B-Cell ELISpot Assay to Quantify Antigen-Specific Antibody-Secreting Cells in Human Peripheral Blood Mononuclear Cells.

Peripheral blood is commonly used to assess the cellular and humoral immune responses in clinical studies. It is a convenient sample to collect for immunological research as compared to the surgically excised and biopsied lymphoid specimens. To determine the functional status of immune system from peripheral blood, the enzyme-linked immunospot (ELISpot) assay is a popular method of choice owing to its high sensitivity, great accuracy, and easy performance. The ELISpot allows detection and quantification of cellular functionality at the single-cell level. Therefore, ELISpot assay is commonly applied to detect cytokines and cytotoxic granules released from T cells as well as to measure antibodies secreted from B cells. Because the ELISpot assay has been increasingly used for evaluation of the vaccine efficacy in clinical trials, standardization and reproducibility are crucial to minimize assay variability amongst samples from different sources. Here we introduce methods to isolate human peripheral blood mononuclear cells (PBMCs) for quantification of the antigen-specific antibody-secreting cells using the ELISpot assay.

The Lupus-Associated Fcγ Receptor IIb-I232T Polymorphism Results in Impairment in the Negative Selection of Low-Affinity Germinal Center B Cells Via c-Abl in Mice.

OBJECTIVE: Fcγ receptor IIb (FcγRIIb) is an essential negative regulator of B cells that blocks B cell receptor (BCR) signaling and triggers c-Abl-dependent apoptosis of B cells. FcγRIIb-deficient mice display splenomegaly with expansion of B cells, leading to lupus. FcγRIIb-I232T is a hypofunctional polymorphism associated with lupus susceptibility in humans, an autoimmune disease linked to diminished deletion of autoreactive B cells. In the context of the FcγRIIb-I232T polymorphism, we investigated the role of FcγRIIb in the deletion of low-affinity germinal center (GC) B cells, an important mechanism for preventing autoimmunity. METHODS: We generated FcγRIIb232T/T mice to mimic human FcγRIIb-I232T carriers and immunized mice with chicken gamma globulin (CGG)-conjugated NP, a T cell-dependent antigen, to examine the response of GC B cells. RESULTS: Compared to wild-type (WT) mice, FcγRIIb232T/T mice showed increased numbers of low-affinity NP-specific IgG and NP-specific B cells and plasma cells; additionally, the expression of a somatic mutation (W33L) in their VH 186.2 genes encoding high-affinity BCR was reduced. Notably, FcγRIIb232T/T mice had a higher number of GC light zone B cells and showed less apoptosis than WT mice, despite having equivalent follicular helper T cell numbers and function. Moreover, phosphorylation of c-Abl was reduced in FcγRIIb232T/T mice, and treatment of WT mice with the c-Abl inhibitor nilotinib during the peak of GC response resulted in reduced affinity maturation reminiscent of FcγRIIb232T/T mice. CONCLUSION: Our findings provide evidence of a critical role of FcγRIIb/c-Abl in the negative selection of GC B cells in FcγRIIb232T/T mice. Importantly, our findings indicate potential benefits of up-regulating FcγRIIb expression in B cells for treatment of systemic lupus erythematosus.

Dual immuno-renal targeting of 7-benzylidenenaltrexone alleviates lupus nephritis via FcγRIIB and HO-1.

Known as a selective δ1 opioid receptor (DOR1) antagonist, the 7-benzylidenenaltrexone (BNTX) is also a DOR1-independent immunosuppressant with unknown mechanisms. Here we investigated if BNTX could be beneficial for diseased MRL/lpr lupus mice. We treated mice with 0.5, 2, 5 or 10 mg/kg/day of BNTX for 2 weeks. At as low as 2 mg/kg/day, BNTX significantly improved splenomegaly and lymphadenopathy. Notably, B cell numbers, particularly autoreactive plasma cells, were preferentially reduced; moreover, BNTX enhanced surface expression of FcγRIIB, an immune complex (IC)-dependent apoptotic trigger of B cells. Consequently, serum autoantibody concentrations were significantly decreased, leading to diminished glomerular IC deposition and renal fibrosis, thereby improving proteinuria. Microarray and pathway analyses revealed heme oxygenase-1 (HO-1) and p38 MAPK as key mediators of BNTX-induced upregulation of FcγRIIB. Moreover, HO-1 expression was also induced by BNTX via p38 MAPK at renal proximal tubules to further cytoprotection. Taken together, we demonstrate that BNTX can alleviate lupus nephritis by reducing autoreactive B cells via FcγRIIB and by augmenting renal protection via HO-1. Accordingly, we propose a new strategy to treat lupus nephritis via such a dual immuno-renal targeting using either a single agent or combined agents to simultaneously deplete B cells and enhance renal protection. KEY MESSAGES: 7-Benzylidenenaltrexone (BNTX) alleviates lupus nephritis in diseased MRL/lpr mice. BNTX reduces autoreactive plasma cell numbers and serum autoantibody titers. BNTX upregulates FcγRIIB levels via p38 MAPK and HO-1 to reduce B cell numbers. Reduction of immune complex deposition and fibrosis by BNTX improves proteinuria. BNTX induces HO-1 via p38 MAPK to enhance protection of renal proximal tubules.