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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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Immunohistology of in vivo PBL engraftment in NOD rag2−/− Prf1−/− mice.On day of sacrifice, day 21 post PBL transfer, spleens were harvested and fixed for immunohistological analysis of B and T cell engraftment. Sections were visualized and photographed using a nikon microscope. All images were taken at 20× magnification. Rows A and B are PBS treated mice; C from mice treated 7 days with BLyS and, D and E from mice treated for 14 days with BLyS (10 ug/mouse/day). All mice were untreated days 14–21 prior to sacrifice. Serial sections were stained with hemotoxylin and eosin, anti-human CD45 or anti-human CD20. Data is representative of 6 experiments.
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pone-0003192-g004: Immunohistology of in vivo PBL engraftment in NOD rag2−/− Prf1−/− mice.On day of sacrifice, day 21 post PBL transfer, spleens were harvested and fixed for immunohistological analysis of B and T cell engraftment. Sections were visualized and photographed using a nikon microscope. All images were taken at 20× magnification. Rows A and B are PBS treated mice; C from mice treated 7 days with BLyS and, D and E from mice treated for 14 days with BLyS (10 ug/mouse/day). All mice were untreated days 14–21 prior to sacrifice. Serial sections were stained with hemotoxylin and eosin, anti-human CD45 or anti-human CD20. Data is representative of 6 experiments.

Mentions: To test whether huBLyS facilitated human B cell engraftment in immunodeficient mice, we transferred 20×106 human PBL by intrasplenic injection into NOD rag1−/−Ppf−/− mice. Recipients were given human recombinant BLyS (10 ug/mouse/day) or PBS i.p. for 7–14 days and sacrificed for analysis on day 21. Spleen samples were assessed by immunohistochemical staining. Figure 4 shows representative serial sections of spleens from PBS or BLys treated mice stained with H&E, anti-human CD45 and anti-human CD20. In PBS treated animals, the H&E staining shows relatively uniform reticular tissue, with few scattered CD45+ cells and no obvious formation of typical splenic architecture; importantly, cells staining with anti-CD20 were not readily observed (Figure 4, A and B). In marked contrast, BLyS treated mice showed a different morphology by H&E staining. Numerous follicle-like collections of human lymphocytes were observed and these stained strongly with anti-CD45 (both T and B cells) and with anti-CD20 (B cells) antibodies (Figure 4, C–E, CD45 and CD20). Higher magnification of the serial sections shown in Figure 5 clearly demonstrates CD45+ cells in areas not staining with CD20 suggesting the presence of T cells (see below). For comparison, we estimate the extent of human B cell engraftment by averaging the area of CD20+ staining cells within a number of microscopic fields. Among mice treated for 14 days with human BLyS, B cells comprise 30–50% of the spleen section areas (Figures 4 and 5, D and E), whereas, mice receiving BLyS for only 7 days generally had smaller areas of B cell engraftment, approximately 20–25% of the spleen sections (Figures 4 and 5, C). We evaluated the possibility that the marked B cell engraftment we observed was the result of an EBV-mediated lymphoproliferative disorder, however, the B cells in BLyS treated recipients were negative for EBV when examined by EBER-1 in situ hybridization (data not shown). This data is consistent with previous observations suggesting that three-fold higher numbers of PBL or depletion of CD8+ T cells are required for EBV-mediated lymphoproliferation in engrafted immunodeficient mice [13].


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)

Immunohistology of in vivo PBL engraftment in NOD rag2−/− Prf1−/− mice.On day of sacrifice, day 21 post PBL transfer, spleens were harvested and fixed for immunohistological analysis of B and T cell engraftment. Sections were visualized and photographed using a nikon microscope. All images were taken at 20× magnification. Rows A and B are PBS treated mice; C from mice treated 7 days with BLyS and, D and E from mice treated for 14 days with BLyS (10 ug/mouse/day). All mice were untreated days 14–21 prior to sacrifice. Serial sections were stained with hemotoxylin and eosin, anti-human CD45 or anti-human CD20. Data is representative of 6 experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2527131&req=5

pone-0003192-g004: Immunohistology of in vivo PBL engraftment in NOD rag2−/− Prf1−/− mice.On day of sacrifice, day 21 post PBL transfer, spleens were harvested and fixed for immunohistological analysis of B and T cell engraftment. Sections were visualized and photographed using a nikon microscope. All images were taken at 20× magnification. Rows A and B are PBS treated mice; C from mice treated 7 days with BLyS and, D and E from mice treated for 14 days with BLyS (10 ug/mouse/day). All mice were untreated days 14–21 prior to sacrifice. Serial sections were stained with hemotoxylin and eosin, anti-human CD45 or anti-human CD20. Data is representative of 6 experiments.
Mentions: To test whether huBLyS facilitated human B cell engraftment in immunodeficient mice, we transferred 20×106 human PBL by intrasplenic injection into NOD rag1−/−Ppf−/− mice. Recipients were given human recombinant BLyS (10 ug/mouse/day) or PBS i.p. for 7–14 days and sacrificed for analysis on day 21. Spleen samples were assessed by immunohistochemical staining. Figure 4 shows representative serial sections of spleens from PBS or BLys treated mice stained with H&E, anti-human CD45 and anti-human CD20. In PBS treated animals, the H&E staining shows relatively uniform reticular tissue, with few scattered CD45+ cells and no obvious formation of typical splenic architecture; importantly, cells staining with anti-CD20 were not readily observed (Figure 4, A and B). In marked contrast, BLyS treated mice showed a different morphology by H&E staining. Numerous follicle-like collections of human lymphocytes were observed and these stained strongly with anti-CD45 (both T and B cells) and with anti-CD20 (B cells) antibodies (Figure 4, C–E, CD45 and CD20). Higher magnification of the serial sections shown in Figure 5 clearly demonstrates CD45+ cells in areas not staining with CD20 suggesting the presence of T cells (see below). For comparison, we estimate the extent of human B cell engraftment by averaging the area of CD20+ staining cells within a number of microscopic fields. Among mice treated for 14 days with human BLyS, B cells comprise 30–50% of the spleen section areas (Figures 4 and 5, D and E), whereas, mice receiving BLyS for only 7 days generally had smaller areas of B cell engraftment, approximately 20–25% of the spleen sections (Figures 4 and 5, C). We evaluated the possibility that the marked B cell engraftment we observed was the result of an EBV-mediated lymphoproliferative disorder, however, the B cells in BLyS treated recipients were negative for EBV when examined by EBER-1 in situ hybridization (data not shown). This data is consistent with previous observations suggesting that three-fold higher numbers of PBL or depletion of CD8+ T cells are required for EBV-mediated lymphoproliferation in engrafted immunodeficient mice [13].

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