Foxp3-dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein

Abstract

Foxp3(+) regulatory T (Treg) cells limit pathogenic immune responses to self-antigens and foreign antigens. An essential role for microRNA (miRNA) in the maintenance and function of Treg cells, revealed by the Treg cell-specific Dicer ablation, raised a question as to a specific miRNA contribution. We found that Foxp3 controlled the elevated miR155 expression required for maintaining Treg cell proliferative activity and numbers under nonlymphopenic conditions. Moreover, miR155 deficiency in Treg cells resulted in increased suppressor of cytokine signaling 1 (SOCS1) expression accompanied by impaired activation of signal transducer and activator of transcription 5 (STAT5) transcription factor in response to limiting amounts of interleukin-2. Our studies suggest that Foxp3-dependent regulation of miR155 maintains competitive fitness of Treg cell subsets by targeting SOCS1, and they provide experimental support for a proposed role for miRNAs in ensuring the robustness of cellular phenotypes.

Figure 1. High amounts of miR155 expression in T_R cells are driven by Foxp3

Foxp3⁺ and Foxp3^- DP and CDSP thymocyte subsets were isolated and A, Foxp3 and B, miR155 amounts were assessed by real-time PCR. Data are representative of two independent experiments. C, Diminished amounts of miR155 in T_R cells upon Foxp3 knockdown with a Foxp3-specific shRNA. Foxp3 shRNA and the corresponding scrambled shRNA (control) were expressed in T_R cells using a retroviral vector equipped with a GFP reporter. D and E, GFP⁺ cells were sorted and the expression of Foxp3 and miR155 were measured by real-time PCR analysis 3 days after retroviral infection.

Figure 2. Reduced thymic and peripheral T_R cell subsets in the absence of miR155

A, Flow cytometric analysis of Foxp3⁺T_R cell subsets in 6-8 wk-old miR155-deficient mice and wild-type littermates. The proportions of different thymocyte subsets and of Foxp3⁺ cells within in CD4 SP thymocyte and CD4⁺ splenic T cell subsets are shown. B-D, Cellularity of the thymus and spleen, and the proportion and absolute numbers of thymic and splenic Foxp3⁺ and Foxp3^-CD4⁺ T cells in miR155-deficient and -sufficient mice are shown.

Figure 3. miR155-deficient T_R cells exhibit impaired homeostais

A, Schematic of generation of mixed BM chimeric mice. B, T_R cell frequencies within each donor-derived populations of peripheral blood lymphocytes at various time points after BM transfer. C, The ratios of Ly5.1^-Foxp3⁺ (miR155^-/- or WT littermates) and Ly5.1⁺Foxp3⁺ cells were plotted over time. D, T_R cell frequencies within each donor derived T cell population from both the thymus and spleen 120 days after BM transfer. E, The ratio of thymic and splenic Ly5.1^-Foxp3⁺and Ly5.1⁺Foxp3⁺cells 120 days after BM transfer. The data in C and E represent three independent experiments.

Figure 4. miR155 deficiency does not affect T_R cell susceptibility to activation induced cell death

Sorted CD4⁺CD25^- (T_E) and CD4⁺CD25^hi (T_R) cells from miR155-deficient mice or littermate controls were cultured in vitro with plate-bound CD3 (1μg/ml) antibodies. Apoptotic cells were labeled with the active caspase 3 probe FITC-VAD-FMK at indicated time points and enumerated using flow cytometric analysis. Data are representative of two independent experiments (n=4).

Figure 5. Diminished proliferative activity of T_R cell in the absence of miR155 in the competitive environment of lympho-replete, but not lymphopenic mice

A, Expression of Ki67 in Ly5.1⁺ (C57/B6) or Ly5.1- (miR155^-/- or WT littermates) SP thymocytes 120 days after BM reconstitution. The frequency of Ki67⁺ cells in either Foxp3⁺ or Foxp3^- population is indicated. B, The ratios of Ly5.1^-Ki67⁺ (miR155^-/- or WT littermates) and Ly5.1⁺Ki67⁺ cells within either Foxp3⁺ or Foxp3^- CD4SP thymocyte subset. C, Expression of Ki67 in Ly5.1⁺ or Ly5.1^- splenic CD4⁺ T cells. The frequency of Ki67⁺ cells within either Foxp3⁺ or Foxp3^- CD4 T cell subsets is indicated. D, The ratios of Ly5.1^-Ki67⁺ and Ly5.1⁺Ki67⁺ cells within Foxp3⁺ and Foxp3^- splenic CD4⁺ T cell subsets. Data are representative of two independent experiments. E, Expression of Ki67 within each donor-derived population of peripheral blood lymphocytes at various time points after BM transfer. The ratios of Ki67⁺Ly5.1^-Foxp3⁺ (miR155^-/- or WT littermates) and Ki67⁺Ly5.1⁺Foxp3⁺ cells were analyzed over time. F, Ly5.1^-T cells (miR155^-/- or WT littermates) isolated from mixed BM chimeric mice 120 days after BM reconstitution were re-transferred into RAG-deficient mice. G, Expression of Ki67 within Foxp3-CD4⁺ and Foxp3⁺CD4⁺ T cell subsets from miR155^-/- (black line) or WT littermates (tinted) 14 days after transfer were assessed by flow cytometric analysis. H, The frequencies of Ki67⁺ cells in either miR155-sufficient or -deficient population were plotted. Data are representative of two independent experiments.

Figure 6. miR155 modulates sensitivity of T_R cells to IL-2 through targeting SOCS1

A, Flow cytometric analysis of Stat5 phosphorylation in miR155-deficient and -sufficient T_R cells induced upon stimulation with IL-2 at the indicated concentrations. B, Expression of the SOCS1 transcript was measured by real-time PCR. T_N: naïve CD4⁺CD25^-CD62L^hi cells; T_E: non-T_R cells activated with anti-CD3/anti-CD28 for 48hrs; T_R: CD4⁺CD25⁺ cells. C, Western blot analysis of the SOCS1 protein expression. Densitometric SOCS1 expression values normalized based on β-actin expression values are indicated below the corresponding lanes as well as fold increase in normalized SOCS1 expression in the absence of miR155 in the indicated T cell subsets. D, Multiple species sequence alignment of the SOCS1 3′ UTR including the predicted miR155 target site sequence (in bold). Mutation of the miR155 target site sequence is shown below. E, 293T cells were co-transfected with WT or mutated SOCS1 3′UTR and miR155 and assessed for luciferase activity 24hrs after transfection. MiR150 was used as a control miRNA in these experiments. F, The proportion and G, absolute numbers of thymic Foxp3⁺ T_R cells in WT littermate control mice, SOCS1 Tg mice as well as SOCS1 cKO mice. The data shown in every panel are representative of two or more independent experiments (n=3∼6); values represent the mean +/- s.d., *P<0.005.

Figure 7. A model for miR155 function in T_R cell homeostasis

In addition to CD25, Foxp3 induces high level of miR155 expression to ensure increased IL-2 responsiveness through miR155-mediated down-regulation of the SOCS1 protein (left panel). In the absence of miR155 (right panel), increased amounts of the SOCS1 protein attenuate IL-2R signaling leading to diminished STAT5 phosphorylation and diminished competitive fitness.