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injection of adeno-Cre virus. Hepatocytes with

p21 overexpression (p21-OE) were cell-cycle

arrested at d4 after injection and exhibited

signs of senescence by d8 after injection, as

evidenced by loss of lamin B1 and nuclear ex-

trusion of HMGB1 (fig. S13, A to C). RNA ex-

tracted from d4 Tom+hepatocytes collected

by fluorescence-activated cell sorting (FACS)

and used for RT-qPCR analysis of PASP fac-

tor gene transcripts indicated that p21-OE

induces a PASP in vivo (Fig. 3D and fig. S13D).

Cxcl14was among the up-regulated PASP fac-

tors, prompting us to test whether p21-OE

hepatocytes attract macrophages. Nearly 40%

of p21-OE Tom+hepatocytes were surrounded

by three or more macrophages as early as

d2 after adeno-Cre injection versus ~10% of

Tom+hepatocytes without p21-OE (Fig. 3E).

Macrophage recruitment to p21-OE hepato-

cytes was CXCL14 dependent as assessed by

injection of antibodies against CXCL14 (Fig.

3F). Lymphocytes were also recruited but

later, with B and T cells surrounding p21-OE

hepatocytes at d4 and d8, respectively (Fig. 4,

A and B). By contrast, p21-OE did not prompt

recruitment of natural killer (NK) cells (fig.

S13E). The number of p21-OE hepatocytes

declined sharply by d8, which coincided with

a marked increase in dying p21-OE hepato-

cytes in the presence of M1-differentiated

macrophages in addition to the presence of

both CD4+and CD8+T lymphocytes (Fig. 4,

C to F, and figs. S13F and S14, A and B). Ad-

ministration of a CD8a-neutralizing anti-

body fully prevented the observed decline in

d8 p21-OE hepatocytes (fig. S14, C to G), in-

dicating that their elimination is mediated by

cytotoxic T cells.

We performed a comparative analysis for

overexpression ofp16(p16-OE), a more selec-

tive cyclin-dependent kinase (CDK) inhibitor,

which, unlike p21, only targets G 1 -CDK activ-

ity. At d4, p16-OE MEFs were characterized by

growth inhibition, normalp21levels, and a

secretome of 197 factors, 183 of which overlap

with the PASP of d4 p21-OE MEFs (fig. S15, A

to F, and table S8). Pathway enrichment analy-

ses on the p16-associated secretory phenotype

suggested a high degree of similarity in bio-

logical properties with the PASP, although the

number of immune-systemÐrelated annota-

tions was markedly reduced (fig. S15, G and

H). CM of d4 p16-OE MEFs failed to promote

migration of macrophages in our transwell

assay, which correlated with a lack ofCxcl14

induction (fig. S15I). Likewise, using the same

transgenic approach as used for p21-OE in mice,

we found that p16-OE in hepatocytes triggered

cell-cycle arrest but not immunosurveillance,

which coincided with a lack of p21 and PASP

factor induction, includingCxcl14(fig. S16).

Corresponding analyses of MEFs overexpress-

ing p27, a CDK inhibitor that enables cell-cycle

withdrawal during terminal differentiation,

Sturmlechneret al.,Science 374 , eabb3420 (2021) 29 October 2021 4 of 15

0

1

2

3

4

5

10

15

20

30

40

D 50

Relative expression

A

Migrating macrophages (fold)

CM d4 EV

+IgG

* **

n=6n=6

n=6

d4 Tom+hepatocytes

d4 Tom+p21-OE hepatocytes

p21 Cxcl14 Igfbp3 Adam15 Ssc5d App Fn1 Psap

*

C pCAG STOP p21-Myc-Flag WPRE L-p21

Migrating macrophages (fold)

n=5n=5n=5n=5

CM d4 p21-OE

+IgG

CM d4 p21-OE

+CXCL14

CM d4

B

Ai14;L-p21

or

Ai14 mice

Tom+;p21-OE

or

Tom+ hepatocytes

Adeno-Cre

Ai14

* *

**

n=3

n=4

n=3

n=4

n=4 n=4

n=4

n=4 n=4 n=4 n=4

n=4

pCAG STOP tdTomato WPRE

*

*

* *

*

* *

n=4n=4n=4n=4

Tom

+

hepatocytes joined

by

3 F4/80

+

cells (%)

***

***

ns

ns

***

***

n=6

n=5

n=6

n=4 n=5

n=4

MAb730 MAb866

F

To

m

+

hepatocytes joined

by

3 F4/80

+

cells (%)

***

***

***

Ai14

Ai14;L-p21

E d2d4d8

n=4

n=4n=5

n=5

n=4n=5

F4/80

Tom

DNA

0

1

2

3

4

0

1

2

3

5

4

0

10

20

30

50

40

60

0

10

20

30

d2 Ai14 IgG

d2 Ai14;L-p21 IgG

d2 Ai14;L-p21 CXCL14

50

40

CM d4 p21-OE

CM d4 p21-OE

CM d4 p21-OE

Fig. 3. p21-induced immunosurveillance requires PASP factor CXCL14.(A) Transwell migration assay
with CM in the presence of CXCL14-neutralizing or IgG antibodies. (B) as in (A) but with CM from
shRNA-transduced MEFs. (C) Schematic of the transgenes used for Cre-inducible expression of Myc-Flag–
tagged p21 (L-p21forLoxP/Stop/LoxP-p21) and tdTomato (Ai14) (top) and the experimental setup for
p21-OE induction in hepatocytes via Cre-encoding adenovirus injection (bottom). (D) RT-qPCR on flow-sorted
Tom+hepatocytes. (E) Representative image and quantification of Tom+hepatocytes joined by≥3 F4/80+
cells (arrowheads). (F) As in (E) but assessing livers from mice treated with CXCL14-neutralizing or
IgG control antibodies. Scale bars: 10mm (E). Data represent means ± SEM. Experiments in the above
panels were performed once. ns, not significant. P< 0.05; P< 0.01; P< 0.001. One-way ANOVA
with Sidak’s correction [(A), (B), (E), and (F)] and unpaired two-tailedttests (D).

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