mTORC1 and mTORC2 differentially promote natural killer cell development
- Blood Center of Wisconsin, United States
- Medical College of Wisconsin, United States
Figures
Figure 1 with 1 supplement
mTORC1 is essential for NK cell homeostasis and maturation.
(A) Raptor expression in freshly-isolated NK cells from WT and Rptor cKO mice was evaluated via western blot. (B) CD3, NK1.1 staining of cells from various organs (left) and quantification of NK …
Figure 1—figure supplement 1
mTORC1 is required for NK cell maturation.
(A) BM and spleen lymphocytes count in WT and Rptor cKO mice. n = 4 pooled from two independent experiments. (B) The viability of freshly-isolated NK cells from BM and spleen of WT and Rptor cKO …
Figure 2 with 1 supplement
mTORC2 is pivotal for NK cell homeostasis and terminal maturation.
(A) Rictor expression in IL-2-cultured NK cells isolated from WT and Rictor cKO mice was evaluated via western blot. A representative of three independent experiments. (B) CD3, NK1.1 staining of …
Figure 2—figure supplement 1
Terminal NK cell maturation was impaired in Rictor cKO mice.
(A) BM and spleen lymphocytes count in WT and Rictor cKO mice. n = 4 pooled from two independent experiments. (B) The viability of freshly isolated NK cells from BM and spleen of WT and Rictor cKO …
Figure 3
mTORC1 and mTORC2 differentially regulate the expression of Eomes and T-bet.
(A–D) Histogram of Eomes (A, C) and T-bet (B, D) expression on each NK cell population gated by CD27 and CD11b of Rptor (A, B) or Rictor (C, D) cKO mice and their corresponding WT control. The …
Figure 4 with 1 supplement
mTORC1 and mTORC2 differentially regulate the expression of CD122 and STAT5 activation.
(A) Histogram of CD122 expression on each NK population gated by CD27 and CD11b of WT and Rptor cKO mice (left). MFI of CD122 was normalized to WT CD27 SP population (right). n = 3 pooled from three …
Figure 4—figure supplement 1
mTORC1 activity, Eomes and CD122 expression were concurrently reduced during the transition from DP to CD11b SP stage.
(A) The MFI of CD132 in NK cells from BM and Spleen of Rptor cKO mice were normalized to WT control demonstrated as a bar graph. n = 4 pooled from two independent experiments. (B) Phosphorylation of …
Figure 5 with 1 supplement
Transcriptome analyses of Raptor- or Rictor-deficient NK cells.
RNAseq was conducted using CD11b− BM NK cells from littermate WT and Rptor cKO mice or CD27+ BM NK cells from littermate WT and Rictor cKO mice. (n = 3 per group). (A) Venn diagram demonstrating the …
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Figure 5—source data 1
Differentially expressed genes (FDR < 0.05) that belong to eIFs, eEFs and ribosome protein families in Rptor cKO NK cells was listed in the table.
This is related to Figure 5D.
- https://doi.org/10.7554/eLife.35619.011
Figure 5—figure supplement 1
Altered oxidative phosphorylation and integrin signaling in Raptor-deficient NK cells indicated by RNA sequencing.
(A, B) IPA analyses revealed enrichment of oxidative phosphorylation (A) and integrin signaling (B) in Rptor cKO NK cells as demonstrated via the heatmap. (C) mRNA level of key transcription factors …
Figure 6 with 1 supplement
mTORC2 is required for T-bet expression through regulation of FoxO1 during NK cell development.
(A) Enrichment of T-bet target genes in Rictor cKO NK cells as shown via the heatmap. (B) Histogram demonstrating phosphorylation of AktS473 (top) and FoxO1T24/FoxO3aT32 (bottom) in NK cells from BM …
Figure 6—figure supplement 1
Cell surface expression of IL7Rα and Ccr7 are increased in Rictor cKO NK cells.
(A, B) Cell surface expression of IL7Rα (A) and Ccr7 (B) in CD27+ NK cells from the spleen of WT and Rictor cKO mice were detected by flow cytometry as shown in the histogram on the left. Percentage …
Figure 7
Disruption of mTORC2 does not affect mTORC1 activation.
(A, B) Splenocytes from WT, Rptor cKO (left) or Rictor cKO mice (right) were stimulated with either medium or 100 ng/mL IL-15 for 1 hr. Phosphorylation of rpS6S240/244 (A) and AktS473 (B) were …
Figure 8
Impaired antitumor activity in Rptor or Rictor cKO mice.
(A−D) 200,000 B16F10 tumor cells were intravenously injected into Rptor (A, B) or Rictor (C, D) cKO mice and their corresponding WT control mice. 14 days post-injection, the lungs were perfused with …
Figure 9 with 1 supplement
mTORC1 is critical for the effector function of NK cells.
In vivo Poly (I:C)-activated splenocytes were co-cultured with RMA/s target cells or stimulated with IL-12/IL-18 or PMA/Ionomycin for 6 hr. Medium and RMA target cells were used as the control …
Figure 9—figure supplement 1
mTORC1 is essential for the IFN-γ generation downstream of NKG2D.
(A) Standard 51Cr-release assay using IL-2-cultured WT NK cells against different target cells at various effector to target ratios in the presence of DMSO or Rapamycin (100 nM). n = 6 pooled from …
Tables
Table 1
mRNA level of key transcription factors governing NK cell development in Rptor or Rictor cKO NK cells.
https://doi.org/10.7554/eLife.35619.012| Raptor | ||||||||
|---|---|---|---|---|---|---|---|---|
| List | Gene | WT mean | WT se | cKO mean | cKO se | log2FC | Adjusted p value | Significant at FDR 0.05 |
| key TF | Id2 | 1 | 0.0116 | 0.7037 | 0.0393 | −0.492 | 1.01E-06 | Y |
| Nfil3 | 1 | 0.024 | 0.7521 | 0.1014 | −0.379 | 0.0369946 | Y | |
| Eomes | 1 | 0.0246 | 0.758 | 0.0111 | −0.392 | 1.77E-06 | Y | |
| Gata3 | 1 | 0.0195 | 0.9258 | 0.0542 | −0.105 | 0.5441197 | N | |
| Ets1 | 1 | 0.0247 | 0.6935 | 0.0272 | −0.515 | 1.37E-08 | Y | |
| Tox | 1 | 0.0549 | 1.0158 | 0.0409 | 0.02 | 0.9239868 | N | |
| Tbx21 | 1 | 0.0295 | 0.7811 | 0.0306 | −0.346 | 0.0009024 | Y | |
| Zeb2 | 1 | 0.0826 | 1.5004 | 0.1377 | 0.546 | 0.0005179 | Y | |
| Prdm1 | 1 | 0.0925 | 1.1062 | 0.2453 | 0.102 | 0.8261435 | N | |
| Rictor | ||||||||
|---|---|---|---|---|---|---|---|---|
| List | gene | WT mean | WT se | cKO mean | cKO se | log2FC | Adjusted p value | Significant at FDR 0.05 |
| key TF | Id2 | 1 | 0.1133 | 1.0699 | 0.0271 | 0.088 | 0.8182466 | N |
| Nfil3 | 1 | 0.0354 | 0.9495 | 0.0635 | −0.069 | 0.8687849 | N | |
| Eomes | 1 | 0.0408 | 1.0366 | 0.0362 | 0.049 | 0.8800234 | N | |
| Gata3 | 1 | 0.0118 | 1.0105 | 0.0283 | 0.014 | 0.9839612 | N | |
| Ets1 | 1 | 0.0516 | 0.8081 | 0.0529 | −0.287 | 0.0117066 | Y | |
| Tox | 1 | 0.0457 | 0.7144 | 0.0379 | −0.442 | 0.0001058 | Y | |
| Tbx21 | 1 | 0.0562 | 0.7603 | 0.073 | −0.357 | 0.0065806 | Y | |
| Zeb2 | 1 | 0.0084 | 0.7995 | 0.0323 | −0.298 | 0.0137732 | Y | |
| Prdm1 | 1 | 0.1397 | 1.0292 | 0.1234 | 0.026 | 0.9839612 | N | |
Additional files
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Supplementary file 1
The expression of Id2 target genes in Rptor cKO NK cells.
- https://doi.org/10.7554/eLife.35619.019
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Transparent reporting form
- https://doi.org/10.7554/eLife.35619.020
