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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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Nuclear localization of NF-κB p52.Purified CD19+ human B cells were A.) incubated with 100 ng/106 cells of FLAG-huBLyS or FLAG-muBLyS followed by biotinylated anti-FLAG and strep-avidin PerCP on the day of isolation prior to FACS analysis. Control stain with anti-FLAG and PerCP (dash/dot line); huBLyS (solid line) and muBLyS (dark dashed line) B.) B cells were cultured unstimulated or with 100 ng/ml of human or murine BLyS for 48 hours. Cells were harvested, nuclear extracts prepared and Western blots prepared following protein separation on a 4–12% SDS gel. Blots were probed with anti-p52 antibody then stripped and reprobed with anti-TATAbpα antibody. Blots were developed by ECL. Data representative of 3 separate expts.
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pone-0003192-g003: Nuclear localization of NF-κB p52.Purified CD19+ human B cells were A.) incubated with 100 ng/106 cells of FLAG-huBLyS or FLAG-muBLyS followed by biotinylated anti-FLAG and strep-avidin PerCP on the day of isolation prior to FACS analysis. Control stain with anti-FLAG and PerCP (dash/dot line); huBLyS (solid line) and muBLyS (dark dashed line) B.) B cells were cultured unstimulated or with 100 ng/ml of human or murine BLyS for 48 hours. Cells were harvested, nuclear extracts prepared and Western blots prepared following protein separation on a 4–12% SDS gel. Blots were probed with anti-p52 antibody then stripped and reprobed with anti-TATAbpα antibody. Blots were developed by ECL. Data representative of 3 separate expts.

Mentions: Murine BLyS has been shown to bind to human BLyS receptors [39], [40]. When assayed by FACS, we find that human B cells bind both hu and mu BLyS, tested at the optimal concentration used in our survival assays (100 ng) (Figure 3A) and also at lower concentrations (1 and 10 ng, data not shown). These studies did not determine which of the BLyS receptors were occupied. BLyS signaling induces both the canonical (NF-κB1) and non-canonical (NF-κB2) pathways; activation of the non-canonical NF-κB2 pathway is important for B cell survival [18], [19], [41]–[43]. Studies using murine B cells have demonstrated that nuclear localization of NF-κB p52 is sustained for at least 48–72 hours following BLyS stimulation (laboratory observations; [18], [19], [44]). Our in vitro survival data (Figure 1) shows that by 48 hours human B cell survival in cultures supplemented with muBLyS is significantly lower (p = 0.008) than cultures supplemented with huBLyS, accordingly, we choose this time point to initially examine differences in nuclear localization of NF-kB p52. Western analysis of nuclear extracts prepared from cells after 48 hours of culture demonstrates that nuclear accumulation of NF-kB p52 in B cells stimulated with huBLyS was 3-fold higher than in B cells cultured with muBLyS and 7-fold higher than unstimulated B cells (Figure 3B, representative example). An analysis of p52 nuclear localization in three separate B cell preparations showed huBLyS induced on averaged 7.1±1.6 fold increase over unstimulated B cells verses an average 2.4±0.9 fold increase with muBLyS. While, muBLyS stimulation does result in higher nuclear accumulation of p52 compared to unstimulated B cells, this is insufficient to support in vitro 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)

Nuclear localization of NF-κB p52.Purified CD19+ human B cells were A.) incubated with 100 ng/106 cells of FLAG-huBLyS or FLAG-muBLyS followed by biotinylated anti-FLAG and strep-avidin PerCP on the day of isolation prior to FACS analysis. Control stain with anti-FLAG and PerCP (dash/dot line); huBLyS (solid line) and muBLyS (dark dashed line) B.) B cells were cultured unstimulated or with 100 ng/ml of human or murine BLyS for 48 hours. Cells were harvested, nuclear extracts prepared and Western blots prepared following protein separation on a 4–12% SDS gel. Blots were probed with anti-p52 antibody then stripped and reprobed with anti-TATAbpα antibody. Blots were developed by ECL. Data representative of 3 separate expts.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2527131&req=5

pone-0003192-g003: Nuclear localization of NF-κB p52.Purified CD19+ human B cells were A.) incubated with 100 ng/106 cells of FLAG-huBLyS or FLAG-muBLyS followed by biotinylated anti-FLAG and strep-avidin PerCP on the day of isolation prior to FACS analysis. Control stain with anti-FLAG and PerCP (dash/dot line); huBLyS (solid line) and muBLyS (dark dashed line) B.) B cells were cultured unstimulated or with 100 ng/ml of human or murine BLyS for 48 hours. Cells were harvested, nuclear extracts prepared and Western blots prepared following protein separation on a 4–12% SDS gel. Blots were probed with anti-p52 antibody then stripped and reprobed with anti-TATAbpα antibody. Blots were developed by ECL. Data representative of 3 separate expts.
Mentions: Murine BLyS has been shown to bind to human BLyS receptors [39], [40]. When assayed by FACS, we find that human B cells bind both hu and mu BLyS, tested at the optimal concentration used in our survival assays (100 ng) (Figure 3A) and also at lower concentrations (1 and 10 ng, data not shown). These studies did not determine which of the BLyS receptors were occupied. BLyS signaling induces both the canonical (NF-κB1) and non-canonical (NF-κB2) pathways; activation of the non-canonical NF-κB2 pathway is important for B cell survival [18], [19], [41]–[43]. Studies using murine B cells have demonstrated that nuclear localization of NF-κB p52 is sustained for at least 48–72 hours following BLyS stimulation (laboratory observations; [18], [19], [44]). Our in vitro survival data (Figure 1) shows that by 48 hours human B cell survival in cultures supplemented with muBLyS is significantly lower (p = 0.008) than cultures supplemented with huBLyS, accordingly, we choose this time point to initially examine differences in nuclear localization of NF-kB p52. Western analysis of nuclear extracts prepared from cells after 48 hours of culture demonstrates that nuclear accumulation of NF-kB p52 in B cells stimulated with huBLyS was 3-fold higher than in B cells cultured with muBLyS and 7-fold higher than unstimulated B cells (Figure 3B, representative example). An analysis of p52 nuclear localization in three separate B cell preparations showed huBLyS induced on averaged 7.1±1.6 fold increase over unstimulated B cells verses an average 2.4±0.9 fold increase with muBLyS. While, muBLyS stimulation does result in higher nuclear accumulation of p52 compared to unstimulated B cells, this is insufficient to support in vitro 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