Parity-induced changes to mammary epithelial cells control NKT cell expansion and mammary oncogenesis

Abstract

Pregnancy reprograms mammary epithelial cells (MECs) to control their responses to pregnancy hormone re-exposure and carcinoma progression. However, the influence of pregnancy on the mammary microenvironment is less clear. Here, we used single-cell RNA sequencing to profile the composition of epithelial and non-epithelial cells in mammary tissue from nulliparous and parous female mice. Our analysis indicates an expansion of γδ natural killer T-like immune cells (NKTs) following pregnancy and upregulation of immune signaling molecules in post-pregnancy MECs. We show that expansion of NKTs following pregnancy is due to elevated expression of the antigen-presenting molecule CD1d on MECs. Loss of CD1d expression on post-pregnancy MECs, or overall lack of activated NKTs, results in mammary oncogenesis. Collectively, our findings illustrate how pregnancy-induced changes modulate the communication between MECs and the immune microenvironment and establish a causal link between pregnancy, the immune microenvironment, and mammary oncogenesis.

Keywords: Brca1 KO; CD1d; NKT cells; mammary development; pregnancy.

Figure 1.. Identification of transcriptional programs and immune cellular heterogeneity in mammary tissue from parous female mice

(A) UMAP of mammary epithelial cells from pre- and post-pregnancy mammary glands. (B) mRNA levels of senescence-associated, immune communication genes Cxcl1, Ccl2, Il6, Cxcl5, Mhc-ii, and Cd1d in pre- and post-pregnancy MECs. (C) UMAP of T cells (CD3e⁺ cells) from pre- and post-pregnancy mammary glands. (D) Feature plots showing the expression of T cell markers Cd4, Cd8, Klrk1, and Gzma. (E) Dendrogram clustering and dot plot showing the molecular signature and lineage identity of pre- and post-pregnancy mammary resident CD3⁺ immune cells. See also Figures S1-S4.

Figure 2.. Pregnancy induces the expansion of a specific population of NKTs

(A) Flow cytometry analysis of resident CD45⁺ NK1.1⁺CD3⁺ NKTs from pre- and post-pregnancy mammary tissue. n = 5 nulliparous and 5 parous female mice. *p = 0.0004. (B) Flow cytometry analysis of the classic NKT cell markers T-bet, CD335, and IFN-γ in NKTs (CD45⁺NK1.1⁺CD3⁺) from pre- and post-pregnancy mammary tissue. For Tbet analysis, n = 4 nulliparous and 4 parous female mice. *p = 0.016. For CD335 analysis, n = 7 nulliparous and 7 parous female mice. *p = 0.03. (C) Flow cytometry analysis of β and γδ TCRs of pre- and post-pregnancy mammary NKTs. n = 5 nulliparous and 5 parous female mice. *p = 0.005. (D) Gene set enrichment analysis of differentially expressed genes in FACS-isolated NKTs from pre- and post-pregnancy mammary tissue. (E) Venn diagram demonstrating the number of shared and exclusive ATAC-seq peaks of FACS-isolated NKTs from pre- (blue circle) and post-pregnancy (orange circle) mammary tissue. (F) Genome browser tracks showing distribution of MACS-called, ATAC-seq peaks at the Pdk4, Maged1, and Lypla1 genomic loci from pre- and post-pregnancy NKTs. For all analyses, error bars indicate standard error of mean across samples of the same experimental group. Statistically significant differences were considered with Student’s t test p < 0.05. See also Figures S5 and S6 and Table S3.

Figure 3.. NKT expansion depends on CD1d expression on post-pregnancy MECs

(A and B) Flow cytometry analysis and quantification of CD1d⁺ MECs harvested from pre-pregnancy (black bars, n = 8) and post-pregnancy (pink bars, n = 10) mammary tissue. *p = 0.0036 for luminal MECs and **p = 0.0006 for myoepithelial MECs. (C) Genome browser tracks showing MACS-called, H3K27ac ChIP-seq peaks at the Cd1d genomic locus in FACS-isolated, pre- and post-pregnancy luminal MECs. (D) H&E-stained histological images and duct quantification from mammary glands harvested from nulliparous (top left, n = 6) and parous (bottom left, n = 7) CD1d^WT female mice and nulliparous (top right, n = 6) and parous (bottom right, n = 7) CD1d^KO female mice. p = 0.86 for pre-pregnancy glands and p = 0.78 for post-pregnancy glands. Scale: 7 mm. Zoom-in panels, scale 500 μm. (E) Flow cytometry analysis of mammary resident NKTs from pre- and post-pregnancy CD1d^KO mammary tissue. n = 4 nulliparous and n = 4 parous female mice. *p = 0.3. (F) Flow cytometry analysis of α and γδ TCRs of mammary resident NKTs from pre- (n = 3) and post-pregnancy (n = 3) CD1d^KO mammary tissue. *p = 0.5. For all analyses, error bars indicate standard error of mean across samples of the same experimental group. Statistically significant differences were considered with Student’s t test p < 0.05. See also Figure S7.

Figure 4.. Lack of mammary oncogenesis is marked by NKT expansion and CD1d⁺ MECs

(A) Flow cytometry analysis of mammary resident NKTs (CD45⁺NK1.1⁺CD3⁺) from DOX-treated, nulliparous (left panel, n = 5) and parous (right panel, n = 5) CAGMYC female mice. *p = 0.002. (B) Flow cytometry quantification of CD1d⁺ luminal and myoepithelial MECs from DOX-treated, nulliparous (left panel, n = 16) and parous (right panel, n = 11) CAGMYC female mice. *p = 0.02. (C) Mammary tumor-free survival plot of nulliparous (black line, n = 5) and parous (pink line, n = 5) Brca1^KO female mice. (D) H&E-stained histological images from mammary tissue and tumors from nulliparous (top panels) and parous (bottom panels) Brca1^KO female mice. Scale: 5 mm. Zoom-in panels, scale: 500 μm. (E) Flow cytometry quantification of CD1d⁺CD24^high luminal MECs from Brca1^KO pre-pregnancy mammary tumors (black bar, n = 3), Brca1^KO post-pregnancy healthy mammary tissue (pink bar, n = 4), and Brca1^KO post-pregnancy mammary tumor (blue bar, n = 1). *p = 0.02. (F) Flow cytometry analysis of NKTs in normal mammary tissue from nulliparous, tumor-bearing, Brca1^KO female mice (left panel, n = 4) and normal mammary tissue from healthy parous Brca1^KO female mice (right panel, n = 4). *p = 0.003. (G) Quantification of γδNKTs in normal mammary tissue from nulliparous, tumor-bearing, Brca1^KO female mice (black bar panel, n = 4), in mammary tumor tissue from nulliparous Brca1^KO female mice (blue bar, n = 3), and in normal mammary tissue from healthy parous Brca1^KO female mice (black bar panel, n = 2). *p = 0.023 and **p = 0.008. For all analyses, error bars indicate standard error of mean across samples of the same experimental group. Statistically significant differences were considered with Student’s t test p < 0.05. See also Figures S8-S12.

Figure 5.. Functionally active NKTs are required to block malignant progression of post-pregnancy MECs

(A) H&E-stained images of mammary tissue harvested from DOX-treated (DD5), nulliparous CAGMYC-CD1d^WT (far-left panels), nulliparous CAGMYC-CD1d^KO (left panels), parous CAGMYC-CD1d^WT (right panels), and parous CAGMYC-CD1d^KO (far right panels) female mice. Green arrows indicate signs of malignant lesions/mammary hyperplasia. Green asterisks indicate normal-like ductal structures. Scale: 1 mm. (B) H&E-stained images of DOX-treated, CD1d^WT mammary tissue transplanted with pre-pregnancy CAGMYC-CD1d^WT MECs (blue font, top far left panel), pre-pregnancy CAGMYC-CD1d^KO MECs (black font, top panel), post-pregnancy CAGMYC-CD1d^WT (blue font, bottom far left panel), or post-pregnancy CAGMYC-CD1d^KO MECs (black font, bottom panel). Green arrows indicate signs of malignant lesions/mammary hyperplasia. Green asterisks indicate normal-like ductal structures. Scale: 500 μm. See also Figure S13.