Science - USA (2020-08-21)

functional data, half of the CD8+T cells labeled
with PSMB4-H-2Kbtetramers shared clono-
types with the much wider TCR repertoire of
T cells labeled with the TMP1-H-2Kb–specific
tetramers (Fig. 4D and tables S4 and S5), but
not with the negative fraction (fig. S6H). There-
fore, T cells recognizing the TMP1 epitope of
immunogenicE. hiraecan cross-react with a
peptide contained in the oncogenic driver
PSMB4 and vice versa.
Some bacteriophages have the potential to
transfer immunogenic sequences to other

strains in the host ecosystem ( 20 – 22 ). To in-

vestigate the capacity of theE. hirae 13144

prophage to lysogenize other bacterial spe-

cies in vivo, we performed culturomic analy-

ses of the ileal content from C57BL/6 mice

subjected to oral gavage withE. hirae 13144

and systemic CTX therapy, followed by poly-

merase chain reaction (PCR) analyses seek-

ing TMP sequences (fig. S8, A and B). We

tested 7 to 18 bacterial colonies from each ani-

mal and a total of 76 colonies. We only found

lysogenic conversion ofEnterococcus gallinarum

by theE. hirae–temperate phage in vivo, as

confirmed by sequencing of the phage ge-

nome in the second host (Fig. 4E and fig. S8,

B and C). By contrast, none of the 90 colonies

(mostly ofE. gallinarum)isolated from naive

mice harbored the TMP sequence (fig. S8A)

and table S6. Similarly, in vitro coculture of TMP+

E. hirae13144 together with TMP-E. gallinarum

spp. at a 1:1 ratio uncovered a significant (~15%)

rate of lysogenic conversion (fig. S8D). Exam-

ination of a preparation admixingE. hirae

13144 andE. gallinarumat a 1:10 ratio by

Fluckigeret al.,Science 369 , 936–942 (2020) 21 August 2020 5of7

Fig. 4. Phage tail length TMP cross-reacts with

the PSMB4 cancer epitope.(A) Flow cytometry

analysis of CD8+tumor-infiltrating lymphocytes

(from tumors treated as outlined in Fig. 1A) after

costaining with two different tetramers (H-2Kb/

TMP1 and H-2Kb/PSMB4, sequences in Fig. 3A).

Each data point indicates one tumor, and error

bars indicate SEM. The graphs represent compiled

results of three independent experiments with

five mice per group. (BandC) Purified CD3+

T splenocytes from animals treated with CTX and

E. hirae 13144were restimulated ex vivo with

bone marrow–derived DCs (BM-DC) loaded with

TMP1 or PSMB4 peptide. One week after

ex vivo restimulation, peptide-specific CD8+

T cells were purified after staining with the

corresponding tetramer to measure IFNgsecretion

in response to DC loaded with peptides (TMP1,

PSMB4, SIINFEKL as negative control) or heat-

inactivatedE. hirae13144. These results were

performed in parallel on the tetramer-binding

versus nonbinding fraction and were normalized to

the PBS controls (Ctrl). Each dot represents one

culture, and error bars represent SEM. Mann-

Whitney test or ANOVA statistical analyses

(Kruskal-Wallis test) were used: *P< 0.05, ***P<

0.001. (D) Venn diagram of TCRaandbchains

from tetramer-positive CD8+T cells specific for

PSMB4 (yellow) or TMP1 (green). (E) Lysogenic

conversion ofE. gallinarumby theE. hirae

Siphoviridaephage in vivo. Ileal content was

obtained from naive mice or from mice receiving

E. hiraetogether with CTX, followed by cultivation

and isolation of bacterial colonies, MALDI-TOF

identification, and PCR-based detection of TMP.

Results are from five mice per group, and

SEMs within the five ilea are indicated for the

CTX+E. hirae13144 group. (F) Transmission

electron microscopy of the phage produced by

E. hirae13144. (G) Kaplan-Meier survival plots of

76 patients with NSCLC or renal cell cancer

subjected to PD-1–targeting immunotherapy,

stratified according to the presence or absence

of TMP in at least fiveE. faecalisorE. hirae

colonies per patient. Univariate log-rank

(Mantel-Cox) analysis was used. The statistical

report is in the supplementary materials.

mOS, mean overall survival; Neg, TMP phage

negative; Pos, TMP phage positive.

A TMP1+ PSMB4+ TMP1+ PSMB4+

C E

F

D

TCR

β

TCR

α

G

PSMB4-

specific

TMP1-

specific

37 34 13808

64 71 24986

% H-2K

b- TMP1

+ PSMB4

• 
  

tetramer

+ in CD3

+ CD8

+

% H-2K

b- TMP1

• PSMB4

+

tetramer

+ in CD3

+ CD8

+

% H-2K

b- TMP1

+ PSMB4

+

tetramer

+ in CD3

+ CD8

+

0

0.5

1.5

1.0

0

0.5

1.5

1.0

0

0.5

1.5

1.0

PBS CTX

CTX + E.hirae 13144CTX + E.hirae 13144

PBS CTX

CTX + E.hirae 13144CTX + E.hirae 13144

PBS CTX

CTX + E.hirae 13144CTX + E.hirae 13144

ATB +++ ATB +++ ATB + + +

Naïve Mice treated

E. hirae 13144

44/44

24/53

0/0 0/71

E.hirae

E.gallinarum

0

20

40

60

80

100

% of bacteria withTMP sequence

20

40

60

80

100

% of bacteria withTMP sequence

H-2Kb TMP1
tetramer+
TMP1PSMB4
SIINFEKL

13144

Fold ratio (IFN

γ spots)

0

2

6

4

++++

D0 D4D3

ATB

Gavage

+CTX

CD3+ Splenocytes

Gavage

D5 D11

Spleen

B

Cell sorting

of CD3+

Tetramer-based

cell sorting

D18

In vitro

stimulation

D11

D18

Restimulation

IFNγ ELISpot

assay

D19

CD8+ H-2Kb/TMP1+

CD8+ H-2Kb/TMP1-

CD8+ H-2Kb/PSMB4+

CD8+ H-2Kb/PSMB4-

TMP1-pulsed

BM-DC

PSMB4-pulsed

BM-DC

BM-DC

Ctrl

TMP1

PSMB4

SIINFEKL

E.hirae13144

+

0102030

29

47

24

30

18

18

9

6

logrank P = 0.04

Pos: mOS 27.02 months

Neg: mOS 15.09 months

0

20

40

60

80

100

Overall survival

* * *

*** *** *** *

*

mice with CTX +

100 nm

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