Bohling SD, Allison KHImmunosuppressive regulatory T cells are associated with aggressive breast cancer phenotypes: a potential therapeutic target. Mod Pathol 21(12): 1527-1532

Department of Anatomic Pathology, University of Washington Medical Center, Seattle, WA 98195-6100, USA.
Modern Pathology (Impact Factor: 6.19). 10/2008; 21(12):1527-32. DOI: 10.1038/modpathol.2008.160
Source: PubMed


FoxP3 is a marker for immunosuppressive CD25+CD4+ regulatory T cells. These regulatory T cells are thought to play a role in inducing immune tolerance to antigens and may be selectively recruited by carcinomas. We investigated whether breast carcinomas had significant numbers of FoxP3-positive regulatory T cells by immunohistochemistry, and if their presence was associated with other prognostic factors, such as Nottingham grade, hormone receptor immunohistochemical profile, tumor size, or lymph node metastases. Ninety-seven needle core or excisional breast biopsies with invasive breast carcinoma diagnosed at the University of Washington were stained with antibodies to FoxP3, estrogen receptor, and Her2/neu. The numbers of FoxP3-positive cells present within the neoplastic epithelium, and immediately adjacent stroma were counted manually in three high-powered fields (HPFs; × 400) by two independent pathologists. The average scores were then correlated with the parameters of interest. A threshold of ≥15 FoxP3-positive cells/HPF was used to define a FoxP3-positive case in some analyses. Higher average numbers of FoxP3-positive cells present significantly correlated with higher Nottingham grade status (P=0.000229). In addition, the presence of significant numbers (≥15/HPF) of FoxP3-positive cells in breast carcinoma was positively associated with higher Nottingham grade (P=0.00002585). Higher average numbers of FoxP3-positive cells were also significantly associated with larger tumor size (>2.0 cm; P=0.012824) and trended toward an association with estrogen receptor negativity. Interestingly, 'triple-negative' (estrogen and progesterone receptor negative and Her2/neu negative) Nottingham grade III cases were also significantly associated with high numbers of FoxP3 cells. These results argue that regulatory T cells may play a role in inducing immune tolerance to higher grade, more aggressive breast carcinomas, and are a potential therapeutic target for these cancers.

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    • "Analysis of the CD4 population both postmagnetic cell sorting and following activation revealed a negligible contaminating population of CD4 − cells. Thus, studies investigating the T REG subset must take cognizance that without a definitive phenotype , isolated populations may contain contaminating effector cells which in turn may contribute to the plethora of conflicting evidence regarding the role of T REG lymphocytes in breast cancer (Yu and Fu, 2006; Bohling and Allison, 2008; Cimino-Mathews et al., 2013; West et al., 2013). Flow cytometry analysis of magnetically isolated NK cells demonstrated four distinct subsets based on the total lymphocyte population. "
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    ABSTRACT: Three-dimensional (3D) culture approaches to investigate breast tumour progression are yielding information more reminiscent of the in vivo microenvironment. We have established a 3D Matrigel system to determine the interactions of luminal phenotype MCF-7 cells and basal phenotype MDA-MB-231 cells with regulatory T lymphocytes and Natural Killer cells. Immune cells were isolated from peripheral blood using magnetic cell sorting and their phenotype validated using flow cytometry both before and after activation with IL-2 and phytohaemagglutinin. Following the establishment of the heterotypic culture system, tumour cells displayed morphologies and cell-cell associations distinct to that observed in 2D monolayer cultures, and associated with tissue remodelling and invasion processes. We found that the level of CCL4 secretion was influenced by breast cancer phenotype and immune stimulation. We further established that for RNA extraction, the use of proteinase K in conjunction with the Qiagen RNEasy MiniKit and only off-column DNA digestion gave the best RNA yield, purity and integrity. We also investigated the efficacy of the culture system for immunolocalisation of the biomarkers oestrogen receptor-α and the glycoprotein mucin 1 in luminal phenotype breast cancer cells; and epidermal growth factor receptor in basal phenotype breast cancer cells, in formalin-fixed, paraffin-wax embedded cultures. The expression of these markers was shown to vary under immune mediation. We thus demonstrate the feasibility of using this co-culture system for downstream applications including cytokine analysis, immunolocalisation of tumour biomarkers on serial sections and RNA extraction in accordance with MIQE guidelines. Copyright © 2015. Published by Elsevier B.V.
    No preview · Article · Jul 2015 · Journal of immunological methods
    • "In 2003, the transcription factor forkhead box P3 (Foxp3), was shown to control the generation and function of murine Tregs [11] [12] [13] and later, in humans, FOXP3 was shown to be expressed predominantly by the CD4 pos cells expressing the highest level of CD25 [14]. Since then, elevated Treg frequencies have been reported in patients with various forms of neoplasia, such as breast cancer [15] [16] [17] [18] [19], melanoma [20] [21] [22] and leukaemia [23] [24] [25], where they are believed to dampen anti-tumour immune responses and promote malignancy. Similarly, Tregs can limit anti-pathogen immunity: high Treg frequency and function are associated with the persistence of infection with hepatitis C virus [26] [27], mycobacterium tuberculosis [28] [29] [30] and the malarial parasite [31] [32] [33]. "
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    ABSTRACT: Regulatory T cells (Tregs) are central to the maintenance of self-tolerance and tissue homeostasis. Markers commonly used to define human Tregs in the research setting include high expression of CD25, FOXP3 positivity and low expression/negativity for CD127. Many other markers have been proposed, but none unequivocally identifies bona fide Tregs. Tregs are equipped with an array of mechanisms of suppression, including the modulation of antigen presenting cell maturation and function, the killing of target cells, the disruption of metabolic pathways and the production of anti-inflammatory cytokines. Treg impairment has been reported in a number of human autoimmune conditions and includes Treg numerical and functional defects and conversion into effector cells in response to inflammation. In addition to intrinsic Treg impairment, resistance of effector T cells to Treg control has been described. Discrepancies in the literature are common, reflecting differences in the choice of study participants and the technical challenges associated with investigating this cell population. Studies differ in terms of the methodology used to define and isolate putative regulatory cells and to assess their suppressive function. In this review we outline studies describing Treg frequency and suppressive function in systemic and organ specific autoimmune diseases, with a specific focus on the challenges faced when investigating Tregs in these conditions.
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    • "In both the tumor periphery and tumor bed, unfavorable pathological parameters (a Ki-67 LI of ≥14%, a worse histologic grade and nuclear grade, tumor recurrence, and aggressive biologic subtype [TNBC]) were associated with more Foxp3 Treg infiltration. These results are in agreement with those of previous studies reporting the correlation between increased Foxp3 Treg infiltration and unfavorable pathologic phenotypes in breast cancer [12,13,14]. "
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    ABSTRACT: Forkhead box P3 (Foxp3) is known as the most specific marker for regulatory T lymphocytes, which play an important role in immune tolerance to disturb antitumor immunity. The present study aimed to investigate the prognostic significance of Foxp3 regulatory T lymphocyte (Foxp3 Treg) infiltration in breast cancer. Immunohistochemical studies with Foxp3, CD4, and CD8 were performed on representative full tissue sections from 143 patients with invasive ductal carcinoma, not otherwise specified. Foxp3 Treg infiltration and the ratios between Foxp3 Treg and CD4 or CD8 T cells were separately analyzed for the tumor bed and tumor periphery to evaluate their association with different clinicopathological parameters and patients' outcome. The tumor periphery was considerably more densely infiltrated by Foxp3 Treg, CD4, and CD8 T cells than the tumor bed. Unfavorable clinicopathological parameters (a Ki-67 labeling index of ≥14%, a worse histologic grade, a worse nuclear grade, hormone receptor negativity, human epidermal growth factor receptor 2 positivity, and tumor recurrence) were associated with increased Foxp3 Treg infiltration and a high ratio between Foxp3 Treg and CD4/CD8 T cells. In the tumor periphery, as Foxp3 Treg infiltration and the Foxp3 Treg/CD8 ratio increased, patients' 5-year disease-free survival rate decreased. The infiltration densities of Foxp3 Treg, CD4, and CD8 T cells were markedly different between the tumor bed and periphery. Besides the absolute count of Foxp3 Treg, the ratio between Foxp3 Treg and effector T cells was a significant prognostic factor in breast cancer.
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