Fig. 4

Breast epithelial carcinoma cells present microbial products to mucosal associated invariant T (MAIT) cells via MR1. a MDA-MB-231 breast carcinoma cells were exposed to killed Escherichia coli or mock-treated, then fixed and permeabilized and stained for MR1 (black line) compared with isotype control (gray histogram). b Left plots: Flow cytometric analysis showing tumor necrosis factor (TNF)-α production by primary human MAIT cells from peripheral blood in response to E. coli-treated MDA-MB-231 breast carcinoma cells. Cells expressing CD161 were magnetically sorted from freshly isolated peripheral blood mononuclear cells (PBMCs) to enrich for MAIT cells and to remove potential antigen-presenting cell (APC) populations. MDA-MB-231 cells were pulsed with fixed E. coli or mock-treated (no E. coli) and used to stimulate the CD161-selected population in the presence or absence of an anti-MR1 blocking antibody. Expression of TNF-α was measured by intracellular cytokine staining after ~ 24 hours. Graph on right: Quantification of the percentage of the MAIT cell population showing positive TNF-α staining in the indicated conditions. Each symbol represents the result of an independent analysis. c Intracellular cytokine staining showing TNF-α production by the indicated in vitro-expanded MAIT cells after exposure to mock-treated or E. coli-pulsed MDA-MB-231 cells in the presence or absence of an anti-MR1 blocking antibody. d T-cell receptor (TCR) cell surface staining of MAIT cells after exposure to E. coli-pulsed MDA-MB-231 cells in the presence or absence of an anti-MR1 blocking antibody. MAIT cell TCR expression (geometric mean fluorescence intensity [gMFI]) is expressed as a percentage of that for the corresponding MAIT cells exposed to mock-treated APCs. Each symbol represents the result of an independent analysis. Statistical results are for a two-tailed, one-sample t test comparison to a theoretical mean of 100%. n.s. Not significant