Ubiquitin-specific peptidase 18 regulates the differentiation and function of Treg cells

Abstract

Ubiquitin-specific peptidase 18 (USP18) plays an important role in the development of CD11b⁺ dendritic cells (DCs) and Th17 cells, however, its role in the differentiation of other T cell subsets, especially in regulatory T (Treg) cells, is unknown. In our study, we used Usp18 KO mice to study the loss of USP18 on the impact of Treg cell differentiation and function. We found that USP18 deficiency upregulates the differentiation of Treg cells, which may lead to disrupted homeostasis of peripheral T cells, and downregulates INF-γ, IL-2, IL-17A producing CD4⁺ T cells and INF-γ producing CD8⁺ T cells. Mechanistically, we also found that the upregulation of Tregs is due to elevated expression of CD25 in Usp18 KO mice. Finally, we found that the suppressive function of Usp18 KO Tregs is downregulated. Altogether, our study was the first to identify the role of USP18 in Tregs differentiation and its suppressive function, which may provide a new reference for the treatment of Treg function in many autoimmune diseases, and USP18 can be used as a new therapeutic target for precise medical treatment.

Keywords: CD25; Foxp3; ICOS; Regulator T cell; USP18.

Figure 1

USP18 deficiency alters the homeostasis of peripheral T cells, but not the development of T cells in thymus. Flow cytometry analyzing the expression of CD4 and CD8 in lymphocytes of thymus, spleen and LN derived from Usp18 KO (n = 8) and WT (n = 8) mice. Shown are representative dot plots (A) as well as percentages and absolute numbers of CD4⁺(B) and CD8⁺ T cells (C). Expression of CD44 and CD62L on CD4⁺TCR⁺ or CD8⁺TCR⁺ T cells of spleen and LN derived from Usp18 KO (n = 6) and WT (n = 6) mice was analyzed by flow cytometry. Shown are representative dot plots and percentages of CD62L^hiCD44^lo naïve CD4⁺ T cells and CD62L^loCD44^hi activated CD4⁺ T cells (D–E). Dot plots and percentages of naïve and activated CD8⁺ T cells are shown in (F–G). Data are representative of three independent experiments and values are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ns, not significant.

Figure 2

USP18 deficiency upregulates the differentiation of Treg cells. Representative figures shown the expression of CD25 in CD4⁺ T cells from the spleen, thymus and LN of WT and Usp18 KO mice (A) Percentages and absolute numbers of CD4⁺CD25⁺ T cells from the spleen, thymus and LN of WT (n = 6) and KO littermates (n = 6) (C) Representative figures, percentages and absolute numbers of CD4⁺Foxp3⁺ T cells from the spleen, thymus and LN of WT (n = 6) and KO littermates (n = 6) as indicated in (B–D) Flow cytometry analyzing expression of CD25 in CD4⁺CD25⁺ T cells (E–G) and Foxp3 in CD4⁺Foxp3⁺ T cells (F–H) from the spleen, thymus and LN of WT (n = 6) and Usp18 KO littermates (n = 6). Flow cytometry analyzing the apoptosis and proliferation of CD4⁺CD25⁺ and CD4⁺Foxp3⁺ T cells from the spleen, thymus and LN of WT (n = 4) and KO littermates (n = 4) (I–L). Shown are representative dot plots from one of three independent experiments. Flow cytometry analysis of CD25 and Foxp3 expression in CD45.2⁺CD4⁺T cells of spleen, thymus and LN in mixed bone marrow chimeras (n = 5) eight weeks after transfer (M−O) ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns, not significant.

Figure 3

USP18 deficiency decreases the ability of cytokine production by effector T cells. Flow cytometry analyzing the levels of IFN-γ, IL-4 and IL-17A secreted by stimulated CD4⁺ and CD8⁺ T cells from the spleen, thymus and LN of WT (n = 5) and Usp18 KO mice (n = 5) (A–H). Shown are representative dot plots and mean values (±SD) of percentages from three independent experiments. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns, not significant.

Figure 4

USP18 deficiency downregulates the suppression function of Treg cells in vitro. Purified Treg cells of spleens from WT and Usp18 KO mice were cultured with CellTrace Violet-labeled naïve CD4⁺ T cells from WT mice and anti-CD3/CD28 antibodies at the indicated ratio for 72 h in vitro. The proliferation of naïve CD4⁺ T cells was measured by flow cytometry. Shown are representative images from at least two independent experiments (A). Flow cytometry analyzing the expression of ICOS (B–C), CTLA-4 (D–E), PD-1 (F–G) and neuropilin-1(Nrp1) (H–I) in CD4⁺CD25⁺ and CD4⁺Foxp3⁺ Treg cells from the spleen, thymus and LN of WT (n = 5) and KO littermates (n = 5). Shown are representative images and mean values (±SD) from three independent experiments. ∗P < 0.05; ns, not significant.