Background
Most breast cancers initially arise in the epithelial ducts [
1]. Although epithelial surfaces of the gastrointestinal and urogenital tracts are well known to be colonized by intricate microbial communities, it has only recently become clear that the breast ducts also contain a complex microbiota [
2‐
8]. Because T cells that are specific for microbial antigens are now known to play key roles in the progression of tumors arising in epithelial layers of the intestine [
9], the observation that the breast ducts contain microbial colonists raises the question whether antimicrobial T cells may also contribute, either positively or negatively, to the genesis of breast cancers. Consistent with this possibility, the presence of cancerous tissue has been found to be associated with alterations to the microbiome of the local breast tissue [
8]. Hence, dysbiosis of the breast duct microbiome might lead to increased or altered T-cell activation. A central obstacle to assessing the role of microbe-specific T cells in breast cancer is that little is known about the T-cell compartment found within human breast ducts, and particularly, the presence of T cells that recognize microbial antigens has not yet been established.
We recently investigated the intraepithelial lymphocyte (IEL) compartment from isolated human breast epithelial duct organoids and observed that it includes T cells with Vα7.2
+ T-cell receptors (TCRs) [
10]. TCR use of the Vα7.2 segment (T-cell receptor alpha variables 1 and 2 [TRAV1–2]) is one of the central characteristics of a distinctive subset called
mucosal associated invariant T cells (MAIT cells) [
11]. MAIT cells are innate T cells that recognize specific microbially synthesized precursors of riboflavin as antigens presented by the nonclassical antigen-presenting molecule MR1 [
12,
13] and are thus microbially reactive T cells. They typically coexpress CD161, promyelocytic leukemia zinc finger protein (PLZF), and interleukin (IL)-18Rα and can be readily detected using MR1 tetramers loaded with 5-(2-oxoprophylideneamino)-6-
d-ribitylaminouracil (5RU) [
12,
14‐
16]. MAIT cells are comparatively abundant in human peripheral blood, typically comprising 0.5–10% of the T-cell population [
16]. MAIT cells have also been detected in a variety of other tissues, including liver, lung, kidney, intestine, female genital tract, prostate, and ovary [
14,
17‐
22]. MAIT cells from blood mainly produce interferon (IFN)-γ and tumor necrosis factor (TNF)-α upon activation, and they efficiently mediate cytolytic responses [
23]. In contrast, compared with those from the blood, MAIT cells from the female genital tract expressed higher levels of T-helper 17 cell (Th17) cytokines (IL-17A and IL-22) and lower levels of Th1 cytokines (IFN-γ and TNF-α) in response to
Escherichia coli [
20]. Thus, MAIT cells from distinct anatomical locations may have important functional differences.
Intriguingly, recent studies suggest that MAIT cells may play a role in the etiology of colon adenocarcinomas. MAIT cells were found to accumulate at tumor sites in patients with colon cancer, and the tumor-associated MAIT cells produced lower levels of IFN-γ than those obtained from healthy intestinal tissue from the same donor [
24]. In another study, circulating MAIT cells from patients with colorectal cancer were found to have reduced expression of IFN-γ and TNF-α and elevated levels of IL-17A compared with MAIT cells from the blood of healthy control subjects [
25]. It is not yet clear whether the apparent Th17 bias of tumor-associated and blood MAIT cells observed in patients with colon cancer is due to a functional skewing that occurs in the context of malignancy or whether it is a result of the expansion of a MAIT cell subset that is normally present only within select mucosal epithelial sites. Similarly, the role of microbial stimulation and/or dysbiosis in the MAIT cell response during colon cancer is as yet unknown. Nevertheless, the observation that Th17-biased MAIT cells are recruited to the sites of colon adenocarcinomas raises the possibility that these T cells also play a role in breast carcinomas. Therefore, in this analysis, we sought to investigate the phenotypes and functional characteristics of breast epithelium-derived MAIT cells, as well as to determine the ability of microbially exposed breast carcinoma cells to elicit responses from human MAIT cells.
Methods
Breast tissue acquisition and preparation
Noncancerous breast tissue from reduction mammoplasties or prophylactic mastectomies was obtained from the Cooperative Human Tissue Network (a National Cancer Institute-supported resource) or from the UW Translational Science BioCore-BioBank, in accordance with an institutional review board (IRB)-approved protocol. Human breast epithelial organoids were isolated as previously described [
10]. Briefly, breast tissue was minced and digested overnight in a 37 °C shaker with 1× collagenase/hyaluronidase in Complete EpiCult B Human Media (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with 5% FBS (HyClone; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). After incubation, digested tissue was spun for ≤ 1 minute at 80–100 ×
g to produce a pellet enriched for epithelial ductal organoids. The pellet was washed, and organoids were collected on a 40-μm filter. Organoids were cryopreserved in 50% FBS/6% dimethyl sulfoxide and stored in liquid nitrogen. Single-cell suspensions from organoids were prepared for all experiments by trypsinizing the organoids using 2-3 ml of 0.1% ethylenediaminetetraacetic acid (EDTA)/trypsin solution (diluted in PBS from 0.5%; Life Technologies, Carlsbad, CA, USA) for ≥ 3 minutes. EDTA/trypsin reaction was quenched using serum-containing media and spun. Pellets were resuspended and filtered using 40–70-μm filters. Cells were spun, supernatants discarded, and breast organoid-derived cells resuspended for experimentation.
Peripheral blood mononuclear cell isolation
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors according to an IRB-approved protocol. Written informed consent was obtained from all donors. PBMCs were isolated from blood using Ficoll-Paque PLUS (GE Healthcare Bio-Sciences) as previously described [
10].
Flow cytometric analyses
For surface stains, cells were washed with PBS, blocked with 20% human AB serum (Atlanta Biologicals, Flowery Branch, GA, USA) for 15 minutes, stained with fluorochrome-conjugated antibodies for 30 minutes at 4 °C, washed, resuspended in PBS, and analyzed by flow cytometry (BD LSR II cytometer; BD Biosciences, San Jose, CA, USA) with FlowJo analysis software (version 9.3.1; FlowJo, Ashland, OR, USA). MR1 tetramers (5RU and 6FP provided by National Institutes of Health, Bethesda, MD, USA) were used at a 1:100 final dilution. In general, optimal staining was seen when tetramers were stained for 40 minutes in the dark at room temperature prior to surface staining. Intracellular cytokine staining was performed according to the manufacturer’s recommendations using the BD Cytofix/Cytoperm kit in the presence of BD GolgiStop or BD GolgiPlug protein transport inhibitors (BD Biosciences). PLZF staining was performed with the BD Cytofix/Cytoperm kit.
The following fluorochrome-conjugated flow cytometry antibodies were used for analysis: CD45 (clone HI30), CD3 (OKT3), Vα7.2 (3C10), CD161 (HP-3G10), IL-18Rα (H44), TNF-α (Mab11), IFN-γ (4S.B3), IL-17A (BL168), natural killer group 2 member D (NKG2D) (1D11), CD31 (WM59), epithelial cell adhesion molecule (9C4), CD49f (GoH3), major histocompatibility complex class I-related chains A and B (MICA/B) (6D4), CD56 (HCD56), MR1 (26.5), and NKp46 (9E2) (all from BioLegend, San Diego, CA, USA); Vβ2 (MPB2D5), Vβ8 (56C5.2), Vβ13.1 (IMMU 222), Vβ13.2 (H132), and Vβ13.6 (JU74.3) (all from Beckman Coulter Life Sciences, Indianapolis, IN, USA); UL16-binding protein 1 (ULBP1) (170818), ULBP2/5/6 (165903), ULBP3 (166510), ULBP4 (709116), immunoglobulin G2A (IgG2A) (20102), and IgG2B (133303) (R&D Systems, Minneapolis, MN, USA); and PLZF (R17-809; BD Pharmingen, San Diego, CA, USA).
Short-term in vitro expansion of MAIT cells
Single-cell suspensions prepared from breast duct organoids or freshly isolated PBMCs were stained using 5RU-loaded MR1 tetramer and antibodies against CD45, CD3, Vα7.2 TCR, and CD161. MAIT cells (1–1000 cells/well) were sorted into 96-well round-bottomed plates. Irradiated PBMCs were added at a density of 1 × 105 cells/well in T-cell medium (RPMI 1640, 15% heat-inactivated bovine calf serum, 3% human AB serum, 1% penicillin/streptomycin [P/S], 200 U/ml recombinant human IL-2) containing 5 μg/ml phytohemagglutinin (PHA; Sigma-Aldrich, St. Louis, MO, USA), and the cultures were maintained at 37 °C in a humidified incubator with 5% CO2. If necessary, the cells were restimulated after 4–6 weeks to induce another round of proliferation by adding irradiated PBMCs in T-cell medium containing 5 μg/ml PHA. The MAIT cell composition of the expanded cells was assessed by flow cytometry after ~ 8 weeks of in vitro expansion using 5RU-loaded MR1 tetramer staining, and lines that were comprised of ≥ 95% MAIT cells were used for functional analyses.
Phorbol 12-myristate 13-acetate and ionomycin stimulation
Cells were washed and resuspended in culture medium (RPMI 1640, 15% HI-BCS, 3% human AB serum, 1% P/S) containing a final concentration of 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of monensin or brefeldin A (BD Biosciences). The cells were stimulated for ~ 6 hours at 37 °C. Unstimulated control cells were incubated in parallel in culture medium alone. Because we observed that effector cytokine production decreased as cell density increased (data not shown), we used a maximum density of 2.5 × 105 cells/well for stimulation. After stimulation, cells were harvested and prepared for flow cytometry.
Breast carcinoma cells and bacterial exposure
The breast carcinoma cell line MDA-MB-231 was obtained from the American Type Culture Collection (Manassas, VA, USA) as an authenticated cell line and maintained in DMEM/F-12 medium (Corning, Corning, NY, USA) supplemented with 10% HI-BCS or 10% FBS and 1% P/S; Mediatech, Manassas, VA, USA). To prepare carcinoma cells for functional assays, the carcinoma cells were plated at a subconfluent density in flat-bottomed tissue culture plates and allowed to adhere for ~ 3 hours at 37 °C. E. coli strain K12 was cultured in Luria-Bertani (LB) broth in a 37 °C shaker overnight and then stored frozen in 40% glycerol/LB broth. Aliquots were thawed prior to use, and E. coli was washed in PBS three times. Bacteria were fixed in 1% formalin for 3 minutes, then washed three times with PBS and resuspended in the DMEM/F-12 medium used to culture the tumor cells. Carcinoma cells were exposed to E. coli overnight at a multiplicity of infection of 400–500 or incubated in medium alone (mock). The wells were then washed with PBS, and fresh DMEM culture medium containing 10% serum and 1% P/S was added.
Enrichment of primary MAIT cells from PBMCs
We performed a magnetic sorting step to remove potential MR1
+ antigen-presenting cell (APC) types (e.g., monocytes, B cells, dendritic cells) from PBMC samples prior to using them to test the responses of primary MAIT cells to MDA-MB-231 cells as APCs. CD161
+ cells were positively selected using indirect magnetic bead separation (Miltenyi Biotec, Bergisch Gladbach, Germany). The PBMCs were incubated first with an anti-CD161-phycoerythrin (PE) antibody and then with anti-PE microbeads, followed by passage over a Miltenyi Biotec LS column. Purity of the resulting cell preparations was assessed by flow cytometric analysis, as shown in Additional file
1: Figure S4.
Functional assays
Cell preparations containing primary MAIT cells (CD161+ PBMCs or breast epithelial organoid cells) or in vitro-expanded MAIT cultures were added to wells containing E. coli-exposed or mock-treated MDA-MB-231 breast carcinoma cells. CD161+ PBMCs and in vitro-expanded MAIT cells were added at a 1:1 ratio to breast carcinoma cells, whereas the breast epithelial organoid cells (which are composed of both IELs and epithelial cells) were added at a 3:1 ratio. Where indicated, the following blocking antibodies were added to the cocultures: 20 μg/ml anti-MR1 (clone 26.5; BioLegend) or 5 μg/ml anti-NKG2D (1D11; BioLegend). The carcinoma and effector cells were coincubated at 37 °C for ~ 18 hours, then monensin (GolgiStop) or brefeldin A (GolgiPlug) was added to all cultures, and the cells were coincubated for an additional 6 hours. After ~ 24 hours of coincubation, the effector cells were resuspended using cold EDTA (500 mM) in PBS, washed, and analyzed by flow cytometry.
Statistical analysis
To assess statistical significance, samples from different tissues (e.g., PBMC vs. breast duct organoids) were analyzed using a Mann-Whitney U test. Different populations of cells within the same sample (e.g., MAIT cells vs. non-MAIT cells from breast duct organoid preparations) were evaluated using a Wilcoxon matched pairs analysis. Where indicated, a two-tailed, one-sample t test was used to assess whether individual treatment groups showed a significant difference compared with a hypothetical value of 100%.
Discussion
We show in the present study that MAIT cells are present in the epithelial ducts of human breast tissue and that MAIT cells mediate effector responses to breast carcinoma cells that have been exposed to microbial compounds. The role that the breast duct microbiome plays in breast cancer initiation and progression is currently completely uncharted, because the composition and dynamics of the breast duct microbiota are just beginning to be explored. However, intriguing hints are starting to emerge that dysbiosis of the normal breast duct microbiota may be associated with breast cancer, because recent studies have documented differences in the composition of the microbial taxa associated with breast cancer tissue compared with healthy breast tissue [
2‐
4,
6‐
8]. Given that MAIT cells specifically recognize riboflavin metabolites as antigens presented by MR1 molecules, it is of particular interest that riboflavin-producing bacterial species have been found to be present at higher relative abundance in women with breast cancer, including
Enterobacteriaceae (the family that includes
E. coli),
Bacillus, and
Staphylococcus species [
5,
6]. Thus, understanding the phenotypic and functional properties of the MAIT cells found within breast ducts is an important step in determining whether these cells play a role during the carcinogenesis of breast duct epithelial cells.
In contrast to MAIT cells from peripheral blood, which show almost exclusively a Th1 phenotype, we observed that about one-third of the breast duct MAIT cell population produced IL-17A. Similarly, MAIT cells from the female genital tract were recently shown to be enriched for IL-17A and IL-22 production [
20]. Hence, Th17 polarization may be a common feature of MAIT cells from ductal and skin epithelial tissues. Because IL-17A enhances epithelial barrier integrity, MAIT cells with Th17 polarization might contribute to the maintenance of a healthy epithelial surface. It is not clear whether secretion of IL-17A promotes or hinders the initial stages of epithelial carcinogenesis. However, there is a growing body of evidence that after tumors are established, IL-17A has protumorigenic functions, because IL-17A has been shown to be produced by breast cancer tumor-infiltrating lymphocytes and thereby to promote chemoresistance, proliferation, and migration of breast cancer cells [
38]. Thus, it will be of great interest to determine whether IL-17A-producing MAIT cells are selectively expanded within breast carcinoma tissue, as has been observed recently for colon carcinomas [
24].
In contrast, the IFN-γ-producing subset of breast duct MAIT cells might be expected to have antitumor functions. However, we found that breast MAIT cells that were exposed to
E. coli-pulsed breast carcinoma cells produced only IL-17A and TNF-α. It is not clear whether the breast carcinoma cells somehow selectively suppress MAIT cell IFN-γ production. Alternatively, it is possible that cancerous cells that have taken up enterobacterial antigens selectively activate only the Th17-polarized subset of breast duct MAIT cells. One mechanism that might explain the selective activation of a particular functional subset of MAIT cells is the observation that the Vβ chain use of the TCR influences the ability of MAIT cells to respond to microbial species, presumably as a result of structural or quantitative variations in the antigenic compounds produced by different types of bacteria [
30]. We found that the breast duct MAIT cell population appeared to have a comparatively low frequency of TCRs that use the two Vβ chains that are most common in the blood MAIT cell population (i.e., Vβ2 and Vβ13.2). Because these Vβ chains have been shown to confer stronger reactivity to
E. coli [
30], this observation would be consistent with the possibility that the TCR repertoire of the breast duct MAIT cell population is shaped by a distinct composition of microbial colonists. If this is the case, an intriguing extension of this hypothesis is the possibility that specific functional subsets of MAIT cells (e.g., Th1- vs. Th17-polarized) bear distinct TCR Vβ uses and thus differ in their microbial reactivity.
It is also interesting that in contrast to the blood, where more than half of the Vα7.2
+ T cells typically are stained by the MR1-5RU tetramer, only about one-third of the breast duct Vα7.2
+ T cells were tetramer-positive. One potential explanation for this is that the tetramer-negative Vα7.2
+ T cells recognize completely distinct antigens presented by MR1 and therefore are not bound by MR1 tetramers loaded with the 5RU compound. Alternatively, these cells may simply not be MR1-restricted. If this is the case, it is intriguing to speculate that they may belong to another conserved Vα7.2
+ T-cell population that is now known as germline-encoded mycolyl-reactive T cells. This subset of T cells also uses Vα7.2 and has been shown to be restricted by CD1b and to recognize mycolate antigens produced by mycobacterial species [
39].
A further question of interest relates to the features of breast duct epithelial cells that are required to activate the responses of breast duct MAIT cells. We found that MAIT cells are readily activated by breast carcinoma cells and that this activation is dependent on prior bacterial exposure of the carcinoma cells and can be blocked by an anti-MR1 antibody. It is not yet clear whether normal (i.e., nontransformed) breast duct epithelial cells are also able to activate MAIT cells via MR1-mediated antigen presentation or whether their activation is normally mediated by other immune cell types present in the local tissues (e.g., monocytes, macrophages, or dendritic cells). However, because neoplastic cells often upregulate ligands for NKG2D, and because the addition of an NKG2D blocking antibody resulted in a partial reduction of MAIT cell effector responses, it is possible that the NKG2D pathway promotes the ability of MAIT cells to detect and respond to breast carcinomas.
Conclusions
Together, these findings underscore the likelihood that there exists a tripartite interaction among MAIT cells, breast duct epithelial cells, and the breast microbiome that may play a role in breast carcinogenesis or during the progression of established tumors. Because breast duct MAIT cells appear to use distinct TCR sequences and include multiple functional subsets (e.g., Th1- or Th17-polarized), the breast-resident MAIT cell population likely mediates divergent responses to different types of challenge. Our data suggest that microbially exposed breast carcinoma cells may selectively activate only Th17-polarized MAIT cells from breast ducts and not the Th1-polarized subset. Therefore, a critical question for future analysis is to investigate whether Th17-polarized MAIT cells are enriched in breast carcinomas and how this impacts prognosis.