Early activation and interferon-γ production of tumor-infiltrating mature CD27 high natural killer cells

Abstract

Natural killer (NK) cells are known to be critically involved in the control of tumors through their direct cytotoxic function, but have also been proposed as an initial source of interferon (IFN)-γ that primes subsequent adaptive tumor-specific immune responses. Although mounting evidence supports the importance of NK cells in antitumor immune responses, the immunological characteristics of NK cells infiltrating the tumor microenvironment and the mechanisms that regulate this process remain unclear. In the present study, we found that NK cells infiltrate early developing MCA205 tumors, and further showed that mature CD27(high) NK cells were the predominant subpopulation of NK cells accumulating in the tumor microenvironment. The tumor-infiltrating NK cells displayed an activated cell surface phenotype and provided an early source of IFN-γ. Importantly, we also found that host IFN-γ was critical for NK cell infiltration into the local tumor site and that the tumor-infiltrating NK cells mainly suppressed tumor growth via the IFN-γ pathway. This work implicates the importance of IFN-γ as a positive regulatory factor for NK cell recruitment into the tumor microenvironment and an effective antitumor immune effector response.

Figure 1

Natural killer (NK) cell controls MCA205 tumor growth. (A) The expression of MHC class I (H‐2Kb) or NKG2D ligand (pan‐Rae‐1) was determined by flow cytometry (shaded histogram: control). (B) B6 mice were inoculated s.c. with MCA205 fibrosarcoma (4 × 10⁵) with or without anti‐AGM1 treatment. Data are represented as the mean ± SEM.

Figure 2

Early natural killer (NK) cell infiltration to MCA205 tumor. B6 mice were inoculated s.c. with MCA205 fibrosarcoma (4 × 10⁵) and tumors were harvested at the indicated times after inoculation. Tumor‐infiltrating lymphocytes (TIL) were isolated and then subjected to flow cytometry analysis. The representative FACS plots from the indicated groups of mice are shown and the numbers represent the percentage of cells in the different gates (A). The proportion of NK cells (B, NK1.1⁺ CD3⁻ cells) or NK cell subsets (Mac‐1^lo CD27^high, Mac‐1^high CD27^high, Mac‐1^high CD27^lo, electronically gated on NK1.1⁺ CD3⁻ cells) from the indicated groups of mice are presented. The data represent the mean ± SEM and are representative of two experiments.

Figure 3

Characterization of early MCA205 tumor‐infiltrating natural killer (NK) cells. B6 mice were inoculated s.c. with MCA205 fibrosarcoma (4 × 10⁵) and tumors were harvested 1 week after inoculation. Tumor‐infiltrating lymphocytes (TIL) were isolated and then subjected to flow cytometry analysis. The histogram plots shown are the expression levels for the indicated cell surface markers on the NK cell subsets in spleen or TIL electronically gated on the relevant population. Data are representative of two experiments (n = 4–5 mice).

Figure 4

Early interferon (IFN)‐γ production of natural killer (NK) cells in MCA205 tumor. B6 mice were inoculated s.c. with MCA205 fibrosarcoma (4 × 10⁵) and tumors were harvested 1 week after inoculation. Tumor‐infiltrating lymphocytes (TIL) were isolated and then incubated with Golgistop for 2 h without any in vitro stimulation. Cells were subjected to flow cytometry analysis to determine the intracellular level of IFN‐γ. (A) The dot plots show the population of IFN‐γ‐producing cells in TIL electronically gated on NK cells (NK1.1⁺ CD3⁻) or T cells (NK1.1⁻ CD3⁺). (B) The proportion of IFN‐γ‐producing cells from NK cells or T cells is shown. The data represent the mean ± SEM and are representative of two experiments (n = 5 mice). FSC, forward light scatter.

Figure 5

Importance of endogenous interferon (IFN)‐γ for natural killer (NK) cell MCA205 tumor infiltration and early antitumor immune responses. B6 wild‐type or IFN‐γ^−/− mice were inoculated s.c. with MCA205 fibrosarcoma (4 × 10⁵) and tumors were harvested 1 week after inoculation. Tumor‐infiltrating lymphocytes (TIL) were isolated and then subjected to flow cytometry analysis. The representative FACS plots from the indicated groups of mice are shown and numbers represent the percentage of cells in the different gates (A). The proportion of NK cells (NK1.1⁺ CD3⁻ cells) from the indicated groups of mice are presented (B). The data represent the mean ± SEM and are representative of two experiments. (C) B6 mice or IFN‐γ^−/− mice were inoculated s.c. with MCA205 fibrosarcoma (4 × 10⁵) with or without anti‐NK1.1 treatment. The data are represented as the mean ± SEM on day 7 after tumor inoculation. *P < 0.05 compared with untreated wild‐type (WT) mice. NS, not significant.