Tumor microenvironment macrophage inhibitory factor directs the accumulation of interleukin-17-producing tumor-infiltrating lymphocytes and predicts favorable survival in nasopharyngeal carcinoma patients

Abstract

The accumulation of an intratumoral CD4(+) interleukin-17-producing subset (Th17) of tumor-infiltrating lymphocytes (TILs) is a general characteristic in many cancers. The relationship between the percentage of Th17 cells and clinical prognosis differs among cancers. The mechanism responsible for the increasing percentage of such cells in NPC is still unknown, as is their biological function. Here, our data showed an increase of Th17 cells in tumor tissues relative to their numbers in normal nasopharynx tissues or in the matched peripheral blood of NPC patients. Th17 cells in tumor tissue produced more IFNγ than did those in the peripheral blood of matched NPC patients and healthy controls. We observed high levels of CD154, G-CSF, CXCL1, IL-6, IL-8, and macrophage inhibitory factor (MIF) out of 36 cytokines examined in tumor tissue cultures. MIF promoted the generation and recruitment of Th17 cells mediated by NPC tumor cells in vitro; this promoting effect was mainly dependent on the mammalian target of rapamycin pathway and was mediated by the MIF-CXCR4 axis. Finally, the expression level of MIF in tumor cells and in TILs was positively correlated in NPC tumor tissues, and the frequency of MIF-positive TILs was positively correlated with NPC patient clinical outcomes. Taken together, our findings illustrate that tumor-derived MIF can affect patient prognosis, which might be related to the increase of Th17 cells in the NPC tumor microenvironment.

FIGURE 1.

Percentage of Th17 cells was decreased in peripheral blood and increased in tumor tissues of NPC patients. A, representative FACS plots of circulating Th17 cells from two NPC patients and two healthy donors (HD). The number displayed is the percentage of the labeled cell population in this study. B, graph of the Th17 cell percentage in PBMCs from NPC patients (n = 21) and healthy donors (n = 21). C, representative FACS plots of circulating and tumor-derived Th17 cells in two matched NPC patients. D, graph of the percentage of Th17 cells among PBMCs and TILs from individual NPC patients (n = 21). Error bars represent S.E. Significance was determined using a χ² test or paired t test. (*, p < 0.001).

FIGURE 2.

Distribution of different lymphocyte subsets in PBMC and in normal nasopharynx (NILs) and nasopharynx tumor tissues (TILs) from individual NPC patients. Representative FACS plots of Th17 cells and Foxp3⁺ Treg (A) and IFNγ-producing cells in total CD4⁺ cells from two NPC patients out of five studied (B) are shown. Graphs for total CD4⁺ cells of the percentage of Th17 cells (C), CD4⁺ Foxp3⁺ cells (D), and IFNγ-producing cells in samples from NPC patients (n = 5) (E) are shown. Significance was determined using a paired t test (p < 0.05).

FIGURE 3.

Characterization and cytokine expression profiles of the Th17 cell subset among PBMCs and TILs from NPC patients. A, T cell surface markers were detected in Th17 cells from NPC patients. T cells were stimulated with phorbol 12-myristate 13-acetate/ionomycin for 4 h, then stained with fluorescence-conjugated antibodies against the markers shown, and analyzed by flow cytometry. B, graph of the percentage of cytokine-secreting Th17 cells among PBMCs and TILs from NPC patients (n = 5) and PBMCs from healthy donors (n = 5). Error bars represent the S.E. Significance was determined by the χ² test or paired t test. (*, p < 0.001).

FIGURE 4.

Generation and migration of Th17 cells mediated by NPC tumor cell lines. A, NPC tumor cell lines induce the differentiation of naive T cells into Th17 cells in vitro. Purified CD4⁺ naive T cells from healthy donors were stimulated with OKT3 and then co-cultured with the irradiated NPC cell line C666 (EBV⁺) or CNE2 (EBV⁻) in IL-2-containing medium for 7 days. The NP69 and LCL lines and the cytokines IL-1β and TGFβ were used as controls. All cultured cells were stained for Foxp3 and IL-17 for FACS analysis after stimulation of phorbol 12-myristate 13-acetate/ionomycin. The data represent one of three independent experiments. B, mean percentage of cytokine-secreting Th17 cells induced from CD4⁺ naive T cells from three experiments. C, migration of Th17 cells was increased in response to cultured supernatants from the NPC cell lines C666 and CNE2, relative to that from the normal NP cell line NP69 or with medium alone. The data represent one of three independent experiments. D, mean percentage of cytokine-secreting Th17 cells in the inner well and outer well after migration induced by supernatants from NP69, C666, or CNE2 cells from three experiments. Error bars represent the S.E. Significance was determined by the χ² test or paired t test. (*, p < 0.05).

FIGURE 5.

Role of MIF in the generation and migration of Th17 cells. A, generation of Th17 cells was decreased by the presence of either ISO-1 or siRNA against MIF. B, graph of IL-17-positive cells as a percentage of Th17 cells; data are from three independent experiments. Error bars represent S.E.; *, p < 0.05. C, ISO-1 noticeably inhibited the migration of Th17 cells exposed to the supernatant of the C666 cell line, and the migration of Th17 cells exposed to supernatant from MIF-siRNA-treated C666 cells was also decreased; data are representative of three independent experiments. D, percentage of Th17 cells migrating to the outer well; data are from three independent experiments. Error bars represent S.E.. *, p < 0.05. Significance was determined by performing paired t test.

FIGURE 6.

Signaling pathways of MIF involved in the generation and migration of Th17 cells. A, generation of Th17 cells induced by cytokines or C666 cells and MIF was decreased by the presence of rapamycin or CsA in the culture media at different levels; data are representative of three independent experiments. B, MIF promoting the generation of Th17 cells in vitro was dependent on mTOR signaling pathway. C, expression of CXCR4 on IL-17-positive and -negative cell population. D, specific neutralizing antibody against CXCR4 significantly inhibited the migration of Th17 cells exposed to the supernatant of the C666 cell line; data are representative of three independent experiments. E, percentage of Th17 cells migrating to the outer well; data are from three independent experiments. Error bars represent S.E. *, p < 0.05. Significance was determined by performing paired t test.

FIGURE 7.

Increased expression of MIF in TILs predicts improved patient survival. A, immunohistochemical staining shows varied intensities of MIF expression in the cytoplasm of tumor cells or tumor-associated lymphocytes (×40). B, percentage of MIF-positive lymphocytes around tumor cells was increased with the expression levels of MIF in tumor cells (R = 0.69, p < 0.0001). C and D, samples from NPC patients (n = 108) were divided into two groups based on positive or negative expression of MIF in NPC tumor cells. Disease-free survival and overall survival were significantly increased with increased expression of MIF in TILs, as displayed in Kaplan-Meier plots of overall survival.