Supplementary Table 1. Ingenuity pathway analysis reveals four significantly enriched biological pathways associated with the genes that are differentially expressed between high and low intra-tumoral Treg tumors. In total, they are 9 genes which are APOH, CXCR3, EGF, FASLG, ITGB7, PLA2g2D, RORA, S1PR1 and SERPINE1 that are involved in the pathways shown in the following.

Pathway and genes / p value
Inflammatory Response:
APOH, CXCR3, FASLG, PLA2g2D, S1PR1 and SERPINE1 / 1.71E-03
Hematological System Development and Function:
APOH, CXCR3, EGF, FASLG, ITGB7, PLA2g2D, RORA, S1PR1 and SERPINE1 / 1.71E-03
Immune Cell Trafficking:
APOH, CXCR3, FASLG, ITGB7, PLA2g2D, S1PR1 and SERPINE1 / 1.71E-03
Cell-mediated Immune Response:
CXCR3, ITGB7, RORA and S1PR1 / 4.76E-03

Supplementary Table 2. Housekeeping genes used for NanoString normalization.

AGK
AMMECR1L
CC2D1B
CNOT10
CNOT4
COG7
DDX50
DHX16
DNAJC14
EDC3
EIF2B4
ERCC3
FCF1
GPATCH3
HDAC3
MRPS5
MTMR14
NOL7
NUBP1
PRPF38A
SAP130
SF3A3
TLK2
TMUB2
TRIM39
USP39
ZC3H14
ZKSCAN5
ZNF143
ZNF346

Supplementary Fig. 3. Univariate analysis showed that bearing a high intra-tumoral Treg tumor was significantly associated with longer DFS and OS, compared to tumors bearing a low intra-tumoral Treg, in triple negative breast cancer.

Overall survival (OS)
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 0% iTregs) / Hazard Ratio / 95% Confidence Interval / p value
0.50 / 0.28-0.90 / 0.022*
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 2% iTregs) / 0.65 / 0.37-0.95 / 0.038*
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 5% iTregs) / 0.75 / 0.38-1.51 / 0.422
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 10% iTregs) / 0.94 / 0.42-2.10 / 0.886
Intratumoral Treg TNBCs
(Every 1% of iTregs) / 0.98 / 0.94-1.03 / 0.424
Disease-free survival (DFS)
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 0% iTregs) / Hazard Ratio / 95% Confidence Interval / p value
0.55 / 0.32-0.95 / 0.034*
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 2% iTregs) / 0.47 / 0.28-0.80 / 0.006*
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 5% iTregs) / 0.55 / 0.28-1.09 / 0.085
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 10% iTregs) / 0.69 / 0.32-1.53 / 0.364
Intratumoral Treg TNBCs
(Every 1% of iTregs) / 0.96 / 0.92-1.00 / 0.072

Supplementary Fig. 4. Multivariate analysis showed that bearing a high intra-tumoral Treg tumor was significantly associated with longer DFS and OS, compared to tumors bearing a low intra-tumoral Treg, in triple negative breast cancer.

Overall survival (OS)
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 0% iTregs) / Hazard Ratio / 95% Confidence Interval / p value
0.45 / 0.24-0.84 / 0.012*
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 2% iTregs) / 0.49 / 0.25-0.95 / 0.034*
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 5% iTregs) / 0.88 / 0.41-1.89 / 0.750
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 10% iTregs) / 1.01 / 0.42-2.43 / 0.985
Intratumoral Treg TNBCs
(Every 1% of iTregs) / 0.98 / 0.94-1.05 / 0.555
Disease-free survival (DFS)
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 0% iTregs) / Hazard Ratio / 95% Confidence Interval / p value
0.49 / 0.27-0.87 / 0.015*
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 2% iTregs) / 0.33 / 0.17-0.66 / 0.002*
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 5% iTregs) / 0.59 / 0.29-1.23 / 0.160
Intratumoral Treg TNBCs
High Vs. Low (Cut-off: 10% iTregs) / 0.70 / 0.30-1.65 / 0.412
Intratumoral Treg TNBCs
(Every 1% of iTregs) / 0.95 / 0.91-1.01 / 0.085

Supplementary Fig. 1. Differentially expressed gene signatures of high and low intra-tumoral Treg TNBCs partially overlap with those of basal-like immune-activated (BLIA) and basal-like immune suppressed (BLIS) triple negative breast cancer subtypes.

Seven of the 31 genes that were differentially expressed in high and low intra-tumoral Treg TNBCs were also significantly differentially expressed in BLIA and BLIS triple negative breast cancer subtypes (p = 0.028). All seven genes are more highly expressed in BLIA tumors, similar to their higher expression in high intra-tumoral Treg TNBCs.

Supplementary Fig. 2. Expression levels of Treg-associated genes correlate with those of CD8+ T cell- and CD20+ B cell - associated genes in TNBC samples.

(A) Analysis of gene expression in the 4 molecular subtypes of triple negative breast cancer defined by Burstein et al. revealed differences in expression of molecules associated with Tregs, CD8+ T cells and CD19+ B cells. (B-C) Relative expression level of the IL2Ra gene is significantly positively correlated with that of CD8A (B) and CD19 (C) in triple negative breast cancers.

Supplementary Fig. 3. Tregs aggregate with CD8+ T-cells and CD20+ B cells in the stromal microenvironment of TNBCs.

H&E and multiplex Immunofluorescent labeling of 2 consecutive sections of a representative triple negative breast cancer shows that Foxp3+ Tregs, CD8+ T-cells and CD20+ B cells are aggregated in close proximity within the stromal tumor microenvironment. (A) H&E staining. (M) Multiplex immunofluorescent labeling for Foxp3 (red), CD8 (green), CD20 (white) and DAPI (blue).