The orphan nuclear receptor Ear-2 (Nr2f6) is a novel negative regulator of T cell development  Christine V. Ichim, Džana D. Dervović, Juan Carlos Zúñiga-Pflücker, Richard A. Wells  Experimental Hematology  Volume 42, Issue 1, Pages 46-58 (January 2014) DOI: 10.1016/j.exphem.2013.09.010 Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 1 EAR-2 expression decreases with T cell differentiation. Expression of EAR-2 relative to the L32 housekeeping gene was determined by quantitative PCR in HSCs and thymic T cell subsets isolated by FACS. Data shown are the mean and SD from analysis of three independent animals. Experimental Hematology 2014 42, 46-58DOI: (10.1016/j.exphem.2013.09.010) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 2 Sustained expression of EAR-2 blocks thymocyte development. Competitive BM transplant were conducted with EAR-2 retrovirally transduced BM cells or GFP vector control. (A) Bone marrow that overexpresses EAR-2 does not engraft the thymus. Shown are flow cytometry plots from the indicated organs from representative mice with indicated surface markers. Although both GFP vector control and EAR-2++ transduced cells engrafted well in the BM (18.9% and 40.0%, respectively), only the vector control animals demonstrated engraftment in the thymus as well (22.0% engraftment for vector control versus 0.4% engraftment for EAR-2++). (B) Pooled data showing engraftment in the thymus relative to the engraftment in the BM as depicted in (A) is shown from three independent experiments (n = 22 at 4 weeks; n = 7 at 12 weeks). **p < 0.001. (C) EAR-2 overexpressing cells do not contribute to peripheral T cells in the spleen or peripheral blood. Chimerical BM transplantation experiments were conducted. Representative animals from the GFP vector control and EAR-2++ cohorts show that although untransduced competitor cells (GFP negative) were able to generate CD3+ cells in the spleen and peripheral blood, only cells transduced with control vector and not the EAR-2–transduced vector were able to generate CD3+ T cells. Data representative of three independent experiments are shown. (D, E) Pooled data from the three independent competitive BM transplantation experiments described in (C) are shows. The x axis lists first whether the animal belongs to the vector control or EAR-2++ cohort, followed by whether the untransduced competitors (GFP negative) or whether the transduced (GFP positive) fraction is represented. The percentages of either GFP-positive or -negative CD3+ T cells are shown as a percentage of all the GFP-positive or -negative cells respectively in either (D) the peripheral blood or (E) the spleen. We observe that while although competitor cells from both the vector control (vector GFP−) and EAR-2++ (EAR-2 GFP−) cohort were able to generate CD3+ T cells in both (D) the spleen and (E) peripheral blood, only cells transduced with the GFP vector control (vector GFP+) and not EAR-2 transduced cells (GFP EAR-2+) vector were able to generates CD3+ T cells. Bars represent the mean ± SEM (n = 20 at 6–8 weeks). (E) EAR-2 overexpressing cells do not contribute to peripheral T cells in the spleen. Pooled data from two independent experiments are shown; bars represent the mean ± SEM (n = 35). **p < 0.001. Experimental Hematology 2014 42, 46-58DOI: (10.1016/j.exphem.2013.09.010) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 3 EAR-2 overexpression results in thymic involution. Recipients transplanted with sorted grafts containing 100% EAR-2–transduced BM cells that were sacrificed 3–4 weeks after transplant showed a dramatic reduction in (A) thymic size and (B) cellularity. (C) The majority of thymocytes recovered from the EAR-2 cohort did not express the EAR-2-GFP transgene. (D) Histologic sections revealed an abnormal thymus (E) tingible body macrophages in the medulla were observed, indicative of apoptosis (F) the thymic cortex showed decreased cellularity while the medulla showed a starry-sky appearance in animals transduced with grafts that overexpressed EAR-2 (n = 4 organs examined in each group). **p < 0.001. Experimental Hematology 2014 42, 46-58DOI: (10.1016/j.exphem.2013.09.010) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 4 EAR-2 inhibits proliferation of immature T cells at the DN1 stage. KSL cells were induced to differentiate to T cells on OP9-DL1 cells. (A) A 59.1% decrease in cell number was observed at day 4, increasing to a 97.4% decrease at day 19. Cumulative cell numbers are shown. (B) Expression of GFP by cells in the cultures was assessed by flow cytometry at day 19. In the control culture, 90.6% of cells expressed GFP and 9.4% were GFP-negative. The increase in the percentage of GFP-negative cells represents an outgrowth of untransduced cells or the loss of transgene expression by transduced cells, or both. In the EAR-2++ culture on day 19, only 18.1% of cells expressed GFP, indicating impaired growth or survival of cells expressing the transgene. (C, D) Proliferation at various times after induction of differentiation was assessed by pulse labeling with BrdU for 2 hours followed by flow cytometry on CD45+GFP+ gated cells using an APC–anti-BrdU antibody and 7-amino-actinomycin D, analyzed with the gates shown here. Pooled data from three to five independent samples per time point are shown; bars represent the mean ± SEM. *p < 0.05; **p < 0.01 (Holm-Sidak). Experimental Hematology 2014 42, 46-58DOI: (10.1016/j.exphem.2013.09.010) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 5 Examination of gene expression mediating the EAR-2++ induced proliferative arrest of DN1 cells. EAR-2 or empty vector–transduced BM-HSCs were seeded in OP9-DL1 cultures and induced to differentiate under conditions that favored differentiation in to the T cell lineage. At (A) 24 hours or (B) 48 hours after induction of differentiation, cells with the immunophenotype GFP+, CD45+, c-kit+, Sca-1−, lineage− were FACS sorted, and gene expression was analyzed using quantitative PCR. Pooled data from two independent experiments are shown, bars represent the mean ± SEM. *p < 0.05; **p < 0.01. Experimental Hematology 2014 42, 46-58DOI: (10.1016/j.exphem.2013.09.010) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 6 EAR-2 inhibits differentiation and increases apoptosis at the DN4 to DP transition. (A) Analysis of T cell development in OP9-DL1 cultures was performed by flow cytometry for expression of CD44 and CD25 from days 5 to days 24, and for CD4 and CD8 expression at days 24 and 28 of culture. All dot plots were gated for CD45+GFP+ cells. Control cultures showed the expected progression from DN1 through DP stages, with 23.7% DP cells at day 24 and 79.2% DP cells at day 28. In contrast, development of EAR-2++ cells was impaired, with 4.06% DP cells and 53.4% DP cells at days 24 and 28, respectively. Representative dot plots from three independent experiments conducted in triplicates are shown. (B, C) Apoptosis and cell death were assessed in the cultures by flow cytometry for Annexin V and propidium iodide. EAR-2++ cultures exhibited an increase in cell death on day 24 of culture, coinciding temporally with the observed block in appearance of DP cells. Three independent experiments were conducted in triplicate. Bars represent the mean ± SEM. *p < 0.05; **p < 0.01. Experimental Hematology 2014 42, 46-58DOI: (10.1016/j.exphem.2013.09.010) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions

Figure 7 Examination of gene expression changes mediating the increase in apoptosis observed in EAR-2++ cells at day 24. EAR-2 or empty vector transduced BM-HSCs were seeded in OP9-DL1 cultures and induced to differentiate under conditions that favored differentiation in to the T cell lineage. After 24 days, DN4 and DP subpopulations were FACS sorted, and gene expression was analyzed using quantitative PCR. Pooled data from two independent experiments are shown; bars represent the mean ± SEM. *p < 0.05; **p < 0.01. Experimental Hematology 2014 42, 46-58DOI: (10.1016/j.exphem.2013.09.010) Copyright © 2014 ISEH - Society for Hematology and Stem Cells Terms and Conditions