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. 2021 Oct 1;12:5764. doi: 10.1038/s41467-021-26091-4

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — a Suppressive capacity of CD177⁺ or CD177⁻ TI Treg cells isolated from breast cancer specimens. CD4 Tconv cells were stimulated by anti-CD3/CD28 co-stimulation. CD4⁺CD25⁺CD127^low total, CD177⁺ or CD177⁻ TI Treg cells were purified from 3 individual breast cancer specimens and combined for the suppression assay. Left: Histograms showing CFSE dilution peaks indicating Teff cell proliferation and Right: Percentages of proliferative CD4 Teff cells co-cultured with total, CD177⁺ or CD177⁻ TI Treg cells at ratios of 2:1, 4:1, 8:1 or no Treg (Combined data from a and b: n = 3 biological replicates for CD177⁺ TI Treg cells and n = 2 for CD177⁻ TI Treg cells at 2:1 ratio; n = 1 biological replicate for other data point). P value is for comparison between CD177⁺ or CD177⁻ TI Treg cells at 2:1 ratio Teff/Treg, using two-sided T-test. b Impact of CD177 blockade on the immune suppressive function of CD177⁺ TI Treg cells, using a monoclonal antibody (MEM166). Similar suppression experiments were performed as in a. CD177⁺ or CD177⁻ TI Treg cells were purified from fresh human breast cancer specimens (combined from 5 patients) using flow cytometry and co-cultured with effector CD4 T (CD4⁺CD25⁻) cells from PBMC for ex vivo suppression assay, with or without the addition of 2 μg/ml isotype control (IgG) or anti-CD177 antibody (MEM166). Percent of proliferating cells were included and the total number of CD4 Tconv cells were enumerated after 4 days of incubation (n = 2 biological replicates for CD177⁺ Treg group, n = 1 for other data points). c Suppressive capacity of CD177⁺ or CD177⁻ TI Treg cells isolated from renal cell cancer specimens (RCC). CD4 Tconv cells were stimulated by anti-CD3 and APC (monocyte derived dendritic cells). A total of 7 RCC specimens were combined (n = 1 for NS, non-stimulated or no Treg; n = 4 for CD177⁺ TI Treg cells; n = 3 biological replicates for CD177⁻ TI Treg cells). The total number of CD4 Tconv cells were counted after 4 days of incubation. d Suppressive capacity of CD177⁺ or CD177⁻ TI Treg cells isolated from mouse MC38 tumors. Effector CD4 T cells were stimulated by anti-CD3 and APC (derived from T-cell depleted splenocytes of tumor bearing mice) and combined with TI Treg cells at different ratios. Left: Histograms showing the CFSE dilution as an indicator of T cell proliferation and right: Percent proliferation as defined by the histogram (n = 3 biological replicates except n = 6 for no Treg group). Two-way ANOVA was used. e Impact of Treg-specific Cd177-KO on the suppressive capacity of TI Treg cells from MC38 tumors similar as shown in Fig. 5c. Upper left: post-sorting Treg purify was determined by intracellular staining of FoxP3; Lower left: histogram showing CFSE dilution peaks indicating CD4⁺ Teff cell proliferation; right: summary of percent proliferating cells (n = 4 biological replicates from 14 tumors from cKO group and 20 tumors from WT group, with 4 tumors combined in one biological replicate). All data are presented mean ± s.t.d. c, e one-way ANOVA was used.