Lys63-polyubiquitination by the E3 ligase casitas B-lineage lymphoma-b (Cbl-b) modulates peripheral regulatory T cell tolerance in patients with systemic lupus erythematosus

Abstract

T cells from systemic lupus erythematosus (SLE) patients display a wide array of anomalies in peripheral immune tolerance mechanisms. The role of ubiquitin ligases such as Cbl-b has been described recently in these phenomena. However, its role in resistance to suppression phenotype in SLE has not been characterized, which was the aim of the present study. Thirty SLE patients (20 with active disease and 10 with complete remission) and 30 age- and sex-matched healthy controls were recruited. Effector (CD4⁺ CD25^- ) and regulatory (CD4⁺ CD25⁺ ) T cells (T_regs ) were purified from peripheral blood mononuclear cells (PBMCs) by magnetic selection. Suppression assays were performed in autologous and allogeneic co-cultures and analysed by a flow cytometry assay. Cbl-b expression and lysine-63 (K63)-specific polyubiquitination profile were assessed by Western blotting. We found a defective Cbl-b expression in T_regs from lupus patients in contrast to healthy controls (1·1 ± 0·9 versus 2·5 ± 1·8, P = 0·003), which was related with resistance to suppression (r = 0·633, P = 0·039). Moreover, this feature was associated with deficient K63 polyubiquitination substrates and enhanced expression of phosphorylated signal transducer and activation of transcription 3 (pSTAT-3) in T_regs from lupus patients. Our findings support that Cbl-b modulates resistance to suppression by regulating the K63 polyubiquitination profile in lupus T_regs . In addition, defective K63 polyubiquitination of STAT-3 is related to increased pSTAT-3 expression, and might promote the loss of suppressive capacity of T_regs in lupus patients.

Keywords: Cbl-b; K63-polyubiquitination; regulatory T cells; resistance to suppression; systemic lupus erythematosus.

Figure 1

Effector T cells from systemic lupus erythematosus (SLE) patients display a resistance to suppression by regulatory T cells (T_regs). CD4⁺CD25⁺ T_regs from SLE patients and healthy controls were tested for their ability to suppress CD4⁺CD25^– effector T cell proliferation in vitro. CD4⁺CD25^– effector and CD4⁺CD25⁺ T_regs were studied in autologous and allogenic co‐cultures (effector : T_regs ratio, 1 : 1) stimulated previously with plate‐bound anti‐CD3 antibody (5 µg/ml) and soluble anti‐CD28 antibody (2·5 µg/ml) for 48 h. Before incubation, only the CD25‐depleted effector T cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE‐DA; Biochemika‐Fluka). Cells were then harvested for proliferation assays by flow cytometry according to the CFSE‐DA dilution protocol. These are the pooled data from 30 SLE patients and 30 age‐ and sex‐matched healthy control proliferation assays. Ctl = healthy controls; T_eff = effector T cells. *P < 0·05.

Figure 2

Cbl‐b expression is decreased in regulatory T cells (T_regs) from systemic lupus erythematosus (SLE) patients and is associated with resistance to suppression. Cbl‐b protein expression was assessed in CD4⁺CD25⁺ T_reg lysates from SLE patients and healthy controls by Western blotting. A representative image of a Western blot for Cbl‐b from an active and a remitted SLE patient versus healthy control is shown (a), as well as the pooled data from 30 SLE patients and 30 age‐ and sex‐matched healthy controls normalized Cbl‐b protein expression (b). There was a positive correlation between percentage of CD4⁺CD25^– effector T cell proliferation suppression and Cbl‐b expression in T_regs from SLE patients (c). Ctl = healthy controls; AU = arbitrary units.

Figure 3

Regulatory T cells (T_regs) from systemic lupus erythematosus (SLE) patients are deficient in K63 ubiquitinated substrates. Lys63 (K63) protein expression was assessed in CD4⁺CD25⁺ T_reg lysates from SLE patients and healthy controls by Western blotting. A representative image of a Western blot for K63 from an SLE patient versus healthy control is shown (a), as well as the pooled data from 30 SLE patients and 30 age‐ and sex‐matched healthy controls normalized K63 protein expression (b). Ctl = healthy controls; AU = arbitrary units.

Figure 4

Deficient Cbl‐b expression and K63 polyubiquitination are associated with higher levels of activated signal transducer and activator of transcription 3 (STAT‐3) in regulatory T cells (T_regs) from systemic lupus erythematosus (SLE) patients. The CD4⁺CD25⁺ T_reg lysates from SLE patients and healthy controls were immunoprecipitated with anti‐STAT‐3 antibody and then subjected to Western blotting with the anti‐STAT‐3 or anti‐pSTAT‐3 antibodies. This is a representative image of a Western blot for pSTAT‐3 from an SLE patient versus healthy control. Ctl = healthy controls.