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

Khodadadi L, Cheng Q, Alexander T, Sercan-Alp Ö, Klotsche J, Radbruch A, Hiepe F, Hoyer BF, Taddeo A - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: German Rheumatism Research Center Berlin (DRFZ) - a Leibniz Institute, Berlin, Germany; Department of Rheumatology and Clinical Immunology, Charité University Hospital Berlin, Berlin, Germany.

ABSTRACT

Methods: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

Effects of short-term depletion treatments on plasma cell numbers in bone marrow and spleen.(A) 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) bone marrow and (C) 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 (n = 5–6 mice per each group). Values are mean±SEM; 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.
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pone.0135081.g001: Effects of short-term depletion treatments on plasma cell numbers in bone marrow and spleen.(A) 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) bone marrow and (C) 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 (n = 5–6 mice per each group). Values are mean±SEM; 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.

Mentions: 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 enumerated by flow cytometry (Fig 1A). In the bone marrow, total plasma cells (CD138+, intracellular κ+) were significantly depleted by the treatments containing plasma cell-targeting agents (anti-CD20/LFA1/VLA4, anti-CD20/Bz and anti-CD20 plus anti-LFA1/anti-VLA4/Bz) 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 (Fig 1B). Of note, combination therapy with anti-CD20, bortezomib and anti-LFA1/anti-VLA4 did not deplete plasma cells better than anti-CD20 plus bortezomib. Bone marrow SLPCs (CD138+, intracellular κ+, BrdU+) and LLPCs (CD138+, intracellular κ+, BrdU-) 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 (anti-CD20/Bz and anti-CD20/LFA1/VLA4/Bz), which decreased the average percentage of remaining cells to 15% and 26% respectively (Fig 1B). The effects of the different treatments in the spleen were comparable to those observed in the bone marrow (Fig 1C). Anti-CD20 plus anti-LFA1/anti-VLA4, anti-CD20 plus Bz and anti-CD20 plus anti-LFA1/anti-VLA4/Bz decreased splenic SLPC numbers significantly, to an average of 10%, 16% and 3% remaining cells, respectively (Fig 1C). Conversely, only the bortezomib-based regimens (anti-CD20 plus Bz and anti-CD20 plus anti-LFA1/anti-VLA4 and Bz) achieved significant depletion of splenic LLPCs, which were reduced to an average 16% and 9% respectively, of the original numbers (Fig 1C). Notably, combination therapy with anti-CD20, bortezomib and anti-LFA1/anti-VLA4 led to greater depletion of plasma cells, especially SLPCs in spleen. Anti-CD20 therapy alone showed a trend to deplete only bone marrow and splenic SLPCs, probably by blocking the new generation of SLPCs from B cells. Anti-LFA1/anti-VLA4 plus anti-CD20 antibodies decreased the number of bone marrow and splenic SLPCs significantly, but did not affect LLPCs in spleen. Of all the regimens tested, anti-CD20 in combination with bortezomib was the most effective in depleting both short- and long-lived plasma cells as compared to the other regimens tested.


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

Khodadadi L, Cheng Q, Alexander T, Sercan-Alp Ö, Klotsche J, Radbruch A, Hiepe F, Hoyer BF, Taddeo A - PLoS ONE (2015)

Effects of short-term depletion treatments on plasma cell numbers in bone marrow and spleen.(A) 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) bone marrow and (C) 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 (n = 5–6 mice per each group). Values are mean±SEM; 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.
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Related In: Results  -  Collection

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pone.0135081.g001: Effects of short-term depletion treatments on plasma cell numbers in bone marrow and spleen.(A) 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) bone marrow and (C) 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 (n = 5–6 mice per each group). Values are mean±SEM; 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.
Mentions: 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 enumerated by flow cytometry (Fig 1A). In the bone marrow, total plasma cells (CD138+, intracellular κ+) were significantly depleted by the treatments containing plasma cell-targeting agents (anti-CD20/LFA1/VLA4, anti-CD20/Bz and anti-CD20 plus anti-LFA1/anti-VLA4/Bz) 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 (Fig 1B). Of note, combination therapy with anti-CD20, bortezomib and anti-LFA1/anti-VLA4 did not deplete plasma cells better than anti-CD20 plus bortezomib. Bone marrow SLPCs (CD138+, intracellular κ+, BrdU+) and LLPCs (CD138+, intracellular κ+, BrdU-) 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 (anti-CD20/Bz and anti-CD20/LFA1/VLA4/Bz), which decreased the average percentage of remaining cells to 15% and 26% respectively (Fig 1B). The effects of the different treatments in the spleen were comparable to those observed in the bone marrow (Fig 1C). Anti-CD20 plus anti-LFA1/anti-VLA4, anti-CD20 plus Bz and anti-CD20 plus anti-LFA1/anti-VLA4/Bz decreased splenic SLPC numbers significantly, to an average of 10%, 16% and 3% remaining cells, respectively (Fig 1C). Conversely, only the bortezomib-based regimens (anti-CD20 plus Bz and anti-CD20 plus anti-LFA1/anti-VLA4 and Bz) achieved significant depletion of splenic LLPCs, which were reduced to an average 16% and 9% respectively, of the original numbers (Fig 1C). Notably, combination therapy with anti-CD20, bortezomib and anti-LFA1/anti-VLA4 led to greater depletion of plasma cells, especially SLPCs in spleen. Anti-CD20 therapy alone showed a trend to deplete only bone marrow and splenic SLPCs, probably by blocking the new generation of SLPCs from B cells. Anti-LFA1/anti-VLA4 plus anti-CD20 antibodies decreased the number of bone marrow and splenic SLPCs significantly, but did not affect LLPCs in spleen. Of all the regimens tested, anti-CD20 in combination with bortezomib was the most effective in depleting both short- and long-lived plasma cells as compared to the other regimens tested.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: German Rheumatism Research Center Berlin (DRFZ) - a Leibniz Institute, Berlin, Germany; Department of Rheumatology and Clinical Immunology, Charité University Hospital Berlin, Berlin, Germany.

ABSTRACT

Methods: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus