Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses
- Institute of Industrial Science, The University of Tokyo, Japan
- Max Planck-The University of Tokyo Center for Integrative Inflammology, Japan
- Core Research for Evolution Science and Technology, Japan
- Yokohama City University Graduate School of Medicine, Japan
- Medical Mycology Research Center, Chiba University, Japan
- Research Institute for Biomedical Sciences, Tokyo University of Science, Japan
Figures
Figure 1 with 4 supplements
Critical contribution of IRF5 to the enhancement of NK cell-mediated anti-tumor responses.
(A) Selective contribution of IRF5 in the suppression of lung metastasis of B16F1 cells. Number of metastasized colonies in lungs from wild-type (WT), Irf3−/−, Irf5−/−, or Irf7−/− mice 14 days after …
Figure 1—figure supplement 1
Critical role of IRF5 in the suppression of tumor growth.
Tumor growth in WT and Irf5−/− mice. Tumor volume of WT or Irf5−/− mice was monitored at the indicated days after subcutaneous injection of 1 × 105 of B16F1 cells. Represented as means ± SD.
Figure 1—figure supplement 2
Requirement of IRF5 in myeloid cells for the suppression of tumor metastasis.
(A) Tumor metastasis in bone marrow chimera mice. Chimera mice were generated using WT, Irf5−/−, and C57BL/6-Ly5.1 (Ly5.1) mice. Numbers of metastasized colonies in lungs from the bone marrow …
Figure 1—figure supplement 3
Involvement of IRF5 in CD11b+ and CD11c+ cells to the enhancement of NK cell-mediated anti-tumor responses.
Requirement of IRF5 in myeloid cells for the enhancement of NK cell's in vitro killing activity. In vitro killing activities of purified NK cells (WT; 1 × 105 cells) against B16F1 cells were …
Figure 1—figure supplement 4
Contribution of DCs and macrophages to the NK cell-mediated tumor killing.
The effect of various myeloid cells on the enhancement of NK cell killing activities. In vitro killing activities of purified NK cells (WT; 1 × 105 cells) were monitored in the absence or presence …
Figure 2 with 8 supplements
Critical role of Dectin-1 signaling in DCs and macrophages for IRF5 activation by and NK cell-mediated killing against B16F1 cells.
(A) Nuclear translocation of IRF5 in WT or Dectin-1−/− splenocytes (5 × 107 cells) co-incubated with B16F1 cells (5 × 105 or 1 × 106 cells) or stimulated with curdlan (30 µg/ml) for 6 hr. Nuclear …
Figure 2—figure supplement 1
Dispensable role of MyD88 in anti-tumor killing activity of NK cells.
In vitro killing activity of WT or Myd88−/− splenocytes against B16F1 cells. Represented as means ± SD. E/T: effector/target cell ratio. In in vitro killing assays, 1 × 104 of 51Cr-labeled B16F1 …
Figure 2—figure supplement 2
Effect of FK506 treatment on IRF5 activation in splenocytes.
Nuclear translocation of IRF5 of WT splenocytes (5 × 107 cells) co-incubated with B16F1 cells (1 × 106 cells) for 6 hr in the absence or presence of 50 or 500 nM of FK506. Nuclear protein extracts …
Figure 2—figure supplement 3
Minor effects of Dectin-1 expressed in NK cells on the tumor killing activity.
(A) In vitro killing activity of purified NK cells from WT or Dectin-1−/− mice against B16F1 cells. Represented as means ± SD. E/T: effector/target cell ratio. In in vitro killing assays, 1 × 104 of …
Figure 2—figure supplement 4
Essential role of Dectin-1 in the suppression of tumor growth.
Tumor growth in WT and Dectin-1−/− mice. Tumor volume of WT or Dectin-1−/− mice was monitored at the indicated days after subcutaneous injection of 1 × 105 of B16F1 cells. Represented as means ± SD.
Figure 2—figure supplement 5
Contribution of Dectin-1 and IRF5 to the control of tumor metastasis.
Number of metastasized colonies in the lungs from WT, Irf5−/−, or Dectin-1−/− mice. Mice were intravenously injected with 1 × 106 of B16F1 cells and, 14 days later, the number of metastasized …
Figure 2—figure supplement 6
Dispensable role of Dectin-1 in NK-cell-independent tumor suppression.
NK-depleted WT, or NK-depleted Dectin-1−/− mice were intravenously injected with 1 × 106 of B16F1 cells. After 14 days, the numbers of metastasized colonies were counted in the lungs of each mouse. …
Figure 2—figure supplement 7
Normal population and functions of Dectin-1-deficient NK cells.
(A) The population of splenic NK cells in Dectin-1−/− mice. Splenocytes were isolated from WT and Dectin-1−/− mice. The NK cell population (percentage of total cells) was then analyzed by flow …
Figure 2—figure supplement 8
Expression levels of cytotoxic mediators and inflammatory cytokines in co-culture system.
NK cells (WT; 1 × 105 cells) and WT or Dectin-1−/− splenic CD11c+ cells (3 × 105 cells) were co-cultured with B16F1 cells (1 × 104 cells). Total RNA was isolated at time zero and 4 hr after the …
Figure 3 with 6 supplements
Recognition of N-glycan structures on B16F1 cells by Dectin-1 and its requirement for the enhancement of NK cell-mediated killing activity.
(A) Binding of sDectin-1 to B16F1 cells (left panel) and primary mouse embryonic fibroblasts (MEFs; right panel). The cells (4 × 105 cells) were incubated with human IgG1 Fc (control Fc) or …
Figure 3—figure supplement 1
Binding of sDectin-1 to various mouse primary cells.
The sDectin-1 binding to splenic CD11b+ cells, CD11c+ cells, NK cells, liver cells, or lung cells were examined as described in Figure 3A.
Figure 3—figure supplement 2
Effect of neuraminidase treatment on the sDectin-1 binding to B16F1 cells.
B16F1 cells (4 × 105 cells) were treated with or without neuraminidase (250 mU/ml) for 1 hr and then subjected to the sDectin-1 binding assay.
Figure 3—figure supplement 3
Effect of N-glycosidase treatment of B16F1 cells on in vitro killing activity of purified NK cells.
B16F1 cells were treated with or without N-glycosidase (25 U/ml) for 1 hr in RPMI medium and then used as target cells for in vitro killing assay with purified NK cells from WT mice. 51Cr …
Figure 3—figure supplement 4
Mass spectrometric analysis of N-glycosidase-treated B16F1 cells.
The supernatant of B16F1 cells treated with N-glycosidase for 1 hr was collected and incubated with protein G-conjugated sepharose bound without (blue peaks) or with (green peaks) sDectin-1. …
Figure 3—figure supplement 5
Proposed N-glycan structures detected by mass spectrometric analysis.
Proposed structures of N-glycans detected by the mass spectrometric analysis (Figure 3—figure supplement 4) are depicted.
Figure 3—figure supplement 6
No differences in the amount of each N-glycan structure between samples with and without sDectin-1 treatment.
Effect of the incubation of sDectin-1 on the relative amount of N-glycan structures released from N-glycosidase-treated B16F1 cells. Relative amounts of each N-glycan structure to total N-glycan …
Figure 4 with 1 supplement
Requirement of cell-to-cell contact between NK cells and DCs for the enhancement of NK cell-mediated killing activity.
(A) Effect of the supernatant of myeloid cells after incubation with tumor cells. NK cell killing activity against B16F1 cells was assessed in the presence of supernatants from the co-culture of …
Figure 4—figure supplement 1
Induction of Il15 or Il15ra mRNAs in DCs co-cultured with B16F1 cells.
Il15 (left panel) or Il15ra (right panel) mRNA were monitored by qRT-PCR analysis as described in Figure 4D. Results are presented relative to the expression of Gapdh mRNA. Represented as means ± …
Figure 5 with 6 supplements
Dectin-1 binding on other tumor cells and its role in tumor suppression.
(A) Binding of sDectin-1 to 3LL (left panel) or SL4 cells (right panel). The sDectin-1 binding to these cell lines was examined as described in Figure 3A. (B) In vitro killing activity of WT or Decti…
Figure 5—figure supplement 1
Binding of sDectin-1 to various mouse cancer cell lines.
The sDectin-1 binding to YAC-1 (left panel), Meth-A (middle panel), or B16F10 (right panel) cells were examined as described in Figure 3A.
Figure 5—figure supplement 2
Pull-down analysis of sDectin-1 binding on cancer cells.
B16F1, 3LL, B16F10 and MEF cells (4 × 107 cells) treated with or without N-glycosidase (25 U/ml) for 1 hr were incubated with sDectin-1 (tagged with HA and human IgG1 Fc region), and then the …
Figure 5—figure supplement 3
In vitro killing activity of WT or Dectin-1−/− splenocytes against YAC-1 cells.
In vitro killing activity of WT or Dectin-1−/− splenocytes against YAC-1 cells as indicated E/T ratio. 51Cr radioactivity released from the target cells was measured. Represented as means ± SD. In …
Figure 5—figure supplement 4
Contribution of Dectin-1 signaling to anti-tumor killing activity against B16F10 cells.
Number of metastasized colonies in lungs from WT or Dectin-1−/− mice intravenously injected with 5 × 105 of B16F10 cells. Means are indicated as black bars. NS, not significant.
Figure 5—figure supplement 5
Binding of sDectin-1 to various human cancer cell lines.
Binding of human sDectin-1 to HepG2, Huh7, HBC4, HeLa, T98, U251, A549, HCT116, MKN45, PC-3, K562, or G-361 cells was examined as described in Figure 3A.
Figure 5—figure supplement 6
Induction of Inam mRNAs in DCs co-cultured with human cancer cells.
Dectin-1-dependent induction of Inam (Fam26f) mRNA by a human cancer cell line. Mouse splenic CD11c+ cells (3 × 105 cells), either from WT mice or Dectin-1−/− mice, were co-cultured with HBC4 cells …
Figure 6
Schematic view of the orchestration of innate immune cells for NK cell-mediated tumor killing.
Dectin-1 expressed by DCs and macrophages recognizes N-glycan structures on tumor cells and signals to activate IRF5 pathway and other pathways, thereby activating NK cells. Thus, NK cells require …
Tables
Table 1
Induction of mRNAs in DCs co-cultured with B16F1 cells via Dectin-1 signaling
| Unigene | Gene symbol | Fold change |
|---|---|---|
| Mm.275426 | Amy2a1///Amy2a2///Amy2a3///Amy2a4///Amy2a5 | 1995.76 |
| Mm.45316 | Cela2a | 173.36 |
| Mm.383263 | Try4///Try5 | 88.72 |
| Mm.475541 | Cela3b///Gm13011 | 61.79 |
| Mm.20407 | Pnlip | 21.28 |
| Mm.142731 | Reg1 | 16.65 |
| Mm.34374 | Ctrb1 | 16.42 |
| Mm.21160 | Clps | 14.85 |
| Mm.276926 | Prss2 | 13.67 |
| Mm.34692 | Cpb1 | 10.25 |
| Mm.450553 | Tpte | 9.99 |
| Mm.10753 | Pnliprp1 | 8.31 |
| Mm.46360 | Reg2 | 6.86 |
| Mm.1825 | Tff2 | 5.19 |
| Mm.867 | Ccl12///LOC100862578 | 4.96 |
| Mm.464256 | Tcl1b3 | 3.97 |
| Mm.2745 | Ctrl | 3.72 |
| Mm.14874 | Gzmb | 3.46 |
| Mm.159219 | Batf2 | 3.45 |
| Mm.439927 | Nol7 | 3.00 |
| Mm.4662 | Irg1 | 2.72 |
| Mm.4922 | Csf2 | 2.70 |
| Mm.2319 | Stmn3 | 2.70 |
| Mm.24375 | Ttpa | 2.70 |
| Mm.41416 | Rilp | 2.65 |
| Mm.159575 | Cdyl2 | 2.57 |
| Mm.261140 | Iigp1 | 2.44 |
| Mm.26730 | Hp | 2.32 |
| Mm.203866 | Ahnak | 2.30 |
| Mm.34520 | Gtpbp8 | 2.30 |
| Mm.32368 | Krit1 | 2.29 |
| Mm.116997 | Hmmr | 2.26 |
| Mm.208125 | Adamts6 | 2.17 |
| Mm.271830 | Dhx58 | 2.10 |
| Mm.131098 | Golga1 | 2.10 |
| Mm.131723 | Cxcl11 | 2.09 |
| Mm.5022 | Mmp13 | 2.09 |
| Mm.291595 | Klf9 | 2.09 |
| Mm.33691 | Reg3d | 2.08 |
| Mm.130 | Socs1 | 2.06 |
| Mm.10948 | Slfn1 | 2.05 |
| Mm.22213 | Glipr2 | 2.01 |
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The genes identified in the microarray analysis in Figure 4C are listed in the order of fold change (WT 4 hr/Dectin-1−/− 4 hr). Genes encoding a membrane-bound protein are indicated in red letters.
