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. 2016 Feb 3:6:20358.
doi: 10.1038/srep20358.

Circulating and tumor-infiltrating mucosal associated invariant T (MAIT) cells in colorectal cancer patients (VSports注册入口)

Limian Ling  1 , Yuyang Lin  1 , Wenwen Zheng  1 , Sen Hong  1 , Xiuqi Tang  1 , Pingwei Zhao  1 , Ming Li  2 , Jingsong Ni  2 , Chenguang Li  1 , Lei Wang  1 , Yanfang Jiang  2   3
Affiliations

Circulating and tumor-infiltrating mucosal associated invariant T (MAIT) cells in colorectal cancer patients

Limian Ling et al. Sci Rep. .

Abstract

Mucosal associated invariant T (MAIT) cells are important for immune defense against infectious pathogens and regulate the pathogenesis of various inflammatory diseases. However, their roles in the development of colorectal cancer (CRC) are still unclear. This study examined the phenotype, distribution, clinical relevance and potential function of MAIT cells in CRC patients. We found that the percentages of circulating memory CD8(+) MAIT cells were significantly reduced while tumor infiltrating MAIT cells were increased, especially in patients with advanced CRC. The serum CEA levels were positively correlated with the percentages of tumor infiltrating MAIT cells in CRC patients, but negatively correlated with the percentages of circulating MAIT in advanced CRC patients. Activated circulating MAIT cells from CRC patients produced lower IFN-γ, but higher IL-17. Furthermore, higher levels of Vα7. 2-Jα33, IFN-γ and IL-17A were expressed in the CRC tissues. Co-culture of activated MAIT cells with HCT116 cells enhanced IL-17 expression and induced HCT116 cell cycle arrest at G2/M phase in a contact- and dose-dependent manner, which was abrogated by treatment with anti-MR1 VSports手机版. Therefore, MAIT cells preferably infiltrate into the solid tumor in CRC patients and may participate in the immune surveillance of CRC. .

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Figure 1
Figure 1. Characterization of circulating MAIT cells.
PBMC were isolated from 22 healthy controls (HC) and 48 CRC patients and stained with fluorescent antibodies against CD3, CD161, TCRγδ, and TCRVα7.2. The frequency of CD3+TCRγδ−Vα7.2+CD161+ MAIT cells was characterized by flow cytometry. The cells were first gated on living lymphocytes and then on CD3+TCRγδ− T cells. The percentages of CD3+TCRγδ−Vα7.2+CD161+ MAIT cells in αβ T cells were determined. The different subsets of circulating MAIT cells were gated on CD3+TCRγδ−Vα7.2+CD161+ cells and the percentages of CD4+ CD8+ or CD4−CD8− (DN) MAIT cells were determined. Subsequently, the percentages of CD45RO+IL-18Rα+ MAIT cells in total CD8+ MAIT cells was further analyzed. Data are representative charts or expressed as the mean values of individual subjects. (a) Flow cytometry analysis of MAIT. (b) The percentages of circulating MAIT cells in CD3+TCRγδ− T cells. (c) The percentages of circulating γδ T cells. (d) Flow cytometry analysis of the different subsets of MAIT cells. (e) The percentages of CD4+, CD8+ or DN MAIT cells. (f) The percentages of CD45RO+IL-18Rα+ MAIT cells.
Figure 2
Figure 2. Accumulation of MAIT cells in colorectal neoplasms.
A total of 32 freshly surgical CRC tumor tissues were dissected to separat the central neoplasms from the surrounding non-tumor tissues. The tumor-infiltrating lymphocytes (TIL) and non-tumor infiltrating lymphocytes (NIL) were isolated from individual surgical tissues. In addition, 13 lamina propria tissues were obtained from non-tumor patients and their lamina propria mononuclear cells (LPMC) were isolated. Subsequently, the frequency of different subsets of MAIT cells in LPMC, NIL and TIL was determined by flow cytometry. Moreover, the numbers of CD3+CD161+MDR1+ MAIT cells in the central tumors and surrounding non-tumor tissues were determined by fluorescent assays. Data are representative charts, images or expressed as the mean ± SD of individual groups as well as the mean values of individual subjects. (a) Flow cytometry analysis. (b) The percentages of MAIT cells. (c) The frequency of different subsets of MAIT cells. (d) Immunofluorescent analysis of MAIT cells in the tumor tissues. (e) Quantitative analysis of the frequency of CD3+CD161+MDR1+ cells in total CD3+ cells in tissue sections.
Figure 3
Figure 3. Stratification analysis of the percentages of MAIT cells.
The CRC patients were stratified as early (I/II, n = 24) or advanced stage (III/IV, n = 24) and the percentages of MAIT cells in PBMC, NIL and TIL were compared with that in PBMC from 22 healthy controls (HC) and in LPMC from 13 non-tumor patients. Furthermore, the relationship among the percentages of MAIT cells in PBMC, NIL and TIL and the potential association between the percentages of MAIT cells in PBMC or TIL with the levels of serum CEA in individual groups of patients were analyzed. Finally, the percentages of circulating MAIT cells in five advanced CRC patients were characterized longitudinally by flow cytometry following different cycles of chemotherapies. Data are the mean values of individual subjects. (a) The frequencies of circulating MAIT cells. (b) The frequencies of MAIT cells in LPMC, NIL and TIL from early (n = 16) and advanced (n = 16) stage of CRC patients. (c) The percentages of circulating and tissue MAIT cells. (d) The relationship between the percentages of circulating and tissue MAIT cells. (e) The frequency of MAIT cells following chemotherapies. (f) The correlation between the levels of serum CEA and the percentages of MAIT cells in PBMC or TIL in the CRC patients.
Figure 4
Figure 4. The cytokine profile of MAIT cells in CRC patients.
CD3+CD161+Vα7.2+ MAIT cells were purified from six healthy controls or CRC patients and stimulated with, or without, PMA/ionomycin for 48 h. The levels of TNF-α, IFN-γ, IL-2, IL-4, IL-10, and IL-17 in the supernatants of cultured cells were determined by CBA. In addition, the relative levels of Vα7.2-Jα33, TNF-α, IFN-γ and IL-17A mRNA transcripts to the control GAPDH in 10 paired central neoplasm and surround non-tumor tissues were determined by quantitative RT-PCR. The relationship between the levels of Vα7.2-Jα33 and cytokine mRNA transcripts was further analyzed. Data are the mean values of individual subjects or expressed as the mean ± SD of individual groups. (a) The levels of cytokines. The dashy lines indicate the detection limits for these cytokines by the CBA. (b) Quantitative real-time PCR analysis of the relative levels of Vα7.2-Jα33, TNF-α, IFN-γ and IL-17A mRNA transcripts in the tumor tissues. (c) The correlation analysis of the relative levels of Vα7.2-Jα33, TNF-α, IFN-γ or IL-17A in the colorectal neoplasms.
Figure 5
Figure 5. MAIT cells induce the cell cycle arrest of HCT116 in a cell-cell contact-dependent manner.
MAIT cells were purified from five healthy controls and stimulated with, or without, PMA/ionomycin for 48 h. The unstimulated or activated MAIT cells were co-cultured with GFP+ HCT116 cells in a cell-cell contact or non-contact manner. The MAIT cells or HCT116 cells in medium alone served as the controls, as illustrated in (a). Subsequently, the levels of TNF-α, IFN-γ and IL-17 in the supernatants of cultured cells were determined by CBA and the cell cycle status of HCT116 cells was examined by flow cytometry. Data are representative histograms of cell cycling or expressed as the mean values or the mean ± SD of each group from three separate experiments. (b) The levels of cytokines. The dashy lines indicate the detection limits for these cytokines by the CBA. (c) Analysis of cell cycling in HCT116 cells following co-culture with MAIT cells with or without anti-MR1 (α-MR1). (d) Dose curves of MAIT cell activity.
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