Elsevier

Clinical Immunology

Volume 126, Issue 3, March 2008, Pages 291-302
Clinical Immunology

Differential expression of CCR7 defines two distinct subsets of human memory CD4+CD25+ Tregs

https://doi.org/10.1016/j.clim.2007.11.008Get rights and content

Abstract

Natural Tregs play an essential role in controlling self-tolerance but the in vivo sites of Treg-mediated suppression remain controversial. We have previously reported the identification of distinct naïve and memory Treg populations in human circulating lymphocytes. Here we show that memory Tregs contain high proportions of inflammatory chemokine-expressing cells and comprise two populations that differ in the expression of the lymphoid chemokine receptor CCR7 and represent the counterparts of conventional CCR7+ central memory (CM) and CCR7 effector memory (EM) T cells. CM and EM Tregs exert comparable ex vivo suppressor functions but the EM population is more prominent among Tregs as compared to conventional CD4+ T cells, and is the main Treg subset found in ovarian tumors. Our data suggest that a division of labor between CM and EM Tregs ensures tolerance at lymphoid and peripheral locations including tumor sites.

Introduction

Suppression of T cell proliferation and effector functions by specialized subsets of lymphocytes is essential for immunoregulation. Natural CD4+CD25+ regulatory T cells (Tregs), in particular, are essential for the maintenance of T cell homeostasis and self-tolerance and also play a general role in the regulation of immune responses to alloantigens, pathogens and tumors. Natural Tregs are generated in the thymus as a distinct lineage of anergic CD4+ T cells bearing self-reactive TCR and constitutively express high levels of the IL-2 receptor α-chain (CD25), along with other molecules such as CTLA-4 and FOXP3, involved in their anergic state and suppressive functions. Many reports in different model systems have underlined the importance of the ability of natural Tregs to home into lymphoid tissues and in particular to draining lymph nodes in order to exert their function [1], [2], [3]. Insights into the mechanisms of suppression by Tregs in lymphoid organs have recently been provided by Tang et al. in a NOD mouse model of diabetes. By using two-photon laser scanning microscopy, these authors were able to show that Tregs can inhibit the formation of stable clusters of diabetogenic T cells with DC in pancreatic lymph nodes [4]. The results of this and other studies support the concept that Tregs mainly suppress immune responses in draining lymph nodes by preventing fruitful interactions of responder cells with DC. Recently, however, this view has been challenged by other reports indicating that in other models or even in the same model under different experimental conditions, Tregs may migrate and exert their function within non-lymphoid sites. For example, using the same mouse model of diabetes used by Tang et al., Chen et al. have shown that islet antigen-specific Tregs exerted their inhibitory function not in pancreatic lymph nodes but at the site of the effector phase of the autoimmune reaction, namely in the pancreatic islets [5]. Similarly, Belkaid et al. have shown that Tregs in the skin of BALB/c mice can dampen the response that controls cutaneous infection by Leishmania major[6].

With regards to the nature of the molecules that may be involved in the trafficking of Tregs to lymphoid and non-lymphoid sites, the involvement of chemokine receptors has been demonstrated by several studies. In humans, a study from Iellem and colleagues initially reported expression of CCR4 and CCR8 and vigorous chemotactic responses to the chemokines CCL22, CCL17, CCL1, and to the virokine vMIP-I by human circulating Tregs [7]. More recently Zou et al. have reported accumulation of Tregs in the bone marrow where they are retained through CXCR4/CXCL12 signals [8]. However, the same authors have also reported that tumor-infiltrating CCR4-expressing Tregs accumulate in human ovarian tumors attracted by tumor environmental CCL22, whereas CXCL12, that is produced at high levels by ovarian tumors and mediates the trafficking of plasmacytoid dendritic cells at the tumor site [9] was not involved in Treg homing to tumors [10]. Lim et al. have described a Treg cell subset in human tonsils that displays potent suppressive activities toward germinal center Th cell-dependent B cell responses [11]. These Tregs are CD4+CD25+CD69 mainly localized in the T cell zone, attracted by the CCR7 ligand CCL19, and reacted poorly to CXCL13, the ligand for CXCR5, expressed in the B cell zone. Upon activation, however, these Tregs readily changed their chemotactic behavior by switching the expression of CXCR5 and CCR7 and trafficking toward B cell follicles. Overall the results obtained in these different experimental systems indicate the existence of distinct Treg subsets that may be selectively recruited to different locations in response to different signals. The nature, origin and mechanisms of function of these subsets, however, remain to be elucidated [12].

We have recently reported that, similar to conventional CD4+CD25 T cells, human circulating CD4+CD25+ Tregs can be distinguished according to their in vivo differentiation stage into naïve and memory populations [13]. In this study, we have identified and characterized two different functional subpopulations of memory Tregs, distinguishable by expression of CCR7. Taken together, our data support the concept that, similar to conventional CD4+ T cells, the memory Treg compartment contains a CM-like population, that controls T cell homeostasis and the onset of immune responses in lymphoid organs, and an EM-like population that controls the effector phase of immune responses in peripheral tissues, including at tumor sites. At variance with conventional CD4+ T cells, however, EM Tregs retain high surface expression of CD62L and CD27.

Section snippets

Samples preparation, cell sorting and phenotypic analysis

Surgical tumor specimens were obtained at the Roswell Park Cancer Institute, Buffalo, NY from ovarian cancer patients under an Institutional Review Board-approved protocol and upon informed consent. Tumor specimens were finely minced in cell culture medium and single cell suspensions were cryopreserved for further analysis. Peripheral blood samples were obtained from the New York Blood Center. Cord blood samples were obtained from Transfusion Medicine at the Columbia University Medical Center.

Expression of CCR7 defines CM and EM-like subsets in human memory Tregs

We have recently shown the presence in human circulating CD4+CD25+ Tregs of two phenotypically distinct populations of naïve (CD45RA+/RO, NnTreg) and memory (CD45RA/RO+, MTreg) cells [13] (Fig. 1A). In full-term cord blood, the large majority of CD4+CD25+ T cells showed the phenotypic characteristics of NnTregs. In contrast, the large majority of CD4+CD25+ T cells in tumor-infiltrating lymphocytes from ovarian cancer samples which, in agreement with previous reports [10], represented about

Discussion

We have shown here that the human memory (CD45RA/RO+) CD4+CD25+ regulatory T cell population can be divided into CM- and EM-like subsets based on the expression of the chemokine receptor CCR7. CCR7 and its ligands, the chemokines SLC and ELC, that are constitutively expressed in secondary lymphoid tissues, are essential for normal lymphoid architecture and trafficking [19], [20] as CCR7- or SLC-deficient mice have disturbed lymphoid architecture and impaired immune responses [21], [22]. The CM

Acknowledgments

The study was supported by the Cancer Research Institute (CRI) through dedicated grants (Ovarian Cancer Working Group) to K. Odunsi, D. Valmori and M. Ayyoub. V. Tosello was supported by a CRI grant to M. Ayyoub (Cancer Antigen Discovery Collaborative) and by the Italian Federation for Cancer Research (FIRC). M. Ayyoub and D. Valmori are grateful to the Ludwig Institute for Cancer Research and the Cancer Research Institute for their continuous support.

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  • Cited by (0)

    1

    V. T. and K. O. equally contributed to this article.

    2

    Current address: INSERM U601, CLCC René Gauducheau, Nantes-Saint Herblain, France.

    3

    D. V. and M. A. share senior authorship.

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