can be adoptively transferred and can operate
in a T cell–independent fashion during path-
ogen rechallenge.
Although antigen specificity has long been
considered as a cardinal feature of adaptive
B and T cell memory ( 23 ), innate populations
such as myeloid cells ( 24 ), natural killer (NK)
cells ( 25 , 26 ), and ILC2s ( 27 )havebeenshown
to have“memory-like”properties. To investi-
gate pathogen-specific properties of Tr-ILC3s,we analyzed whetherC. rodentium–activated
Tr-ILC3s could protect againstListeria
monocytogenes.L. monocytogenesinfection
(LM) elicits IL-23 release that can trigger ILC3
activation and IL-22 production, which is862 25 FEBRUARY 2022•VOL 375 ISSUE 6583 science.orgSCIENCE
A RorcGFP Il22TdTCR AbxD0 D29CRD-1 D3 D33C CR CRACRACRBCD49a+ ILC3CCR6+ ILC3DG- AA/R/O
020406080IL-22+ ILC3 (%)Tr-ILC3ILC3
Tr-ILC3ILC3-22
0CCR6+ ILC3CD49a+ ILC3Glycolysis TCA cycleOxphosCCR6+ ILC3CD49a+ ILC3CCR6+ ILC3CD49a+ ILC3-22
0NES (p<0.05, FDR 25%)1.0 1.2 1.4DNA replicationFocal adhesionAdherens junctionProteasomeChemokine signalingCytokine/receptor interactionNK cell mediated cytotoxicityCell CyclePathogenic E.Coli infectionOxidative phosphorylationFatty acid metabolismTCA cycleGlycolysisArg and Pro Metabolism1.0 1.2CE F1.4H0 20 40 60 80 1000246810Time (min)
ITr-ILC3ILC3CCR6+ ILC3 CD49a+ ILC3 J
Tr-ILC3ILC30 20 40 60 80 1000246810Time (min)OCR (pmol/min)Oligo FCCPC
CR
CRA
CRACRC
CR
CRA
CRACRR/AECAR (mpH/min)Glucose Oligo 2-DG0246810Max. glycolysis (mpH/min)
Basal Max0246810Respiration (pmole/min)- 2DG/B/E2DG/B/E/N
Ulk4
Rec
114
Sertad1
PcnaNap1l1Ccnyl1Dynll1NaspEgr1FosbIer5Klf43104105Sequence reads (normalized)
Ulk4
Rec114
Sertad1PcnaNap1l1Ccnyl1Dynll1NaspEgr1FosbIer5Klf4020406080100IL-22+ ILC3 (%)Ki-67
CRACRILC3
CR
CRACR024681012cells (% of ILC3)CR CR CRACR10
Ki67*
ns
ns**+Ndufa1Ndufa2Ndufa3Ndufa4Ndufa5Ndufa6Ndufa7Ndufa8Ndufa9Ndufa10Ndufa11Ndufa12Ndufab1Ndufb2Ndufb3Ndufb4Ndufb5Ndufb6Ndufb7Ndufb8Ndufb9Ndufb10Ndufb11Ndufc1Ndufc2Ndufs1Ndufs2Ndufs3Ndufs4Ndufs5Ndufs6Ndufs7Ndufs8Ndufv1Ndufv2Ndufv3SdhaSdhbSdhcSdhdUqcrbUqcrc1Uqcrc2Uqcrfs1UqcrqUqcr10UqcrhUqcr11UqcrbCox7bCox7a2Cox8aCox6b1Cox6a1Cox6cAtp5hAtp5j2Atp5kAtp5f1Atp5dAtp5eAtp5g3Slc2a3PfkpSlc2a1PdhbHk1Gpi1PkmPfklPdha1Hk2AldoaDldAdh5Pfkfb3DlatG6pc3Eno1Akr1a1Pck2LdhaAldh9a1Aldh3a2Pgm1Aldh2LdhbGalm Acsl1Acsl3Acsl4Acsl5Aco2Idh2Idh3aIdh3bIdh3gOgdhSuclg1SdhaSdhbSdhcSdhdFh1Mdh1Mdh22.1 7.2***Fig. 4. Gene expression analysis and metabolic profiles of Tr-ILC3s.(A) CCR6+
and CD49a+ILC3 from naïve (C) and infected mice (CR, CRA, and CRACR) were
sorted for RNA-seq analysis. (BandC) A heatmap showing the relative expression
levels of differentially expressed genes is shown in (B). In (C), pathway analysis
was performed and gene pathways were organized into clusters to compare CR,
CRA, and CRACR conditions to C (n= 3). FDR, false discovery rate; NES, normalized
enrichment score. (D) Heatmap with clustering of differentially expressed
metabolism-associated genes (n= 3). (EandF) Seahorse analysis of freshly
sorted ILC3s (C) and Tr-ILC3s (CRACR) in response to a mitochondrial uncoupler
[fluoro-carbonylcyanide phenylhydrazone (FCCP)], oligomycin (oligo), rotenone and
antimycin (R/A), and 2-DG. Representative metabolic profiles (OCR and ECAR),
glycolysis, and respiration were determined (three independent experiments;n=3to
4). (G) Effect of OXPHOS inhibition (18 hours) on IL-22 production by intestinal
ILC3s (n= 5). AA, antimycin; R, rotenone; O, oligomycin. (H) Effect of metabolic
pathway inhibition (2 hours) on IL-22 production by intestinal ILC3s (two
independent experiments;n= 3 to 8). B, BPTES; E, etomoxir; N, Nor-NOHA.
(I) Differentially expressed genes in CR and CRACR ILC3s (n= 3). (J) Frequency
of Ki67+ILC3 from infected (CR) and reinfected (CRACR) mice (two independent
experiments;n= 5). Each graph corresponds to the mean ± SEM of the
values obtained. ns indicates not significant, *P< 0.05, **P< 0.01, and ***P<
0.001 using two-tailed unpaired StudentÕsttest [(E) and (F)], two-tailed Mann-
Whitney test [(G) and (J)], or Krustal-Wallis test (H).RESEARCH | REPORTS