cells. We found that SIRT6 was
up-regulated in exhausted infiltrating NK cells in the murine CRC tissue.
SIRT6 knockdown promoted NK cell cytotoxicity both in vitro and in vivo,
suggesting SIRT6 is a negative regulator in murine NK cells. Therefore,
we discovered a novel molecular factor essential for NK cell exhaustion
in murine CRC.
2. Materials and methods
2.1. CRC induction in mice
This animal research was approved by the Wuhan Fourth Hospital
Animal Research Committee and conducted under the Animal Research:
Reporting of In Vivo Experiments (ARRIVE) guidelines. Male C57BL/6J
mice (8 weeks old) were purchased from Wuhan Wonderjourney Animal
Technology Inc. and housed in a pathogen-free condition. Azoxy­
methane (AOM, Cat# A5486) and dextran sulfate sodium (DSS, Cat#
D8906–50 G) were purchased from Sigma-Aldrich. AOM was diluted in
sterile saline at a concentration of 2.5 mg/ml. 2 % dextran sodium sul­
fate (DSS) solution was prepared in sterile water. The model was
established according to previous reports with minor modifications
(Arnesen et al., 2021; Parang et al., 2016). Briefly, on day 0, AOM was
intraperitoneally injected into each mouse (4 µl/g body weight). The
mice were then fed with regular water for 6 days. On day 7, the mice
were fed with 2 % DSS solution for consecutive 7 days, followed by two
weeks of regular water. The 7-day DSS plus 2-week regular water
treatment was repeated twice (Supplemental Fig. 1 A). About 75 % of
mice survived sixteen weeks after the AOM injection. The mice were
then euthanized with CO2, followed by examining CRC formation. Mice
with visible tumor(s) in their colons and rectums were recruited for the
study (Supplemental Fig. 1B). About 80 % of mice had CRC. The control
mice were fed with regular water for the same period.
2.2. Cell isolation from murine mesenteric lymph nodes (mLNs), spleen,
and CRC tissue
mLNs and spleens were harvested and immersed in ice-cold phos­
phate-buffered saline (PBS). Single cells were prepared by gently
grinding the mLNs or spleens through a 70-µm cell strainer. Erythrocytes
were removed by suspending cells in 1 ml of red blood cell lysis buffer
(Beyotime Inc) for 3 min at room temperature. After centrifugation at
200 g for 5 min, the cells were resuspended in PBS before further pro­
cessing. To isolate cells from the CRC tissue, the tumors were minced
into small pieces and digested in RPMI1640 containing 500 µg/ml
collagenase IV, 100 µg/ml DNase I, 5 mM ethylenediaminetetraacetic
acid (EDTA), and 2.5 mM CaCl2 (The reagents were purchased from
Sigma-Aldrich) at 37 ◦ C for 30 min. The tissue and the digestion buffer
were then filtered through a 70-µm cell strainer to prepare a single-cell
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Molecular Immunology 158 (2023) 68–78
suspension. The single-cell suspension was loaded onto the mouse
lymphocyte isolation buffer (Beyotime Biotech) and centrifuged at 400 g
for 20 min. Mononuclear cells were then harvested and washed with PBS
before flow cytometry. Cell isolation from normal colons/rectums fol­
lowed the same procedures. In some experiments, cells from 5 to 20 mice
were pooled for analysis.
2.3. Flow cytometry
Antibodies were shown in Supplemental Table 1. In surface marker
detection, 1 × 106/ml cells were incubated with 5 µg/ml of each anti­
body on ice for 15 min. In intracellular protein detection, cells were
fixed in 200 µl of Cytofix/Cytoperm solution (BD Biosciences) for 20 min
on ice. Cells were then permeabilized in 200 µl of perm/wash solution
(BD Biosciences) with 7.0 % goat serum for 30 min on ice, followed by
staining with 5 µg/ml antibodies for 1 h on ice. In some experiments,
cells were treated with 2.5 mg/ml brefeldin A and 2 μM monensin for 4 h
before staining. In apoptosis detection, cells were incubated with an APC
Annexin V Apoptosis Detection Kit with propidium iodide (Cat#
640932, Biolegend) according to the manufacturer’s instructions. Cells
were analyzed on a BD FACSCalibur™ cytometer (BD Biosciences). Cell
sorting was performed on a BD Influx cell sorter (BD Biosciences).
2.4. Lentiviral transduction
NK cells were isolated from normal mouse spleens using an NK cell
isolation kit (Solarbio Life Sciences) following the vendor’s instructions.
SIRT6 shRNA lentivector (Cat# 438110940495) and scramble shRNA
lentivector (Cat# 438110940490) were obtained from Applied Biolog­
ical Materials Inc. The SIRT6 shRNA sequence is 5’-ATGGCCCTGGTT­
CAGCTAGAA-3’, targeting all potential transcript variants of mouse
SIRT6. The scramble shRNA sequence is 5’-GCAGTCGATCAAACGAT­
GAGA-3’. The lentivectors also encode the green fluorescence protein
(GFP). Lentivirus packaging and titration were accomplished by Bio­
favor Biotech Inc. 1 × 106/ml NK cells were suspended in RPMI 1640
supplemented with 10 % FCS. NK cells were stimulated with 500 U/ml
recombinant mouse IL-2 (Beyotime Inc) for 24 h. After that, polybrene
(Maokang Biotech) was added at a final concentration of 6 μg/ml.
Lentiviral particles were then added at the multiplicity of transduction
(MOI) of 50. NK cells were centrifuged at 700g for 1 h at 32 ◦ C and
incubated for 18 h. The next day the supernatant was replaced by fresh
medium supplemented with 10 % FCS and 200 U/ml IL-2. NK cells were
incubated for additional three or five days before further experiments.
The transduction efficiency was determined by assessing GFP+ cells.
2.5. NK cell cytotoxicity
YAC-1, an NK cell-sensitive murine lymphoma cell line, was pur­
chased from Procell Inc. The murine CRC cell line MC38 was purchased
from Immocell Ltd. The cell lines were maintained in DMEM plus 10 %
FCS. Primary NK cells were co-cultured with YAC-1 cells at a ratio of 5:1
in 200 µl of supplemented RPMI 1640 for 6 h in a 96-well plate (Corn­
ing). After that, the co-cultured cells were centrifuged at 200g for 5 min
and then stained with 2 µg ml-1 APC/Cy7 anti-NK1.1 antibody for 15
min on ice. After PBS wash, cells were stained with the APC Annexin V
apoptosis detection kit, followed by quantifying the apoptosis and ne­
crosis of NK1.1-negative YAC-1 cells by flow cytometry.
Lentivirus-transduced GFP+ NK cells were co-cultured with MC38
cells at a ratio of 2:1 in 200 µl of supplemented RPMI 1640 medium for 6
h. The co-cultured cells were directly stained with the APC Annexin V
apoptosis detection kit, followed by quantifying apoptosis and necrosis
of GFP- MC38 cells by flow cytometry.
2.6. Quantitative RT-PCR
All kits and reagents were purchased from ThermoFisher Scientific.
F. Xiao et al.
RNAs were harvested using the Arcturus PicoPure RNA Isolation Kit.
cDNAs were prepared using the RevertAid First Strand cDNA Synthesis
Kit. cDNAs were mixed with the PowerUp™ SYBR® Green Master Mix
and quantitative PCR was carried out on a CFX Connect Real-Time PCR
Detection System (Bio-Rad). The primers were listed in Supplemental
Table 2. The SIRT6 primer pair targets all potential transcript variants of
mouse SIRT6. The relative levels of mRNAs of interest were normalized
to β-actin and computed according to the 2-ΔΔCt formula.
2.7. Immunoblotting
Cells were lysed in RIPA buffer (ThermoFisher Scientific) containing
protease inhibitors (Sigma-Aldrich). A total of 10 µg whole-cell protein
was loaded per lane. The polyclonal anti-SIRT6 antibody (Cat# 13572-
1-AP, 1:500) and monoclonal anti-GAPDH antibody (Cat# MA1–16757,
1:1000) were purchased from ThermoFisher Scientific. Protein signals
were recorded on a Biospetrum 300 system (UVP Ltd). The optical
densities of target proteins were normalized to GAPDH.
2.8. Adoptive transfer
C57BL/6 J mice were subjected to AOM/DSS treatment as described
above. From week 10 after the initial AOM/DSS treatment, 1 × 106
lentivirus-transduced GFP+ NK cells (post-transduction day 3) were
suspended in 100 µl of saline and intraperitoneally injected into each
AOM/DSS-treated mouse. The injection was conducted once a week for
4 consecutive weeks. At week 16 after the initial AOM/DSS treatment (i.
e. 3 weeks after the last transfer), the recipients were euthanized and
their mLNs and CRC tissues were collected.
2.9. Cell cycle assay
NK cells were fixed in 70 % ethanol-PBS for 3 h, followed by 5-min­
ute centrifugation at 350g at 4 oC. After removing the supernatants, NK
cells were resuspended in 500 µl of ice-cold Hank’s balanced salt solu­
tion (HBSS) containing Mg2+ and Ca2+. The cell suspension was added
into an equal volume of HBSS containing 4 μg/ml Hoechst 33342
(Sigma-Aldrich) and 8 μg/ml Pyronin Y (Sigma-Aldrich) and incubated
for 45 min at 37 ◦ C. The cell cycle was analyzed on the flow cytometer.
2.10. Isolation of murine primary CRC cells
The reagents were purchased from Sigma-Aldrich. Mouse colons and
rectums were harvested. Cancer masses were excised and minced into
about 1-mm3 pieces, followed by digestion in 0.5 ml of RPMI 1640
medium containing 10 % fetal calf serum (FCS), 500 µg/ml collagenase
IV, 100 µg/ml DNase I, 5 mM EDTA, and 2.5 mM CaCl2 for 30 min at 37
o
C. The digested tissues were gently pressed through a 70-µm cell
strainer. The single-cell suspension was incubated with 5 μg/ml APC
anti-CD45 antibody, APC anti-CD31 antibody, and unconjugated anti-
MCT4 antibody for 30 min at 4 oC. After three washes with PBS, cells
were stained with 5 μg/ml APC goat anti-rabbit IgG for 30 min at 4 oC.
CD45-positive leukocytes, CD31-positive vascular endothelial cells, and
MCT4-positive fibroblasts were excluded while CD45-CD31-MCT4- cells
were gated by flow cytometry for further analysis.
2.11. Histology and Immunochemistry
The murine colon and CRC tissue were subjected to fixation in 10 %
formalin, paraffin embedding, and 5-µm sectioning. Hematoxylin and
eosin staining was conducted based on the standard protocol. The his­
tology score was assigned according to the severity of inflammation,
ulceration, and hyperplasia of the mucosa as described in previous
research (Zaki et al., 2011) (Supplemental Table 3). For immunochem­
istry, the sections were stained with the Ki67 antibody (11F6, Bio­
Legend, 1:100) to analyze tumor proliferation or GFP antibody (ab290,
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Molecular Immunology 158 (2023) 68–78
Abcam, 1:500) to locate exogenous NK cells.
2.12. Statistics
Each experiment was repeated two or three times. The data were
expressed as mean ± SD. Student’s t-test or Welch ANOVA was applied
to compare mean values among different groups. A P value less than
0.05 was significant.
3. Results
3.1. Infiltrating NK cells up-regulate SIRT6 in murine CRC
CRC was induced by feeding mice with AOM and DSS as described in
Materials and methods (Supplemental Fig. 1). At post-induction week
16, mLNs and colons/rectums were harvested from the healthy control
mice whereas mLNs and tumors were harvested from CRC-bearing mice,
followed by the preparation of single-cell suspensions from these tissues
for flow cytometry. Single cells and viable cells were sequentially gated
among total cells (Fig. 1 A), followed by gating TCRβ-NK1.1+ cells in
viable cells (Fig. 1 A). Within TCRβ-NK1.1+ cells, a CD49a-CD49b+
population was recognized as NK cells and sorted (Fig. 1 A). As shown in
Fig. 1B, in healthy mice, the frequency of NK cells in the mLNs was
higher than that in the colon/rectum. Similarly, in CRC-bearing mice,
the frequency of NK cells in the mLNs was higher than that in the CRC
tissue. Next, we quantified SIRT6 expression in sorted NK cells. SIRT6
was increased by about 2 folds in NK cells in the CRC tissue, compared
with the other three groups (Fig. 1 C and D), suggesting SIRT6 up-
regulation in infiltrating NK cells.
3.2. SIRT6 is up-regulated in exhausted infiltrating NK cells in murine
CRC
To identify exhausted NK cells, the expression of T cell immunor­
eceptor with Ig and ITIM domains (TIGIT) and lymphocyte-activation
protein 3 (LAG3), both of which are NK cell exhaustion markers
(Judge et al., 2020), was determined on the surface of NK cells. As
shown in Fig. 2 A, TIGIT and LAG3 were almost negative on healthy
mLN NK cells, healthy colonic/rectal NK cells, and CRC-associated mLN
NK cells. However, TIGIT and LAG3 were substantially expressed on
infiltrating NK cells in the CRC tissue, resulting in a TIGIT+LAG3+
subset. The TIGIT+LAG3+ NK subset also expressed high T-cell immu­
noglobulin and mucin domain 3 (TIM3, another NK cell exhaustion
marker) whereas the TIGIT-LAG3- NK subset did not (Fig. 2B). The fre­
quency of the TIGIT+LAG3+ NK subset in total NK cells reached 40 %
(Fig. 2 C). Interestingly, SIRT6 mRNA levels were comparable in healthy
mLN TIGIT-LAG3- NK cells, healthy colonic/rectal TIGIT-LAG3- NK cells,
CRC-associated mLN TIGIT-LAG3- NK cells, and infiltrating TIGIT-LAG3-
NK cells (Fig. 2D). In contrast, SIRT6 was remarkably up-regulated in
infiltrating TIGIT+LAG3+ NK cells in the CRC tissue (Fig. 2D).
To further substantiate exhausted NK cells, the expression of gran­
zyme B and perforin was determined in these NK subsets. As shown in
Fig. 3 A–C, healthy mLN NK cells, healthy colonic/rectal NK cells, and
CRC-associated mLN NK cells expressed very low granzyme B and per­
forin. However, infiltrating TIGIT-LAG3- NK cells and infiltrating
TIGIT+LAG3+ NK expressed abundant granzyme B and perforin.
Notably, infiltrating TIGIT+LAG3+ NK cells expressed significantly
lower granzyme B and perforin than infiltrating TIGIT-LAG3- NK cells
(Fig. 3 A–C). To test cytotoxicity, each NK subset was co-cultured with
YAC-1 cells (E: T = 5:1) for 6 h. TIGIT+LAG3+ NK cells induced less
YAC-1 cell death than TIGIT-LAG3- NK cells (Fig. 3D and E and Sup­
plemental Fig. 2), implying that TIGIT+LAG3+ NK cells were function­
ally exhausted.
F. Xiao et al. Molecular Immunology 158 (2023) 68–78

Fig. 1. SIRT6 expression in NK cells. (A) Dot plots show single cells, viable cells, TCRβ-NK1.1+ cells, and CD49a-CD49b+ NK cells in mLNs. HLN: mLNs of healthy
mice. HCR: colons and rectums of healthy mice. CLN: mLNs of CRC-bearing mice. CRC: CRC tissue. (B) NK cell frequency in each organ/tissue. N = 6 mice per group
in three experiments. (C & D) SIRT6 protein in NK cells. Representative Immunoblot images are shown in (C) and statistics are shown in (D). N = 4 samples per group
in three experiments. Each sample contains NK cells pooled from 20 mice. * : P < 0.05. Welch ANOVA.

Fig. 2. SIRT6 up-regulation in exhausted infiltrating NK cells. (A) Dot plots show the expression of TIGIT and LAG3 on NK cells. HLN: mLNs of healthy mice. HCR:
colons and rectums of healthy mice. CLN: mLNs of CRC-bearing mice. CRC: CRC tissue. (B) TIM3 expression on TIGIT-LAG3- NK cells and TIGIT+LAG3+ NK cells.
Isotype: isotypic antibody control. The histograms represent two independent experiments. (C) Frequencies of TIGIT-LAG3- NK cells and TIGIT+LAG3+ NK cells. (D)
SIRT6 mRNA levels in TIGIT-LAG3- and TIGIT+LAG3+ NK cells. Note that TIGIT+LAG3+ NK cells in HLN, HCR, and CLN are not shown due to their rarity. N = 5 mice
per group in three experiments. * ** : P < 0.001. Welch ANOVA.

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Fig. 3. Functions of TIGIT-LAG3- NK cells and TIGIT+LAG3+ NK cells. (A) Dot plots show the expression of perforin and granzyme B in NK cells. HLN: mLNs of
healthy mice. HCR: colons and rectums of healthy mice. CLN: mLNs of CRC-bearing mice. CRC: CRC tissue. TIGIT-LAG3-: TIGIT-LAG3- NK cells. TIGIT+LAG3+:
TIGIT+LAG3+ NK cells. Note that TIGIT+LAG3+ NK cells in HLN, HCR, and CLN are not shown due to their rarity. (B & C) Frequencies of perforin+ and granzyme B+
cells in each population. (D) Dot plots show apoptosis and necrosis of YAC-1 cells. Alone: YAC-1 alone. TIGIT-LAG3-: YAC-1 with infiltrating TIGIT-LAG3- NK cells.
TIGIT+LAG3+: YAC-1 with infiltrating TIGIT+LAG3+ NK cells. (E) Frequencies of necrotic plus apoptotic YAC-1. N = 3 or 5 samples per group in three experiments.
Each sample contains cells pooled from 5 mice. * *: P < 0.01. * ** : P < 0.001. Welch ANOVA.

3.3. SIRT6 knockdown boosts murine NK cell function in vitro
To determine the role of SIRT6, NK cells were sorted from normal
mouse spleens and transduced with a lentivirus encoding both SIRT6
shRNA and GFP. On day 3 after transduction, 25–30 % of NK cells
became GFP+, suggesting that they were successfully transduced
(Fig. 4 A). GFP+ NK cells were then sorted by flow cytometry for further
assays. SIRT6 knockdown was confirmed by quantitative RT-PCR and
Immunoblotting (Fig. 4B). GFP+ NK cells transduced with the scramble
shRNA virus were termed control NK cells, while GFP+ NK cells trans­
duced with the SIRT6 shRNA virus were termed SIRT6-KD NK cells. Cell
cycle evaluation demonstrated that in comparison with control NK cells,
more SIRT6-KD NK cells entered the G1 phase (for protein synthesis) and
S-G2/M phase (for DNA synthesis and mitotic division) on post-
transduction day 3 and day 5, suggesting a moderately higher prolifer­
ation rate after SIRT6 knockdown (Fig. 4 C and D). SIRT6-KD NK cells
expressed higher IFN-γ, TNF-α, perforin, and granzyme B than control
NK cells (Fig. 5 A–D). To test their tumoricidal effects on CRC, NK cells
were co-cultured with the murine CRC cell line MC38 at a ratio of 2:1 for
6 h, followed by quantification of MC38 cell apoptosis and necrosis. As
demonstrated in Fig. 5E and F, SIRT6-KD NK cells induced more MC38
cell death than control NK cells, implying their higher cytotoxicity.

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Fig. 4. NK cell proliferation after SIRT6 knockdown in vitro. (A) Dot plots show GFP expression in enriched splenic NK cells on day 0 and day 3 after transduction.
LVSC: scramble shRNA lentivirus. LVSS: SIRT6 shRNA lentivirus. (B) SIRT6 mRNA (upper panel) and protein (lower panel) in GFP+ NK cells on day 3 and day 5 after
lentiviral transduction. The Immunoblotting image represents two independent experiments. (C) Dot plots show Hoechst 33342 and Pyronin Y staining in GFP+ NK
cells on day 3 and day 5 after lentiviral transduction. Ctrl: control NK cells (transduced with scramble shRNA lentivirus). KD: SIRT6-KD NK cells. (D) Frequencies of
GFP+ NK cells at the G1 phase and S-G2/M phase on day 3 and day 5, respectively. N = 5 or 6 samples per group in three experiments. * *: P < 0.01. * ** : P < 0.001.
Welch ANOVA for (B) and Student’s t-test for (D).

3.4. SIRT6 knockdown promotes murine NK cell function in vivo
C57BL/6 J mice were subjected to AOM/DSS treatment as described
above. From week 10 after the initial AOM/DSS treatment, the mice
were intraperitoneally injected with 1 × 106 control NK cells or 1 × 106
SIRT6-KD NK cells once a week for 4 consecutive weeks. At week 16
after the initial AOM/DSS treatment (i.e. 3 weeks after the last transfer,
Supplemental Fig. 3), GFP+ NK cells (i.e. exogenous NK cells) were
retrieved from recipients’ mLNs and CRC tissues (Fig. 6 A). Compared
with control NK cells before the transfer, control NK cells in recipients’

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Fig. 5. NK cell cytotoxicity after SIRT6 knockdown in vitro. (A) Dot plots show the expression of IFN-γ and TNF-α in GFP+ NK cells on day 3 after SIRT6 knockdown.
Ctrl: control NK cells. KD: SIRT6-KD NK cells. (B) Frequencies of cytokine-expressing NK cells in (A). (C) Dot plots indicate the expression of perforin and granzyme B
in GFP+ NK cells on day 3 after SIRT6 knockdown. (D) Frequencies of perforin+ and granzyme B+ NK cells. (E) Dot plots indicate MC38 cell apoptosis and necrosis
after 6-hour co-culture with GFP+ NK cells. Alone: MC38 cells alone. Ctrl: MC38 cells with control NK cells. KD: MC38 cells with SIRT6-knockdown NK cells. (F)
Frequencies of necrotic plus apoptotic MC38 cells. N = 3 or 5 independent samples per group in three experiments. * : P < 0.05. * ** : P < 0.001. Student’s t-test for
(B) and (D). Welch ANOVA for (F).

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Fig. 6. The effect of SIRT6-knockdown on NK cells in vivo. (A) Dot plots indicate GFP+ NK cells in recipients’ mLNs and CRC tissues at week 16. Ctrl: control NK cells.
KD: SIRT6-KD NK cells. LN: mLNs. CRC: CRC tissue. (B) Frequencies of GFP+ NK cells. (C) Dot plots indicate the expression of TIGIT and LAG3 on GFP+ NK cells in
recipients’ mLNs and CRC tissues. (D) Frequencies of TIGIT+LAG3+ NK cells in total GFP+ NK cells. (E) Dot plots indicate the expression of perforin and granzyme B
in GFP+ NK cells. (F) Frequencies of perforin+ and granzyme B+ cells in GFP+ NK cells. (G) Dot plots indicate Ki67 expression in GFP+ NK cells. (H) Frequencies of
Ki67+ cells in GFP+ NK cells. N = 4 or 8 mice per group in three experiments. * : P < 0.05. * *: P < 0.01. * ** : P < 0.001. Welch ANOVA.

mLNs expressed similar SIRT6 mRNA levels. However, control NK cells
in recipients’ CRC tissues up-regulated SIRT6 mRNA (Supplemental
Fig. 4). In contrast, SIRT6-KD NK cells expressed very low SIRT6 mRNA
in both mLNs and CRC tissues (Supplemental Fig. 4). Interestingly, the
frequency of SIRT6-KD NK cells was higher than control NK cells in the
CRC tissue but not in the mLNs (Fig. 6B). We then assessed the pheno­
type and function of exogenous NK cells. As shown in Fig. 6 C and D,
control NK cells and SIRT6-KD NK cells expressed scarce TIGIT and
LAG3 in the mLNs. However, part of them became TIGIT+LAG3+ in the
CRC tissue. Of note, SIRT6-KD NK cells generated fewer TIGIT+LAG3+
NK cells than control NK cells in the CRC tissue (Fig. 6 C and D).
Compared with control NK cells in the CRC tissue, SIRT6-KD NK cells
expressed lower activating receptors such as NKG2D and NKp46 but
higher inhibitory receptors such as PD-1, NKG2A, and TIM3 (Supple­
mental Fig. 5). Besides, control NK cells and SIRT6-KD NK cells
expressed very limited perforin and granzyme B in the mLNs, but they
profoundly up-regulated these cytotoxic proteins in the CRC tissue
(Fig. 6E and F). Notably, compared with control NK cells, SIRT6-KD NK
cells produced higher granzyme B and perforin in the CRC tissue (Fig. 6E
and F). Quantification of Ki67 indicated that SIRT6-KD NK cells
expressed more Ki67 than control NK cells in the CRC tissue (Fig. 6 G
and H), suggesting a higher proliferation rate of SIRT6-KD NK cells.
Meanwhile, Ki67 expression was very low in both control NK cells and
SIRT6-KD NK cells in the mLNs (Fig. 6 G and H).
transfer. Compared with mice receiving control NK cells, mice receiving
SIRT6-KD NK cells had smaller tumors (Fig. 7 A) Histological exami­
nation of tumors revealed less severe CRC in mice receiving SIRT6-KD
NK cells compared with mice receiving control NK cells, as evidenced
by a lower histology score representing less inflammation, hyperplasia,
and lesion area (Fig. 7B and Supplemental Fig. 6). To measure CRC cell
death, the tumors were digested into single cells. CD45+ leukocytes,
CD31+ endothelial cells, and MCT4+ cancer-associated fibroblasts were
excluded from total cells by flow cytometry, followed by evaluation of
necrosis and apoptosis of CD45-CD31-MCT4- cells, i.e. CRC cells (Fig. 7 C
and D). Transfer of SIRT6-KD NK cells induced higher necrosis and
apoptosis of CRC cells than the transfer of control NK cells (Fig. 7 C and
D). Moreover, Immunochemistry indicated that the transfer of SIRT6-KD
NK cells significantly reduced Ki67 expression in the CRC tissue
compared with the transfer of control NK cells (Fig. 7E and Supple­
mental Fig. 6). To more accurately measure Ki67, Ki67 staining in iso­
lated CRC cells was determined by flow cytometry. As illustrated in
Fig. 7 F and 7 G, a consistent change in Ki67 expression was observed,
suggesting a more profound suppression of CRC cell growth by SIRT6-
KD NK cells. To locate exogenous NK cells in CRC, GFP staining was
performed on the CRC sections. As outlined in Supplemental Fig. 7,
GFP+ cells were found to be proximal to CRC cells and in the interstitial
tissue. Therefore, GFP+ cells, i.e. exogenous NK cells, could directly
contact CRC cells to exert cytotoxic activity.

3.5. SIRT6-KD murine NK cells are more potent in suppressing murine 4. Discussion
CRC development
In this research, we explored the expression and function of SIRT6 in
CRC development in the recipients was evaluated after NK cell murine NK cells. We found SIRT6 up-regulation only in tumor-

75
F. Xiao et al. Molecular Immunology 158 (2023) 68–78

Fig. 7. CRC development after adoptive transfer. (A) Tumor volume. (B) Histology score. (C & D) Necrosis and apoptosis of CD31-CD45-MCT4- cells (i.e. CRC cells)
after adoptive transfer. Statistics of dead cells are shown in (C). Representative dot plots are shown in (D). (E) The proportion of Ki67+ cells in Immunochemistry. (F
& G) Ki67 expression in CD31-CD45-MCT4- cells determined by flow cytometry. Representative dot plots are shown in (F) and statistics are shown in (G). Ctrl: mice
receiving control NK cells. KD: mice receiving SIRT6-KD NK cells. N = 6–8 mice per group in three experiments. * : P < 0.05. * *: P < 0.01. Student’s t-test.

76
F. Xiao et al.
infiltrating murine NK cells, especially TIGIT+LAG3+ NK cells. TIGIT
and LAG3 are considered NK cell exhaustion makers since they transmit
inhibitory signals that prevent NK cell activation (Wolf et al., 2020;
Graydon et al., 2020; Zhang et al., 2018). Indeed, TIGIT+LAG3+ murine
NK cells expressed lower perforin and granzyme B, and exhibited weaker
cytotoxicity than TIGIT-LAG3- infiltrating NK cells. However, the exact
local factors responsible for murine NK cell exhaustion need to be
identified in future investigations. Interestingly, although the mLNs are
considered a frequent CRC metastasis site, NK cells in the mLNs of
CRC-bearing mice did not exhibit an exhaustion phenotype or SIRT6
up-regulation. Perhaps malignant CRC cells had not substantially
invaded the mLNs in our experimental setting, or the mLN microenvi­
ronment somehow prevented NK cell exhaustion. These possibilities
need to be studied in the future.
The mechanisms resulting in SIRT6 up-regulation in exhausted mu­
rine NK cells remain elusive. To our knowledge, little is known about the
regulation of SIRT6 expression in immune cells. Previous studies indi­
cate that the cAMP signaling pathway reduces SIRT6 expression in lung
cancer cells (Kim and Juhnn, 2015) whereas p53 activates SIRT6
expression in HCT116, a human colon cancer cell line (Zhang et al.,
2014). In the future, it would be necessary to check whether cAMP and
p53 are involved in SIRT6 up-regulation in exhausted NK cells.
Furthermore, since SIRT6 up-regulation was primarily present in
TIGIT+LAG3+ NK cells (also TIM3+), SIRT6 expression is likely modu­
lated by signals related to TIGIT, LAG3, TIM3, or other inhibitory re­
ceptors. In this regard, future studies should also analyze whether
malignant CRC cells, which might express the ligands of NK inhibitory
receptors, trigger SIRT6 up-regulation. Furthermore, a thorough inves­
tigation of the differential signaling pathways between competent NK
cells and exhausted NK cells might answer the question.
SIRT6 is implicated in transcriptional repression dependently or
independently of deacetylase activity (Pillai and Gupta, 2021; Etch­
egaray et al., 2019; Ravi et al., 2019). Therefore, SIRT6 up-regulation
might suppress the expression of genes key to NK cell activation and
cytotoxicity. Our study revealed significant increases in perforin and
granzyme B in SIRT6-knockdown NK cells, suggesting that NK cell
function is repressed by SIRT6. However, whether SIRT6 directly reg­
ulates the transcription of cytotoxic mediators and cytokines should be
further determined. Besides, SIRT6-knockdown NK cells generated
fewer TIGIT+LAG3+ NK cells, i.e. exhausted NK cells in vivo. The inhi­
bition of TIGIT and LAG3 expression is probably not a direct effect of
SIRT6 knockdown because SIRT6 is a transcriptional repressor. We
speculate that SIRT6 knockdown might first promote the transcription of
NK cell activating genes to enhance NK cell activation, followed by in­
hibition of exhaustion markers.
The adoptive transfer assay substantiated the role of SIRT6 as an NK
cell repressor because the transfer of SIRT6-knockdown NK cells decel­
erated murine CRC development. CRC development, as represented by
tumor volume, the extent of inflammation and hyperplasia, as well as
lesion area, was markedly inhibited by exogenous SIRT6-knockdown NK
cells. Consistently, CRC cell death was profoundly induced while CRC
cell expansion was substantially inhibited by exogenous SIRT6-
knockdown NK cells. These findings imply the prospect of using
SIRT6-deficient NK cells in immunotherapy. Furthermore, CRC induc­
tion in NK cell-specific SIRT6-deficient mice would further reveal the
importance of SIRT6 to NK cell function in murine CRC.
In conclusion, this study reveals that SIRT6 up-regulation is essential
for the exhaustion of murine NK cells in the murine CRC tissue. Artificial
SIRT6 down-regulation could boost the function of infiltrating NK cells
to oppress murine CRC progression.
Funding sources
This work was supported by the Wuhan Science and Technology
Bureau Innovation Foundation, China [Grant# 2022020801010559].
77
Molecular Immunology 158 (2023) 68–78
CRediT authorship contribution statement
Fei Xiao: Conceptualization, Methodology, Investigation, Writing-
Original draft preparation. Bo Hu: Methodology, Investigation. Zhilong
Si: Investigation. Huanbin Yang: Investigation. Juan Xie: Conceptuali­
zation, Supervision, Validation, Writing- Reviewing and Editing.
Conflict of Interest
The authors declare no financial, personal, or other conflict interests
with regard to the publication of the study.
Data Availability
Data will be made available on request.
Acknowledgments
None.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.molimm.2023.04.011.
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