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

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.

Figure 1. 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.

Figure 2. FACS analysis of B cell cultures.

B cell populations were FACS analyzed for surface markers associated with resting B cells (CD45, CD19), memory cells (CD27), plasma cells (CD38) and kappa and lambda light chains on the day of isolation and after 4 days of culture either unstimulated or stimulated with human or murine BLyS. All samples were initially gated for lymphocytes by forward and side scatter. Data representative of 4 experiments.

Figure 3. 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.

Figure 4. 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.

Figure 5. Immunohistology.

Higher magnification images (100×) of portions of the sections shown in figure 3. Estimates of percentages of B cell engraftment are done at this magnification by comparing the percentage of the splenic section staining with anti-CD20.

Figure 6. 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.