Additional file 2: Figure S2. of Analyzing pathogenic (double-stranded (ds) DNA-specific) plasma cells via immunofluorescence microscopy

Correlation between dsDNA-specific plasma cells numbers in spleen and bone marrow with the anti-dsDNA antibody titer in the serum. (JPEG 1553 kb

Effects of short-term depletion treatments on bone marrow and splenic T cells.

<p>Percentage of remaining CD3⁺ T cells, CD4⁺ T-helper cells, and CD8⁺ T-cytotoxic cells after one week of treatment in ratio to the mean of control in (<b>A)</b> the bone marrow, and (<b>B)</b> spleen. Values are mean±SEM; ns, non-significant, P>0.05, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, post-hoc test (<i>n</i> = 5–6 mice per group). Abbreviations: Bz, bortezomib; CD20, anti-mouse CD20 antibody; Int, Integrin blocking antibodies, anti-LFA1 and anti-VLA4 antibodies.</p

Effects of short-term depletion treatments on plasma cell numbers in bone marrow and spleen.

<p><b>(A)</b> Representative FACS histogram of bone marrow and splenic CD138⁺ intracellular κ⁺ BrdU⁺ short-lived plasma cells (SLPCs), and CD138⁺ intracellular κ⁺ BrdU^- long-lived plasma cells (LLPCs) from each treatment group. Percentage of remaining cell numbers relative to the control mean of (<b>B)</b> bone marrow and (<b>C)</b> splenic CD138⁺ intracellular κ⁺ total plasma cells (PCs), SLPCs, and LLPCs in mice treated with PBS, anti-CD20, anti-CD20 plus integrin-blocking antibodies (Int; anti-LFA1 and anti-VLA4 antibodies), anti-CD20 plus bortezomib (Bz) and anti-CD20 plus Int and Bz. Total PCs, SLPCs and LLPCs were enumerated by flow cytometry 7 days after the start of treatment (<i>n</i> = 5–6 mice per each group). Values are mean±SEM; ns, non-significant; P>0.05, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><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.</p

B cell depletion (BCD) maintenance therapy after short-term depletion (STD) of B and plasma cells with ant-CD20 and bortezomib improves the disease in NZB/W F1 mice.

<p>Mice (n = 4) were treated with anti-CD20 and bortezomib (STD) alone or continuous B cell depletion without bortezomib (BCD, n = 5) or treated as STD followed by BCD maintenance therapy with anti-CD20 (STD+BCD, n = 4). (<b>A)</b> Serum IgM and IgG anti-dsDNA antibody levels in treated and untreated mice (n = 9), as measured by ELISA. (<b>B)</b> Proteinuria in treated and untreated mice. Statistical differences between treated and untreated mice were analyzed using the post-hoc test (ns, non-significant, P>0.05, *<i>P</i><0.05; **<i>P</i><0.01, ***<i>P</i><0.001). (<b>C)</b> Survival curves for treated and untreated NZB/W F1 mice (Kaplan—Meier log-rank test). Abbreviations: STD, Short-term depletion (anti-CD20 and bortezomib); BCD; B cell depletion (anti-CD20).</p

Effects of short-term depletion treatments on the numbers of different B-cell subsets in bone marrow and spleen.

<p>Percentage of remaining B cell subsets in the bone marrow and spleen in ratio to the mean of control. (<b>A)</b> Bone marrow B-cell subsets identified by flow cytometry: total B cells (BCs) (CD19⁺), bone marrow pro-B cells (CD93⁺CD117⁺), pre-B cells (CD24⁺IgM^-IgD^-), immature B cells (CD24⁺IgM⁺IgD^-), and mature B cells (CD24^-IgM⁺IgD⁺). (<b>B)</b> Splenic B-cell subsets identified by flow cytometry: follicular (FO) B cells (CD23⁺CD21⁺IgM⁺), marginal zone (MZ) B cells (CD23^- CD21⁺IgM⁺), germinal center (GC) B cells (IgD^-GL7⁺), and B1 B cells (CD23^-CD21^-IgM⁺). Values are mean±SEM; ns, non-significant, P>0.05, *<i>P</i><0.05; **<i>P</i><0.01, ***<i>P</i><0.001, post-hoc test (<i>n</i> = 5–6 mice per group). Abbreviations: Bz, bortezomib; CD20, anti-mouse CD20 antibody; Int, Integrin blocking antibodies; anti-LFA1 and anti-VLA4 antibodies.</p