Mucosal Immunity in the Female Murine Mammary Gland

Abstract

The mammary gland is not classically considered a mucosal organ, although it exhibits some features common to mucosal tissues. Notably, the mammary epithelium is contiguous with the external environment, is exposed to bacteria during lactation, and displays antimicrobial features. Nonetheless, immunological hallmarks predictive of mucosal function have not been demonstrated in the mammary gland, including immune tolerance to foreign Ags under homeostasis. This inquiry is important, as mucosal immunity in the mammary gland may assure infant and women's health during lactation. Further, such mucosal immune programs may protect mammary function at the expense of breast cancer promotion via decreased immune surveillance. In this study, using murine models, we evaluated mammary specific mucosal attributes focusing on two reproductive states at increased risk for foreign and self-antigen exposure: lactation and weaning-induced involution. We find a baseline mucosal program of RORγT⁺ CD4⁺ T cells that is elevated within lactating and involuting mammary glands and is extended during involution to include tolerogenic dendritic cell phenotypes, barrier-supportive antimicrobials, and immunosuppressive Foxp3⁺ CD4⁺ T cells. Further, we demonstrate suppression of Ag-dependent CD4⁺ T cell activation, data consistent with immune tolerance. We also find Ag-independent accumulation of memory RORγT⁺ Foxp3⁺ CD4⁺ T cells specifically within the involution mammary gland consistent with an active immune process. Overall, these data elucidate strong mucosal immune programs within lactating and involuting mammary glands. Our findings support the classification of the mammary gland as a temporal mucosal organ and open new avenues for exploration into breast pathologic conditions, including compromised lactation and breast cancer.

FIGURE 1.

Evidence for coordinated mammary epithelial and immune cell regulation across a reproductive cycle. (A) H&E and IHC for CK18 and CD45 in human nulliparous breast tissue showing close physical proximity (original magnification ×40, scale bar, 50 μm). (B) H&E and IHC for E-cadherin and CD45 in nulliparous mouse mammary tissue (original magnification ×40, scale bar, 50 μm). (C) IHC for CD45 in BALB/c mouse mammary tissue across a reproductive cycle. Color deconvolution using an algorithm on Aperio ScanScope was used, for which blue indicates negative epithelium and stroma, and yellow, orange, and red indicate increasing intensities of DAB signal (CD45) in nulliparous (Nullip), pregnant (Preg, D16), lactation (Lac), involution (InvD2, InvD4, InvD6, and InvD8), and regressed at 6 wk (Reg) mice (original magnification ×40, scale bar, 50 μm). (D) Representative flow cytometry gating schema to identify live CD45⁺ cells in mammary digests in a nulliparous host. (E) Quantitation of abundance of CD45⁺ cells normalized to milligrams of mammary tissue weight from distinct reproductive hosts. n = 3–7 per group, ****p ≤ 0.0001 by one-way ANOVA with Tukey multiple comparison test. (F–H) Expression of mucosal-associated mRNAs at various reproductive states, (F) IgA H chain, (G) mucins 3, 10, and 1 (Muc), and (H) orosomucoid 1 (Orm1) and serum amyloid A1 (Saa1). Data were obtained from a publicly available microarray data set and normalized to expression in Nullip, pregnant (D8.5 and D14.5), lactation (LacD1, LacD3, and LacD7), involution (InvD1, InvD2, InvD3, and InvD4), and regressed (InvD20) mice.

FIGURE 2.

Mammary gland dendritic cell abundance and activation are altered by reproductive state. (A) Representative flow gating schema to identify mammary dendritic cells in nulliparous mouse mammary digest, with inguinal LN removed. Gating scheme starts with live CD45⁺ cells (Fig. 1D). (B) Quantitation of total dendritic cells (CD45⁺ Ly6C⁻ Ly6G⁻ B220⁻ F480⁻ CD11c⁺ MHCII⁺) normalized to the milligram of mammary tissue weight or (C) as a percentage of total CD45⁺ cells. (D–F) Flow histograms and quantitation of dendritic cell maturation markers CD86, MHCII, and CD80 as average fluorescence (gMFI) at various reproductive states. Groups: nulliparous (Nullip), lactation, involution (InvD2, InvD4, InvD6, and InvD8), and regressed. n = 3–7 per group, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 by one-way ANOVA with Tukey multiple comparison test, ^ǂp ≤ 0.05 by two-tailed unpaired Student t test.

FIGURE 3.

CD11b and CD103 dendritic cell subtypes are dynamically regulated during lactation and involution. (A) Representative flow plots from nulliparous, lactation, InvD2, InvD6, and regressed hosts showing CD11b and CD103 subsets vary by reproductive state. (B) Quantitation of CD11b⁻/CD103⁻ dendritic cells (CD45⁺ Ly6C⁻ Ly6G⁻ B220⁻ F480⁻ CD11c⁺ MHCII⁺CD11b⁻CD103⁻), (C) CD11b⁺ dendritic cells (CD45⁺ Ly6C⁻ Ly6G⁻ B220⁻ F480⁻ CD11c⁺ MHCII⁺CD11b⁺CD103⁻), and (D) CD103⁺ dendritic cells (CD45⁺ Ly6C⁻ Ly6G⁻ B220⁻ F480⁻ CD11c⁺ MHCII⁺CD11b⁻CD103⁺) as percentage of total dendritic cells. (E) Pie charts depicting average abundance of dendritic cell populations at various reproductive stages and color coded to match Fig. 3B–D. Groups: nulliparous (Nullip), lactation, involution (InvD2, InvD6, and InvD8), and regressed mice. n = 3–7 per group, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 by one-way ANOVA with Tukey multiple comparison test, ^ǂp ≤ 0.05 by two-tailed unpaired Student t test.

FIGURE 4.

Dendritic cell Ag cross-presentation is enhanced during involution. (A) Schematic of ex vivo Ag cross-presentation assay using presence of processed Ag in MHC1 complex (MHCI-SIINFEKL) as readout for Ag uptake, processing, and presentation. (B) Representative flowcharts showing MHCI-SIINFEKL levels on ex vivo cultured mammary dendritic cells (DCs) as detected by the 25-D1 Ab in InvD6 sample without 25-D1 Ab staining (fluorescence minus one [FMO] control, left), lactation (middle), and InvD6 (right). Positive staining is shaded red. (C) Quantitation of the percentage of total dendritic cells positive for 25-D1 staining. Groups: nulliparous (Nullip), lactation, and involution (InvD2 and InvD6) mice. n = 4–7 per group, *p ≤ 0.05 by one-way ANOVA with Tukey multiple comparison test, ^ǂp ≤ 0.05 by unpaired two-tailed Student t test.

FIGURE 5.

In presence of mammary Ag, lactation and involution hosts have reduced ability to activate naive CD4⁺ T cells. (A) Schematic of experiment in which CD4⁺ T cells specific for OVA Ag (DO11.10) were adoptively transferred into BALB/c hosts, then OVA Ag or PBS was introduced locally into either the left or right no. 4 mammary gland. (B) DO11.10 CD4⁺ T cells were detected in the inguinal (mammary tissue draining) LN, and absolute cell counts were calculated by the addition of counting beads (top left panel). Representative flow data from inguinal draining LNs of mammary glands that received OVA Ag or PBS. (C) DO11.10 T cell count in inguinal draining LNs of mammary tissue by reproductive state. Each mouse received PBS in one mammary gland to serve as an internal control, and the contralateral gland received OVA Ag. Each control (red dots)–experimental (blue dots) pair of mammary glands within one animal is connected by a black line. Two-tailed paired T tests were performed, *p ≤ 0.05, **p ≤ 0.01. n = 25 for nulliparous, n = 15 for lactation, n = 18 for InvD2, n = 7 for InvD6, and n = 5 for regressed hosts. (D) The ratio of T cell counts in the inguinal LNs that drain mammary glands that received OVA Ag to those that received PBS. One way ANOVA with Tukey multiple comparison test was performed, *p ≤ 0.05, **p ≤ 0.01. Statistical outliers were calculated using GraphPad Prism outlier test, resulting in three data points being removed. n = 25 for nulliparous, n = 14 for lactation, n = 17 for InvD2, n = 7 for InvD6, and n = 4 for regressed. Combined results of four independent experiments are shown. Groups: nulliparous (Nullip), lactation, involution (InvD2 and InvD6), and regressed mice.

FIGURE 6.

Phenotyping of mammary gland CD4⁺ T cells across a reproductive cycle. (A) Identification of CD4⁺ T cells in nulliparous mammary gland digest (live, CD45⁺ CD4⁺ CD11b⁻). (B) CD4⁺ T cell abundance as the percentage of total CD45⁺ cells. (C) Representative flow plots of RORγT and Foxp3. (D) Abundance of RORγT⁺ (Foxp3⁻) and (E) Foxp3⁺ (RORγT⁻) CD4⁺ T cells by reproductive stage. (F) Representative flow plots of Gata3. (G) Incidence of Gata3⁺ CD4⁺ T cells in the mammary gland. (H) Representative flow plots of PD-1. (I) Incidence of PD-1⁺ CD4⁺ T cells in the mammary gland by reproductive state. For (B), (D), (E), (G), and (I), there are n = 4–10 mice per group. (J) Average incidence of 16 variants of CD4⁺ T cells are shown for each reproductive stage. Pie slices that are exploding and outlined in red indicate PD-1 positivity. Each pie slice represents the average of 4–10 animals in that reproductive group. Groups: nulliparous (Nullip), lactation, involution (InvD2 and InvD6), and regressed mice. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 by one-way ANOVA with Tukey multiple comparison test, ^ǂp ≤ 0.05 by unpaired two-tailed Student t test.

FIGURE 7.

Involution hosts support Ag-independent memory CD4⁺ T cell accumulation in mammary tissue. (A) Experimental schema in which memory RORγT⁺ Foxp3⁺ CD4⁺ T cells specific for OVA Ag (DO11.10) were generated in vitro and then adoptively transferred into BALB/c nulliparous and involution hosts, followed by injection of OVA Ag into the mammary gland and PBS into the contralateral mammary gland as the internal control. The absolute abundance of transferred T cells in various tissues was calculated by the addition of counting beads in flow cytometry. (B) Number of DO11.10 transgenic memory T cells in mammary glands of nulliparous and involution hosts. Two-tailed unpaired Student t test was performed, ****p ≤ 0.0001. (C) Data from (B) separated by delivery of OVA or PBS to mammary tissue. Two-way ANOVA with Tukey multiple comparison test, **p ≤ 0.01. For data in (B) and (C), n = 8 for nulliparous and n = 6 for InvD2 in which one mammary gland received PBS and the other mammary gland received OVA. n = 8 for nulliparous and n = 9 for InvD2 hosts in which both mammary glands received PBS. (D) Schema to identify memory T cell abundance in LN and spleen in the presence or absence of mammary-delivered OVA Ag or PBS. (E) Number of DO11.10 transgenic memory CD4⁺ T cells in LNs of nulliparous and InvD2 hosts with or without mammary OVA Ag introduction. Each mouse received PBS (blue) in one mammary gland and OVA Ag (green) in the other, with individual mouse data joined by a line. Two-tailed paired t tests were performed, ^{ʄ ʄ}p ≤ 0.01. n = 8 for nulliparous and n = 6 for InvD2. (F) Number of DO11.10 transgenic CD4⁺ T cells in spleens. n = 8 for nulliparous and n = 6 for InvD2 animals in which one mammary gland received OVA Ag and the other mammary gland received PBS. n = 8 for nulliparous and n = 6 for InvD2 animals in which both mammary glands received PBS. Combined results of two independent experiments. Groups: nulliparous (Nullip) and involution (InvD2) mice.

FIGURE 8.

Model of dynamic regulation of mucosal immune features in the female murine mammary gland. Dendritic cell subsets, T cell infiltrates, mucins, and IgA Igs are greatly altered by reproductive state. A baseline RORγT⁺ CD4⁺ T cell infiltrate in the nulliparous hosts greatly expands during lactation and early involution (InvD2), along with increased mucin and IgA expression, suggesting enhanced barrier function. InvD6 is a unique immunologic state characterized by a peak in immune infiltrates and the appearance of regulatory immune cells, which are observations consistent with the known role of Tregs in tissue remodeling. The regressed host shares similarities with the nulliparous host, suggesting a return to a baseline mucosal immune state.