Background/Purpose: Lymphopenia has been associated with the development of autoimmune diseases. Particularly, in patients with Systemic Lupus Erythematosus (SLE) is a common clinical feature. However, the molecular mechanisms related to lymphopenia in SLE have not been fully addressed. Besides, the E3 ligase Cbl-b has been shown to regulate T cell responsiveness and its deficiency has been documented in SLE. The aim of this study was to assess CD4⁺ T cell number and proliferation regulated by cell cycle inhibitors ubiquitination by the E3 ligase Cbl-b in SLE patients.

Methods: We included 20 patients with SLE (8 in remission and 12 with active untreated disease) according to the classification criteria of the American College of Rheumatology and 20 age and gender-matched healthy controls. Absolute peripheral lymphocyte count was obtained for each subject. PBMCs were isolated by density gradient and effector (CD4⁺CD25^–) T cells were purified by magnetic selection and stimulated with anti-CD3+anti-CD28 beads. The expression of total ubiquitin, Cbl-b, p27^kip1 and p21^cip1 was analyzed by Western blotting. Interaction between Cbl-b, p27^kip and p21^cip1 was addressed by immunoprecipitation (IP). Proliferative responses were assessed by CFSE. Differences were assessed by t Student test and Spearman correlation coefficient was also used. p<0.05 was considered as statistically significant.

Results: We found decreased Cbl-b expression in effector T cells from SLE patients in comparison to healthy controls (1.6±0.2 vs 3.8±1.3, p=0.003), which was associated with lower proliferation upon TCR stimulation vs healthy controls (p=0.022). Decreased proliferation and absolute number of effector T cells correlated with Cbl-b expression (r=0.553, p=0.020; r=0.680, p=0.003, respectively). Moreover, this phenomenon was associated with diminished ubiquitination of the cell cycle inhibitor p27^kip1 in effector T cells from SLE patients when compared to healthy controls. We did not find differences in ubiquitination and expression of another cell cycle inhibitor, p21^cip1 in comparison to healthy controls. Interestingly, we also found by IP assays, that p27^kip1 and p21^cip1interact with Cbl-b. We found no significant differences regarding to disease activity.

Conclusion: Our data suggest that the deficiency of the E3 ligase Cbl-b is associated with the presence of lymphopenia in SLE patients. This phenomenon is related to decreased ubiquitination of the cell cycle inhibitor p27^kip1 in effector T cells from SLE patients, which triggers its sustained expression as well as decreased proliferation of lupus lymphocytes. Furthermore, this effect is specific to p27^kip1, since even though, p21^cip1 also interacts with Cbl-b, its ubiquitination and expression was not related to this E3 ligase in SLE patients. To our knowledge, this is the first evidence that suggest deficient ubiquitination as a molecular mechanism of lymphopenia in SLE patients.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cd4-t-cell-lymphopenia-is-associated-with-deficient-ubiquitination-of-the-cell-cycle-inhibibitor-p27kip1-by-the-e3-ligase-casitas-b-lineage-lymphoma-b-in-patients-with-systemic-lupus-erythematosus/