BackgroundSystemic lupus erythematosus (SLE) is a B-cell-driven autoimmune disease that leads to long-term chronic inflammation. The B-cell stimulatory cytokine B-cell activating factor (BAFF) and IL-21 are implicated in the development of SLE, and both are elevated in the serum of SLE patients.1,2 B-cell differentiation signals through CD40 and the B-cell receptor (BCR) mediate B-cell differentiation and antibody production. The Celgene clinical candidate CC-220 is being developed for the treatment of SLE.ObjectivesDetermine the requirements for BAFF, IL-2, IL-21, CD40, and BCR in plasmablast differentiation from naive and memory B cells in T-cell contact-dependent and -independent models of plasmablast differentiation and assess the impact of the SLE clinical drug candidate CC-220.MethodsPeripheral blood mononuclear cells (PBMC) were isolated from healthy and SLE donors for flow cytometry analysis of B-cell subtypes. For B-cell differentiation experiments, naive and memory B cells were purified from healthy donors and treated with DMSO or CC-220 (1, 10, and 100 nM) 1 hour before culturing 5 days with combinations of IL-2, IL-21, BAFF, CD40L, and anti-IgM. B cells were assessed for proliferation, activation, and plasmablast generation by flow cytometry. IgM and IgG secretion in the culture supernatants were measured by ELISA, and gene expression was measured by qRT-PCR.ResultsBAFF combined with IL-2 and IL-21 was required to induce proliferation and plasmablast formation from memory B cells, but did not induce naive B-cell differentiation. Naive B-cell differentiation required CD40 signaling. Anti-IgM combined with IL-21 induced extensive cell death. Treatment of naive B cells with BAFF, IL-2, and IL-21 increased naive B-cell basal survival, but could not counter the cell death observed when IL-21 was present in combination with anti-IgM. In all cases, induced proliferation, plasmablast differentiation, and antibody production were inhibited by CC-220.ConclusionsThe soluble factors IL-2, IL-21, and BAFF in combination were sufficient to induce memory B-cell differentiation and antibody secretion in the absence of CD40L, but were insufficient to induce differentiation of naive B cells. Plasmablast generation from naive B cells required CD40L. Regardless of the stimulus used to induce B-cell proliferation and differentiation, CC-220 had a dominant inhibition effect. This has implications for the potential use of CC-220 in the treatment of B-cell-mediated pathology in SLE, regardless of whether B-cell proliferation and differentiation are occurring in secondary lymphoid organs in contact with T cells or differentiating at sites of inflammation in the presence of soluble factors such as IL-2, BAFF, and IL-21.ReferencesElbirt D, et al. BLyS levels in sera of patients with systemic lupus erythematosus: clinical and serological correlation. IMAJ 2014;16:491.Wang L, et al. Increased interleukin 21 and follicular helper T-like cells and reduced interleukin 10+ B cells in patients with new-onset systemic lupus erythematosu...
BackgroundProtein Kinase C theta (PKC-θ), a member of the PKC family of serine/threonine kinases, is essential in T cell receptor (TCR) signaling and T cell activation [1]. Inhibition of PKC-θ activity may provide new therapeutic options for autoimmune diseases with a T effector cell dependent pathology. CC-90005 is a highly selective small molecule inhibitor of PKC-θ.ObjectivesEvaluate the impact of specific PKC-θ inhibition on T-cell activation and anergy using the highly selective inhibitor CC-90005.MethodsHuman peripheral blood mononuclear cell (PBMC) or isolated T cell cultures were pre-treated with CC-90005 (0.1–10 μM) and stimulated for various times up to 24 h with anti-CD3 and anti-CD28 activation of the TCR, followed by a washout incubation period (1 h-24 h). Cells were then re-stimulated in the presence or absence of CC-90005 and cultured a further 24–96 h. T cell activation was assessed by CD25 and CD69 cell surface expression, thymidine incorporation, and cytokine production.ResultsUpon TCR stimulation, CC-90005 significantly inhibited CD25 and CD69 expression and T cell proliferation in PBMC and isolated T cells. Similarly, CC-90005 inhibited the production of Th1, Th2, Th17 and other pro-inflammatory cytokines. Furthermore, the inhibitory effects of CC-90005 on T cell activation persisted after washout in stimulated T cells, indicating an induction of an anergic cell state. CC-90005 anergy induction was shown to require only short initial exposures of as little as 2 h and to maintain anergy for as long as 48–96 h post CC-90005 washout. However, T cells pre-treated with the inhibitor in the absence of concomitant TCR stimulation were able to respond to stimulus after as little as 1 h washout, indicating a rapid recovery of PKC-θ activity and T-cell function. CC-90005 upregulated an anergy-related E3 ubiquitin ligase, Gene related to anergy in lymphocytes (GRAIL) in T cells. Elevated expression level of GRAIL was maintained in unresponsive T cells after washout, suggesting that GRAIL was a possible downstream mediator of CC-90005 effect on T cell anergy.ConclusionsCC-90005, a specific inhibitor of PKC-θ, significantly inhibits TCR-mediated T cell activation, proliferation, and cytokine production. Moreover, inhibition of these T-cell functions persists after drug withdrawal and restimulus of T-cells, but only if the primary T cell activation event occurrs in the presence of CC-90005. Thus, CC-90005 induces a functional unresponsiveness anergic state in T cells if present during TCR activation, which may have long-term therapeutic benefit in the treatment of T-cell mediated autoimmune and allergic inflammatory conditions.References Zhang EY, Kong K-F, Altman A. The yin and yang of protein kinase C-theta (PKCθ): a novel drug target for selective immunosuppression. Elsevier Inc. 2013. doi:10.1016/B978–0-12–404717–4.00006–8. Disclosure of InterestNone declared
BackgroundSystemic Lupus Erythematosis (SLE) is an autoimmune disease characterized by alterations in B-cells, autoantibody production, and elevations in circulating B-cell activating factor (BAFF). Aiolos single nucleotide polymorphisms and elevations in Aiolos mRNA are associated with SLE susceptibility. CC-220 is a Cullin Ring Ligase 4 Cereblon (CRL4CRBN) E3 Ubiquitin Ligase Modulator that induces ubiquitination and degradation of Aiolos and Ikaros transcription factors. We investigated the effects of CC-220 in B-cells from SLE patients to assess its impact on Aiolos protein levels and B-cell proliferation, differentiation, and antibody production.ObjectivesDetermine the protein levels of the B-cell transcription factors Aiolos and Ikaros in B-cell subtypes of SLE patients and assess the impact of the cereblon modulator CC-220 on Aiolos and Ikaros protein degradation, proliferation, and plasmablast differentiation.MethodsPeripheral blood mononuclear cells (PBMC) were measured for levels of circulating B-cell subtypes, B-cell activation state, and Aiolos and Ikaros protein levels by flow cytometry. Cell types were defined as: CD27-IgD+ naive, CD27+IgD- switched memory, CD27+IgD+ nonswitched memory, and CD27-IgD- double negative B-cells and CD20-CD38+ plasmablasts. Measurements of BAFF, IL-2 and IL-21 from plasma and IgG and IgM were done by ELISA. Plasmablast differentiation was induced by BAFF, IL-2, and IL-21 co-stimulation of B-cell cultures for 5 days in the absence and presence of CC-220.ResultsPlasma samples from SLE patients were found to have higher circulating BAFF, similar IL-2 levels, and reduced IL-21 levels relative to healthy individuals. Alterations in circulating B-cell subtypes occur in SLE patients including reduction of memory and elevations of naive and double negative B-cells. Alterations in biomarkers associated with chronic B-cell activation, including elevation of CD95 and reduction of CD21 and CD23 were also observed. Assessment of the B-cell differentiation transcription factors Aiolos and Ikaros in SLE B-cells showed that Aiolos, but not Ikaros is elevated in naive, switched memory, nonswitched memory, and double negative B-cells. CC-220 treatment reduced Aiolos and Ikaros protein levels in both SLE and healthy individuals in all four B-cell subtypes measured. CC-220 treatment was associated with reduced B-cell proliferation, plasmablast differentiation, and secretion of IgG and IgM in cells co-stimulated with BAFF, IL-2 and IL-21. Similarly, CC-220 reduced the expression of genes involved in B-cell differentiation including IRF-4, Xbp-1, Blimp-1, and IgJ indicating an early blockade in B-cell differntiation.ConclusionsOur observations that SLE is associated with alterations of circulating B-cell subtypes and their activation state, Aiolos overexpression in B-cells, and increases in circulating BAFF support the hypothesis that B-cells play a significant role in SLE pathology. Moreover, these observed changes suggest that B-cells may be predisposed towards plasmablast differentiation an...
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