Stability of the regulatory T cell lineage in vivo

Abstract

Tissue maintenance and homeostasis can be achieved through the replacement of dying cells by differentiating precursors or self-renewal of terminally differentiated cells or relies heavily on cellular longevity in poorly regenerating tissues. Regulatory T cells (T(reg) cells) represent an actively dividing cell population with critical function in suppression of lethal immune-mediated inflammation. The plasticity of T(reg) cells has been actively debated because it could factor importantly in protective immunity or autoimmunity. By using inducible labeling and tracking of T(reg) cell fate in vivo, or transfers of highly purified T(reg) cells, we have demonstrated notable stability of this cell population under physiologic and inflammatory conditions. Our results suggest that self-renewal of mature T(reg) cells serves as a major mechanism of maintenance of the T(reg) cell lineage in adult mice.

Fig. 1

Inducible labeling of Foxp3⁺ cells reveals stability of Treg cell subset under physiologic conditions. (A) Representative flow-cytometric analyses demonstrate stability of Foxp3 expression at 14 days and 5 months after tamoxifen-induced labeling. YFP⁺, YFP⁻GFP⁺, and YFP⁻GFP^-subsets of CD4⁺ T cells and proportion of GFP⁺ cells among YFP-tagged cells are shown. Numbers represent percentages of cells within the indicated gates. (B) Percentages of YFP⁺ cells expressing GFP in the indicated organs analyzed as in (A) at 5 months post tamoxifen administration (bottom). Each symbol represents an individual mouse. (C) Flow cytometric analysis of CD25 expression in CD4⁺ cell subsets at 5 months post initial tamoxifen treatment. Expression of CD25 within the indicated subset is shown for individual mice. (D) Splenocytes from Foxp3^{eGFP-Cre-ERT2} x R26Y mice were isolated 5 months after tamoxifen administration, stimulated with PMA and ionomycin in vitro and production of IFNγ and IL2 was assessed by flow-cytometry. Expression of IFNγ and IL2 in YFP⁺Foxp3⁺, YFP⁻Foxp3⁺, and YFP⁻Foxp3⁻subsets is shown. (E) Proportion of YFP-labeled cells among Foxp3⁺ cells in the periphery of thymectomized and non-thymectomized Foxp3^{eGFP-Cre-ERT2} x R26Y mice at 14 days and 5 months post tamoxifen treatment. ATx: adult thymectomy. Data are representative of three independent experiments (n≥3 per group). Mice were 6–8 weeks of age at the time of labeling.

Fig. 2

IL-2 deprivation results in a small, but detectable decrease in Foxp3 expression. (A–C) Tamoxifen-treated Foxp3^{eGFP-Cre-ERT2} x R26Y mice were injected with IL2 blocking antibody or control IgG and Foxp3 expression in FACS-sorted YFP⁺ cells was assessed 9 days later. (A) Intracellular Foxp3 staining of splenic YFP⁺ cells. Numbers represent percentages of cells in the indicated gate. (B) Percentages of YFP⁺ cells expressing Foxp3. Each symbol represents an individual mouse. (C) Relative expression of Foxp3 in YFP⁺ cells from spleens of mice treated with IL2 blocking antibody. Data are representative of three independent experiments (n≥2 per group). Mice were 6–8 weeks of age at the time of antibody administration. (D–F) Treg cells maintain stable expression of Foxp3 under lymphopenic conditions. Lymphopenia was induced in tamoxifen treated Foxp3^{eGFP-Cre-ERT2} x R26Y mice upon irradiation (600 rad) and mice were analyzed 14 days later. (D) Representative flow-cytometric analysis of Foxp3 expression among cells isolated from spleens of control and irradiated mice 14 days post irradiation. Numbers represent percentages of cells in the indicated gate. (E) Percentages of GFP⁺ cells among YFP⁺ cells in spleens 14 days post irradiation. Each symbol represents an individual mouse. (F) Representative flow-cytometric analysis of Foxp3 and Ki67 expression by YFP⁺ splenocytes on day 14 post-irradiation. Similar data were obtained from cells isolated from lymph nodes of irradiated mice. Data are representative ≥2 independent experiments, each with ≥2 mice per group. Mice were 8–12 weeks of age at the time of irradiation.

Fig. 3

Th1 type inflammation does not induce loss of Foxp3. Tamoxifen treated Foxp3^{eGFP-Cre-ERT2} x R26Y mice were infected i.v. with 5 × 10³ CFU of Listeria monocytogenes and analyzed 9 days later. (A) Flow-cytometric analysis demonstrates up-regulation of T-bet and CXCR3 by splenic YFP⁺ CD4⁺ cells in infected mice. (B) Percent of T-bet positive cells among Foxp3⁻⁻and YFP⁺ splenocytes as determined in (A). Each symbol represents an individual mouse. (C) Antigen specific production of IFNγ in splenocytes re-stimulated with LLO190-201 peptide. CD4⁺ T cell gate is shown; numbers represent percentages of cells in corresponding quadrants. (D) Foxp3 expression in sorted YFP⁻GFP⁺ (top) or YFP⁺ cells (bottom) from L. monocytogenes infected or control mice 9 days post infection. Data are representative of three independent experiments with ≥2 mice per group each. Mice were 6–8 weeks of age at the time of infection.

Fig. 4

Foxp3 expression is stable in Treg cells during autoimmune inflammation. (A–C) Highly purified GFP⁺ cells from BDC2.5.Foxp3^eGFP mice were transferred into 12-week-old prediabetic NOD.Thy1.1 recipients, and the spleen, pancreatic lymph nodes (PLN) or pancreatic infiltrates were analyzed 4 weeks later. (A) Representative Foxp3 staining of Thy1.2⁺CD4⁺ donor cells. (B) Tabulation of individual mice in three independent experiments. (C) Intracellular IFNγ⁺ and IL-17⁺ staining of host and donor CD4⁺ cells (numbers represent percentage of cells in the gate ± SD; n=3–6 per group). (D–F) Highly purified CD4⁺GFP⁺ cells from 20 day-old K/BxN.Foxp3^gfp mice were transferred into age-matched K/BxN.CD45.2 mice. (D) Spleen, joint-draining lymph node and small intestine lamina propria (LP) were collected from arthritic 35 day-old recipients and CD45.1⁺CD4⁺ donor cells analyzed for Foxp3-GFP expression. (E) Tabulation of individual mice in two independent experiments. (F) Expression of IL-17a by host and donor CD4⁺ cells from the lamina propria (numbers represent percentage of cells in the gate ± SD; n=3 per group). Data are representative of two independent experiments.