Selective deletion of Eos (Ikzf4) in T-regulatory cells leads to loss of suppressive function and development of systemic autoimmunity

Abstract

Eos (lkzf4) is a member of the Ikaros family of transcription factors and is preferentially expressed in T-regulatory (Treg) cells. However, the role of Eos in Treg function is controversial. One study using siRNA knock down of Eos demonstrated that it was critical for Treg suppressor function. In contrast, Treg from mice with a global deficiency of Eos had normal Treg function in vitro and in vivo. To further dissect the function of Eos in Tregs, we generated mice with a conditional knock out of Eos in Treg cells (lkzf4^fl/fl X Foxp3^YFP-cre, Eos cKO). Deletion of Eos in Treg resulted in activation of CD4⁺Foxp3^- and CD8⁺ T cells at the age of 3 months, cellular infiltration in non-lymphoid tissues, hyperglobulinemia, and anti-nuclear antibodies. While Tregs from Eos cKO mice displayed normal suppressive function in vitro, Eos cKO mice developed severe Experimental Autoimmune Encephalomyletis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG) and Eos cKO Treg were unable to suppress Inflammatory Bowel Disease (IBD). Eos cKO mice had decreased growth of the transplantable murine adenocarcinoma MC38 tumor accompanied by enhanced IFN-γ/TNF-α production by CD8⁺ T cells in tumor draining lymph nodes. Mice with a global deficiency of Eos or a deficiency of Eos only in T cells developed autoimmunity at a much older age (12 months or 7-8 months, respectively). Taken together, Eos appears to play an essential role in multiple aspects of Treg suppressor function, but also plays an as yet unknown role in the function of CD4⁺Foxp3^- and CD8⁺ T cells and potentially in non-T cells.

Keywords: EAE; Eos; Foxp3; T effector cells; T regulatory cells; Transcription factor.

Fig. 1.

Eos cKO mice display an activated phenotype at 3 months of age. A) CD4⁺Foxp3⁻, B) CD4⁺Foxp3⁺, and C) CD8⁺ T cells in spleens from Eos^fl/fl or Eos cKO mice were stained with anti-CD44 and anti-CD62L. D) Splenocytes were gated on total CD4⁺ T cells from Eos^fl/fl (left) or Eos cKO(right) mice and percentages of Foxp3⁺ T cells were analyzed by flow cytometry. E) Splenocytes from Eos^fl/fl and Eos cKO mice were cultured with cell stimulation cocktail for 4 h and then stained for IFN-γ and IL-17 production among CD4⁺Foxp3⁻ and CD8⁺ T cells.

Fig. 2.

Eos cKO mice develop organ infiltration as early as 3 months of age. Lungs, small intestine, kidneys and salivary glands of 3 month old Eos^fl/fl or Eos cKO mice were sectioned and stained with H&E. Scale bars are 20 and 30 μm, respectively.

Fig. 3.

Eos cKO mice have hypergammaglobulinemia and decreased Tfr function. A) Sera from 3 month old Eos^fl/fl and Eos cKO mice were analyzed by ELISA for the indicated Ig isotype and the presence of ANA. B) Eos cKO mice have increased percentages of Tfh cells and decreased percentages of Tfr cells both before and after immunization. Top: Eos^fl/fl mice and Eos cKO mice were either not immunized or immunized with SRBCs and analyzed 7d post immunization. Splenocytes were gated on CD4⁺CD19⁻ T cells and analyzed for CXCR5 and PD-1 expression. Bottom: CXCR5⁺PD-1⁺ cells (Tf cells) were then analyzed for Foxp3 expression to determine the percentages of Tfh and Tfr cells.

Fig. 4.

Eos cKO mice have enhanced percentages of germinal center B cells and larger and more numerous germinal centers. A) Eos^fl/fl or Eos cKO mice were not immunized or immunized with SRBCs and stained for GL7⁺CD95⁺ GC B cells 7 days post immunization. B) 3-month old Eos^fl/fl or cKO mice were unimmunized or immunized with SRBC. Spleen sections were frozen, sectioned, and stained for IgD (blue), CD4 (green), and GL7 (red). Quantitation of active germinal centers is shown in the right panel. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5.

CD4⁺Foxp3⁻ T cells from Eos cKO proliferate normally and Eos cKO Treg exhibit normal suppressive function in vitro. A) Eos^fl/fl or Eos cKO naive CD4⁺Foxp3⁻CD44^hiCD62L^lo T cells (5 × 10⁴) were stimulated with soluble anti-CD3 and irradiated T cell-depleted splenocytes (5 × 10⁴) for 3 days. Cultures were pulsed with [³H]-thymidine for the last 6 h. B) Naïve Eos^fl/fl T cells were stimulated with anti-CD3 and T cells-depleted splenocytes in the presence of graded numbers of CD4⁺CD25⁺ Treg from either Eos^fl/fl or cKO mice. [³H]- thymidine was added for the last 6 h of culture.

Fig. 6.

Eos cKO Treg fail to suppress IBD. A) CD4⁺CD25⁻CD45RB^hi T cells from 8-week-old mice C57BL/6 mice were injected (4 × 10⁵ cells/mouse) into 8–10-week-old RAG^−/− recipients. CD4⁺CD25⁺ cells (2 × 10⁵ cells/mouse) from 3-month old Eos^fl/fl or Eos cKO mice were co-injected where indicated. Mice were monitored weekly for weight loss. Data is plotted as percent weight change from original weight and is representative of two independent experiments (n = 5 per group). Mice were sacrificed on day 35 post transfer and mesenteric lymph node cells analyzed for: B) percentages of CD4⁺Foxp3⁻ producing IFN-γ, IL-17, and IFN-γ/IL-17 double producers, and C) absolute number of CD4⁺Foxp3⁻ T cells, and D) percentage of CD4⁺Foxp3⁺ T cells were shown.

Fig. 7.

Eos cKO mice develop more severe EAE. A) Mice were immunized with MOG/CFA and injected with pertussis toxin on day 0 and 2. B) Percentages of CD4⁺Foxp3⁻ (left panel) CD4⁺Foxp3⁺(right panel) T cells in the spleen, dLNs, spinal cord and brain on day 14 were shown. C) Percentage of CD4⁺IL-17⁺ D) CD4⁺IFN-γ⁺ T cells, and E) CD4⁺IFN-γ⁺IL-17⁺ T cells in the spleen, DLNs, spinal cord and brain of WT and Eos cKO mice on day 14 were shown. The cells were harvested and stimulated with PMA/Ionomycin for 4 h before measurement of cytokine production.

Fig. 8.

Eos cKO mice have enhanced tumor immunity. A) Foxp3^YFP–cre and Eos cKO mice were injected with MC38 (2 × 10⁵) cells subcutaneously; tumor volumes were determined daily. B) TIL were isolated on d18 after tumor implantation and the frequencies of CD4⁺Foxp3⁺ Tregs, C) CD8⁺ T cells and D) the ratio of CD8⁺ to CD4⁺Foxp3⁺ cells within TIL were determined. E) TIL from d18 post tumor implantation were stimulated with cell stimulation cocktail for 5 h at 37 °C. CD8⁺CD44^hi T cells from TIL from d18 post tumor implants were stained for IFN-γ and TNF-α producing cells. *P < 0.05, **P < 0.01 and ***P < 0.0005 (unpaired two-tailed Student’s t-test). Data is a representative of two independent experiments (A-D) involving three to five mice per group (mean ± SEM).