Fig 2: 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) CD1dWT female mice and nulliparous (top right, n = 6) and parous (bottom right, n = 7) CD1dKO 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 CD1dKO mammary tissue. n = 4 nulliparous and n = 4 parous female mice. *p = 0.3.(F) Flow cytometry analysis of a and ?d TCRs of mammary resident NKTs from pre- (n = 3) and post-pregnancy (n = 3) CD1dKO 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.

Fig 3: 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.

Fig 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) Brca1KO female mice.(D) H&E-stained histological images from mammary tissue and tumors from nulliparous (top panels) and parous (bottom panels) Brca1KO female mice. Scale: 5 mm. Zoom-in panels, scale: 500 µm.(E) Flow cytometry quantification of CD1d+CD24high luminal MECs from Brca1KO pre-pregnancy mammary tumors (black bar, n = 3), Brca1KO post-pregnancy healthy mammary tissue (pink bar, n = 4), and Brca1KO 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, Brca1KO female mice (left panel, n = 4) and normal mammary tissue from healthy parous Brca1KO female mice (right panel, n = 4). *p = 0.003.(G) Quantification of ?dNKTs in normal mammary tissue from nulliparous, tumor-bearing, Brca1KO female mice (black bar panel, n = 4), in mammary tumor tissue from nulliparous Brca1KO female mice (blue bar, n = 3), and in normal mammary tissue from healthy parous Brca1KO 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.