screened the differential capacity ofE. hirae
strains to elicit memory T cell responses after
priming of the host, measured as the ex vivo
recall response (IFNgsecretion) of splenic
CD8+T cells against variousE. hiraestrains
loaded onto dendritic cells (DCs) (fig. S2A).
WhereasE. hirae13144 triggered specific CD8+
T cell responses (that were not cross-reactive
against irrelevant enterococci),E. hirae 708
and 13344 (two prototypic inefficient strains)
( 6 ) failed to do so (fig. S2A).
To identify relevant T cell epitopes, we
aligned the sequences of bacterial genes en-
coding putative cell wall and secreted proteins
for immunogenic (13144) versus nonimmu-
nogenic (708 and 13344)E. hiraestrains,
followed by the in silico identification of
13144-specific nonapeptides with strong af-
finity (<50 nM) for the MHC class I H-2Kb
protein (table S2). Subsequently, we recov-
ered splenic CD8+T cells from mice that had
been exposed toE. hirae13144 and CTX (Fig.
1C), restimulated them in vitro with pools of
potentially immunogenic nonapeptides from
E. hirae13144 to measure IFNgproduction
(table S2 and fig. S2B), and then split the
most efficient pool (no. 7) into individual
peptides (Fig. 1D). This approach led to the
identification of one dominant epitope (one-
letter amino acid code: TSLARFANI, abbre-
viation TMP1) in position 187 to 197 of the
amino acid sequence of the bacteriophage
tail length tape measure protein (TMP, 1506
amino acids) from a 39.2-kb prophage of
E. hirae13144 (Fig. 1D, fig. S3, and tables S2
and S3). Temperate and lytic bacteriophages
are bacterial viruses that transfer virulence,
antimicrobial resistance genes, and immu-
nogenic sequences to new bacterial hosts
with a strict specificity ( 20 ). The TMP protein,
which contains a variable number of tandem
repeats with highly conserved tryptophan
and phenylalanine residues at fixed positions,
is encoded by the genome ofSiphoviridae
phages ( 21 , 22 ).
The 39.2-kb prophage (i.e., bacteriophage
genome) encodes 65 genes, including one
shared between all 18E. hiraegenomes and
38 specific toE. hirae13144 (fig. S1B), encod-
ing capsid, portal, and tail structures charac-
teristic ofSiphoviridaebacteriophages. The
TMP1 epitope of the 39.2-kb prophage from
E. hirae13144 and the prophage fragment
contained inE. hiraeIGR11 showed 100% se-quence identity (figs. S3 and S4A). Accordingly,
E. hiraeIGR11 was as efficient asE. hirae 13144
in reducing the growth of MCA205 sarcomas
treated with CTX (Fig. 1, A and B). By con-
trast, the absence of a bona fide TMP1 epitope
(observed inE. hirae708 and 13344) (fig. S1B)
and a mutation in position 3 of the TSLARFANI
peptide (L→F observed inE. hiraeATCC9790)
(fig. S4A) correlated with the lack of anticancer
effects of theseE. hiraestrains (Fig. 1B) ( 6 ).
Enzyme-linked immunosorbent spot (ELISpot)
assays designed to detect peptide-specific IFNg-
producing T cells revealed that mice gavaged
withE. hirae13144 or IGR11 mounted a CD8+
T cell response against TMP1 (but not against
the control peptides TMP2 and TMP3), but
mice receivingE. hiraestrains lacking TMP1
(strains 708 and 13344) or a strain possessing
a mutated TMP1 (strain ATCC9790) were un-
able to stimulate a response (Fig. 1D). We used
a fluorescent H-2Kb/TSLARFANI tetrameric
complex [and its negative control H-2Kb/
SIINFEKL binding to ovalbumin (OVA) spe-
cific CD8+T cells] to detect the frequency
and distribution of TMP1-specific cytotoxic
T lymphocytes (CTLs) in naive and MCA205
sarcoma-bearing C57BL/6 mice. We observedFluckigeret al.,Science 369 , 936–942 (2020) 21 August 2020 2of7
% H-2Kb-TMP1+tetramer+ CD8+AD0 D4D3ATBGavage
+CTX
GavageD5 D11 D12 D18 D19Tumor
inoculationD25Gavage
+CTX
GavageGavage
+CTX
GavageTumor size
at sacrificeD0 D4D3ATBGavage
+CTX
GavageD5 D11Tumor
inoculationSpleenEx vivo
recall responses
& flow cytometryCCTXCTX+++++Tumor size (mm2 )IFNγ spots / 2x105 CD8+ T cellsBD E20406080CTX+E.hirae
13144CTX+E.hirae
IGR11CTX+E.hirae
ATCC9790CTX +E.hirae
13344CTX +E.hirae
708CTX- +++++- ++
0100200E.hirae13144 E.hirae IGR11^300
E.hirae ATCC9790 E.hirae 13344E.hirae ATCC9790 E.hirae 13344E.hirae13144 E.hirae IGR11E.hirae 708E.hirae ATCC9790 E.hirae 1334400.51.01.52.0No peptide
TMP1
TMP2TMP3VSTNHYGLLNopeptideTMP1TMP2TMP3
NopeptideTMP1TMP2TMP3
No peptide
TMP1TMP2TMP3
No peptideTMP1TMP2
TMP3
No peptideTMP1TMP2TMP3VSTNHYGLL VSTNHYGLLVSTNHYGLL VSTNHYGLLVSTNHYGLLPBS E.hirae13144 E.hirae IGR11Tetramer
TMP1+Tetramer
SIINFEKL+******
*** ********* **
***nsns
nsnsFig. 1. Phage tail length TMP is a specific antigenic sequence inE. hirae
13144.(AandB) C57BL/6 mice bearing MCA205 sarcomas were conditioned
with broad-spectrum antibiotics (ATB) (streptomycin, colistin, ampicillin,
vancomycin) for 3 days before performing oral gavages withE. hiraestrain
13144 and intraperitoneal injectionsof CTX. (B) Tumor size was recorded for
each mouse at sacrifice on day 25. Mean tumor sizes at sacrifice are
depicted. (CtoE). Naive C57BL/6 mice were conditioned with antibiotics,
gavaged with indicatedE. hiraestrains and treated with CTX (C). (D) Day 11
purified CD8+T splenocytes [from (C)] were restimulated ex vivo in a recall
assay with bone marrow–derived DCs loaded with the indicated peptides
(group 7 in table S2) to quantify IFNg-secreting CD8+T cells. The number of
IFNg-producing cells per 2 × 10^5 CTL is depicted for three independent
experiments. (E) H-2Kb/TMP1 (TSLARFANI) or H-2Kb/SIINFEKL tetramer-
binding CD8+splenocytes [from (C)] were detected by cytofluorometry at
day 11. The percentages of tetramer-binding CTL in the CD8+T cell gate are
depicted for three independent experiments. Also, fig. S2D presents tetramer
stainings in tumor-draining LNs. Each graph assembles results from two to
three independent experiments containing groups of five to six mice.
ANOVA statistical analyses (Kruskal-Wallis test): *P< 0.05, **P< 0.01,
***P< 0.001. The statistical report is in the supplementary materials.RESEARCH | RESEARCH ARTICLE