Advertisement
Research Article

Human BLyS Facilitates Engraftment of Human PBL Derived B Cells in Immunodeficient Mice

  • Madelyn R. Schmidt mail,

    Madelyn.Schmidt@umassmed.edu

    Affiliation: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America

    X
  • Michael C. Appel,

    Affiliation: National Institutes of Health, The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Bethesda, Maryland, United States of America

    X
  • Lisa J. Giassi,

    Affiliation: Department of Medicine, Division of Diabetes, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America

    X
  • Dale L. Greiner,

    Affiliation: Department of Medicine, Division of Diabetes, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America

    X
  • Leonard D. Shultz,

    Affiliation: The Jackson Laboratory, Bar Harbor, Maine, United States of America

    X
  • Robert T. Woodland

    Affiliation: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America

    X
  • Published: September 11, 2008
  • DOI: 10.1371/journal.pone.0003192

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-κB 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.

Introduction

The development of a small animal model that reproducibly supports human lymphocyte development and/or peripheral lymphocyte survival and function should lead to improved treatment strategies for human tumors, autoimmune diseases and new vaccines. Immunodeficient mice, such as NOD-Prkdcscid, NOD-rag1−/− Prf1−/−, NOD-scid IL-2Rγ−/−, Balb/c-rag1−/−IL-2Rγ/ and H2d-rag1−/−IL2Rγ−/− have been used as recipients of human peripheral blood lymphocytes (PBL) or human hematopoietic stem cells (HSC) (reviews [1], [2]). Chimeric mice engrafted with human HSC can support B and T cell development and the survival of peripheral T and B cell populations [3][12], however lymphoid development following engraftment is slow (3–6 months) and is of varying efficiency even when the same HSC preparation is used to engraft multiple mice.

Conceptually, the engraftment of mature human peripheral blood lymphocytes (PBL) into immunodeficient murine recipients overcomes the need for long reconstitution times required when using human CD34+ cord blood derived HSC and should allow for a rapid assessment of immune responsiveness of diverse individuals, for example, young and aged humans. In addition, the potential to establish primary lymphoid tumors, such as leukemias and lymphomas, in a chimeric host would provide opportunities to investigate the efficacy of novel therapeutic strategies tailored to individual patients. T cell engraftment is frequently observed when PBL are transferred into NOD rag2−/− Prf1−/− and NOD rag2−/−IL-2rγ−/− mice, whereas B cell homeostasis is abnormal. Mature B cells persist early after PBL transfer and can produce recall and polyclonal immunoglobulin (Ig) responses, however, these B cells are lost within 2 weeks and this loss is accelerated if CD4+ T cells are removed from the initial inoculum [13]. These data suggest that the murine environment may not provide the critical growth factors and/or signaling ligands necessary for B cell homeostasis.

Mature B cells are actively maintained in vivo by survival signals received through the B cell antigen receptor (BCR) and a receptor for the TNF family ligand B lymphocyte stimulator, BLyS, also known as BAFF, TALL-1, THANK, TNFSF13B and zTNF4 [14][17]. BLyS is a type II protein produced in both membrane-bound and soluble forms by stromal cells, macrophages, dendritic cells and neutrophils [18]. BLyS has two proposed mechanisms of action; to facilitate the differentiation of short-lived immature B cells into mature recirculating long-lived B cells, and to actively maintain mature B cells in the periphery by facilitating their survival through non-canonical NF-κB mediated signals [17], [19][21]. BLyS dependent survival signals are delivered through the BLyS receptor, BR3 (BAFF-R) that is expressed on late immature and mature peripheral B cells [18], [22][24]. Two other receptors for BLyS are also found on B cells, TACI and BCMA, and their expression is associated with different maturation and differentiation states [18], [25]. In humans, BLyS has been shown to be critical for B cell survival, the generation of lymphoid follicles and survival of plasmablasts formed from human memory B cells [24], [26], [27]. Overexpression of BLyS has been correlated with autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis [28][30], whereas lower levels of BLyS are associated with antibody immunodeficiency [31], [32]. In addition, some human lymphoid tumors (non-Hodgkins lymphoma and multiple myeloma) may produce BLyS as an autocrine growth factor promoting tumor survival [33][35].

Given the critical role of BLyS in normal B cell homeostasis, we proposed that the failure of efficient human B cell engraftment and survival in xenochimeras may be due to a BLyS deficiency. This failure could be due to species differences between human and murine BLyS that affect survival signaling. Consistent with this view, there is a single amino acid difference between the human and murine BLyS proteins in the portion of the molecule recognized by the BR3 receptor [18], [22], [23], [36].

In this report, we show human recombinant BLyS improves human peripheral blood B cell survival in vitro whereas murine BLyS is ineffective. Moreover, engraftment of both B and T cells is markedly enhanced in immunodeficient NOD rag2−/− Prf1−/− mice supplemented with recombinant human BLyS. All B cell subpopulations are maintained, follicular-like structures develop and B cells secrete antibody and respond to challenge with thymus-independent pneumococcal antigens. Taken together, the data demonstrate the requirement for human BLyS for efficient engraftment of human B cells in immunodeficient mice.

Results

Human BLyS enhances human B cell survival in vitro

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.

thumbnail

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.

doi:10.1371/journal.pone.0003192.g001

To determine if huBLyS conferred a selective survival advantage to a particular subpopulation of PBL derived human B cells, input populations and cells remaining after 4 days of culture were compared by FACS using antibodies to CD19 (all B cells), CD27 (memory B), kappa and lambda light chains, CD10 (immature B), and CD38 (immature and plasma cells). Immature B cells (CD10+, CD27, CD38+) represented 1–3% of input B cells from the various donors (our data and [37], [38]) and were undectable on day 4 analysis under all culture conditions. No other significant change in the character of the surviving B cell population relative to the input population as assessed by these markers was observed, Figure 2 data shown from CD19 and CD27 analysis.

thumbnail

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.

doi:10.1371/journal.pone.0003192.g002

muBLyS binds to human B cells but does not mobilize NF-kB p52 as effectively as huBLyS

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.

thumbnail

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.

doi:10.1371/journal.pone.0003192.g003

To further investigate the notion that the amount of nuclear NF-κB p52 correlated with B cell survival, we cultured murine B cells with varying concentrations of huBLyS and assessed cell survival and p52 nuclear localization. We found the survival of murine B cells cultured with 1 ng/ml of huBLyS was similar to that observed in unstimulated cultures (figure S1A). In contrast, both 10 and 100 ng/ml of huBLyS supported murine B cell survival (figure 1 and figure S1A). After 48 hours of culture, nuclear extracts were made from all cultures, western blotted for NF-κB p52 and quantified. We observe that, similar to human B cells, murine B cells cultured with a sub-optimal huBLyS concentration mobilize low levels of p52 to the nucleus (supplemental figure 1B). It is noteworthy that the amount of nuclear p52 induced by the non-protective dose of huBLyS is similar to that seen with human cells cultured with 100 ng of muBLyS. Taken together, the data show a strong correlation between survival and the amount of p52 localized to the nucleus.

Engraftment of human B cells in immunodeficient mice

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].

thumbnail

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.

doi:10.1371/journal.pone.0003192.g004
thumbnail

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.

doi:10.1371/journal.pone.0003192.g005

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.

thumbnail

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.

doi:10.1371/journal.pone.0003192.g006

A summary of in vivo engraftment efficiency is shown in Table 1. Of the 16 mice treated with PBS, only 7 engrafted T cells and one of those 7 also engrafted B cells, overall 43% of the mice contained human lymphocytes. The PBS treated mice that were positive for human T or B cells had far fewer engrafted cells as determined by the sparse immunohistological staining; and reduced viable spleen cell yields. In comparison, 88% of the BLyS treated mice showed engraftment of both B and T cells with significantly higher numbers of donor splenocytes being isolated from these mice.

thumbnail

Table 1. Summary of in vivo engraftment data.

doi:10.1371/journal.pone.0003192.t001

Human BLyS is required for maintenance of the engrafted B cells

Human BLyS is required to initiate engraftment of B cells from human PBL in NOD rag1−/−Ppf−/− mice and we noted that mice receiving BLyS for only 7 days had lower amounts of B cells compared with mice receiving 14 days of treatment (20–25% vs 30–60%, determined by comparison of overall CD20 staining of histology sections, Table 2). To examine whether human B cells required the continued presence of huBLyS for in vivo survival as we would predict from murine B cell studies, a group of animals was treated for 7 days with huBLyS followed by injections of the soluble BLyS decoy receptor TACI-Ig protein (10 ug/mouse/day) for the next 7 days. TACI-Ig will bind both human and murine BLyS creating a BLyS deficient environment. Mice were sacrificed at day 21 and immunohistological analysis carried out on the spleens. We found that B cells were reduced when BLyS was withdrawn during PBS injections (about 2-fold lower) and were further reduced when mice received the BLyS decoy receptor (10-fold lower, Table 2, summary of histology results). Immunohistological analysis showed that T cells were still present (data not shown). Thus, maintenance of peripheral human B cell populations in reconstituted immunodeficient mice still require huBLyS.

thumbnail

Table 2. BLyS decoy receptor decreases B cell engraftment in NOD rag1−/− Prf1−/− mice.

doi:10.1371/journal.pone.0003192.t002

In vivo antibody production

To determine if the B cells in recipient mice were functional, we assessed human antibody production in mice treated with PBS or huBLyS for 14 days. NOD rag1−/−Ppf−/− mouse serum had undetectable levels of antibody. PBS treated recipients averaged 5 ug/ml of IgM and 60 ug/ml of IgG suggesting some early activation of transferred B cells prior to their loss from the animals. Recipients treated 14 days with BLyS had 40 fold more IgM, average 200 ug/ml, and 10 fold more IgG, 570 ug/ml consistent with the levels of B cell engraftment found (Table 3). To determine if antibody production was sensitive to BLyS depletion, mice were treated for 7 days with BLyS or 7 days with BLyS and then 7 days of TACI-Ig decoy receptor. Mice treated with TACI-Ig had approximately 8-fold lower amounts of IgM, 28 ug/ml, and 2-fold lower IgG compared to recipients receiving 14 days of BLyS treatment; consistent with TACI-Ig depletion of B cells and with the different half-lives of the antibody classes.

thumbnail

Table 3. Human serum Ig.

doi:10.1371/journal.pone.0003192.t003

To assess the antigen responsiveness of transferred B cells, PBL recipients were immunized with pneumovax23, to test whether engrafted B cells could produce a de novo antibody response to a thymus-independent type 2 antigen. Recipient mice were vaccinated on the day of PBL transfer and serum collected at day 21 and analyzed with a serotype specific ELISA. Shown in Table 4 is the data for Streptococcus pneumoniae serotype 14, one of the 23 pneumococcal polysaccharide strains in the vaccine. Serum from PBL engrafted mice receiving PBS, huBLyS or PBS plus vaccine had a similar low quantity of anti-polysaccharide antibody, ranged between 0.7–1.4 ng/ml for IgM and 2.4–3.3 ng/ml for IgG. In striking contrast, recipients of huBLyS and vaccine averaged 10-fold higher amounts, 9 ng/ml IgM and 45 ng/ml IgG, of serotype 14 specific antibody. Similar levels of IgG and IgM were observed for pneumococcal serotype 4 (data not shown). These data demonstrate that the human B cells in PBL recipients can respond de novo to challenge with T cell independent antigens.

thumbnail

Table 4. Induction of a thymus-independent immune response in human PBL engrafted NOD rag1−/− Prf1−/− mice.

doi:10.1371/journal.pone.0003192.t004

Discussion

These experiments demonstrate the requirement for human BLyS to efficiently reconstitute human B cells in NOD rag1−/−Ppf−/− immunodeficient mice. A species specific BLyS restriction is demonstrated for human B cells in culture and for B cells transferred to murine hosts. The NOD rag1−/−Ppf−/− environment is not BLyS deficient, per se, as these mice readily support the engraftment of murine B cells. Species specificity in the action of survival and growth promoting cytokines is not novel to members of the TNF family; IL-2, IL-5, IL-6, and gamma interferon have all demonstrated species restrictions [18], [48][52]. Moreover, amino acid differences between murine and human BLyS in the BR3 receptor binding region and/or differences in the BLyS binding region of the BR3 receptor are known to have marked effects on survival signaling for human B cells [18], [22], [23], [53]. While we favor the notion that the basis for species restriction is in the interaction between BLyS and BR3, human B and T cells also express the TACI receptor for BLyS and differences in huBLyS and muBLyS binding to and signaling through TACI may also contribute to our results.

In vitro, human BLyS supported significantly better human B cell survival than did murine BLyS, which was indistinguishable from unsupplemented cultures. In contrast, human and murine BLyS were equally effective at promoting the in vitro survival of murine B cells. Concurrent with the survival effect, BLyS stimulation leads to a prolonged induction of NF-κB p100 processing and nuclear localization of NF-κB p52 (laboratory observations and [19], [44]). While freshly isolated human B cells bind FLAG-tagged murine BLyS as efficiently as human BLyS, p52 nuclear localization in human B cells induced by human BLyS was more robust than that induced by murine BLyS. This shows a dissociation between BLyS binding and survival signaling which may be the result of a species difference in the binding requirement for activation of BR3/TACI receptors on human B cells. It is noteworthy that in titration of human BLyS on murine B cells diminished B cell survival was correlated directly with the extent of p52 nuclear localization (supplemental figure 1). Our laboratory has recently defined the survival pathways induced by BLyS in murine B cells [54] and further experiments are in progress to define these pathways in human B cells.

Engraftment of human B cells is efficient in huBLyS treated NOD rag1−/−Ppf−/− recipients. Human BLyS is continually required to maintain the B cells over the 3 week period used in these studies since treatment with a BLyS decoy receptor, TACI-Ig, which depletes both human and murine BLyS from the animals, resulted in loss of B cells as assessed by immunohistological analysis and resulted in lower serum IgM and IgG compared with mice treated for 14 days with BLyS. Our engraftment data using huBLyS supplementation contrasts from that observed in other PBL xenochimera models where few, if any, human B cells are observed after cell transfer and those B cells do not survive beyond one week [10], [13], [55][60].

The B cells are functional as we find significantly higher amounts of human IgM and IgG in chimeras receiving huBLyS compared with PBS treated controls. Human Ig production could result from the homeostatic activation of naïve or memory B cells. Homeostatic B cell proliferation is seen when murine B cells are transferred into B cell deficient environments [45], [61], [62], however this mechanism has not been evaluate in xenochimeras. It is also possible that activation by xenoantigens can contribute to B cell activation and serum Ig. The antigen responsiveness of engrafted human B cells and the ability to induce a de novo immune response is demonstrated by the production of anti-pneumococcal antibodies following immunization with the thymus-independent antigens in pneumovax23. There was a 10–20 fold increase, respectively in IgM and IgG, in pneumococcal antibodies in BLyS treated recipients compared to BLyS treated and unimmunized and PBS treated immunized controls. This xenochimera model with enhanced B cell engraftment provides a unique opportunity to test vaccine responses using PBL samples from a variety of individuals, including neonates and the aged who frequently exhibit weak immune responsiveness.

Immunohistological analysis of recipient mice receiving only PBS revealed only scattered human T cells but these cells were readily identified by FACS analysis of collagenase digested spleens. It was noted, however, that there was a marked improvement of T cell engraftment with human BLyS supplementation. Both T and B cell yields from BLyS treated recipients was much higher than that observed with PBS treated recipients. Recent publications demonstrate that BLyS can act as a co-stimulator for T cells acting through BR3 and/or TACI [63][66]. T cells, like B cells, will under go homeostatic proliferation when introduced to a T cell deficient environment [45], [46], [67][69]. This proliferation causes induction of early activation markers, including expression of BR3 mRNA and protein on T cells [63]. Human T cells may also undergo activation to xenoantigens in the murine environment, again upregulating the BLyS receptor. In this regard, it is also possible that the human B cells themselves can present antigen to the T cells, thereby facilitating T cell expansion and survival. TACI-BLyS signaling in B cell-dendritic cell interactions have been shown to increase the expansion of antigen responsive CD8+ T cells [70] whereas BR3-BLyS costimulates CD4+ T cell alloresponses [66]. We did note that use of the TACI decoy receptor in vivo resulted in loss of both B and T cells, although B cells were affected to a greater degree. Taken together, these data suggest that human T cell engraftment may also be enhanced directly or indirectly by human BLyS treatment. Further studies using purified human T cells and human BLyS supplementation should address whether human BLyS acts directly on T cells to facilitate engraftment.

Since huBLyS enhances engraftment of both B and T cells, we considered the possibility that prolonged exposure to huBLyS may increase the rate of graft verses host disease (GVH) in the PBL recipient mice. No GVH was seen by observation or histological analysis at the 3 week time points assessed in these experiments even in recipients with the robust B and T cell engraftment, however, other investigators have found evidence of GVH by 4–6 weeks post-PBL engraftment in a variety of immunodeficient murine hosts [1], [71]. Regulatory T cells have recently been shown to express BLyS receptors and it is possible that huBLyS treated animals engraft sufficient regulatory cells to delay or prevent GVH [63], [65].

Our data suggest recombinant human BLyS has a significant enhancing effect on the engraftment of both T and B cell populations in PBL.

Materials and Methods

Human lymphocytes

Human blood was obtained from healthy volunteers and blood donors under signed consent in accordance with the Declaration of Helsinki and approval from the Institutional Review Board of the University of Massachusetts Medical School. Total human peripheral blood mononuclear cells (PBL) were purified by Ficoll gradient separation, quantified and viability assessed by trypan blue exclusion. Human CD19+ B cells were purified from PBL by negative selection using RosetteSep (StemCell Technologies, Vancouver BC, Canada).

Murine lymphocytes

Murine B cells were prepared by anti-thy1.2 and complement treatment of splenocytes followed by purification of resting B cells using a step percoll gradient harvesting cells at the 60–70% interface [72].

Mice

NOD/Cg-Rag1tm1MomPpftm1Sclz/SzJ (abbreviated NOD rag1−/−Ppf−/−, stock # 004848) and C57BL/6 mice were obtained from Jackson Laboratories and NCI, respectively. Mice were housed at the University of Massachusetts Medical School under specific pathogen free conditions in accordance with federal and institutional IACUC guidelines. Immunodeficient mice received acidified (HCl; pH 2.8–3.2) water containing trimethoprim-sulfamethoxazole (Goldline Laboratories, Ft. Lauderdale, FL) ad librium for 7 consecutive days every other week.

BLyS

Two sources of human BLyS were used in these experiments. Purified recombinant human BLyS (huBLyS, Human Genome Sciences, Rockville, MD 20850) or recombinant human FLAG-tagged BLyS (FL-BLyS) purified in our laboratory from transfected CHO cell supernant (cell line generously provided by Dr. Randolph Noelle, Dartmouth Univ. Lebanon, NH). Briefly, stably transfected CHO cells were grown in DMEM (7%FCS, 2 mM glutamine, 1× MEM non-essential amino acids, 10 units/ml penicillin and 10 ug/ml streptomycin) and supernatants collected. Supernatants were dialyzed against 50 mMTris pH 8.0, 50 mM NaCl, 0.02% sodium azide prior to running over an anti-FLAG M2 agarose column (Sigma-Aldrich Biochemicals). FL-BLyS was eluted with 0.1 M glycine HCl pH 3.0 and dialyzed against PBS, incubated with polymyxin B-agarose beads (Sigma-Aldrich, 1 ml of 50% suspension per 15 mls) for I hour, sterile filtered and quantitated by spectrophotometry (1 OD280 = 1.15 mg/ml). Purity was assessed by SDS-PAGE and Western blot. All human BLyS preparations tested negative for endotoxin and mycoplasma. Both huBLyS and FL-BLyS performed equivalently and were used interchangeably in these experiments. FLAG-tagged murine BLyS (muBLyS) (#522-052-C010) was obtained from Alexis biochemicals (Axxora, San Diego, CA 92121).

Cell culture

Purified CD19+ B cells (94–98% pure by FACS analysis) were cultured at 5×106 cells per ml in RPMI 1640 - complete media (CM: 10% FCS, 2 mM glutamine, 1× non-essential DMEM amino acids, 10 units/ml penicilin, 1 ug/ml streptomycin, 5×10−5 M 2-mercaptoethanol) alone or in the presence of 100 ng/ml of human or murine BLyS for 4 days. Preliminary experiments established the dose of BLyS sufficient for optimal cell survival: 1, 10, 100, 250 and 500 ng/ml were tested and no significant differences on cell survival were found at doses of 10 ng/ml or greater. BLyS was readded to the cultures on day 2. Cell survival was determined by counting daily using trypan blue exclusion for viability. Input populations and cells remaining after 4 days of culture were stained for human CD19, CD27, CD38, CD10, kappa and lambda light chains and analyzed by FACS.

In vivo engraftment

In preparation for engraftment NOD rag1−/−Ppf−/− received a single i.p. injection of 1.0 mg of TMβ1 (anti-CD122) monoclonal antibody for depletion of NK cells and 10 ug of purified human recombinant BLyS (huBLyS). Recipients received 20×106 PBL by intrasplenic injection followed by daily i.p. injection of 10 ug huBLyS or endotoxin free PBS for 7–14 days. Spleens were harvested 14–21 days post PBL transfer and processed for immunohistochemisry. In some expts, spleens were bisected and each half processed for either immunohistochemistry or FACS analysis.

Decoy receptor

To deplete BLyS in vivo, groups of 3 mice that had previously received huBLyS for 7 days were given soluble TACI-Ig (10 ug i.p./mouse/day, Human Genome Sciences) for 7 subsequent days.

Antibodies

Sources of anti-human antibodies used for FACS staining. Caltag: CD3-FITC, CD45-APC, CD45-PE, CD20-APC, CD19-APC, CD19-PE, IgD-FITC, CD38-PE; ebioscience: CD27-FITC, CD138-FITC and PE, CD10-biotin, kappa-biotin, lambda-biotin: BD Pharmingen: streptavidin-PerCP, IgM-APC, and CD45-PECy5.5. For immunohistochemistry BD Pharmingen: CD45, CD3, CD20, Ki67, and EBNA1.

Immunohistochemistry

At sacrifice, spleens were fixed in 10% neutral buffered formalin, embedded in paraffin, and 5 um tissue sections were cut. Immunohistochemical staining was performed with human specific mABs (BD Pharmingen, San Diego, CA). Prior to staining sections were incubated in 0.1 M citrate buffer (pH 6.0) for 15 mins and then stained on a Dako (Carpinteria, CA) automated immunostainer using the EnVision (Dako) staining procedure. The sections were incubated with the EnVision plus Dual Link reagent (a polymer conjugated with goat anti-rabbit Ig or goat anti-mouse Ig and horseradish peroxidase) for 30 min. The sections were washed and reacted with 3-diaminobenzidine and hydrogen peroxide and counterstained with hemotoxylin for visualization by light microscopy.

Flow cytometry

For in vitro experiments, purified PBL B cells were tested for purity and subpopulations characterized after initial isolation and 4 days of culture using CD45, CD19, CD20, CD27 and CD38 antibodies conjugated with APC, Fitc, PE or PerCp, described below. BLyS binding was determined by incubating 106 purified B cells with 100 ng of murine or human FL-BLyS, followed by biotinylated anti-FLAG antibody (#F9291, SigmaAldrich) and strept-avidin PerCP (BD). FACS analysis of human lymphocyte engrafted mice was performed as follows: half of each recipient spleen was minced and incubated in 0.5ml HBSS containing 2.4 mg/ml collagenase XI (SigmaAldrich), 1 mg/ml DNAse I and 2% FCS for one hour at 37°C in a shaking water bath. Cell suspensions were then washed twice with CM, resuspended in 1 ml CM and incubated for one hour at 37°C in a CO2 incubator prior to FACS staining. Single cell suspensions were washed into FACS buffer (PBS, 3%FCS, 0.02% sodium azide) and106 cells in 100 ul kept on ice for staining. Nonspecific staining was blocked by incubating cells with rabbit and mouse IgG (3ug) or 10 ul of C57BL/6 mouse serum for 10 mins on ice prior to adding fluorchrome-labeled antibodies. Cells were stained for 30–45 mins, washed and fixed with 2% paraformaldehyde prior to analysis. Engrafted cell numbers were determined from total cell counts isolated by collagenase multiplied by 2 (half of each spleen digested) and by the percentage of human CD45+ cells. Samples were analyzed using BD FACScalibur or FACSVantage machines and data analyzed using FlowJo software (Tree Star Inc., Ashland, OR).

Western blotting

Purified CD19+ B cells were cultured unstimulated or with 100 ng/ml of mu FL-BLyS or hu FL-BLyS for 48 hours. Cells were harvested and nuclear and cytoplasmic extracts prepared as described [73]. Protein content was determined using Commassie protein assay reagent (Pierce Biotechnology, Rockland, IL) Samples were run on 4–12% NuPAGE MOPS gels (Invitrogen) and transferred to nitrocellulose membrane overnight. Membranes were blocked with 5% BSA, PBS, 0.2% Tween-20 buffer, anti-p100/p52 antibody (#4882, Cell Signaling Technology, Danvers, MA) or TATA binding protien (#AB818, Abcam, Cambridge Science Park, UK) as nuclear loading control added and incubated overnight at 4°C. Filters were washed with PBS+0.2%Tween-20, incubated with goat anti-rabbit HRP (Cell Signaling Technology), washed and developed using Amersham ECL plus detection system. Quantitation of signal was performed using a Molecular Dynamics Densitometer and BioRad Multi-analyst software.

Determination of serum Ig

To assess polyclonal Ig production, sera were collected from cell recipients at the time of sacrifice and assayed for human Ig by ELISA. ELISA plates were coated overnight with 3 ug/ml F(ab′)2 goat anti-human IgG or IgM (Jackson Immunologicals) in PBS, 100 ul/well. Plates were washed with PBS, blocked for one hour with 0.5% BSA in PBS and serum dilutions added and incubated 1–2 hours. After washing, biotinylated anti-human kappa and/or lambda antibodies were added followed by washing and addition of streptavidin-HRP. The assay was developed with tetramethylbenzidine dihydrochloride (Sigma-Aldrich), stopped with sulfuric acid and read at 450 nm. Limits of detection were 3 ng/ml for IgM and 2 ng/ml for IgG.

Immunizations

In two experiments, engrafted mice were immunized with pneumovax 23 (Merck, Whitehouse Station, NJ), 20 ul/mouse s.c. (containing 1 ug of each of 23 pneumococcal serotypes) on the day of cell transfer.

Pneumovax ELISA

Immune responses to pneumovax23 were examined for 2 of the 23 pneumococcal serotypes, 4 and 14 (Danish strain designation) in the vaccine. ELISA analysis was performed according to WHO protocol (www.vaccine.uab.edu). Briefly, polystyrene ELISA plates were coated with 100 ul of 1 ug/ml serotype specific polysaccharides (type 4, ATCC #18-X and type 14, ATCC #23-X) for 5 hours at 37°C. Control human serum (89SF-2, US reference standard generously provided by Dr. Carl Frasch, CBER/FDA, Rockville, MD) with known concentrations of IgG and IgM to each serotype was used as a standard. Control serum and serial dilutions of unknowns were added to the plates, incubated overnight at room temperature. Plates were washed and incubated with anti-human IgG biotin or anti-human IgM biotin (#2040-08 and #2020-08, respectively, Southern Biotech, Birmingham, AL) for 2 hours, washed and strep-avidin-alkaline phosphatase (Southern Biotech) added for 1 hour. After washing the assay was developed with 1 mg/ml p-nitrophenyl phosphate in diethanolamine substrate for 2 hours, fixed with 3 M NaOH and read at OD 405 nm and 690 nm. Concentration determined as OD405-OD690 and read off the standard curve.

Supporting Information

Figure S1.

doi:10.1371/journal.pone.0003192.s001

(0.15 MB TIF)

Acknowledgments

We thank Sarah Kenward, Jean Leif and Linda Paquin for their technical assistance. We also thank Human Genome Sciences for providing recombinant human BLyS.

Author Contributions

Conceived and designed the experiments: MRS MCA RTW. Performed the experiments: MRS MCA. Analyzed the data: MRS MCA LJG. Contributed reagents/materials/analysis tools: LJG DLG LDS RTW. Wrote the paper: MRS RTW.

References

  1. 1. Shultz LD, Ishikawa F, Greiner DL (2007) Humanized mice in translational biomedical research. Nat Rev Immunol 7: 118–130.
  2. 2. Manz MG (2007) Human-Hemato-Lymphoid-System Mice: Opportunities and Challenges. Immunity 26: 537–541.
  3. 3. Macchiarini F, Manz MG, Palucka AK, Shultz LD (2005) Humanized mice: are we there yet? J Exp Med 202: 1307–1311.
  4. 4. Di Ianni M, Terenzi A, Falzetti F, Bartoli A, Di Florio S, et al. (2002) Homing and survival of thymidine kinase-transduced human T cells in NOD/SCID mice. Cancer Gene Ther 9: 756–761.
  5. 5. Goldman JP, Blundell MP, Lopes L, Kinnon C, Di Santo JP, et al. (1998) Enhanced human cell engraftment in mice deficient in RAG2 and the common cytokine receptor gamma chain. Br J Haematol 103: 335–342.
  6. 6. Hiramatsu H, Nishikomori R, Heike T, Ito M, Kobayashi K, et al. (2003) Complete reconstitution of human lymphocytes from cord blood CD34+ cells using the NOD/SCID/gammacnull mice model. Blood 102: 873–880.
  7. 7. Kerre TC, De Smet G, De Smedt M, Zippelius A, Pittet MJ, et al. (2002) Adapted NOD/SCID model supports development of phenotypically and functionally mature T cells from human umbilical cord blood CD34(+) cells. Blood 99: 1620–1626.
  8. 8. Kikuchi K, Lian ZX, He XS, Ansari AA, Ishibashi M, et al. (2003) Appearance of human plasma cells following differentiation of human B cells in NOD/SCID mouse spleen. Clin Dev Immunol 10: 197–202.
  9. 9. Shultz LD, Lang PA, Christianson SW, Gott B, Lyons B, et al. (2000) NOD/LtSz-Rag1null mice: an immunodeficient and radioresistant model for engraftment of human hematolymphoid cells, HIV infection, and adoptive transfer of NOD mouse diabetogenic T cells. J Immunol 164: 2496–2507.
  10. 10. Shultz LD, Banuelos S, Lyons B, Samuels R, Burzenski L, et al. (2003) NOD/LtSz-Rag1nullPfpnull mice: a new model system with increased levels of human peripheral leukocyte and hematopoietic stem-cell engraftment. Transplantation 76: 1036–1042.
  11. 11. Traggiai E, Chicha L, Mazzucchelli L, Bronz L, Piffaretti JC, et al. (2004) Development of a human adaptive immune system in cord blood cell-transplanted mice. Science 304: 104–107.
  12. 12. Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, et al. (2005) Human Lymphoid and Myeloid Cell Development in NOD/LtSz-scid IL2R{gamma}null Mice Engrafted with Mobilized Human Hemopoietic Stem Cells. J Immunol 174: 6477–6489.
  13. 13. Wagar EJ, Cromwell MA, Shultz LD, Woda BA, Sullivan JL, et al. (2000) Regulation of human cell engraftment and development of EBV-related lymphoproliferative disorders in Hu-PBL-scid mice. J Immunol 165: 518–527.
  14. 14. Kraus M, Alimzhanov MB, Rajewsky N, Rajewsky K (2004) Survival of resting mature B lymphocytes depends on BCR signaling via the Igalpha/beta heterodimer. Cell 117: 787–800.
  15. 15. Mackay F, Browning JL (2002) BAFF: a fundamental survival factor for B cells. Nat Rev Immunol 2: 465–475.
  16. 16. Mackay F, Schneider P, Rennert P, Browning J (2003) BAFF AND APRIL: a tutorial on B cell survival. Annu Rev Immunol 21: 231–264.
  17. 17. Rolink AG, Tschopp J, Schneider P, Melchers F (2002) BAFF is a survival and maturation factor for mouse B cells. Eur J Immunol 32: 2004–2010.
  18. 18. Bossen C, Schneider P (2006) BAFF, APRIL and their receptors: structure, function and signaling. Semin Immunol 18: 263–275.
  19. 19. Claudio E, Brown K, Park S, Wang H, Siebenlist U (2002) BAFF-induced NEMO-independent processing of NF-kappa B2 in maturing B cells. Nat Immunol 3: 958–965.
  20. 20. Sasaki Y, Derudder E, Hobeika E, Pelanda R, Reth M, et al. (2006) Canonical NF-kappaB activity, dispensable for B cell development, replaces BAFF-receptor signals and promotes B cell proliferation upon activation. Immunity 24: 729–739.
  21. 21. Sasaki Y, Casola S, Kutok JL, Rajewsky K, Schmidt-Supprian M (2004) TNF family member B cell-activating factor (BAFF) receptor-dependent and -independent roles for BAFF in B cell physiology. J Immunol 173: 2245–2252.
  22. 22. Gordon NC, Pan B, Hymowitz SG, Yin J, Kelley RF, et al. (2003) BAFF/BLyS receptor 3 comprises a minimal TNF receptor-like module that encodes a highly focused ligand-binding site. Biochemistry 42: 5977–5983.
  23. 23. Kim HM, Yu KS, Lee ME, Shin DR, Kim YS, et al. (2003) Crystal structure of the BAFF-BAFF-R complex and its implications for receptor activation. Nat Struct Biol 10: 342–348.
  24. 24. Hase H, Kanno Y, Kojima M, Hasegawa K, Sakurai D, et al. (2004) BAFF/BLyS can potentiate B-cell selection with the B-cell coreceptor complex. Blood 103: 2257–2265.
  25. 25. Shulga-Morskaya S, Dobles M, Walsh ME, Ng LG, MacKay F, et al. (2004) B cell-activating factor belonging to the TNF family acts through separate receptors to support B cell survival and T cell-independent antibody formation. J Immunol 173: 2331–2341.
  26. 26. Zhang X, Park CS, Yoon SO, Li L, Hsu YM, et al. (2005) BAFF supports human B cell differentiation in the lymphoid follicles through distinct receptors. Int Immunol 17: 779–788.
  27. 27. Avery DT, Kalled SL, Ellyard JI, Ambrose C, Bixler SA, et al. (2003) BAFF selectively enhances the survival of plasmablasts generated from human memory B cells. J Clin Invest 112: 286–297.
  28. 28. Stohl W (2002) B lymphocyte stimulator protein levels in systemic lupus erythematosus and other diseases. Curr Rheumatol Rep 4: 345–350.
  29. 29. Collins CE, Gavin AL, Migone TS, Hilbert DM, Nemazee D, et al. (2006) B lymphocyte stimulator (BLyS) isoforms in systemic lupus erythematosus: disease activity correlates better with blood leukocyte BLyS mRNA levels than with plasma BLyS protein levels. Arthritis Res Ther 8: R6.
  30. 30. Stohl W (2006) Therapeutic targeting of B lymphocyte stimulator (BLyS) in the rheumatic diseases. Endocr Metab Immune Disord Drug Targets 6: 351–358.
  31. 31. Stewart DM, McAvoy MJ, Hilbert DM, Nelson DL (2003) B lymphocytes from individuals with common variable immunodeficiency respond to B lymphocyte stimulator (BLyS protein) in vitro. Clin Immunol 109: 137–143.
  32. 32. Losi CG, Salzer U, Gatta R, Lougaris V, Cattaneo G, et al. (2006) Mutational analysis of human BLyS in patients with common variable immunodeficiency. J Clin Immunol 26: 396–399.
  33. 33. He B, Chadburn A, Jou E, Schattner EJ, Knowles DM, et al. (2004) Lymphoma B cells evade apoptosis through the TNF family members BAFF/BLyS and APRIL. J Immunol 172: 3268–3279.
  34. 34. Baker KP (2004) BLyS–an essential survival factor for B cells: basic biology, links to pathology and therapeutic target. Autoimmun Rev 3: 368–375.
  35. 35. Novak AJ, Darce JR, Arendt BK, Harder B, Henderson K, et al. (2004) Expression of BCMA, TACI, and BAFF-R in multiple myeloma: a mechanism for growth and survival. Blood 103: 689–694.
  36. 36. Oren DA, Li Y, Volovik Y, Morris TS, Dharia C, et al. (2002) Structural basis of BLyS receptor recognition. Nat Struct Biol 9: 288–292.
  37. 37. Cuss AK, Avery DT, Cannons JL, Yu LJ, Nichols KE, et al. (2006) Expansion of functionally immature transitional B cells is associated with human-immunodeficient states characterized by impaired humoral immunity. J Immunol 176: 1506–1516.
  38. 38. Sims GP, Ettinger R, Shirota Y, Yarboro CH, Illei GG, et al. (2005) Identification and characterization of circulating human transitional B cells. Blood 105: 4390–4398.
  39. 39. Day ES, Cachero TG, Qian F, Sun Y, Wen D, et al. (2005) Selectivity of BAFF/BLyS and APRIL for binding to the TNF family receptors BAFFR/BR3 and BCMA. Biochemistry 44: 1919–1931.
  40. 40. Thompson JS, Schneider P, Kalled SL, Wang L, Lefevre EA, et al. (2000) BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population. J Exp Med 192: 129–135.
  41. 41. Patke A, Mecklenbrauker I, Tarakhovsky A (2004) Survival signaling in resting B cells. Curr Opin Immunol 16: 251–255.
  42. 42. Kanakaraj P, Migone TS, Nardelli B, Ullrich S, Li Y, et al. (2001) BLyS binds to B cells with high affinity and induces activation of the transcription factors NF-kappaB and ELF-1. Cytokine 13: 25–31.
  43. 43. Ramakrishnan P, Wang W, Wallach D (2004) Receptor-specific signaling for both the alternative and the canonical NF-kappaB activation pathways by NF-kappaB-inducing kinase. Immunity 21: 477–489.
  44. 44. Enzler T, Bonizzi G, Silverman GJ, Otero DC, Widhopf GF, et al. (2006) Alternative and classical NF-kappa B signaling retain autoreactive B cells in the splenic marginal zone and result in lupus-like disease. Immunity 25: 403–415.
  45. 45. Cabatingan MS, Schmidt MR, Sen R, Woodland RT (2002) Naive B lymphocytes undergo homeostatic proliferation in response to B cell deficit. J Immunol 169: 6795–6805.
  46. 46. Woodland RT, Schmidt MR (2005) Homeostatic proliferation of B cells. Semin Immunol 17: 209–217.
  47. 47. Woodland RT, Schmidt MR, Thompson CB (2006) BLyS and B cell homeostasis. Semin Immunol 18: 318–326.
  48. 48. Tavernier J, Devos R, Van der Heyden J, Hauquier G, Bauden R, et al. (1989) Expression of human and murine interleukin-5 in eukaryotic systems. DNA 8: 491–501.
  49. 49. McKenzie AN, Barry SC, Strath M, Sanderson CJ (1991) Structure-function analysis of interleukin-5 utilizing mouse/human chimeric molecules. Embo J 10: 1193–1199.
  50. 50. Williams IR, Unanue ER (1991) Characterization of accessory cell costimulation of Th1 cytokine synthesis. J Immunol 147: 3752–3760.
  51. 51. Yoshioka M, Mori Y, Miyazaki S, Miyamoto T, Yokomizo Y, et al. (1999) Biological functions of recombinant bovine interleukin 6 expressed in a baculovirus system. Cytokine 11: 863–868.
  52. 52. Kumar CS, Muthukumaran G, Frost LJ, Noe M, Ahn YH, et al. (1989) Molecular characterization of the murine interferon gamma receptor cDNA. J Biol Chem 264: 17939–17946.
  53. 53. Smirnova AS, Andrade-Oliveira V, Gerbase-Delima M (2008) Identification of new splice variants of the genes BAFF and BCMA. Mol Immunol 45: 1179–1183.
  54. 54. Woodland RT, Fox CJ, Schmidt MR, Hammerman PS, Opferman JT, et al. (2008) Multiple signaling pathways promote B lymphocyte stimulator dependent B-cell growth and survival. Blood 111: 750–760.
  55. 55. Depraetere S, Verhoye L, Leclercq G, Leroux-Roels G (2001) Human B cell growth and differentiation in the spleen of immunodeficient mice. J Immunol 166: 2929–2936.
  56. 56. Tournoy KG, Depraetere S, Pauwels RA, Leroux-Roels GG (2000) Mouse strain and conditioning regimen determine survival and function of human leucocytes in immunodeficient mice. Clin Exp Immunol 119: 231–239.
  57. 57. Turgeon NA, Banuelos SJ, Shultz LD, Lyons BL, Iwakoshi N, et al. (2003) Alloimmune injury and rejection of human skin grafts on human peripheral blood lymphocyte-reconstituted non-obese diabetic severe combined immunodeficient beta2-microglobulin-null mice. Exp Biol Med (Maywood) 228: 1096–1104.
  58. 58. van Rijn RS, Simonetti ER, Hagenbeek A, Hogenes MC, de Weger RA, et al. (2003) A new xenograft model for graft-versus-host disease by intravenous transfer of human peripheral blood mononuclear cells in RAG2−/− gammac−/− double-mutant mice. Blood 102: 2522–2531.
  59. 59. Berney T, Molano RD, Pileggi A, Cattan P, Li H, et al. (2001) Patterns of engraftment in different strains of immunodeficient mice reconstituted with human peripheral blood lymphocytes. Transplantation 72: 133–140.
  60. 60. Cao T, Lazdina U, Desombere I, Vanlandschoot P, Milich DR, et al. (2001) Hepatitis B virus core antigen binds and activates naive human B cells in vivo: studies with a human PBL-NOD/SCID mouse model. J Virol 75: 6359–6366.
  61. 61. Agenes F, Freitas AA (1999) Transfer of small resting B cells into immunodeficient hosts results in the selection of a self-renewing activated B cell population. J Exp Med 189: 319–330.
  62. 62. Freitas AA, Rocha B (2000) Population biology of lymphocytes: the flight for survival. Annu Rev Immunol 18: 83–111.
  63. 63. Mackay F, Leung H (2006) The role of the BAFF/APRIL system on T cell function. Semin Immunol 18: 284–289.
  64. 64. Huard B, Schneider P, Mauri D, Tschopp J, French LE (2001) T cell costimulation by the TNF ligand BAFF. J Immunol 167: 6225–6231.
  65. 65. Ng LG, Mackay CR, Mackay F (2005) The BAFF/APRIL system: life beyond B lymphocytes. Mol Immunol 42: 763–772.
  66. 66. Ye Q, Wang L, Wells AD, Tao R, Han R, et al. (2004) BAFF binding to T cell-expressed BAFF-R costimulates T cell proliferation and alloresponses. Eur J Immunol 34: 2750–2759.
  67. 67. Prlic M, Jameson SC (2002) Homeostatic expansion versus antigen-driven proliferation: common ends by different means? Microbes Infect 4: 531–537.
  68. 68. Goldrath AW, Luckey CJ, Park R, Benoist C, Mathis D (2004) The molecular program induced in T cells undergoing homeostatic proliferation. Proc Natl Acad Sci U S A 101: 16885–16890.
  69. 69. Marleau AM, Sarvetnick N (2005) T cell homeostasis in tolerance and immunity. J Leukoc Biol 78: 575–584.
  70. 70. Diaz-de-Durana Y, Mantchev GT, Bram RJ, Franco A (2006) TACI-BLyS signaling via B-cell-dendritic cell cooperation is required for naive CD8+ T-cell priming in vivo. Blood 107: 594–601.
  71. 71. King M, Pearson T, Shultz LD, Leif J, Bottino R, et al. (2008) A new Hu-PBL model for the study of human islet alloreactivity based on NOD-scid mice bearing a targeted mutation in the IL-2 receptor gamma chain gene. Clin Immunol 126: 303–314.
  72. 72. Woodland RT, Schmidt MR, Korsmeyer SJ, Gravel KA (1996) Regulation of B cell survival in xid mice by the proto-oncogene bcl-2. J Immunol 156: 2143–2154.
  73. 73. Tam WF, Wang W, Sen R (2001) Cell-specific association and shuttling of IkappaBalpha provides a mechanism for nuclear NF-kappaB in B lymphocytes. Mol Cell Biol 21: 4837–4846.