RESUMEN
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 (Tregs ) 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 Tregs 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 Tregs from lupus patients. Our findings support that Cbl-b modulates resistance to suppression by regulating the K63 polyubiquitination profile in lupus Tregs . In addition, defective K63 polyubiquitination of STAT-3 is related to increased pSTAT-3 expression, and might promote the loss of suppressive capacity of Tregs in lupus patients.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Tolerancia Inmunológica , Inmunomodulación , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Adulto , Femenino , Humanos , Masculino , Factor de Transcripción STAT3/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , UbiquitinaciónRESUMEN
CD5 functions as a negative regulator of TCR signaling during thymocyte development, however, the molecular mechanisms involved in this process remain elusive. A key molecule involved in the down modulation of TCR signaling is c-Cbl, an ubiquitin ligase that physically associates with CD5. Crosslinking of TCR in thymocytes leads to ubiquitylation and lysosomal/proteasomal degradation of TCR downstream signaling effectors and CD5 itself. The present report shows that co-engagement of CD3 with CD5 enhanced c-Cbl phosphorylation, which was not affected by the deletion of the pseudo-ITAM domain of CD5, the putative binding site for c-Cbl. However, amino acids present in the carboxy-terminal region of CD5, were necessary for this effect, indicating that ITAM-independent sites were involved in the interaction of c-Cbl with CD5. The carboxy-terminal region of CD5 was also required for Vav degradation, a well-known target for c-Cbl-dependent ubiquitylation. These results support the notion that the distal cytoplasmic domain of CD5, including Y463, plays a relevant role in the downmodulation of TCR signals in thymocytes via c-Cbl.
Asunto(s)
Antígenos CD5/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Timocitos/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD5/química , Antígenos CD5/genética , Células Cultivadas , Regulación hacia Abajo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fosforilación , Estructura Terciaria de Proteína , Eliminación de Secuencia , Transducción de SeñalRESUMEN
OBJECTIVE: To analyze whether the expression and modulation of T cell receptor (TCR) signaling is dependent on Casitas B lineage lymphoma b (Cbl-b) in T cells from patients with systemic lupus erythematosus (SLE) upon stimulation with a tolerogenic substance. METHODS: Peripheral blood mononuclear cells were obtained from 20 patients with SLE (active disease or in remission) and 20 healthy controls. Levels of Cbl-b expression were measured using reverse transcription-polymerase chain reaction and Western blotting in peripheral CD4+ T cells from SLE patients and healthy controls upon anergy induction. Cell proliferation was measured using the carboxyfluorescein diacetate succinimidyl ester dilution method. Cytokine production was analyzed by luminometry, and surface expression of activation markers was assessed by flow cytometry. Transfection assays were performed to induce overexpression of Cbl-b, and phosphorylation of TCR-associated kinases was evaluated. RESULTS: CD4+ T cells from SLE patients displayed resistance to anergy (as evidenced by increased cell proliferation, interleukin-2 production, and expression of activation and costimulatory markers), and this was associated with altered Cbl-b expression. Upon ionomycin treatment, primary T cells showed enhanced MAPK activity and decreased Akt phosphorylation, which was representative of the anergic state. In T cells from lupus patients, Cbl-b overexpression led to increased expression of phosphorylated MAPK, thus indicating the reversibility of anergy resistance. CONCLUSION: These findings suggest that abnormal peripheral tolerance in SLE is caused by a deficiency in Cbl-b, and that this ubiquitin ligase plays a key role in regulating TCR signaling during the induction of peripheral tolerance.