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. 2018 Sep 28;9:2259.
doi: 10.3389/fimmu.2018.02259. eCollection 2018.

Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment

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Free PMC article

Lack of FcRn Impairs Natural Killer Cell Development and Functions in the Tumor Microenvironment

Diana Cadena Castaneda et al. Front Immunol. .
Free PMC article

Abstract

The neonatal Fc receptor (FcRn) is responsible for the recycling and transcytosis of IgG and albumin. FcRn level was found altered in cancer tissues and implicated in tumor immunosurveillance and neoplastic cell growth. However, the consequences of FcRn down-regulation in the anti-tumor immune response are not fully elucidated. By using the B16F10 experimental lung metastasis model in an FcRn-deficient microenvironment (FcRn-/- mice), we found lung metastasis associated with an abnormal natural killer (NK) cell phenotype. In FcRn-/- mice, NK cells were immature, as shown by their surface marker profile and their decreased ability to degranulate and synthesize interferon γ after chemical and IL-2 or IL-12, IL-15 and IL-18 activation. These new findings support the critical role of FcRn downregulation in the tumor microenvironment in anti-tumor immunity, via NK cell maturation and activation.

Keywords: CD107a; FcRn; IFN-γ; NK cells; anti-tumor immunity; fcgrt knock-out.

Figures

Figure 1
Figure 1
Role of neonatal Fc receptor (FcRn) in experimental lung metastasis development. Wild-type (WT) (n = 14) and FcRn−/− (n = 12) mice were intravenously injected in the tail vein with 1 × 105 B16-F10 tumor cells in 100 μl medium. 18 days after tumor injection, animals were sacrificed and lung tumor lesions were blindly counted. (A) Representative photographs of WT and FcRn−/− lungs after 18 days. (B) Nodule counts in WT and FcRn−/− mice. Data are expressed as median ± Min to Max from three independent experiments. ***p ≤ 0.0001 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.
Figure 2
Figure 2
Flow cytometry of leukocytes in lungs of WT (n = 8) and FcRn−/− (n = 8) mice injected, in the tail vein, with 1 × 105 B16-F10 tumor cells in 100 μl medium. Lungs were excised and dissociated by combining mechanical dissociation with enzymatic degradation of the extracellular matrix to obtain a single-cell suspension and cells were resuspended at 107 cells/ml in 1X PBS containing 5% FBS and 2 mM EDTA for flow cytometry staining. Results correspond to the proportion (top panel: A–H) and number (bottom panel: I–P) of (A,I) conventional dendritic cells, (B,J) macrophages/monocytes, (C,K) neutrophils, (D,L) B lymphocytes, (E,M) T lymphocytes, (F,N) CD4 T lymphocytes, (G,O) CD8 T lymphocytes, and (H,P) natural killer (NK) cells (See Figure S1 for gating strategy). Data are expressed as median ± Min to Max from one out of three independent experiments with similar results. ns = not significant, *p ≤ 0.05, **p ≤ 0.005, and ***p ≤ 0.0001 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.
Figure 3
Figure 3
Flow cytometry of leukocytes in the spleen of WT (n = 5) and FcRn−/− (n = 5) mice injected, in the tail vein, with 1 × 105 B16F10 tumor cells in 100 μl medium. Spleens were recovered and dissociated by combining mechanical dissociation with enzymatic degradation of the extracellular matrix to obtain a single-cell suspension. Then, cells were resuspended at 107 cells/ml in 1X PBS containing 5% FBS and 2 mM EDTA for flow cytometry staining. Results correspond to the proportion (top panel: A–H) and number (bottom panel: I–P) of (A,I) conventional dendritic cells, (B,J) macrophages/monocytes, (C,K) neutrophils, (D,L) B lymphocytes, (E,M) T lymphocytes, (F,N) CD4 T lymphocytes, (G,O) CD8 T lymphocytes, and (H,P) NK cells. Data are expressed as median ± Min to Max from one of two independent experiments with similar results. ns = not significant, *p ≤ 0.05 and **p ≤ 0.005 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.
Figure 4
Figure 4
Flow cytometry of NK subtypes based on CD11b and CD27 markers in (A) lungs and (B) spleen from WT (n = 10) and FcRn−/− (n = 10) mice injected, in the tail vein, with 1 × 105 B16F10 cells in 100 μl medium. Lungs and spleen were collected from euthanized animals and dissociated by combining mechanical dissociation with enzymatic degradation of the extracellular matrix to obtain a single-cell suspension. Then, cells were resuspended at 107 cells/ml in 1X PBS containing 5% FBS and 2 mM EDTA for flow cytometry staining. Histograms represent the sum of the percentage of CD11b/CD27, CD11b/CD27+ (CD11b), CD11b+/CD27+, and CD11b+/CD27 NK cell subtypes in WT and FcRn−/− mice. Data are means from two independent experiments. ns = not significant, *p ≤ 0.05 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.
Figure 5
Figure 5
Flow cytometry of NK cell proportion in (A) lungs and (B) spleen from WT (n = 13) and FcRn−/− (n = 12) naive mice. Data are expressed as median ± Min to Max from two independent experiments. Flow cytometry of NK subtypes based on CD11b and CD27 markers in (C) lungs and (D) spleen from WT (n = 5) and FcRn−/− (n = 5) naive mice. After euthanasia of naive animals, lungs and spleen were collected and dissociated by combining mechanical dissociation with enzymatic degradation of the extracellular matrix to obtain a single-cell suspension. Then, cells were resuspended at 107 cells/ml in 1X PBS containing 5% FBS and 2 mM EDTA for flow cytometry staining. Histograms represent the sum of the percentage of CD11b/CD27, CD11b/CD27+ (CD11b), CD11b+/CD27+, and CD11b+/CD27 NK cell subtypes in WT and FcRn−/− mice. Data are means from one experiment. ns = not significant, *p ≤ 0.05, **p ≤ 0.005 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.
Figure 6
Figure 6
Flow cytometry of NK cell development in bone marrow of WT (n = 5) and FcRn−/− (n = 5) naive mice. After euthanasia of naive animals, bone-marrow cells were isolated from the femur and tibia and cells separated by mechanical dissociation. Then, cells were resuspended at 107 cells/ml in 1X PBS containing 5% FBS and 2 mM EDTA for flow cytometry staining. The development of NK cells is initiated with the expression of (A) CD122, then three early immature steps [NK precursor (NKP), stage 1 and stage 2] are identified with the expression of (B) CD122, NK1,1 and NKp46. (C) From stage 2, stage 5 and 6 subtypes are characterized by CD49b and CD11b expression. Acquisition of marker during NK cell development is represented schematically by arrows under the graphics. Data are expressed as median ± Min to Max from one out of two independent experiments with similar results. ns = not significant and *p ≤ 0.05 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.
Figure 7
Figure 7
Influence of FcRn on NK cell functions and in vitro expansion. Purified splenic NK cells were analyzed by flow cytometry for (A) the intracellular measurement of IFN-γ and (B) the surface expression of the late endosomal marker CD107a, after 4-h incubation at 37°C without (Ctr) or with PMA (100 ng/mL)/ionomycine (500 ng/mL). Data are median ± Min to Max analyzed from eight independent experiments using pooled NK cells from 2 mice. Freshly isolated splenocytes were seeded in RMPI 1640 complete medium supplemented with 5,000 U/ml rhIL2 or with 5 ng/ml rhIL12, 50 ng/ml rhIL15, and 10 ng/ml rhIL18 for 4-h (C,D). Within splenocytes, CD3/NK1.1+/NKp46+ cells were analyzed for (C) the intracellular expression of IFN-γ and (D) the surface expression of CD107a by flow cytometry. Data are median ± Min to Max from two independent experiments using pooled spleens from 2 mice. (E) Cytotoxicity assay was performed against CFSE-labeled YAC-1 target cells with different ratios of purified NK cells previously maintained overnight in RPMI 1640 complete medium supplemented with 50 U/ml of rhIL2 (n = 3). The results were expressed as means ± SEM. (F,G) Purified splenic NK cells were plated in complete medium supplemented with 5,000 U/ml rhIL2. (F) The living cell numbers and (G) the percentage of dead cells were determined daily by manual cell counting using trypan blue in Malassez chamber (n = 3). The results were expressed as mean ± SEM (F) and median ± Min to Max (G). ns = not significant *p ≤ 0.05 and **p ≤ 0.005 using two-tailed non-parametric and unpaired Wilcoxon-Mann-Whitney test.

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