Fig 2: The Breast Cancer Immune Landscape(A) Frequencies of selected immune cell types in juxta-tumoral and tumor samples.(B) t-SNE plots of the normalized marker expression of 40,000 T cells from all samples.(C) t-SNE of T cells colored by PhenoGraph cluster.(D) Heatmap of normalized T cell marker expression for 20 T cell clusters. CM, central memory; Eff/Mem, effector and memory; Reg, regulatory; PD-1, PD-1+.(E) Boxplots showing the frequencies of the CD4+ (left) and CD8+ T cell clusters (right) in juxta-tumoral and tumor samples.(F) PD-1+ T cell frequency (top) and mean PD-1 expression (bottom) among tumor-derived CD4+ and CD8+ T cells.(G) Comparison of the PD-1+ T cell frequency and mean PD-1 expression for CD8+ (top) and CD4+ T cells (bottom).(H and I) Frequencies of selected T cell clusters in (H) ER+ and ER- tumors and (I) luminal A and B tumors.(J) t-SNE plots of normalized marker expression of 40,000 myeloid cells from all samples.(K) t-SNE of myeloid cells colored by PhenoGraph cluster.(L) Heatmap of normalized myeloid marker expression for 19 myeloid clusters. Mono, monocyte; T.-res, tissue-resident; E. im., early immigrant; TAM, tumor-associated macrophage; MDSC, myeloid-derived suppressor cell.(M) Frequencies of the myeloid clusters in juxta-tumoral and tumor samples.(N and O) Frequencies of the indicated myeloid clusters in (N) ER+ and ER- tumors and (O) luminal A and B tumors.Wilcoxon rank-sum test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S2.

Fig 3: Subsets of Fetal Skin CD4+ Tconv and CD8+ T Cells Display a Memory Phenotype and Demonstrate Capacity for IFNγ ProductionCells were isolated from second trimester fetal skin (scalp and/or torso) as well as adult (torso) skin and analyzed by flow cytometry.(A) Representative plots demonstrating CD45RO expression by fetal CD8+ T cells (gated on live CD3+CD8+CD4neg) and CD4+ Tconv (gated on live CD3+CD4+CD8negFoxp3negCD25lo).(B and C) Percentage of CD45RO+ (B) CD8+ T cells and (C) CD4+ Tconv in fetal versus adult skin.(D–F) Representative histogram (D) and quantification (E and F) of Nur77 MFI on fetal skin CD45RO+ versus CD45RA+ CD4+ Tconv and CD8+ cells.(G–J) Percentage of CD4+ Tconv producing (G) IL-17A, (H) IL-13, and (I) IFNγ after PMA/ionomycin re-stimulation, and (J) percentage of IFNγ-producing CD8+ T cells.Each point in (B)–(G) represents data from an individual tissue sample; for some fetal samples data from scalp and torso skin from the same fetal donor are included as separate points. ns, not significant (p > 0.05); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig 4: Conventional αβ T Cells in Fetal Skin Largely Demonstrate a Naive, Proliferative PhenotypeLive, CD4+, and CD8+ single-positive cells from 23 weeks fetal skin and adult skin processed for CyTOF were identified by surface markers and analyzed as follows.(A–D) UMAP plots of combined fetal and adult (A) CD4+ and (B) CD8+ T cells colored by skin age. Principal component analysis (PCA) plots demonstrating the distribution of all (C) CD4+ and (D) CD8+ T cells from each individual sample by age.(E) UMAP plots of CD4+ T cells from fetal and adult skin, combined. Cells are labeled and colored by cluster, with clusters A and B constituting conventional CD4+ T cells and cluster C representing Tregs.(F and G) Analogous UMAP plots containing only (F) fetal or (G) adult CD4+ T cells.(H) Heatmap demonstrating relative expression of key markers by CD4+ clusters A, B, and C.(I–K) combined and separated UMAP plots of fetal and adult skin CD8+ T cells, colored by cluster.(L) Heatmap demonstrating relative expression of key markers by each CD8+ cluster.(M–R) Median expression intensity (m.e.i.) of Ki-67 (M and P), CD45RO (N and Q), and CD25 (O and R) for fetal versus adult CD4+ Tconv and CD8+ T cells as revealed by mass cytometric analyses. Each point in (C), (D), and (M)–(R) represents data from an individual donor. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Fig 5: Lymphocytes Progressively Accumulate in Fetal Skin via a Combination of Thymic Egress and Local ProliferationCells were isolated from 17–23 weeks g.a. fetal skin (scalp and/or torso) as well as adult (torso) skin and analyzed by flow cytometry.(A–C) Percentage of live CD3+ lymphocytes (A), percentage of CD4+ T cells (B), and percentage of Foxp3+ Tregs (C) by age.(D–F) Percentage of CD31+ cells among CD4+ Tconv cells (D), Tregs (E), and CD8+ T cells (F) by age.(G–I) Percentage Ki-67+ cells among CD4+ Tconv cells (G), Tregs (H), and CD8+ T (I) cells by age.Points represent data from an individual tissue sample; for some fetal samples data from scalp and torso skin from the same fetal donor are included as separate points. ns, not significant (p > 0.05); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.