MAIT cells reside in the female genital mucosa and are biased towards IL-17 and IL-22 production in response to bacterial stimulation

Abstract

The female genital tract (FGT) mucosa is a critically important site for immune defense against microbes. Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population that recognizes microbial riboflavin metabolite antigens in an MR1-dependent manner. The role of MAIT cells in the FGT mucosa is unknown. Here, we found that MAIT cells and MR1⁺ antigen-presenting cells were present in the upper and lower FGT, with distinct tissue localization of MAIT cells in endometrium vs. cervix. The MAIT cells from the FGT and blood displayed a distinct phenotype with expression of interleukin (IL)-18Rα, CD127, α4β7, PD-1, as well as the transcription factors promyelocytic leukemia zinc finger (PLZF), RORγt, Helios, Eomes, and T-bet. Their expression levels of PLZF and Eomes were lower in the FGT compared with blood. When stimulated with Escherichia coli, MAIT cells from the FGT displayed a bias towards IL-17 and IL-22 expression, whereas blood MAIT cells produced primarily IFN-γ, TNF, and Granzyme B. Furthermore, both FGT- and blood-derived MAIT cells were polyfunctional and contributed to the T-cell-mediated response to E. coli. Thus, MAIT cells in the genital mucosa have a distinct IL-17/IL-22 profile and may have an important role in the immunological homeostasis and control of microbes at this site.

Figure 1

Localization and spatial distribution of MAIT cells in the FGT. Representative immunofluorescence images of (a) endometrial (n=6), (b) endocervical (n=2), (c) transformation zone (n=2), and (d) ectocervical (n=6) tissue sections stained for Vα7.2⁺ (red) and IL-18Rα⁺ (green) cells. To be noted, the 40× pictures of the ectocervix were rotated 90° right from the 10× overview picture. DAPI (blue) was used as a counterstain for visualization of cell nuclei. Double-positive (MAIT) cells are shown in yellow and are indicated by the white arrows. The images were collected with 10× and 40× objectives. The scale bars are 250 μm in the images in the first column and 60 μm in the images in the other three columns.

Figure 2

Localization and spatial distribution of MR1⁺ APCs in the endometrium and ectocervix. Representative immunofluorescence images of endometrial (n=2) and ectocervical (n=2) tissue sections stained for (a) MR1 (red) and HLA-DR (green); the scale bars are 250 μm in the images in the first column and 60 μm in the images in the other three columns. (b) Representative immunofluorescence images of endometrial and ectocervical tissue sections stained for MR1 (red) with CD11c (green) and MR1 (red) with Langerin (green); the scale bars are 60 μm. Double-positive cells are shown in yellow and are indicated by the white arrows. DAPI (blue) was used as a counterstain for visualization of cell nuclei. The images were collected with 10× and 40× objectives.

Figure 3

Enumeration of the frequency of MAIT cells in the FGT and blood. (a) MAIT cell (CD161^highVα7.2⁺) gating strategy and representative scatter plots. (b) Frequencies of MAIT cells out of T cells (CD45⁺CD3⁺) from the endometrium (n=19), cervix (n=14), and blood (n=16). (c) Frequencies of CD8 (CD4^-CD8⁺), CD4 (CD4⁺CD8^-), and DN (CD4^-CD8^-) MAIT cells out of total MAIT cells from the endometrium (n=10), cervix (n=5), and blood (n=10). The cervix samples consisted of pooled endocervical and ectocervical cells. Each dot represents a different patient; endometrium (circle), cervix (triangle), and blood (square). Horizontal lines represent median ± interquartile range. *P<0.05, **P<0.01, ***P<0.001.

Figure 4

Expression of transcription factors in MAIT cells in the FGT and blood. Expression levels of PLZF, RORγt, Helios, Eomes, and T-bet transcription factors in MAIT cells (GMFI, geometric mean intensity fluorescence). (a) Scatter plots represent the expression levels of transcription factors in MAIT cells from the endometrium (n=7), cervix (n=4), and blood (n=6). Each symbol represents a different patient; endometrium (circle), cervix (triangle), and blood (square). Horizontal lines represent the median ± interquartile range, *P<0.05. (b) Representative histograms showing the expression levels of transcription factors in MAIT cells (red), CD4⁺ T cells (blue), and CD8⁺ T cells (green) from the endometrium (n=7), cervix (n=4), and blood (n=6).

Figure 5

Cytokine and GrzB production profile by MAIT cells in the FGT and blood upon E. coli stimulation. (a) Representitive dot plot showing IFN-γ, TNF, IL-17, IL-22 and GrzB production by MAIT cells from FGT and blood samples that were either unstimulated (upper panel; n=8), stimulated with E.coli (middle panel; FGT n=10, blood n=6), or stimulated with PMA (lower panel; n=4). (b) Frequencies of cytokines and GrzB production by MAIT cells from the FGT (n=10) and blood (n=6). Each symbol represents a different patient; FGT (circle) and blood (square). Horizontal lines represent median ± interquartile range. *P<0.05, **P<0.01, ***P<0.001.

Figure 6

Polyfunctional characterization of MAIT cells from the FGT (n=10) and blood (n=6), using SPICE software. (a) Left and right pie charts representing the number of functions expressed by MAIT cells from the FGT and blood respectively (b) Bar and pie charts showing the distribution of GrzB, IFN-γ, IL-17, IL-22 and TNF combinations expressed by MAIT cells from the FGT (blue) and blood (red). Permutation test was performed between the bar charts. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.