Regulatory T (Treg) cells play a major role in the development of an immunosuppressive tumor microenvironment. The origin of intratumoral Treg cells and their relationship with peripheral blood Treg cells remain unclear. Treg cells consist of at least three functionally distinct subpopulations. Here we show that peripheral blood CD45RA−FOXP3hi Treg cells (Treg II cells) are phenotypically closest to intratumoral Treg cells, including in their expression of CCR8. Analyses of T cell antigen receptor repertoires further support the hypothesis that intratumoral Treg cells may originate primarily from peripheral blood Treg II cells. Moreover, the signaling responsiveness of peripheral blood Treg II cells to immunosuppressive, T helper type 1 (TH1) and T helper type 2 (TH2) cytokines reflects intratumoral immunosuppressive potential, and predicts future relapse in two independent cohorts of patients with breast cancer. Together, our findings give important insights into the relationship between peripheral blood Treg cells and intratumoral Treg cells, and highlight cytokine signaling responsiveness as a key determinant of intratumoral immunosuppressive potential and clinical outcome.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
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We would like to thank M. Kirschenbaum for obtaining consent from patients and acquiring samples at City of Hope Comprehensive Cancer Center. We would like to thank D. Dunkley for organizing the collection of blood samples from age-matched healthy controls. This work was supported by the Department of Defense Breast Cancer Research Program, Stand Up to Cancer, the Breast Cancer Research Foundation and the V Foundation. Research reported in this publication included work performed in the Analytical Cytometry Core and Pathology Research Services Core supported by the National Cancer Institute of the National Institutes of Health under award number P30CA33572. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The authors declare no competing interests.
Peer review information: Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
(a) Representative flow plots for peripheral and intratumoral Treg cells. (b-c) Representative flow plots showing the percentages of IL-2Rβ+ (b) and IL-2Rγ+ (c) in peripheral blood Tconv, Treg I, II and III cells. (d) Representative flow plots for peripheral blood Treg cells in phosflow cytometry.
Supplementary Figure 2 Expressions of immune-regulating proteins in peripheral blood Treg cell subpopulations and intratumoral Treg cells.
The percentages of CD73+ (n=40) (a), PD1+ (n=40), ****p<0.0001 (b), Tim3+ (n=40), ****p<0.0001 (c), LAG3+ (n=40), ****p<0.0001; ***p=0.0002 (d), GITR+ (n=20), ****p<0.0001; **p=0.007 (e) or HLA-DR+ (n=40), ****p<0.0001; ***p=0.0003 (f) in peripheral Tconv, Treg I, II and III cells from newly diagnosed patients with BC were determined by flow cytometry. Friedman test. Shown are mean ± s.e.m. Representative flow plots showing the percentages of LAG3+ (d), GITR+ (e), or HLA-DR+ (f) in intratumor Treg cells from untreated primary breast tumors (n=8). Data from intratumoral Treg cells were highlighted with purple box.
Supplementary Figure 3 Flow sort gating strategy of peripheral blood Tconv and Treg cell subpopulations.
Peripheral Treg I (CD45RA+CD25lo), II (CD45RA−CD25hi), III (CD45RA−CD25lo) and Tconv (CD45RA−CD25−) cells were sorted from PBMCs of newly diagnosed patients with BC.
Supplementary Figure 4 Chemokine receptors CCR2, CCR10 and CXCR3 not differentially expressed in intratumoral Treg cells.
The percentages and representative flow plots of CCR2+, ***p=0.0003; *p=0.03 (a), CCR10+, ****p<0.0001; **p=0.009 (b), CXCR3+, ****p<0.0001; *p=0.025 (c) in peripheral Tconv, Treg I, II and III cells from newly diagnosed patients with BC (n=20) and in intratumor Treg cells from untreated primary breast tumors (n=8). Friedman test. Shown are mean ± s.e.m. Data from intratumoral Treg cells were highlighted with purple box.
Supplementary Figure 5 Intratumoral Treg cells have higher TCR clonal overlap with peripheral Treg II cells.
(a) TCRβ CDR3 regions of flow sorted intratumor Treg cells (CD4+CD25+CD127−) and paired peripheral Treg I, II or III cells from patients with BC (n=3) were sequenced. Pair-wise scatter plots showing the overlapping TCR clones between intratumor Treg cells and peripheral Treg cell subpopulations. (b) The percentages of unique TCR nucleotide clonal overlap between peripheral blood Treg I, II or III cells. Shown are mean ± s.e.m.
Supplementary Figure 6 Representative flow plots showing TGFβ-induced signaling in peripheral blood Treg II cell subpopulation.
TGFβ (25ng/ml for 30 mins)-induced Smad2/3 phosphorylation in peripheral blood Treg II cells from newly diagnosed patients with BC (n=40) was determined by phosphoflow cytometry with anti-p-Smad2 (pS465/pS467)/p-Smad3 (pS423/pS425) antibody. Plots from relapsed patients with BC were highlighted with red box.
Supplementary Figure 7 Representative flow plots showing IL-10-induced signaling in peripheral blood Treg II cell subpopulation.
IL-10 (100ng/ml for 15 mins)-induced STAT1 phosphorylation in peripheral blood Treg II cells from newly diagnosed patients with BC (n=40) was determined by phosphoflow cytometry with anti-p-STAT1 (pY701) antibody. Plots from relapsed patients with BC were highlighted with red box.
Supplementary Figure 8 Representative flow plots showing IL-4-induced signaling in peripheral blood Treg II cell subpopulation.
IL-4 (50ng/ml for 15 mins)-induced STAT6 phosphorylation in peripheral blood Treg II cells from newly diagnosed patients with BC (n=40) was determined by phosphoflow cytometry with anti-p-STAT6 (pY641) antibody. Plots from relapsed patients with BC were highlighted with red box.
Supplementary Figure 9 Representative flow plots showing IFNγ-induced signaling in peripheral blood Treg II cell subpopulation.
IFNγ (50ng/ml for 15 mins)-induced STAT1 phosphorylation in peripheral blood Treg II cells from newly diagnosed patients with BC (n=40) was determined by phosphoflow cytometry with anti-p-STAT1 (pY701) antibody. Plots from relapsed patients with BC were highlighted with red box.
Supplementary Figure 10 Cytokine signaling index of Tconv, Treg I or Treg III cells not correlated with clinical outcome.
Relapse-free survival (RFS) was compared between patients with BC (n=40) with above median CSI and below median CSI in peripheral Tconv (p=0.22) (a), Treg I (p=0.68) (b) or Treg III cells (p=0.07) (c) using Kaplan-Meier estimate and log rank test. All blood were collected from patients with BC at diagnosis before surgery or any therapy who had been clinically followed for at least 36 months. NS, not significant.
Supplementary Figure 11 Cytokine plasma levels in patients with BC at diagnosis do not correlate with clinical outcome.
(a) Plasma levels of TGFβ, IL-10, IL-4 and IFNγ were determined by ELISA and compared between relapse-free (n=25) and relapsed (n=15) BC patients. Shown are mean ± s.e.m. (b) The association between plasma level of TGFβ (p=0.08), IL-10 (p=0.79), IL-4 (p=0.36) or IFNγ (p=0.58) and signaling response in Treg II cells, respectively. Spearman correlation coefficient test. NS, not significant.
(a-b) PBMCs from age-matched healthy donors (HD) (n=10) were stimulated with TGFβ (25ng/ml, 30 mins), IL-10 (100ng/ml, 15 mins), IL-4 (50ng/ml, 15 mins) or IFNγ (50ng/ml, 15 mins). TGFβ-induced p-Smad2/3 and IL-10-induced p-STAT1, ***p<0.001 (a), IFNγ-induced p-STAT1 and IL-4-induced p-STAT6 **p=0.008; *p=0.028 (b) in peripheral Treg II cells were determined by phosphoflow cytometry and were compared between healthy donors (n=10) and relapsed patients with BC (n=15). (c) Treg II CSI was compared between HD (n=10) and relapsed patients with BC (n=15), ****p<0.001, two-tailed, Mann-Whitney test. Shown are mean ± s.e.m.
Supplementary Figure 13 Cytokine signaling index (CSI) of peripheral Treg I or III cells not associated with immunosuppressive potential of intratumoral Treg cells.
Untreated breast primary tumor tissue sections were stained for FoxP3, CD8, CD68, CD123, CK, and DAPI. (a) The density of Treg cells within primary tumors (FoxP3+ cell number per mm2) from patients with BC who later relapsed (n=9) and from patients who remained relapse-free (n=11), p=0.07, two-tailed, Mann-Whitney test, Shown are mean ± s.e.m. (b) The association between Treg cell density and the percentage of Treg cells within 20μm from TAMs (n=20) (p=0.14). (c-d) The association between Treg I CSI (p=0.52) (C) or Treg III CSI (p=0.15) (d) and the percentage of Treg cells within 20μm from TAMs (n=20). Spearman correlation coefficient test. NS, not significant.
Supplementary Figure 14 Consistent cytokine signaling responses in reference PBMCs between discovery and validation runs.
Different vials of reference PBMC from the same healthy donors (n=5) were thawed and run together with samples from patient with BC in the discovery and validation cohorts to determine consistency between runs. (a) TGF-induced Smad2/3 phosphorylation, (b) IL-10-induced STAT1 phosphorylation, (c) IL-4-induced STAT6 phosphorylation, or (d) IFNγ-induced STAT1 phosphorylation in Treg II cells.