The transcription factor Bach2 negatively regulates murine natural killer cell maturation and function

  1. Shasha Li
  2. Michael D Bern
  3. Benpeng Miao
  4. Changxu Fan
  5. Xiaoyun Xing
  6. Takeshi Inoue
  7. Sytse J Piersma
  8. Ting Wang
  9. Marco Colonna
  10. Tomohiro Kurosaki
  11. Wayne M Yokoyama  Is a corresponding author
  1. Division of Rheumatology, Department of Medicine, Washington University School of Medicine, United States
  2. Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, United States
  3. Laboratory of Lymphocyte Differentiation, WPI Immunology Frontier Research Center, Osaka University, Japan
  4. Department of Pathology and Immunology, Washington University School of Medicine, United States
5 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
BTB domain And CNC Homolog 2 (Bach2) expression at different natural killer (NK) cell developmental stages by analysis of Bach2Flag knock-in mouse.

(A) Histogram plots of Bach2Flag expression (red) in common lymphoid progenitor (CLP), pre-NK progenitor (pre-NK), and refined NK progenitor (rNKp) as compared to cells from wild-type (WT) mice …

Figure 1—source data 1

Bach2 expression in different subsets by western blot.

Splenic NK cells were enriched from splenocytes of Bach2Flag mice. Enriched NK cells (CD3-NK1.1+) from Bach2Flag mice were further sorted into CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ subsets. Bach2 expression in the subsets was detected using Anti-FLAG M2-Peroxidase (HRP) antibody by western blot. Expression of Actin was used as an internal control. Splenocytes from wild-type (WT) mice were used as negative control. Splenocytes from Bach2Flag mice were used as positive control. Two individual experiments have been done with one mouse each time.

https://cdn.elifesciences.org/articles/77294/elife-77294-fig1-data1-v2.zip
Figure 1—figure supplement 1
Gating strategy for flow cytometry analysis.

(A) Representative flow cytometry plots show bone marrow (BM) Lin-2B4+CD27+CD127+ cells were subdivided into common lymphoid progenitor (CLP) (Flt3+CD122-), pre-natural killer (NK) progenitor …

Figure 2 with 1 supplement
RNA-seq analysis reveals BTB domain And CNC Homolog 2 (Bach2) deficiency in natural killer (NK) cells promotes the terminal maturation of NK cells with elevated effector function.

(A) Heatmap of differentially expressed genes in NK cells compared between control and Bach2cKO mice in RNA-seq analysis (FDR < 0.01 and log2 fold change >1.5). Each column represents total splenic …

Figure 2—figure supplement 1
BTB domain And CNC Homolog 2 (Bach2) deficiency in natural killer (NK) cells resemble activated effector CD8+ T cells.

(A) Schematic plot shows quantitative PCR (qPCR) primers targeting regions on Bach2 mRNA. Red arrows represent primers targeting exon 1 to exon 2. Blue arrows represent primers targeting exon 3 to …

Figure 3 with 1 supplement
Bach2 deficiency results in increased accessible regions in the genome of natural killer (NK) cells.

(A) Volcano plot shows the differentially accessible regions (DARs) and non-DARs between control and Bach2cKO splenic NK cells. (B) De novo motif enrichment at regions of open chromatin as defined …

Figure 3—figure supplement 1
Potential protein interactions between BTB domain And CNC Homolog 2 (Bach2) and differentially regulated transcription factors in natural killer (NK) cells.

(A and B) Differentially expressed genes in NK cells between Bach2cKO mice and control mice were compared for genes that were recognized as Bach2 target genes in CD8+ T cells (Roychoudhuri et al., …

Figure 4 with 1 supplement
Bach2 deficiency increases natural killer (NK) cells with terminally differentiated phenotype.

Representative flow cytometry plots show NK cells (CD3-CD19-NK1.1+NKp46+) separated into maturation stages by CD27 and CD11b expression. The percentage of CD27+CD11b-, CD27+CD11b+, CD27-CD11b+ NK …

Figure 4—figure supplement 1
BTB domain And CNC Homolog 2 (Bach2) deficiency results in a more mature phenotype.

(A and B) Representative flow cytometry plots and summary of total natural killer (NK) cells (CD3-CD19-NK1.1+NKp46+) maturation stages determined by CD27 and CD11b expression from bone marrow (BM) (A

Figure 5 with 1 supplement
Lack of BTB domain And CNC Homolog 2 (Bach2) expression in natural killer (NK) cells suppresses B16F10 tumor metastasis.

(A) Representative picture of lung metastatic nodules in Bach2cKO mice and control mice under steady-state or anti-NK1.1 (PK136) depletion. (B) Summary of the number of B16F10 metastatic nodules in …

Figure 5—figure supplement 1
Natural killer (NK) cell effector function was unchanged with BTB domain And CNC Homolog 2 (Bach2) deficiency.

(A) Killing of B16F10 tumor cells by lymphokine-activated killer (LAK) cells from spleen of Bach2cKO mice or control mice. Data are shown as of one representative experiment with three mice per …

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)C56BL/6J, wild typeThe Jackson LaboratoriesRRID:IMSR_JAX:000664
Strain, strain background (Mus musculus)RAG1-/-, wild typeThe Jackson LaboratoriesRRID:IMSR_JAX:034159
Strain, strain background (Mus musculus)Bach2FlagTomohiro Kurosaki
Strain, strain background (Mus musculus)Bach2fox/flox, flox-Bach2Tomohiro Kurosaki
Strain, strain background (Mus musculus)NKp46iCre; Ncr1tm1.1(icre)VivEric VivierMGI:5308410
Strain, strain background (Mus musculus)Bach2-/-, Bach2tm1eEuCommBach2tm1e(EUCOMM)WtsiES clone EPD0689_1_H03, ES line JM8A3.N
Cell line (Mus musculus)B16F10ATCCCRL-6475
AntibodyAnti-Mouse CD127 PE-Cyanine7, rat monoclonaleBioscienceCatalog# 25-1271-82Flow cytometry (1:100)
AntibodyAnti-Mouse CD3e FITC, armenian hamster monoclonaleBioscienceCatalog# 11-0031-85Flow cytometry (1:100)
AntibodyAnti-Mouse CD19 FITC, rat monoclonaleBioscienceCatalog# 11-0193-86Flow cytometry (1:100)
AntibodyAnti-Mouse CD49b (Integrin α2) FITC, rat monoclonaleBioscienceCatalog# 11-5971-85Flow cytometry (1:100)
AntibodyAnti-Mouse NK1.1 PE-Cyanine7, mouse monoclonaleBioscienceCatalog# 25-5941-82Flow cytometry (1:100)
AntibodyAnti-Mouse CD27 APC, armenian hamster monoclonaleBioscienceCatalog# 17-0271-82Flow cytometry (1:100)
AntibodyAnti-Mouse CD11b eFluor 450, rat monoclonaleBioscienceCatalog# 48-0112-82Flow cytometry (1:100)
AntibodyAnti-Mouse NKp46 PerCP-eFluor 710, rat monoclonaleBioscienceCatalog# 46-3351-82Flow cytometry (1:100)
AntibodyAnti-Mouse CD39 PE, rat monoclonaleBioscienceCatalog# 12-0391-82Flow cytometry (1:100)
AntibodyAnti-Mouse Granzyme B PE, rat monoclonaleBioscienceCatalog# 12-8898-80Flow cytometry (1:100)
AntibodyAnti-Mouse IFNγ gamma eFluo450, rat monoclonaleBioscienceCatalog# 48-7311-82Flow cytometry (1:100)
AntibodyAnti-Mouse Ly49A/D PE, rat monoclonaleBioscienceCatalog# 12-5783-81Flow cytometry (1:100)
AntibodyAnti-Mouse Ly49D APC, rat monoclonaleBioscienceCatalog# 17-5782-82Flow cytometry (1:100)
AntibodyAnti-Mouse Ly49E/F APC, rat monoclonaleBioscienceCatalog# 17-5848-80Flow cytometry (1:100)
AntibodyAnti-Mouse Ly49I FITC, mouse monoclonaleBioscienceCatalog# 11-5895-82Flow cytometry (1:100)
AntibodyAnti-Mouse CD94 FITC, rat monoclonaleBioscienceCatalog# 11-0941-82Flow cytometry (1:100)
AntibodyAnti-Mouse NKG2A PerCP eFluor 710, mouse monoclonaleBioscienceCatalog# 46-5897-82Flow cytometry (1:100)
AntibodyAnti-Mouse TER-119 Biotin, rat monoclonaleBioscienceCatalog# 13-5921-85Flow cytometry (1:100)
AntibodyAnti-Mouse CD117 APC, rat monoclonaleBioscienceCatalog# 17-1171-81Flow cytometry (1:100)
AntibodyAnti-Mouse CD62L eFluor450, rat monoclonaleBioscienceCatalog# 48-0621-82Flow cytometry (1:100)
AntibodyAnti-Mouse CD122 eFluor 450, rat monoclonaleBioscienceCatalog# 48-1222-82Flow cytometry (1:100)
AntibodyAnti-Mouse CD244.2 FITC, mouse monoclonalBD PharmingenCatalog# 553305Flow cytometry (1:100)
AntibodyAnti-Mouse TCF-7/TCF-1 PE, mouse monoclonalBD PharmingenCatalog# 564217Flow cytometry (1:100)
AntibodyAnti-Mouse Ly49F PE, mouse monoclonalBD PharmingenCatalog# 550987Flow cytometry (1:100)
AntibodyAnti-Mouse CD122 FITC, rat monoclonalBD PharmingenCatalog# 553361Flow cytometry (1:100)
AntibodyAnti-Mouse CD69 PE, armenian hamster monoclonalBD PharmingenCatalog# 553237Flow cytometry (1:100)
AntibodyAnti-Mouse Ly49-G2 APC, rat monoclonalBD PharmingenCatalog# 555316Flow cytometry (1:100)
AntibodyAnti-Mouse CD135 Brilliant Violet 421, rat monoclonalBiolegendCatalog# 135313Flow cytometry (1:100)
AntibodyAnti-DYKDDDDK tag APC, rat monoclonalBiolegendCatalog# 637307Flow cytometry (1:100)
AntibodyAnti- DYKDDDDK tag PE, rat monoclonalBiolegendCatalog# 637310Flow cytometry (1:100)
AntibodyAnti-Mouse KLRG1 (MAFA) Biotin, syrian hamster monoclonalBiolegendCatalog# 138406Flow cytometry (1:100)
AntibodyAnti-Mouse CX3CR1 PE, mouse monoclonalBiolegendCatalog# 149005Flow cytometry (1:100)
AntibodyAnti-Mouse Ly49H Biotin, mouse monoclonalIn-houseFlow cytometry (1:100)
AntibodyAnti-Mouse Ly49C Biotin, mouse monoclonalIn-houseFlow cytometry (1:100)
AntibodyAnti-Mouse CD3 Biotin, armenian hamster monoclonaleBioscienceCatalog# 145–2C11Flow cytometry (1:100)
AntibodyAnti-Mouse CD19 Biotin, rat monoclonaleBioscienceCatalog# 13-0193-85Flow cytometry (1:100)
AntibodyAnti-Mouse
NK1.1 Biotin, mouse monoclonal
BiolegendCatalog# 108704Flow cytometry (1:100)
AntibodyAnti-Mouse CD11b Biotin, rat monoclonalBiolegendCatalog# 101204Flow cytometry (1:100)
AntibodyAnti-Mouse TER-119 Biotin, rat monoclonaleBioscienceCatalog# 13-5921-85Flow cytometry (1:100)
AntibodyAnti-Mouse Ly-6G and Ly-6C Biotin, rat monoclonalBD PharmingenCatalog# 553125Flow cytometry (1:100)
AntibodyAnti-Mouse
CD135 Biotin, rat monoclonal
BD HorizonCatalog# 562537Flow cytometry (1:100)
AntibodyAnti-Human/Mouse IRF8 APC, mouse monoclonaleBioscienceCatalog# 17-9852-82Flow cytometry (1:100)
AntibodyAnti-Mouse Blimp-1 PE, rat monoclonaleBioscienceCatalog# 12-9850-82Flow cytometry (1:100)
AntibodyAnti-Mouse Ki-67 eFluor, rat monoclonaleBioscienceCatalog# 48-5698-82Flow cytometry (1:100)
Peptide, recombinant proteinStreptavidin PerCPBD PharmingenCatalog# 554064Flow cytometry (1:200)
Peptide, recombinant proteinStreptavidin PEBD PharmingenCatalog# 554061Flow cytometry (1:200)
OtherFixable Viability Dye eFluor 506eBioscienceCatalog# 65-0866-14Flow cytometry (1:1000)
AntibodyAnti-FLAG M2-Peroxidase (HRP) mouse monoclonalSigmaCatalog# A8592Western blot (1:1000)
AntibodyBeta-Actin antibody rabbit polyclonalCell SignalingCatalog# 4967SWestern blot (1:1000)
AntibodyAnti-rabbit IgG HRP-linked antibody affinity purified from goatCell SignalingCatalog# 7074SWestern blot (1:1000)
Commercial assay or kitEasySep Mouse NK cell isolation kitSTEMCELL TechnologiesCatalog# 19855NK cell enrichment
Commercial assay or kitPureLink RNA Mini KitAmbionCatalog# 12183018ARNA isolation
Sequence-based reagentTcf7IDT DNAMm.PT.58.13528979Fwd: CTTCAATCTGCTCATGCCCTA
Rev: TGTTCGTAGAGTGGAGAAAGC
Sequence-based reagentGzmbIDT DNAMm.PT.58.42155916Fwd: AAGAGAGCAAGGACAACACTC
Rev: CATGTCCCCCGATGATCTC
Sequence-based reagentKlrg1IDT DNAMm.PT.58.30803964Fwd: GCTCACATCTCCTTACATTTCCG
Rev:
TCCTCAAGCCGATCCAGTA
Sequence-based reagentKitIDT DNAMm.PT.58.33701407Fwd: CGGCTAACAAAGGGAAGGAT
Rev:
GTATAAGTGCCTCCTTCTGTGC
Sequence-based reagentSellIDT DNAMm.PT.58.13849728Fwd: CTTCATTCCTGTAGCCGTCAT
Rev: CCATCCTTTCTTGAGATTTCTTGC
Sequence-based reagentCD69IDT DNAMm.PT.58.32284621Fwd: ACGGAAAATAGCTCTTCACATCT
Rev: ACCACTATTAACACAGCCCAAG
Sequence-based reagentKlrb1bIDT DNAMm.PT.58.41786742Fwd: CTAGCCAGGATCCAAGAACC
Rev: CAATCACGACCAGCACAAGA
Sequence-based reagentBhlhe41IDT DNAMm.PT.31539347Fwd: GGAACATAGGGATTTTATAGGACTGG
Rev:
GCATTCATTAATTCGGTCTCGTC
Sequence-based reagentSox6IDT DNAMm.PT.58.30547031Fwd: CCGTACAGTTCATTCCGTCAA
Rev: GTCACTTATGCCCTTTAGCCT
Sequence-based reagentCcr7IDT DNAMm.PT.58.312575202Fwd:
GAGACAAGAACCAAAAGCACAG
Rev:
GGAAAATGACAAGGAGAGCCA
Sequence-based reagentCD39IDT DNAMm.PT.58.8557349Fwd:
CGAGAAGGAGAATGACACAGG
Rev:
GTATCAGTTCGGTGGACAGTTC
Sequence-based reagentIrf8IDT DNAMm.PT.58.30819027Fwd: TGTCTCCCTCTTTAAACTTCCC
Rev: GAAGACCATGTTCCGTATCCC
Sequence-based reagentCcl5IDT DNAMm.PT.58.43548565Fwd:
GCTCCAATCTTGCAGTCGT
Rev:
CCTCTATCCTAGCTCATCTCCA
Sequence-based reagentCx3cr1IDT DNAMm.PT.58.17555544Fwd:
TCCCTTCCCATCTGCTCA
Rev:
CACAATGTCGCCCAAATAACAG
Sequence-based reagentPrdm1IDT DNAMm.PT.58.10253822Fwd: GAACCTGCTTTTCAAGTATGCTG
Rev:
TTCCCTTCGGTATGTACTCCT
Sequence-based reagentActbIDT DNAMm.PT. 39a.22214843.gFwd: GATTACTGCTCTGGCTCCTAG
Rev: GACTCATCGTACTCCTGCTTG
Sequence-based reagentBach2
Exon1-2
IDT DNAMm.PT.58.12332872Fwd: TGTAGCCTTCTCATCTCTTCCT
Rev: ATCCACAGACATGCCGTTC
Sequence-based reagentBach2
Exon3-4
IDT DNAMm.PT.58.11332004Fwd: GAAGCAGACAGTGAGTCGTG
Rev: ACTGTTCTGAGGTTAGCTTGTG
Sequence-based reagentBach2
Exon4-5
IDT DNAMm.PT.58.11289601Fwd: GAGTTCATCCACGACATCCG
Rev: CAGTTTTTCCTTCTCGCACAC
Chemical compound, drugDNaseSigmaCatalog# DN25
Chemical compound, drugHyaluronidaseSigmaCatalog# H2126
Chemical compound, drugLiberaseRocheCatalog# 05401119001
Peptide, recombinant proteinRecombinant murine IL-15PeproTechCatalog# 210–15
Peptide, recombinant proteinRecombinant murine IL-12 p70PeproTechCatalog# 200–12
Peptide, recombinant proteinRecombinant mouse IL-18MBLCatalog# B002-5
Chemical compound, drugCalcein-AMBiolegendCatalog# 425201
Software, algorithmPrism 9GraphPadhttps://www.graphpad.com/
Software, algorithmFlowJo 10Treestarhttps://www.flowjo.com/

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