induced dysbiosis. Introducing a knock-in
mutation in position 3 of PSMB4 into TC1
lung cancer cells again compromised the anti-
tumor effects of CTX (Fig. 3E). Moreover, in
the setting of PD-1 blockade, administration
ofE. hirae13144 without prior conditioning
with antibiotics reduced the growth of pa-
rental but not PSMB4-mutated MCA205 can-
cers (fig. S6E). These results support the idea
that the TSLARFANI TMP1 peptide encoded
byE. hirae13144 induces T cell responses
against the PSMB4-derived GSLARFRNI pep-
tide across different tumor types and therapy
modalities.
Reinforcing the notion of molecular mim-
icry between phage-encoded and cancer antigens,
flow cytometric analyses using fluorescent-
labeled tetramers H-2Kb/TSLARFANI (from
TMP1) and H-2Kb/GSLARFRNI (from PSMB4)
identified a subset of double-positive CTLs
that infiltrate MCA205 tumors from CTX and
E. hirae 13144–treated mice (fig. S6F) and
that was as frequent as CTLs recognizing the
PSMB4 peptide only (Fig. 4A). We purified
the splenic CD8+T cells using either the
TMP1–H-2Kb–or PSMB4–H-2Kb–specific tet-
ramers and stimulated them with irrelevant
(OVA-derived SIINFEKL) versus relevant(TMP-derived TSLARFANI or PSMB4-derived
GSLARFRNI) peptides (Fig. 4B). CD8+T cells
binding H-2Kb–TMP1 tetramers produced IFNg
not only in response to TMP1 (up to fivefold
increase in IFNg-secreting T cells) but also in
response to the PSMB4 epitope (twofold in-
crease, as much as with heat-killedE. hirae
13144 processed by DCs) (Fig. 4C, fig. S6G).
Similarly, CD8+TcellsbindingH-2Kb–PSMB4
tetramers functionally recognized TMP1, albeit
less efficiently than the PSMB4 epitope (fig.
S6G). We analyzed the T cell receptor (TCR)
repertoire of these two tetramer-reactive
CD8+T cell subsets. In accordance with theFluckigeret al.,Science 369 , 936–942 (2020) 21 August 2020 4of7
clone 2 clone 1 clone 2 clone 3PSMB4-mut2 PSMB4-mut2 PSMB4-mut3 PSMB4-mut3 PSMB4-mut3
clone 1WT
clone 2WTWT PSMB4-mut3clone 1MCA205 WT
polyclonalCDEB123456A TMP1 PSMB4 PSMB4-mut2 PSMB4-mut^3MCA205TC1
MC38Rel. expression of
PSMB4 (fold change)MCA205 WT (clone 1)Tumor size (mm2 )0100150502002500Tumor size (mm2 )01001505020025010 20 30
Days after tumor inoculationPBS CTX
CTX + E.hirae 13144CTX CTX + E.hirae 13144CTX CTX + E.hirae 1314401020 30Tumor size (mm2 )010015050200250MCA205 PSMB4-mut2 (clone 1)Days after tumor inoculationPBS CTX
CTX + E.hirae 13144Tumor size (mm2 )010015050200250MCA205 PSMB4-mut3 (clone 1)01020 30
Days after tumor inoculationPBS CTX
CTX + E.hirae 13144*TC1 WT
PBS CTX
CTX + E.hirae 13144TC1 PSMB4-mut3
PBS CTX
CTX + E.hirae 13144Tumor size (mm2 )0
010020030010 20 30
Days after tumor inoculationTumor size (mm2 )0100200400300Tumor size (mm2 )010020040030001020 30 40
Days after tumor inoculationnsp= 0.0823*
***** **nsnsFig. 3. Molecular mimicry between phage tail length TMP and the
oncogenic PSMB4 epitope in murine tumors.(A) Sequence alignment of the
enterophage TMP1 peptide and a PSMB4 epitope with its two experimental
mutants. (B) Relative (Rel.) expression of PSMB4 mRNA in MCA205 sarcoma,
TC1 lung cancer, and MC38 colon carcinomas as compared with their healthy
tissue of origin (mean ratio ± SEM,n= 3). (CandD) Therapeutic response of
wild type (WT) versus knock-in mutants of MCA205 to CTX alone or in
combination with immunogenicE. hiraestrain 13144 (as outlined in Fig. 1A).
Results are shown as tumor growth kinetics (mean ± SEM) for (C) selected
MCA205 clones or as (D) individual results (one data point corresponds to one
mouse) on day 25. (E) Therapeutic response of WT versus mutated TC1 lung
cancers to CTX alone or in combination withE. hirae13144 (as outlined in Fig. 1A,
but without antibiotic preconditioning), reflected by tumor growth kinetics and
individual tumor sizes at sacrifice. Results are shown as mean ± SEM. Mann-
Whitney test or ANOVA statistical analyses (Kruskal-Wallis test) were used: *P<
0.05, **P< 0.01. The statistical report is in the supplementary materials.RESEARCH | RESEARCH ARTICLE