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The transcription factor Fli1 restricts the formation of memory precursor NK cells during viral infection

Nature Immunology volume 23, pages 556–567 (2022)Cite this article

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Abstract

Natural killer (NK) cells are innate lymphocytes that possess traits of adaptive immunity, such as memory formation. However, the molecular mechanisms by which NK cells persist to form memory cells are not well understood. Using single-cell RNA sequencing, we identified two distinct effector NK cell (NKeff) populations following mouse cytomegalovirus infection. Ly6C– memory precursor (MP) NK cells showed enhanced survival during the contraction phase in a Bcl2-dependent manner, and differentiated into Ly6C+ memory NK cells. MP NK cells exhibited distinct transcriptional and epigenetic signatures compared with Ly6C+ NKeff cells, with a core epigenetic signature shared with MP CD8+ T cells enriched in ETS1 and Fli1 DNA-binding motifs. Fli1 was induced by STAT5 signaling ex vivo, and increased levels of the pro-apoptotic factor Bim in early effector NK cells following viral infection. These results suggest that a NK cell-intrinsic checkpoint controlled by the transcription factor Fli1 limits MP NK formation by regulating early effector NK cell fitness during viral infection.

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Fig. 1: scRNA-seq reveals two clusters of NKeff cells following MCMV infection.
Fig. 2: Ly6C– NKeff cells preferentially persist following MCMV infection.
Fig. 3: Ly6C– NKeff cells differentiate into Ly6C+ memory NK cells.
Fig. 4: MP NK cells are transcriptionally distinct and require Bcl2 for optimal survival during the contraction phase of the response to MCMV.
Fig. 5: MP NK cells share a core epigenetic signature with MP CD8+ T cells.
Fig. 6: Fli1 is induced by IL-15-mediated STAT5 signaling in mature NK cells.
Fig. 7: Fli1 restricts the formation of MP NK cells during viral infection.

Data availability

Sequencing datasets are accessible from GEO with accession number GSE176208. Original western blot scans can be found in source data. Source data are provided with this paper.

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Acknowledgements

We thank members of the O’Sullivan and Sun labs for helpful discussion. The Regents of the University of California have filed a provisional patent application with the United States Patent and Trademark Office towards methods for adoptive cell immunotherapy targeting Fli1 expression in NK cells. L.R., J.H.L. and T.E.O’S. are listed as inventors on this patent application. T.E.O’S. was supported by the NIH (AI145997) and UC CRCC (CRN-20-637105). J.H.L. was supported by the NIH NIGMS (T32GM008042).

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Authors and Affiliations

  1. Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    Luke Riggan, Joey H. Li & Timothy E. O’Sullivan

  2. Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Luke Riggan, Joey H. Li & Timothy E. O’Sullivan

  3. Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA, USA

    Feiyang Ma & Matteo Pellegrini

  4. Institute for Genomics and Proteomics, University of California, Los Angeles, Los Angeles, CA, USA

    Feiyang Ma & Matteo Pellegrini

  5. Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

    Elizabeth Fernandez & David A. Nathanson

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Contributions

L.R. and T.E.O’S. designed the study; L.R. J.H.L. and E.F. performed the experiments; F.M. and M.P. performed bioinformatics analysis; D.A.N. provided reagents; T.E.O’S. and L.R. wrote the manuscript.

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Correspondence to Timothy E. O’Sullivan.

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Extended data

Extended Data Fig. 1 Phenotypic and single-cell sequencing analysis of naive, D7 PI and D14 PI Ly49H+ NK cells.

(a) Gating strategy to sort adoptively transferred TCRβ–CD3ε–NK1.1+KLRG1+Ly49H+ NK cells from the spleen of recipient Ly49H–/– mice on D7 and D14 following MCMV infection. (b-e) Ly49H+ NK cells were adoptively transferred into Ly49H–/– mice and infected with MCMV i.p. 16 hours later. TCRβ–CD3ε–NK1.1+Ly49H+KLRG1+ NK cells were sorted on D7 and D14 PI. Cells were immediately processed for single-cell sequencing using 10x Genomics Chromium droplet single-cell RNA sequencing. (b) Heatmap showing the top differentially expressed genes between the two clusters of D7 PI NK cells (padj < 0.05). (c) Representative flow plots of cell-surface expression of CD27, CD11b, NK1.1 and KLRG1 on naive (left) and D7 PI (right) TCRβ–CD3ε–NK1.1+Ly49H+ NKeff cells. (d) Heatmap showing the top differentially expressed genes between the six clusters from NK cells at D7 and D14 PI (padj < 0.05). (e) GO enrichment analysis of marker genes for each cluster from (d). Terms were considered statistically significantly enriched if -log10(padj)<0.05. For (c-e), differentially expressed genes were identified using Wilcoxon Rank Sum test in Seurat v3.1.2.

Extended Data Fig. 2 Time-resolved putative differentiation pathways of NKeff and MP NK cells during MCMV infection.

(a-d) Ly49H+ NK cells were adoptively transferred into Ly49H–/– mice and infected with MCMV i.p. 16 hours later. TCRβ–CD3ε–NK1.1+Ly49H+KLRG1+ NK cells were sorted on D7 and D14 PI. Cells were immediately processed for single-cell sequencing using 10x Genomics Chromium droplet single-cell RNA sequencing. (a) RNA velocity analysis of D7 and D14 NKeff cell clusters with velocity field arrows projected onto the UMAP plot. (b) Arrows show the local average velocity evaluated on a regular grid and indicate the extrapolated future states of cells. (c) Monocle pseudotime analysis of NK cell clusters, indicating cluster identities (left) and pseudotime (right). (d) Scatter plot displaying relative expression (y-axis) of selected genes along pseudotime (x-axis).

Extended Data Fig. 3 NKeff subsets do not demonstrate differential trafficking or proliferation following MCMV infection.

WT splenic CD45.1 and CD45.2 Ly49H+ NK cells were transferred into Ly49H–/– mice, and infected with MCMV i.p. 16 hours later. 7 days after MCMV infection, TCRβ–CD3ε–NK1.1+Ly49H+KLRG1+CD45.1+CX3CR1+ and TCRβ–CD3ε–NK1.1+Ly49H+KLRG1+CD45.2+CX3CR1– NKeff were sorted and then transferred into naive Ly49H–/– hosts at a 1:1 ratio. Recipient spleens were harvested 12 days post transfer and analyzed by flow cytometry. (a) Quantification of the change in frequency of CX3CR1+ (CD45.1) and CX3CR1– (CD45.2) Ly49H+ NK cells subsets from recipient mice on D12 (post transfer). (b-d) Splenic WT Ly49H+ NK cells were transferred into Ly49H–/– mice and infected with MCMV i.p. 16 hours later. (b) Quantification of Ly6C+ and Ly6C– Ly49H+ NK cells from the indicated peripheral organs at various timepoints PI and (c) D10 PI. (d) Quantification of Ki-67+ Ly49H+ splenic NK cells at D10 PI. (a) Data represent 2 independent experiments with n = 4 mice per group. (b-d) Data represent 2 independent experiments with n = 6 mice per group. Samples were compared using two-tailed Student’s t-test with Welch’s correction, assuming unequal SD, and data points are presented as individual mice with the mean ± SEM (ns = not significant).

Extended Data Fig. 4 Ly6C+ and Ly6C– NKeff cells do not display differences in mitophagy, cell-intrinsic metabolism and memory functionality.

(a-e) Splenic Ly49H+ NK cells were transferred into Ly49H–/– mice i.v. and infected i.p with MCMV 16 hours after adoptive transfer. (a) Single cell metabolic analysis of naive (top) or D7 PI (bottom) NKeff using SCENITH. (b) MFI for tetramethylrhodamine ethyl ester (TMRE) staining (top) or MitoTracker Green staining (bottom) in adoptively transferred Ly49H+ NK cells from recipient spleens at the indicated time points PI. (c) Percentage of IFN-γ+ naive or adoptively transferred D14 PI Ly6C+ or Ly6C– Ly49H+ NK cells following no stimulation or 4 hr stimulation ex vivo with anti-NK1.1 monoclonal plate-bound antibody. Ly6C+ p = 0.000083, Ly6C- p = 0.000101. (d) 2.5 × 103 D7 PI sorted TCRβ–CD3ε–NK1.1+KLRG1+CD45.1+Ly49H+Ly6C+ or TCRβ–CD3ε–NK1.1+KLRG1+CD45.1+Ly49H+Ly6C– NK cells were adoptively transferred i.v. into naive Ly49H–/– mice and infected with MCMV 7 days later. (d) Kaplan-Meier survival curves of Ly49H–/– mice that received PBS or indicated sorted NK cell populations i.v. p = 0.0174. (e) The number of Ly6C+ and Ly6C– Ly49H+ NK cells was quantified in various organs at D28 PI. Lung p = 0.001997, liver p = 0.000495, spleen p = 0.0003. (a-c,e) Data represent 2-3 independent experiments with n = 3 mice per group. Samples were compared using two-tailed Student’s t-test with Welch’s correction, assuming unequal SD, and data are presented as individual points with the mean ± SEM. (d) Data are pooled from 2 independent experiments with n = 4–5 mice per group per experiment. Data points are presented as individual mice. Conditions were compared using the Log-rank (Mantel–Cox) test with correction for testing multiple hypotheses. (**p < 0.01, ***p < 0.001).

Extended Data Fig. 5 MP NK cells are transcriptionally distinct from MP CD8 T cells.

Splenic Ly49H+ NK cells were transferred into Ly49H–/– mice i.v. and infected i.p with MCMV 16 hours after adoptive transfer. Splenic TCRβ–CD3ε–NK1.1+KLRG1+Ly49H+Ly6C+ and TCRβ–CD3ε–NK1.1+KLRG1+Ly49H+Ly6C– NKeff were sorted from recipient mice on D7 PI. Sorted NK cells were immediately processed for mRNA extraction, library preparation and sequencing. (a) Differentially expressed genes common between MP Ly6C– NK and Ly6C+ NKeff cells compared to MP and TE CD8+ T cells (padj < 0.05). Differentially expressed genes were determined using the Wald test in DESeq2. (b) Normalized read counts of selected genes in indicated cell types. Data are representative of n = 3 mice per group, presented as mean ± SEM.

Extended Data Fig. 6 MP NK cells and MP CD8+ T cells display similar chromatin accessibility at specific gene loci.

ATAC sequencing reads from Fig. 5 in the indicated cell subsets mapping to the (a) Bcl2 locus and (b) Cxcr3 locus. Highlighted peaks represent differentially accessible peaks (padj < 0.15). For (a-b), differentially accessible peaks were identified using the Wald test in DESeq2. (c) GO term analysis from differentially accessible peaks shared between MP NK cells and MP CD8+ T cells. Terms were considered statistically significantly enriched if -log10(padj)<0.05. Data are representative of three independent replicates for MP and TE CD8+ T cells, D7 PI Ly6C– NKeff and two independent replicates for D7 PI Ly6C+ NKeff. GO term analysis was calculated using the cumulative hypergeometric distribution in HOMER.

Extended Data Fig. 7 Proposed model of MP NK cell formation and mechanism of Fli1 induction.

(a) IL-15 stimulated NK cells signal through STAT5 to induce Fli1 expression. During infection, Fli1 increases Bim levels to limit the number of early effector NK cells contributing to the D7 effector pool. In the absence of Fli1, Bim levels are reduced while BCL2 is unaffected, allowing more early effector NK cells to persist and form MP NK cells. (b) In the contraction phase post D7, MP NK cells continually give rise to Ly6C+ memory NK cells. The majority of D7 PI Ly6C+ NK cells undergo cell death, while a small number can become Ly6C+ memory cells. Lacking a stem-like transcriptional program, the pool of MP cells is depleted over time.

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Source Data Fig. 7

Unprocessed western blots, annotated in the file.

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Riggan, L., Ma, F., Li, J.H. et al. The transcription factor Fli1 restricts the formation of memory precursor NK cells during viral infection. Nat Immunol 23, 556–567 (2022). https://doi.org/10.1038/s41590-022-01150-0

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