ting-cells, 96-well microtiter plates were pre-coated with methyl-BSA and subsequently coated with calf thymus DNA, as previously described. The spots were developed with 5-bromo-4-chloro-3-indolyphosphate and enumerated using an automated ELISPOT reader and software. Detection of serum antibodies by enzyme-linked immunosorbent assay and detection of proteinuria Serum was collected from treated and untreated mice at different time points, and IgM- and IgG- anti-dsDNA antibodies were measured by ELISA as described previously using biotinlabeled goat anti-mouse IgG and IgM antibodies . Proteinuria was monitored monthly using Albustix. Statistical analysis Survival of the mice was analyzed by Kaplan-Meier curves and the effect of treatment on survival by a Cox proportional hazard model. Pairwise comparisons between controls and different treatments were done using post-hoc tests after fitting linear mixed models to avoid the type one error accumulation of single private statistical tests. Statistical analysis was performed with STATA 12 and figures were created using GraphPad Prism 5.0. All data were expressed as mean SEM. Results Short-term treatment regimens containing bortezomib lead to effective depletion of plasma cells, including the long-lived compartment Neuromedin N biological activity Twenty- to 22-week-old female NZB/W F1 mice were treated with a) PBS, b) anti-mouse CD20, c) anti-mouse CD20 plus anti-LFA-1/anti-VLA-4 blocking antibodies, d) anti-mouse CD20 combined with bortezomib, or e) anti-mouse CD20 together with bortezomib and with anti-LFA-1/anti-VLA-4 antibodies. Seven days after treatment, total plasma cells, SLPC and LLPCs in the bone marrow and spleen were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1972496 enumerated by flow cytometry. In the bone marrow, total plasma cells were significantly depleted by the treatments containing plasma cell-targeting agents to the average of 45%, 9% and 27% respectively, of their original value. In the group receiving anti-CD20 alone, no significant difference in the numbers of total plasma cells was observed. Of note, combination therapy with 4 / 17 Long-Term Plasma Cell Depletion Ameliorates SLE Fig 1. Effects of short-term depletion treatments on plasma cell 
numbers in bone marrow and spleen. Representative FACS histogram of bone marrow PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19724041 and splenic CD138+ intracellular + BrdU+ short-lived plasma cells, and CD138+ intracellular + BrdU- long-lived plasma cells from each treatment group. Percentage of remaining cell numbers relative to the control mean of bone marrow and splenic CD138+ intracellular + total plasma cells, SLPCs, and LLPCs in mice treated with PBS, anti-CD20, anti-CD20 plus integrin-blocking antibodies, anti-CD20 plus bortezomib and anti-CD20 plus Int and Bz. Total PCs, SLPCs and LLPCs were enumerated by flow cytometry 7 days after the start of treatment. Values are meanSEM; ns, non-significant; P>0.05, P<0.05, P<0.01, P<0.001, post-hoc test. Abbreviations: Bz, bortezomib; CD20, anti-mouse CD20 antibody; FMO, Fluorescence-minus-one; Int, Integrin blocking antibodies; anti-LFA1 and anti-VLA4 antibodies. doi:10.1371/journal.pone.0135081.g001 5 / 17 Long-Term Plasma Cell Depletion Ameliorates SLE anti-CD20, bortezomib and anti-LFA1/anti-VLA4 did not deplete plasma cells better than anti-CD20 plus bortezomib. Bone marrow SLPCs and LLPCs were depleted significantly in all groups treated with plasma cell-targeting agents. Interestingly, more significant reduction of bone marrow LLPCs was achieved by the two bortezomib-based regimens, which decreased the aver