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Human BLyS facilitates engraftment of human PBL derived B cells in immunodeficient mice.

Schmidt MR, Appel MC, Giassi LJ, Greiner DL, Shultz LD, Woodland RT - PLoS ONE (2008)

Bottom Line: Although human B cells bound both human and murine BLyS, nuclear accumulation of NF-kappaB p52, an indication of the induction of a protective anti-apoptotic response, following stimulation with human BLyS was more robust than that induced with murine BLyS suggesting a fundamental disparity in BLyS receptor signaling.Human BLyS treated recipients had on average 40-fold higher levels of serum Ig than controls and mounted a de novo antibody response to the thymus-independent antigens in pneumovax vaccine.The data indicate that production of fully immunologically competent humanized mice from PBL can be markedly facilitated by providing human BLyS.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America. Madelyn.Schmidt@umassmed.edu

ABSTRACT
The production of fully immunologically competent humanized mice engrafted with peripheral lymphocyte populations provides a model for in vivo testing of new vaccines, the durability of immunological memory and cancer therapies. This approach is limited, however, by the failure to efficiently engraft human B lymphocytes in immunodeficient mice. We hypothesized that this deficiency was due to the failure of the murine microenvironment to support human B cell survival. We report that while the human B lymphocyte survival factor, B lymphocyte stimulator (BLyS/BAFF) enhances the survival of human B cells ex vivo, murine BLyS has no such protective effect. Although human B cells bound both human and murine BLyS, nuclear accumulation of NF-kappaB p52, an indication of the induction of a protective anti-apoptotic response, following stimulation with human BLyS was more robust than that induced with murine BLyS suggesting a fundamental disparity in BLyS receptor signaling. Efficient engraftment of both human B and T lymphocytes in NOD rag1(-/-) Prf1(-/-) immunodeficient mice treated with recombinant human BLyS is observed after adoptive transfer of human PBL relative to PBS treated controls. Human BLyS treated recipients had on average 40-fold higher levels of serum Ig than controls and mounted a de novo antibody response to the thymus-independent antigens in pneumovax vaccine. The data indicate that production of fully immunologically competent humanized mice from PBL can be markedly facilitated by providing human BLyS.

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In vitro survival of CD19+ human B cells with human or murine BLyS.CD19+ B cells were purified from PBL by negative selection using RosetteSep kit and ficoll hypaque centrifugation. B cells were cultured for 4 days with 100 ng/ml of human or murine BLyS, cultures were resupplemented with BLyS on day 2. Viability was determined daily using cell counting with trypan blue and is represented as percentage of input cell number surviving. Donor 1 data is the average of 2 separate B cell preparations; donors 2–6 represent a single cell preparation. Statistical analysis for significance after 4 days in culture; huBLyS vs. muBLyS, p = 0.0041; huBLyS vs unstimulated, p = 0.0014; muBLyS vs. unstimulate, p = ns.
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pone-0003192-g001: In vitro survival of CD19+ human B cells with human or murine BLyS.CD19+ B cells were purified from PBL by negative selection using RosetteSep kit and ficoll hypaque centrifugation. B cells were cultured for 4 days with 100 ng/ml of human or murine BLyS, cultures were resupplemented with BLyS on day 2. Viability was determined daily using cell counting with trypan blue and is represented as percentage of input cell number surviving. Donor 1 data is the average of 2 separate B cell preparations; donors 2–6 represent a single cell preparation. Statistical analysis for significance after 4 days in culture; huBLyS vs. muBLyS, p = 0.0041; huBLyS vs unstimulated, p = 0.0014; muBLyS vs. unstimulate, p = ns.

Mentions: To establish possible species restrictions on BLyS dependent human B cell survival in vitro, CD19+ B cells from the PBL of normal human donors were cultured unstimulated or with human or murine BLyS and viability determined daily. The results from these determinations, Figure 1, clearly demonstrate that huBLyS enhances human B cell survival relative to either unstimulated or muBLyS supplemented cultures. Indeed, muBLyS provides no more survival advantage for human B cells than is seen in unstimulated cultures. Increasing the dose of muBLyS to 200 or 500 ng/ml did not improve human B cell survival (data not shown). Donor 1 was assayed on two separate occasions, 8 months apart, with similar results as indicated by the error bars in the graph. Statistical analysis of pooled data from 6 donors demonstrated the species dependence of BLyS mediated human B cell survival (at day 4 of culture: huBLyS vs. muBLyS, p = 0.0041; huBLyS vs unstimulated, p = 0.0014; muBLyS vs. unstimulated, p = ns). The species dependence of BLyS mediated human B cell survival was not observed for murine B cells, Figure 1B; both human and murine BLyS were equally effective at supporting murine B cell survival.


Human BLyS facilitates engraftment of human PBL derived B cells in immunodeficient mice.

Schmidt MR, Appel MC, Giassi LJ, Greiner DL, Shultz LD, Woodland RT - PLoS ONE (2008)

In vitro survival of CD19+ human B cells with human or murine BLyS.CD19+ B cells were purified from PBL by negative selection using RosetteSep kit and ficoll hypaque centrifugation. B cells were cultured for 4 days with 100 ng/ml of human or murine BLyS, cultures were resupplemented with BLyS on day 2. Viability was determined daily using cell counting with trypan blue and is represented as percentage of input cell number surviving. Donor 1 data is the average of 2 separate B cell preparations; donors 2–6 represent a single cell preparation. Statistical analysis for significance after 4 days in culture; huBLyS vs. muBLyS, p = 0.0041; huBLyS vs unstimulated, p = 0.0014; muBLyS vs. unstimulate, p = ns.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2527131&req=5

pone-0003192-g001: In vitro survival of CD19+ human B cells with human or murine BLyS.CD19+ B cells were purified from PBL by negative selection using RosetteSep kit and ficoll hypaque centrifugation. B cells were cultured for 4 days with 100 ng/ml of human or murine BLyS, cultures were resupplemented with BLyS on day 2. Viability was determined daily using cell counting with trypan blue and is represented as percentage of input cell number surviving. Donor 1 data is the average of 2 separate B cell preparations; donors 2–6 represent a single cell preparation. Statistical analysis for significance after 4 days in culture; huBLyS vs. muBLyS, p = 0.0041; huBLyS vs unstimulated, p = 0.0014; muBLyS vs. unstimulate, p = ns.
Mentions: To establish possible species restrictions on BLyS dependent human B cell survival in vitro, CD19+ B cells from the PBL of normal human donors were cultured unstimulated or with human or murine BLyS and viability determined daily. The results from these determinations, Figure 1, clearly demonstrate that huBLyS enhances human B cell survival relative to either unstimulated or muBLyS supplemented cultures. Indeed, muBLyS provides no more survival advantage for human B cells than is seen in unstimulated cultures. Increasing the dose of muBLyS to 200 or 500 ng/ml did not improve human B cell survival (data not shown). Donor 1 was assayed on two separate occasions, 8 months apart, with similar results as indicated by the error bars in the graph. Statistical analysis of pooled data from 6 donors demonstrated the species dependence of BLyS mediated human B cell survival (at day 4 of culture: huBLyS vs. muBLyS, p = 0.0041; huBLyS vs unstimulated, p = 0.0014; muBLyS vs. unstimulated, p = ns). The species dependence of BLyS mediated human B cell survival was not observed for murine B cells, Figure 1B; both human and murine BLyS were equally effective at supporting murine B cell survival.

Bottom Line: Although human B cells bound both human and murine BLyS, nuclear accumulation of NF-kappaB p52, an indication of the induction of a protective anti-apoptotic response, following stimulation with human BLyS was more robust than that induced with murine BLyS suggesting a fundamental disparity in BLyS receptor signaling.Human BLyS treated recipients had on average 40-fold higher levels of serum Ig than controls and mounted a de novo antibody response to the thymus-independent antigens in pneumovax vaccine.The data indicate that production of fully immunologically competent humanized mice from PBL can be markedly facilitated by providing human BLyS.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America. Madelyn.Schmidt@umassmed.edu

ABSTRACT
The production of fully immunologically competent humanized mice engrafted with peripheral lymphocyte populations provides a model for in vivo testing of new vaccines, the durability of immunological memory and cancer therapies. This approach is limited, however, by the failure to efficiently engraft human B lymphocytes in immunodeficient mice. We hypothesized that this deficiency was due to the failure of the murine microenvironment to support human B cell survival. We report that while the human B lymphocyte survival factor, B lymphocyte stimulator (BLyS/BAFF) enhances the survival of human B cells ex vivo, murine BLyS has no such protective effect. Although human B cells bound both human and murine BLyS, nuclear accumulation of NF-kappaB p52, an indication of the induction of a protective anti-apoptotic response, following stimulation with human BLyS was more robust than that induced with murine BLyS suggesting a fundamental disparity in BLyS receptor signaling. Efficient engraftment of both human B and T lymphocytes in NOD rag1(-/-) Prf1(-/-) immunodeficient mice treated with recombinant human BLyS is observed after adoptive transfer of human PBL relative to PBS treated controls. Human BLyS treated recipients had on average 40-fold higher levels of serum Ig than controls and mounted a de novo antibody response to the thymus-independent antigens in pneumovax vaccine. The data indicate that production of fully immunologically competent humanized mice from PBL can be markedly facilitated by providing human BLyS.

Show MeSH
Related in: MedlinePlus