T-regulatory cells shift from a protective anti-inflammatory to a cancer-promoting proinflammatory phenotype in polyposis - PubMed (original) (raw)

T-regulatory cells shift from a protective anti-inflammatory to a cancer-promoting proinflammatory phenotype in polyposis

Elias Gounaris et al. Cancer Res. 2009.

Abstract

T-regulatory (Treg) cells play a major role in cancer by suppressing protective antitumor immune responses. A series of observations (from a single laboratory) suggest that Treg cells are protective in cancer by virtue of their ability to control cancer-associated inflammation in an interleukin (IL)-10-dependent manner. Here, we report that the ability of Treg cells to produce IL-10 and control inflammation is lost in the course of progressive disease in a mouse model of hereditary colon cancer. Treg cells that expand in adenomatous polyps no longer produce IL-10 and instead switch to production of IL-17. Aberrant Treg cells from polyp-ridden mice promote rather than suppress focal mastocytosis, a critical tumor-promoting inflammatory response. The cells, however, maintain other Treg characteristics, including their inability to produce IL-2 and ability to suppress proliferation of stimulated CD4 T cells. By promoting inflammation and suppressing T-helper functions, these cells act as a double-edged knife propagating tumor growth.

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Figures

Figure 1

Figure 1

Adoptive transfer of Treg cells in APCΔ468 mice causes polyps to regress and reduces mastocytosis. A, nonlinear regression of the number versus the diameter of polyps remaining after adoptive transfer of nTreg from healthy mice. Blue, control APCΔ468; green, 3 wk after adoptive transfer of 106 Treg cells; red, 6 wkafter adoptive transfer of 106 Treg cells. Adenomatous polyps in mice transferred with Treg cells display increased apoptosis and decreased proliferation. Mice were transferred with 1 × 106 nTreg cells derived from healthy C57BL/6 mice 6 wkbefore analysis. B, frequency of apoptotic nuclei and mitotic cells in polyps of APCΔ468 mice with no transfer (white columns) or after Treg transfer (gray columns). C, chloroacetate esterase staining of mast cells infiltrating the polyps in APCΔ468 (a) and in APCΔ468 6 wkafter adoptive transfer of 106 Treg cells (b). Magnification, ×200. c, mast cells per 200× field. White circles, APCΔ468; gray circles, APCΔ468 6 wkafter adoptive transfer of 106 Treg cells; black circles, wt. D, MCps assay using MNCs prepared from the intestine 6 wkafter adoptive transfer of 106 Treg cells. White column, APCΔ468; gray column, APCΔ468; black column, wt.

Figure 2

Figure 2

Frequencies and numbers of CD4+CD25+Foxp3+ cells are elevated in APCΔ468 mice. A, contour fluorescence-activated cell sorting (FACS) plots of CD4+CD25+Foxp3+ cells prepared from the MLN of (a) control C57BL/6 and (b) age-matched APCΔ468 mice or intestine of (c) control C57BL/6 and (d) microdissected intestinal polyps of APCΔ468 mice or (e) tissue marginal to polyps of APCΔ468 mice. B, frequencies of CD4+CD25+Foxp3+ cells in C57BL/6 (black columns) and APCΔ468 (white columns) mice. Lymph nodes from the neck, spleen, and MLN were analyzed from healthy intestine (black columns), microdissected polyps (white columns), and tissue marginal to polyps (gray column). C, total numbers of CD4+CD25+Foxp3+ cells in the spleen, MLN, and intestine of control C57BL/6 (black columns) and APCΔ468 (white columns) mice. B6 mice (n = 7) and APCΔ468 mice (n = 11).

Figure 3

Figure 3

CD4+CD25+Foxp3+ cells from polyp-bearing APCΔ468 mice do not produce IL-2 and suppress proliferation of helper CD4 T cells. A, FACS contour plots of total T cells derived from 4-mo-old C57BL/6 or polyp-bearing APCΔ468 mice, showing CD4+CD25+Foxp3+ cells stained for intracellular IL-2. B and C, inhibition of proliferation of naive T cells derived from the spleen (B) or small intestine (C). CD4+Foxp3− cells (2 × 104) were cultured alone (0:1) or with increasing numbers of CD4+Foxp3+ T cells as indicated, together with 2 × 105 irradiated spleen cells as feeder cells. In control plates, IL-2 was added to overcome Treg suppression. The results shown are representative of five independent experiments.

Figure 4

Figure 4

CD4+Foxp3+ cells derived from polyp-ridden APCΔ468 mice are deficient in IL-10 but produce IL-17. MNCs isolated from MLN, spleen, intestine, microdissected adenomatous polyps, or healthy tissue from the margin of the polyps were cultured with anti-CD3/CD28 beads for 3 d and then stained for cell surface expression of CD4, CD25, intracellular Foxp3, and IL-10 (A) or IL-17 (B) and analyzed by FACS. All contour plots except for isotype control have been pregated for CD4. Representative contour plots from one of three independent assays are shown, depicting expression of IL-10 or IL-17 and Foxp3. Isotype controls are shown for IL-10 (IgG2b) and IL-17(IgG1).

Figure 5

Figure 5

Elevated levels of proinflammatory cytokines in sera of APCΔ468 mice are corrected after adoptive transfer of nTreg cells from healthy mice. A, adoptively transferred Treg cells were explanted from the intestine of polyp-ridden mice 3 wkafter transfer and analyzed by FACS for expression of IL-10; a representative contour plot is shown. B, summary of three independent experiments; transferred and endogenous Treg cells were distinguished by expression of Ly5.1 versus Ly5.2 and staining for intracellular Foxp3. C, sera from 4-mo-old APCΔ468 and age-matched wt mice were analyzed with multiplex ELISA for 10 cytokines. The results of three independent experiments conducted in triplicate were used to calculate the positive or negative changes using the wt values as baseline. D, standard ELISA used to measure the level of TH17-associated cytokines 3 wk after adoptive transfer of 1×106 nTreg cells from healthy donors to polyp-ridden APCΔ468 mice of 2.5 to 3 mo of age. Sera from untreated age-matched APCΔ468 mice were used as control. White columns, APCΔ468; black columns, C57BL/6; gray columns, Treg-treated APCΔ468.

Figure 6

Figure 6

Healthy Treg cells suppress, whereas diseased Treg cells promote, MCp differentiation and expansion. A, frequency of MCps among total MNCs isolated from the intestine of APCΔ468Rag−/− mice (white column) and cocultured with CD4+CD25+Foxp3+ cells at 1:1 ratio from wt B6, APCΔ468, or IL-10−/− mice. B, frequencies of MCps isolated from the intestine of wt B6 mice (black column), depleted of CD4+ (_B6_-CD4) or CD25+ (_B6_-CD25) cells, or cultured in the presence of anti–IL-10 or from the intestine of Rag−/− or IL-10−/− mice.

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