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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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FACS analysis of collagenase disrupted engrafted spleens.One half of engrafted spleens were collagenase digested and then stained with anti-human CD45, CD20 and CD3 antibodies and analyzed by FACS. All samples were gated on live lymphocytes by forward and side scatter and then on CD45 positive cells. Data representative of 3 experiments. Total lymphocyte number = spleen cell count×percentage of CD45+ cells.
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pone-0003192-g006: FACS analysis of collagenase disrupted engrafted spleens.One half of engrafted spleens were collagenase digested and then stained with anti-human CD45, CD20 and CD3 antibodies and analyzed by FACS. All samples were gated on live lymphocytes by forward and side scatter and then on CD45 positive cells. Data representative of 3 experiments. Total lymphocyte number = spleen cell count×percentage of CD45+ cells.

Mentions: Whereas lymphocyte organization is revealed by immunohistology, more definitive quantitation of the human lymphocyte engraftment was assessed by flow cytometry. Spleens were divided in half upon harvest, with one half prepared for immunohistological analysis and the other prepared for FACS analysis by digestion with collagenase. Human lymphocyte yields (CD45+) from PBS treated recipients ranged between 0.27–5.8×106 cells/spleen, whereas, significantly more cells were isolated from recipients treated with BLyS (7.7–20.2×106 cells/spleen). The human B cell yields from the BLyS treated spleens was usually higher (4.1–9.5×106 cells/spleen) than the number of input B cells (2×106; 5–10% of total PBL) used to reconstitute the mice indicating that the engrafted cells had undergone proliferative expansion, perhaps by homeostatic proliferation induced by lymphocyte deficiency [45]–[47] or antigenic stimulation. Indeed, immunohistological staining of splenic sections with anti-Ki67, a marker for cells that have recently proliferated, was positive for almost all cells within the sections in BLyS-treated recipients whereas only scattered cells were positive in PBS-treated recipients (data not shown). Representative samples of collagenase disrupted spleens from separate experiments were analyzed by FACS (Figure 6) and showed that while T cells were present in some of the PBS treated mice (examples of positive engraftment shown with lymphocyte yield from test spleen), few, if any, B cells were detected in the same spleens. In contrast, both human B and T cells are readily detected in the huBLyS treated mice.


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)

FACS analysis of collagenase disrupted engrafted spleens.One half of engrafted spleens were collagenase digested and then stained with anti-human CD45, CD20 and CD3 antibodies and analyzed by FACS. All samples were gated on live lymphocytes by forward and side scatter and then on CD45 positive cells. Data representative of 3 experiments. Total lymphocyte number = spleen cell count×percentage of CD45+ cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003192-g006: FACS analysis of collagenase disrupted engrafted spleens.One half of engrafted spleens were collagenase digested and then stained with anti-human CD45, CD20 and CD3 antibodies and analyzed by FACS. All samples were gated on live lymphocytes by forward and side scatter and then on CD45 positive cells. Data representative of 3 experiments. Total lymphocyte number = spleen cell count×percentage of CD45+ cells.
Mentions: Whereas lymphocyte organization is revealed by immunohistology, more definitive quantitation of the human lymphocyte engraftment was assessed by flow cytometry. Spleens were divided in half upon harvest, with one half prepared for immunohistological analysis and the other prepared for FACS analysis by digestion with collagenase. Human lymphocyte yields (CD45+) from PBS treated recipients ranged between 0.27–5.8×106 cells/spleen, whereas, significantly more cells were isolated from recipients treated with BLyS (7.7–20.2×106 cells/spleen). The human B cell yields from the BLyS treated spleens was usually higher (4.1–9.5×106 cells/spleen) than the number of input B cells (2×106; 5–10% of total PBL) used to reconstitute the mice indicating that the engrafted cells had undergone proliferative expansion, perhaps by homeostatic proliferation induced by lymphocyte deficiency [45]–[47] or antigenic stimulation. Indeed, immunohistological staining of splenic sections with anti-Ki67, a marker for cells that have recently proliferated, was positive for almost all cells within the sections in BLyS-treated recipients whereas only scattered cells were positive in PBS-treated recipients (data not shown). Representative samples of collagenase disrupted spleens from separate experiments were analyzed by FACS (Figure 6) and showed that while T cells were present in some of the PBS treated mice (examples of positive engraftment shown with lymphocyte yield from test spleen), few, if any, B cells were detected in the same spleens. In contrast, both human B and T cells are readily detected in the huBLyS treated mice.

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