Bortezomib Plus Continuous B Cell Depletion Results in Sustained Plasma Cell Depletion and Amelioration of Lupus Nephritis in NZB/W F1 Mice.

NZB/W F1 mice were treated with: 1) anti-CD20, 2) anti-CD20 plus bortezomib, 3) anti-CD20 plus anti-LFA-1/anti-VLA-4 blocking antibodies, 4) anti-CD20 plus bortezomib and anti-LFA-1/anti-VLA4 blocking antibodies. Short- and long-lived plasma cells including autoreactive cells in the bone marrow and spleen were enumerated by flow cytometry and ELISPOT seven days after treatment. Based on these data in another experiment, mice received one cycle of anti-CD20 plus bortezomib followed by four cycles of anti-CD20 therapy every 10 days and were monitored for its effect on plasma cells and disease.Short-lived plasma cells in bone marrow and spleen were efficiently depleted by all regimens targeting plasma cells. Conversely, LLPCs and anti-dsDNA-secreting plasma cells in bone marrow and spleen showed resistance to depletion and were strongly reduced by bortezomib plus anti-CD20. The effective depletion of plasma cells by bortezomib complemented by the continuous depletion of their precursor B cells using anti-CD20 promoted the persistent reduction of IgG anti-dsDNA antibodies, delayed nephritis and prolonged survival in NZB/W F1 mice.These findings suggest that the effective depletion of LLPCs using bortezomib in combination with a therapy that continuously targeting B cells as their precursors may prevent the regeneration of autoreactive LLPCs and, thus, might represent a promising treatment strategy for SLE and other (auto)antibody-mediated diseases

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

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

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

Memory CD8+ T cells colocalize with IL-7+ stromal cells in bone marrow and rest in terms of proliferation and transcription

It is believed that memory CD8(+) T cells are maintained in secondary lymphoid tissues, peripheral tissues, and BM by homeostatic proliferation. Their survival has been shown to be dependent on IL-7, but it is unclear where they acquire it. Here we show that in murine BM, memory CD8(+) T cells individually colocalize with IL-7(+) reticular stromal cells. The T cells are resting in terms of global transcription and do not express markers of activation, for example, 4-1BB (CD137), IL-2, or IFN-γ, despite the expression of CD69 on about 30% of the cells. Ninety-five percent of the memory CD8(+) T cells in BM are in G(0) phase of cell cycle and do not express Ki-67. Less than 1% is in S/M/G(2) of cell cycle, according to propidium iodide staining. While previous publications have estimated the extent of proliferation of CD8(+) memory T cells on the basis of BrdU incorporation, we show here that BrdU itself induces proliferation of CD8(+) memory T cells. Taken together, the present results suggest that CD8(+) memory T cells are maintained as resting cells in the BM in dedicated niches with their survival conditional on IL-7 receptor signaling

Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

At present, it is not clear how memory B lymphocytes are maintained over time, and whetheronly as circulating cells or also residing in particular tissues. Here we describe distinctpopulations of isotype-switched memory B lymphocytes (Bsm) of murine spleen and bonemarrow, identified according to individual transcriptional signature and B cell receptorrepertoire. A population of marginal zone-like cells is located exclusively in the spleen, while apopulation of quiescent Bsm is found only in the bone marrow. Three further residentpopulations, present in spleen and bone marrow, represent transitional and follicular B cellsand B1 cells, respectively. A population representing 10-20% of spleen and bone marrowmemory B cells is the only one qualifying as circulating. In the bone marrow, all cells individuallydock onto VCAM1+ stromal cells and, reminiscent of resident memory T and plasmacells, are void of activation, proliferation and mobility.Peer Reviewe