7 figures and 1 additional file

Figures

Figure 1 with 2 supplements
NKG2D is engaged and internalized by constitutive interactions with endogenous RAE-1ε in vivo.

(A) NKG2D surface levels measured by flow cytometry of blood NK cells 48 hr after injection of blocking antibody specific for the indicated NKG2D ligand. Data are representative of >4 independent …

https://doi.org/10.7554/eLife.30881.003
Figure 1—figure supplement 1
Blockade of RAE-1ε results in NKG2D upregulation.

(A) Specific blockade of NKG2D binding by anti-RAE-1 mAbs. The indicated cells lines were incubated for 20 min at 4°C with blocking antibody. Subsequently and without washing, biotinylated NKG2D-Fc …

https://doi.org/10.7554/eLife.30881.004
Figure 1—figure supplement 2
RAE-1-deficiency results in NKG2D upregulation in NK cells in bone marrow and liver.

(A) NKG2D surface levels on NK cells from bone marrow and liver. Data are representative of two independent experiments. Statistical significance was determined using two-tailed unpaired Student’s t …

https://doi.org/10.7554/eLife.30881.005
Figure 2 with 1 supplement
Endogenous RAE-1ε negatively regulates NK responsiveness.

(A) WT or RAE-1-KO splenic NK cell IFNγ production and degranulation (CD107a) after 5 hr ex vivo stimulation with platebound control Ig or anti-NKp46. (B and C) Percentage of activated (IFNγ- and …

https://doi.org/10.7554/eLife.30881.006
Figure 2—figure supplement 1
Normal NK cell cellularity and differentiation but enhanced NK-mediated tumor cell killing in RAE-1-knockout mice.

(A, left panel) NK cells from WT and RAE-1-KO mice as percentage of live splenocytes. Data are representative of >4 independent experiments. (A, right panel) CD27 and CD11b 

expression on splenic NK cells from WT and RAE-1-KO mice. Data are representative of three independent experiments. (B) NK cells from WT, RAE-1-KO, or NKG2D-KO mice (n = 3) were pre-activated with a single injection of 200 g Poly I:C. Two days later, peritoneal wash cells were harvested, pooled, and used as effector cells to kill YAC-1 cells in a standard 51Cr in vitro cytotoxicity assay, at the indicated effector: target ratios. Data are representative of two independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t tests (A) or two-way ANOVA (B). Data represent means ± SEM (error bars are not visible because they are small).

https://doi.org/10.7554/eLife.30881.007
Figure 3 with 1 supplement
RAE-1ε contributes to cell-intrinsic NKG2D-mediated regulation of NK responsiveness.

(A and B) Percentage of activated splenic or peritoneal NK cells from WT or NKG2D-KO mice after ex vivo stimulation. Data are representative of >4 independent experiments. (C) NK activation in WT, …

https://doi.org/10.7554/eLife.30881.008
Figure 3—figure supplement 1
NK cell activation with NKG2D stimulation, and composition of NK cell population in mixed NKG2D bone marrow chimeras.

(A) NK activation in WT, RAE-1-KO, and NKG2D-KO peritoneal cells after ex vivo stimulation with platebound anti-NKG2D antibody. Data are representative of three independent experiments. (B) …

https://doi.org/10.7554/eLife.30881.009
Figure 4 with 2 supplements
Lymph node endothelial cells as the endogenous source of RAE-1ε.

(A) NKG2D cell surface levels on blood NK cells 8 weeks after WT or RAE-1-KO mice were lethally irradiated and reconstituted with WT or RAE-1-KO bone marrow. Data are representative of three …

https://doi.org/10.7554/eLife.30881.010
Figure 4—figure supplement 1
Spleen and peritoneal wash NKG2D expression in RAE-1 bone marrow chimeras, and expression of RAE-1 on endothelial cells and high endothelial venules.

(A) NKG2D surface levels on NK cells from spleens or lymph nodes of RAE-1-KO chimeric mice. Data are representative of three independent experiments. (B) Gating strategy for the four populations of …

https://doi.org/10.7554/eLife.30881.011
Figure 4—figure supplement 2
Comparison of RAE-1 expression by endothelial cells in different organs and sites.

RAE-1ε MFI levels on endothelial cells (CD45-neg; Ter119-neg; CD31+) in the indicated tissues.

https://doi.org/10.7554/eLife.30881.012
Figure 5 with 1 supplement
Endothelial RAE-1ε and NKG2D engagement in the tumor microenvironment.

(A) NKG2D surface levels on NK cells infiltrating subcutaneous B16 tumors in mice 48 hr after injection of the indicated antibody. Data are representative of three independent experiments. (B) NKG2D …

https://doi.org/10.7554/eLife.30881.013
Figure 5—figure supplement 1
Analysis of tumor-associated NKG2D levels in RAE-1 chimeras and endothelial RAE-1ε staining in RAE-1-KO mice.

(A) Repetition of the experiment in Figure 5C. Statistical significance was determined using one-way ANOVA with Bonferroni post-tests. (B) RAE-1ε staining on endothelial cells in established B16 or …

https://doi.org/10.7554/eLife.30881.014
Figure 6 with 1 supplement
Endogenous RAE-1ε - NKG2D interactions limit NK responses to tumors.

(A) WT or NKG2D-KO mice (n = 29–30) were challenged with 2 × 104 B16 cells i.v. and monitored for morbidity. Matched groups of each strain (n = 9–10) were depleted of NK cells before implanting the …

https://doi.org/10.7554/eLife.30881.015
Figure 6—figure supplement 1
Endogenous RAE-1-NKG2D interactions limit anti-tumor responses in mice lacking T and B cells.

(A) Rag2-KO NKG2D-WT or Rag2-KO NKG2D-KO mice were challenged with 1 × 104 B16 cells s.c. and monitored for tumor growth. Data are representative of three independent experiments. (B) Rag2-KO …

https://doi.org/10.7554/eLife.30881.016
Author response image 1
NK cells from WT or RAE-1-KO mice were incubated with undiluted serum from WT or RAE-1- KO mice at 37°C for one hour, and NKG2D levels were analyzed.
https://doi.org/10.7554/eLife.30881.019

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