means of transmission electron microscopy
revealed numerous phages with the typical
Siphoviridaemorphology in the medium,
whereas control cultures (bacteria separately)
were free of such phages (Fig. 4F). These results
indicate that the TMP1 peptide–encoding
Siphoviridaeprophage ofE. hirae13144 dis-
seminates within enterococci.
We next explored the possible pathophys-
iological relevance of these findings. We first
screened a total of 3027 adult and mother-
infant metagenomes ( 27 ), validated by a
second independent metagenomic assembly–
based screening of 9428 metagenomes ( 28 ),
to assess the breadth of coverage (BOC) of the
E. hiraegenomeanditsphages(fig.S9A).
E. hiraewas present with 100% confidence
(BOC > 80%) in fewer than 150 fecal samples
from disparate geography, age, and datasets.
This phage (and its host) can be vertically
transmitted from mothers to infants and then
colonizes the neonate. There was an increased
prevalence of the phage (57%) in fecal micro-
biomes from children (representing 16% of all
metagenomes, Fisher’stestPvalue < 0.00001)
(table S7). TheE. hirae13144 phage was de-
tectable in many samples lacking the pres-
ence of theE. hiraecore genome, suggesting
that other bacteria in addition toE. hiraecan
host this phage (table S7). All host genomes be-
longed to theEnterococcusgenus (except two
assigned toCoprobacillus), in particularEnter-
ococcus faecalis(80 genomes),Enterococcus
faecium(23 genomes), andE. hirae(15 ge-
nomes), suggesting that phage 13144 (and
its homologs fromE. hirae708, and 13344)
are genus-specific but not species-specific
(table S8).
Contrasting with metagenomics that has a
low sensitivity to detect poor abundance spe-
cies, culturomics followed by MALDI-TOF
(matrix-assisted laser desorption ionization
time-of-flight mass spectrometry) provides a
technology for detecting rareE. hiraecolo-
nies in the stool of healthy individuals ( 29 )
or cancer patients ( 8 ). PCR analyses of each
single cultivatable enterococcal colony (up to
five per species and individual) from 76 can-
cer patients led to the detection of the TMP
sequence encompassing the TMP1 peptide
in 34% of the patients, only inE. faecalisand
E. hirae(figs. S9B and S10). Advanced renal
and lung cancer patients [cohort described
in Ref. ( 8 )] with detectable fecal TMP at di-
agnosis exhibited prolonged overall survival
after therapy with immune checkpoint inhib-
itors targeting PD-1 (Fig. 4G). Therefore, we
screened 16 TMP-derived nonapeptides pre-
dicted to bind the human MHC class I human
leukocyte antigen (HLA)-A*0201 with high
affinity for their ability to prime naive CD8+
T cells from six healthy volunteers in vitro.
We found 6 out of 16 epitopes capable of trig-
gering significant peptide-specific IFNgrelease
that were located in two distinct regions of the
TMP protein (504 to 708 and 1397 to 1462) (fig.
S11, A and B, and table S9). Using the NCBI
BLASTP suite, we searched the human cancer
peptidome [of the Cancer Genome Atlas (TCGA)
database] for a high degree of homology with
these six HLA-A*0201–restricted immunogenic
nonapeptides. We found that only the TMP-
derived peptide KLAKFASVV (amino acids 631
to 639) shared significant homology (seven out
of nine amino acids, with identical residues at
the MHC anchoring positions 2 and 9) with
a peptide contained in the protein glycerol-3-
phosphate dehydrogenase 1-like (GPD1-L)
(fig. S11C). GPD1-L reportedly counteracts
the oncogenic hypoxia-inducible factor 1a–
dependent adaptation to hypoxia, and its
expression is associated with favorable prog-
nosis in head and neck squamous cell car-
cinomas ( 30 – 32 ). The TCGA transcriptomics
database unveiled that high expression of
GPD1-L is associated with improved over-
all survival in lung adenocarcinoma and
kidney cancers (fig. S11D). Moreover, high
expression of GPD1-L mRNA by tumors at
diagnosis was associated with improved
progression-free survival in three independent
cohorts of non–small cell lung cancer (NSCLC)
patients (n=157)(tableS10)treatedwithanti-
PD1 antibodies (fig. S11, E and F). Expression
of GPD-1L failed to correlate with that of PD-
L1 in NSCLC (fig. S11G). Mutations in or adjacent
to the 631 to 639 amino acid sequence of
GPD-1L gene could rarely be identified in
several types of neoplasia (fig. S12).
We derived an HLA-A*0201–restricted, phage
peptide (KLAKFASVV)–specific T cell line from
peripheral blood mononuclear cells (PBMCs)
of a human volunteer. Clones from this line
also recognized the HLA-A*0201–restricted
GPD-1L epitope (KLQKFASTV) (fig. S13, A to
C). Moreover, we detected CD8+T cells bind-
ing HLA-A*0201/KLAKFASVV tetramers ex-
hibiting hallmarks of effector functions after
in vitro stimulation of PBMCs with the
KLAKFASVV phage epitope in three out of six
NSCLC patients (fig. S13, D to F). In the reverse
attempt, searching for molecular mimicry be-
tween well-known and naturally processed non-
mutated melanoma differentiation antigens
recognized by human T cell clones (such as
HLA-A*0201–bindingMART-1orMELOE
epitopes) and gut commensal antigens, we
found microbial analogs in the public micro-
biome databases (figs. S14 and S15 and tables
S11 and S13). Some of these microbial peptides
arerecognizedbythecorrespondingTCR
(tables S11 and S13) with similar affinities
as the parental (tumoral) epitope.
Altogether, our results suggest that micro-
bial genomes code for MHC class I–restricted
antigens that induce a memory CD8+Tcell
response, which, in turn, cross-reacts with can-
cer antigens. Several lines of evidence plead infavor of this interpretation, as exemplified for
the TMP1 epitope found in a phage that infects
enterococci. First, naturally occurring (mut3 in
E. hiraestrain ATCC9790) or artificial muta-
tions (mut2 or mut3 inE. coli)introducedinto
the TMP1 epitope suppressed the tumor-
prophylactic and therapeutic potential of
bacteria expressing TMP1. Second, trans-
fer of the TMP1-encoding gene intoE. coli
conferred immunogenic capacity to this pro-
teobacterium, which acquired the same anti-
tumor properties as TMP1-expressingE. hirae.
Third, when cancer cells were genetically
modified to remove the TMP1–cross-reactive
peptide in the PSMB4 protein, they formed
tumors that could no longer be controlled
upon oral gavage with TMP1-expressing
E. hirae. Fourth, cancer patients carrying the
TMP phage sequence in fecal enterococci or
the GPD1-L tumoral antigen homologous to
TMP epitopes exhibited a better response to
PD-1 blockade, suggesting that this type of
microbe-cancer cross-reactivity might be clin-
ically relevant.
Recent reports point to the pathological rel-
evance of autoantigen–cross-reactive, microbiota-
derived peptides for autoimmune disorders
such as myocarditis, lupus, and rheumatoid
arthritis ( 15 , 33 , 34 ). Given the enormous rich-
ness of the commensal proteome ( 35 ), we ex-
pect the existence of other microbial antigens
mimicking auto- and tumor antigens. We have
extended these findings to naturally processed
melanoma-specific antigens that have micro-
bial orthologs recognized by the same TCRs
(figs. S14 and 15 and tables S10 to S12). Global
phage numbers have been estimated to reach
as high as 10^31 particles with the potential of
1025 phage infections occurring every second
( 36 , 37 ). Thus, the perspective opens that in the
microbiota, bacteriophages may enrich the
therapeutic armamentarium for modulating
the intestinal flora and for stimulating sys-
temic anticancer immune responses.REFERENCES AND NOTES- P. Sharma, J. P. Allison,Cell 161 , 205–214 (2015).
- L. Galluzzi, A. Buqué, O. Kepp, L. Zitvogel, G. Kroemer,Cancer
Cell 28 , 690–714 (2015). - L. Zitvogel, Y. Ma, D. Raoult, G. Kroemer, T. F. Gajewski,
Science 359 , 1366–1370 (2018). - T. Tanoueet al.,Nature 565 , 600–605 (2019).
- M. Vétizouet al.,Science 350 , 1079–1084 (2015).
- R. Daillèreet al.,Immunity 45 , 931–943 (2016).
- Y. Ronget al.,Exp. Cell Res. 358 , 352–359 (2017).
- B. Routyet al.,Science 359 ,91–97 (2018).
- N. R. Rose,Curr. Opin. Immunol. 49 ,51–55 (2017).
- V. Rubio-Godoyet al.,J. Immunol. 169 , 5696– 5707
(2002). - L. Vujanovic, M. Mandic, W. C. Olson, J. M. Kirkwood,
W. J. Storkus,Clin. Cancer Res. 13 , 6796–6806 (2007). - M. E. Perez-Muñoz, P. Joglekar, Y.-J. Shen, K. Y. Chang,
D. A. Peterson,PLOS ONE 10 , e0144382 (2015). - Y. Yanget al.,Nature 510 , 152–156 (2014).
- J. N. Chaiet al.,Sci. Immunol. 2 , eaal5068 (2017).
- C. Gil-Cruzet al.,Science 366 , 881– 886 (2019).
- Q. Ji, A. Perchellet, J. M. Goverman,Nat. Immunol. 11 , 628– 634
(2010). - V. P. Balachandranet al.,Nature 551 , 512–516 (2017).
Fluckigeret al.,Science 369 , 936–942 (2020) 21 August 2020 6of7
RESEARCH | RESEARCH ARTICLE