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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 the numbers of different B-cell subsets in bone marrow and spleen.Percentage of remaining B cell subsets in the bone marrow and spleen in ratio to the mean of control. (A) 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) 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, *P<0.05; **P<0.01, ***P<0.001, post-hoc test (n = 5–6 mice per group). Abbreviations: Bz, bortezomib; CD20, anti-mouse CD20 antibody; Int, Integrin blocking antibodies; anti-LFA1 and anti-VLA4 antibodies.
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pone.0135081.g003: Effects of short-term depletion treatments on the numbers of different B-cell subsets in bone marrow and spleen.Percentage of remaining B cell subsets in the bone marrow and spleen in ratio to the mean of control. (A) 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) 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, *P<0.05; **P<0.01, ***P<0.001, post-hoc test (n = 5–6 mice per group). Abbreviations: Bz, bortezomib; CD20, anti-mouse CD20 antibody; Int, Integrin blocking antibodies; anti-LFA1 and anti-VLA4 antibodies.

Mentions: Total CD19+ B cells in the bone marrow and spleen were depleted significantly by all treatments; anti-CD20 plus bortezomib achieved the strongest effect (Fig 3A and 3B). Anti-CD20 alone did not significantly affect early-stage B cells (pro- and pre-B cells) in the bone marrow, but depleted immature and mature B cells significantly (Fig 3A). Anti-CD20 plus anti-LFA1/anti-VLA4 achieved similar results. In contrast, the bortezomib-based treatments (anti-CD20 plus bortezomib and anti-CD20 plus anti-LFA1/anti-VLA4/Bz) substantially reduced pro-B cells and significantly decreased pre-, immature and mature B cells (Fig 3A). In the spleen, all treatments significantly reduced the number of follicular (FO) B cells, but anti-CD20 plus anti-LFA1/anti-VLA4 and anti-CD20 plus anti-LFA1/anti-VLA4/Bz achieved the greatest reductions (Fig 3B). Germinal center (GC) B cells were significantly decreased by anti-CD20 plus bortezomib and anti-CD20 plus anti-LFA1/anti-VLA4/Bz. Marginal zone (MZ) B cells and B1 B cells were resistant to all treatments. MZ and B1 B cell numbers were actually higher in the groups treated with anti-LFA1/anti-VLA4 antibodies (Fig 3B). These data show that anti-CD20 antibody can deplete immature and mature B cells in the spleen and bone marrow without affecting early-stage (pro- and pre-B cells), MZ and B1 B cells. The addition of anti-LFA1/anti-VLA4 antibodies to the regimen did not increase the BCD effect, but increased the number of MZ and B1 B cells. Interestingly, the bortezomib-based treatments achieved efficient depletion of pre-B cells and germinal center B cells. The reduction of these cells can be considered a direct effect of bortezomib on cells with high protein synthesis and proliferation rates, as previously shown for germinal center B cells in lupus mice [10].


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 the numbers of different B-cell subsets in bone marrow and spleen.Percentage of remaining B cell subsets in the bone marrow and spleen in ratio to the mean of control. (A) 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) 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, *P<0.05; **P<0.01, ***P<0.001, post-hoc test (n = 5–6 mice per group). Abbreviations: Bz, bortezomib; CD20, anti-mouse CD20 antibody; Int, Integrin blocking antibodies; anti-LFA1 and anti-VLA4 antibodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4529137&req=5

pone.0135081.g003: Effects of short-term depletion treatments on the numbers of different B-cell subsets in bone marrow and spleen.Percentage of remaining B cell subsets in the bone marrow and spleen in ratio to the mean of control. (A) 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) 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, *P<0.05; **P<0.01, ***P<0.001, post-hoc test (n = 5–6 mice per group). Abbreviations: Bz, bortezomib; CD20, anti-mouse CD20 antibody; Int, Integrin blocking antibodies; anti-LFA1 and anti-VLA4 antibodies.
Mentions: Total CD19+ B cells in the bone marrow and spleen were depleted significantly by all treatments; anti-CD20 plus bortezomib achieved the strongest effect (Fig 3A and 3B). Anti-CD20 alone did not significantly affect early-stage B cells (pro- and pre-B cells) in the bone marrow, but depleted immature and mature B cells significantly (Fig 3A). Anti-CD20 plus anti-LFA1/anti-VLA4 achieved similar results. In contrast, the bortezomib-based treatments (anti-CD20 plus bortezomib and anti-CD20 plus anti-LFA1/anti-VLA4/Bz) substantially reduced pro-B cells and significantly decreased pre-, immature and mature B cells (Fig 3A). In the spleen, all treatments significantly reduced the number of follicular (FO) B cells, but anti-CD20 plus anti-LFA1/anti-VLA4 and anti-CD20 plus anti-LFA1/anti-VLA4/Bz achieved the greatest reductions (Fig 3B). Germinal center (GC) B cells were significantly decreased by anti-CD20 plus bortezomib and anti-CD20 plus anti-LFA1/anti-VLA4/Bz. Marginal zone (MZ) B cells and B1 B cells were resistant to all treatments. MZ and B1 B cell numbers were actually higher in the groups treated with anti-LFA1/anti-VLA4 antibodies (Fig 3B). These data show that anti-CD20 antibody can deplete immature and mature B cells in the spleen and bone marrow without affecting early-stage (pro- and pre-B cells), MZ and B1 B cells. The addition of anti-LFA1/anti-VLA4 antibodies to the regimen did not increase the BCD effect, but increased the number of MZ and B1 B cells. Interestingly, the bortezomib-based treatments achieved efficient depletion of pre-B cells and germinal center B cells. The reduction of these cells can be considered a direct effect of bortezomib on cells with high protein synthesis and proliferation rates, as previously shown for germinal center B cells in lupus mice [10].

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