Tumor-derived CSF-1 induces the NKG2D ligand RAE-1δ on tumor-infiltrating macrophages
- University of California, Berkeley, United States
Figures
Figure 1 with 3 supplements
RAE-1δ is induced on tumor-associated macrophages in subcutaneously transferred and spontaneous tumors.
(A) Established B16 S.C. tumors were dissociated and analyzed for NKG2D ligand expression on tumor-associated macrophages. (B) RAE-1δ expression (left) and MFI quantification (right) on the …
Figure 1—figure supplement 1
Gating strategy and RAE1δ expression on tumor-associated macrophages and monocytes in mice with B16 tumors.
(A) Gating strategy to identify tumor-associated macrophages in B16 tumors. (B) Expression of RAE-1δ on TAMs in B16 S.C. tumors in WT and RAE-1-KO mice. (C) RAE-1δ expression on TAMs, splenic …
Figure 1—figure supplement 2
RAE-1 antibody validation and RAE-1δ staining on TAMs in different tumors.
(A) B16 cells transduced to stably express RAE-1δ or RAE-1ε were stained with biotinylated antibodies against RAE-1δ or RAE-1ε, followed by fluorophore-conjugated streptavidin. (B) Expression of …
Figure 1—figure supplement 3
Gating strategies for blood and tumor-associated monocytes and peritoneal macrophages.
Gating strategies for (A) monocytes in B16 tumors, (B) blood monocytes, and (C) peritoneal macrophages.
Figure 2 with 1 supplement
B16 and KP cell line conditioned medium and CSF-1 induces RAE-1δ on macrophages.
(A) Peritoneal wash cells were cultured with a 1:1 mixture of fresh medium plus 20X concentrated fresh medium or 20X concentrated B16 cell culture supernatants, and macrophage RAE-1δ was analyzed by …
Figure 2—figure supplement 1
Peritoneal macrophage CSFR1 expression and dose-dependent RAE-1δ induction by CSF-1, and bone marrow macrophage stimulation with CSF-1 or GM-CSF.
(A) CSF-R1 expression on peritoneal macrophages ex vivo. (B) RAE-1δ expression on peritoneal macrophages cultured 48 hr with the indicated concentration of CSF-1. (C) RAE-1δ expression on …
Figure 3 with 1 supplement
CSF-1 is necessary for macrophage RAE-1δ induction by tumor conditioned media.
(A) The indicated numbers of B16 or RMA-S cells were seeded in 12-well plates, and CSF-1 levels in the supernatants were measured by ELISA 48 hr later. (B) Established B16 or RMA-S tumors were …
Figure 3—figure supplement 1
In vitro and in vivo CSF-1 production in tumors.
(A) CSF-1 concentrations in the indicated cell line culture supernatants. (B) CSF-1 concentrations in B16 tumors or serum of naïve or tumor-bearing mice. Data are representative of 2–3 independent …
Figure 4 with 1 supplement
Blockade of CSF-1 or CSF-1R abrogates RAE-1δ expression by TAMs in vivo.
(A) Mice with established B16 tumors were injected i.p. with 200 ug of the indicated antibody, and RAE-1δ on TAMs was analyzed 48 hr later. (B) KP mice with established sarcomas were injected i.p. …
Figure 4—figure supplement 1
Tumor associated macrophage numbers and RAE-1δ expression after treatments with anti-CSF-1R.
(A) RAE-1δ on TAMs in B16 tumors from mice given control Ig or anti-CSF-1R at days −1, 2, and 5 and harvested at the indicated time point. (B) TAMs as a percent of total live cells in B16 tumors …
Figure 5
Tumor-derived CSF-1 is required for TAM RAE-1δ expression in vivo.
(A) RAE-1δ expression on TAMs in established B16 or B16-Csf1-KO tumors. (B) RAE-1δ on TAMs in mice with established B16 tumors or tumors of a second clone of B16-Csf1-KO cells. (C) RAE-1δ on TAMs in …
Figure 6 with 1 supplement
PI3Kα signals are required for macrophage RAE-1δ induction by CSF-1.
(A) Peritoneal wash cells were stimulated with CSF-1 plus vehicle control or PI3Kα inhibitors at 3 μM, and macrophage RAE-1δ was analyzed at 24 hr. (B). Relative macrophage RAE-1δ MFI 24 hr after …
Figure 6—figure supplement 1
Induction of phospho-S6 by CSF-1.
(A) Phospho-S6 staining (top) and MFI quantification (bottom) in macrophages stimulated with control medium or 10 ng/ml CSF-1 for the indicated times. Data are representative of 3 independent …
Figure 7 with 1 supplement
Co-culture of NK cells with RAE-1δ-expressing macrophages and tumor cells.
(A) Peritoneal macrophages from WT or RAE-1-KO mice or were stimulated with 10 ng/ml CSF-1 for 48 hr and then co-cultured with WT splenocytes for 18 hr, and NKG2D levels were analyzed by flow …
Figure 7—figure supplement 1
Tumor associated macrophage numbers and RAE-1δ expression in RAE-1-KO and NKG2D-KO mice.
(A) TAMs as a percentage of CD45 + cells in established B16 tumors in WT and RAE-1-KO mice. (B) RAE-1δ on TAMs in B16 tumors in WT and NKG2D-KO mice.
Author response image 1
WT mice were injected with 1 x 106 of the indicated tumors.
Volumes were analyzed at day of harvest (day 15).
Tables
Table 1
Cytokine stimulation of macrophages for RAE-1δ induction.
https://doi.org/10.7554/eLife.32919.008| Treatment | Macrophage RAE-1δ induction? |
|---|---|
| IL-1α | |
| IL-1β | |
| IL-4 | |
| IL-6 | |
| IL-12 | |
| IFNβ | |
| IFNγ | |
| TNFα | |
| CSF1 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.32919.018
