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The Journal of Immunology, 2001, 166: 2929-2936.
Copyright © 2001 by The American Association of Immunologists

Human B Cell Growth and Differentiation in the Spleen of Immunodeficient Mice1

Stany Depraetere2,*, Lieven Verhoye{dagger}, Georges Leclercq3,{ddagger} and Geert Leroux-Roels2,{dagger}

* Innogenetics N.V., Ghent-Zwijnaarde, Belgium; and {dagger} Center for Vaccinology and {ddagger} Department of Clinical Chemistry, Microbiology and Immunology, Ghent University and Hospital, Ghent, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human mAbs (HumAbs) have therapeutic potential against infectious diseases and cancer. Heretofore, their production has been hampered by ethical constraints preventing the isolation of Ag-specific activated B cells by in vivo immunization. Alternatively, severe combined immune deficient (SCID) mice, transplanted i.p. with human (Hu)-PBLs, allow the in vivo stimulation of human Ab responses without the usual constraints. Unfortunately, human B cells only represent a minor fraction of the surviving graft, they are scattered all over the animal body, and thus are hard to isolate for subsequent immortalization procedures. To prevent this dispersion and to provide the human B cells with a niche for expansion and maturation, SCID mice were engrafted with Hu-PBL directly into the spleen. Simultaneously endogenous murine NK cell activity was depleted by treatment with an anti-mouse IL-2 receptor {beta}-chain Ab. During engraftment, human B lymphocytes became activated, divided intensely, and differentiated into plasmacytoid cells. In vivo exposure to a recall Ag after cell transfer induced expansion of Ag-specific B cell clones. One week after inoculation, human B cells were abundant in the spleen and could easily be recovered for fusion with a heteromyeloma line. This resulted in the formation of stable hybridoma cell lines that secreted Ag-specific HumAbs. Thus transplantation of human lymphoid cells in the spleens of immune deficient mice represents a model for the study of human T cell-dependent B cell activation and proves to be an excellent tool for the successful production of HumAbs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The C.B.-17 Prkdcscid/Prkdcscid (SCID) mouse expresses a truncated form of the catalytic subunit of the DNA-dependent protein kinase and is unable to properly rearrange the Ig and TCR genes (1, 2). The ensuing severe combined immunodeficiency endows these mice with the capacity to accept xenografts. This quality has been exploited to create a laboratory model for the study of the human immune system. The most common way to construct a human-SCID mouse chimera is by i.p. injection of freshly isolated human (Hu)4-PBL (3). Despite the lack of functional murine T and B cells, a significant xenogeneic host-vs-graft reaction is hampering engraftment of human cells, which results in a large variation of cell recovery and functional heterogeneity of human cells recovered from engrafted mice. The macrophages, polymorphonuclear cells, and especially the NK cells of the SCID mouse have a normal or even enhanced activity, which leads to a rapid destruction of the graft (4, 5). The targeted reduction of the murine NK cell activity with Abs directed toward specific NK cell membrane markers such as anti-asialoGM1, anti-N.K.-1.1, and IL-2R{beta} or against NK cell products such as anti-mouse IFN-{gamma}, improves both the survival of the human graft and the production of human Igs (6, 7, 8, 9). TM{beta}1, a rat mAb (IgG2b) directed against the {beta}-chain of the murine IL-2 receptor (IL-2R{beta}), which is present on a subpopulation of CD8+ T cells and on all NK cells, is of particular interest because this mAb induces a long-lasting depletion of murine NK cell activity in normal and SCID mice (10). Intraperitoneal injection of TM{beta}1 1 day before Hu-PBL transfer has pronounced and long-lasting effects on the survival, distribution, and function of human cells in the SCID mouse (8).

In the past few years, new mouse strains with additional defects of the innate immune system have been developed. Backcrossing the Scid mutation onto the nonobese diabetic (NOD)/Lt strain resulted in the NOD/LtSz-Prkdcscid/Prkdcscid mouse (NOD/SCID), which has a reduced NK activity, macrophage function, and serum hemolytic complement activity in addition to the deficit in mature T and B cells (11). These NOD/SCID mice are better hosts for the Hu-PBL grafts with a concomitant higher human Ig production when compared with SCID mice (12). Hu-PBL engraftment in NOD/SCID mice can be further enhanced by conditioning the host with TM{beta}1 Ab and total body irradiation (13).

Activation of quiescent B lymphocytes in an Ag-specific manner is a prerequisite for the successful generation of clones secreting HumAbs because proliferating B cells can then be immortalized by fusion with human tumor cells (14) or heteromyelomas (15). In vivo immunization of humans is limited by practical and ethical considerations. The Hu-PBL-SCID may prove to be useful to generate these Ag-specific B cell clones needed to generate HumAb-producing hybridomas. When Hu-PBL are transferred i.p., T cells constitute the majority of the human cell population present in the peritoneal cavity or lymphoid organs of the recipient mice, whereas B cells represent only a minor population (6, 8, 12, 13, 16, 17). It is now well established that B cells require more than the mere ligation of surface Ig with Ag to enter the cell cycle and become responsive to growth factors. Additional stimuli or accessory signals are provided by membrane-bound molecules on activated T cells that react with acceptor molecules on B cells (18). To optimize the required T-B cell cooperation in the Hu-PBL-SCID model, we have injected human lymphoid cells directly into the spleen rather than into the peritoneum. We speculated that this might result in enhanced B cell growth and survival. We assumed that apart from the direct T-B cell contacts, the splenic environment might be superior in providing B cell-stimulating cytokines. We noticed that under these engraftment conditions, human B cells become activated, expanded vigorously, and transiently became the most prominent cellular subset among the human leukocytes residing in the spleen.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C.B.-17 Prkdcscid/Prkdcscid (SCID) and NOD/LtSz-Prkdcscid/Prkdcscid (NOD/SCID) mice were bred under sterile conditions and fed ad libitum with autoclaved food and water without addition of prophylactic antibiotics. The NOD/SCID strain was free of Emv 30, an endogenous murine ecotropic retrovirus responsible for induction of lethal thymomas (19). Mice were used between 8 and 12 wk of age.

Pretreatment of mice

Anti-asialoGM1 (Wako Pure Chemical, Osaka, Japan) is a rabbit polyclonal Ab that recognizes murine NK cells and depletes NK activity (20). Mice were pretreated by a single i.p. injection of 20 µl of the anti-asialoGM1 solution.

TM{beta}1 is a rat mAb directed against the murine IL-2 receptor {beta}-chain (10). It was produced in our laboratory as described (8, 13). In vivo NK depletion was achieved by a single i.p. injection of 1 mg purified TM{beta}1 in 500 µl PBS. Sublethal total body irradiation (3 Gy) was administered using a linear accelerator.

Generation of mononuclear subsets and transplantation

Hu-PBL were isolated from buffy coats or heparinized venous blood by Ficoll-Hypaque (Nycomed, Oslo, Norway) centrifugation. Depletion of CD3+ T cells and CD8+ cytotoxic T cells was performed using the appropriate specific Ab-coated immunomagnetic beads according to the manufacturer’s instructions (Dynal, Oslo, Norway). For intrasplenic engraftment, animals were anesthetized and a subcostal incision of the skin was made followed by incisions of the abdominal wall and the peritoneum. The spleen was carefully exposed and injected with a 50-µl cell suspension in PBS. After injection, the spleen was repositioned in the abdominal cavity, and the abdominal wall and skin were sutured separately.

Flow cytometry

Analysis of freshly isolated Hu-PBL and single cell suspensions from Hu-PBL-SCID spleen was conducted on a FACscan flow cytometer (Becton Dickinson, San Jose, CA) as described (8, 13). Cell viability in each cell preparation was estimated by trypan blue exclusion. To overcome the possibility of murine cells staining nonspecifically for human markers, they were gated out together with the dead cells (propidium iodide positive) using CyChrome-conjugated anti-mouse common leukocyte Ag CD45 (30-F11; PharMingen, Hamburg, Germany). Human cells were stained with Abs directly conjugated with FITC or PE. Isotype controls (X40) and Abs recognizing human CD3 (SK7), CD4 (SK3), CD8 (SK1), CD14 (MoP9), CD19 (4G7), CD20 (L27), CD23 (EBVCS-5), and CD45 (2D1) were obtained from Becton Dickinson. Abs specific for human CD16 (3G8), CD38 (HIT2), CD40 (5C3), CD56 (B159), CD86 (2331), and membrane-bound IgD (IA6-2) were supplied by PharMingen. Anti-CD21 (BL13) was obtained from Coulter (Miami, FL) and anti-CD71 (T56/14) and anti-HLA-DR (TU36) were purchased from Caltag Laboratories (San Francisco, CA).

Cell culture and fusion

A Hu-PBL-SCID spleen cell suspension was prepared by gently squeezing the tissue with angled forceps followed by filtration on a sterile gauze to remove larger fragments. Cells were cultured in 96-well flat-bottom microculture plates in 200 µl RPMI 1640 medium supplemented with sodium pyruvate (1 mM), L-glutamine (2 mM), 2-ME (5 x 10-5 M), penicillin (100 U/ml), streptomycin (100 µg/ml), nonessential amino acids (all obtained from Life Technologies, Paisley, U.K.), and 10% Fetal Clone I serum (HyClone, Logan, UT).

For cell fusion, Hu-PBL-SCID spleen cells and K6H6/B5 heteromyeloma cells (15), washed in calcium-free PBS, were mixed at a 5:1 ratio. Polyethylene glycol 1500 (50%; Boehringer Mannheim, Mannheim, Germany) was added for 2 min and washed away. Fused cells (105) were cultured in 200 µl of the culture medium supplemented with human recombinant insulin (10 µg/ml; Boehringer Mannheim), ouabain (1 µM; Sigma, St. Louis, MO), hypoxanthine-aminopterin-thymidine (Life Technologies), and 10% BM Condimed H1 (Boehringer Mannheim). The K6H5/B5 heteromyeloma cell line is hypoxanthine/aminopterin/thymidine sensitive and ouabain resistant. Cultures were replenished with fresh medium every other day.

In vivo immunization and detection of total and Ag-specific Ig

Hepatitis B surface Ag (HBsAg, aluminum hydroxide adsorbed; Engerix-B; SmithKline Biologicals, Rixensart, Belgium) was injected s.c. in the hind leg of SCID mice a few hours after Hu-PBL transfer. Blood was drawn from the retro-orbital plexus and collected in heparinized tubes. The in vivo and in vitro production of specific human Abs against HBsAg (anti-HBsAg) Ig was measured with the ETI-AB-AUK-3 anti-HBs enzyme immunoassay kit (Sorin Biomedica, Saluggia, Italy). Titers are expressed as IU/L (detection limit 5 IU/L).

In vivo and in vitro anti-hepatitis C virus (anti-HCV) Ab production was evaluated with the INNOTEST HCV Ab IV, the INNOTEST HCV E1Ab prototype version, and the confirmatory assay INNOLIA HCV Ab III update (Innogenetics, Ghent, Belgium).

Determination of total human IgG and IgM concentrations in Hu-PBL-SCID plasma was performed by ELISA. Microtiter plates (96-well, Nunc-Immunoplate Maxisorb; Nunc, Roskilde, Denmark) were coated with 100 µl (2 µg/ml PBS) rabbit anti-human IgG (Dako, Glostrup, Denmark) or goat anti-human IgM (Cappel; Organon Teknika, Durham, NC) for 1 h at 37°C and subsequently blocked for 2 h with 300 µl of 1% BSA in PBS at 37°C. In a third step, Hu-PBL-SCID serum or human Ig standards (Behring Diagnostics, Westwood, MA) diluted in PBS containing 0.5% BSA were added for 1 h at 37°C. After four washes, bound Ab was detected by incubating the plates with HRP-conjugated rabbit anti-human IgG (Dako) or goat F(ab')2 anti-human IgM (Tago; BioSource International, Camarillo, CA) for 1 h at 37°C followed by the addition of tetramethylbenzidine (Sigma) for 30 min at room temperature. The enzymatic reaction was stopped with H2SO4, and plates were read at 450 nm. The lower detection limits were 10 and 1 ng/ml for IgG and IgM, respectively. Sera from SCID, NOD/SCID, or immune competent mice (BALB/c) were not reactive in these ELISAs.

Statistical analysis

The statistical package SPSS 6.1.2 (SPSS, Chicago, IL) was used. Different groups were compared using the Kruskal-Wallis H test. When the Kruskal-Wallis significance level was p < 0.05, Mann-Whitney U tests were applied as post hoc analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mouse conditioning regimen and mouse strain determine survival of intrasplenically engrafted Hu-PBL

In a first series of experiments, we examined the effects of different conditioning regimens on the short term engraftment of Hu-PBL following injection in the spleen of SCID and NOD/SCID mouse strains. All animals within a single experiment comparing different pretreatment regimens were transplanted with 2 x 107 Hu-PBL derived from the same donor. Results are depicted in Fig. 1Go.



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  		FIGURE 1. Human B cell survival and Ig production following intrasplenic transplantation of Hu-PBL in immune deficient mice. A, SCID or NOD/SCID mice were left untreated or were conditioned with TM{beta}1 with or without additional total body irradiation (rad, 3 Gy). The following day, 2 x 107 Hu-PBL were injected directly into the spleens of the animals. Human cell survival and the distribution of the different lymphocyte subsets (T cells {square} and B cells ) was assayed by flow cytometry 7 days after cell transfer. Data represent mean ± SEM of three inoculated mice. To allow for the easy comparison of engraftment outcome between different mouse strains and conditioning regimens, all animals within one experiment were reconstituted with PBL from the same donor. Results of engraftment with Hu-PBL from one donor are shown. Very similar results were obtained with Hu-PBL from three different blood donors. B, TM{beta}1-pretreated and irradiated SCID mice were transplanted in the spleen with 2 x 107 Hu-PBL. At the indicated time points, leukocytes were isolated from the spleen, counted, and phenotyped by FACS analysis. The number of human CD3+ T cells ({circ}) and human CD19+ B cells ({blacktriangleup}) is shown. Data represent mean values of three transplanted mice. All mice were transplanted with Hu-PBL from the same donor. Identical results were obtained with Hu-PBL from four different blood donors. C, TM{beta}1-pretreated and irradiated SCID mice were injected either i.p. or intrasplenically with 2 x 107 Hu-PBL derived from the same blood donor. At indicated time points, T ({square}) and B () cell numbers in the peritoneal cavity and the spleen, respectively, were determined and expressed as percentages of the total human cell number isolated. Data represent mean ± SEM of three transplanted mice. Identical results were obtained with Hu-PBL from four different blood donors. D, Untreated SCID ({blacksquare}), TM{beta}1-pretreated/irradiated SCID (), and TM{beta}1-pretreated/irradiated NOD/SCID ({square}) mice were transplanted in the spleen with 2 x 107 Hu-PBL, isolated from the same blood donor. At 7 and 14 days postinoculation, human IgG and IgM concentrations were determined in the mouse plasma. Data represent the mean ± SEM of six mice. Very similar results were obtained using PBL derived from five other blood donors.

 
One week after inoculation of Hu-PBL in untreated SCID mice, ~105 human leukocyte (CD45+) cells (0.6 ± 0.1% of total spleen leukocytes; data not shown) still resided at the site of injection (Fig. 1GoA). The presence of the cells was 10- to 100-fold higher upon pretreatment of the SCID host with anti-asialoGM1 (data not shown) or TM{beta}1, respectively. In the case of TM{beta}1, 25–30% of the leukocytes residing in the murine spleen were of human origin (data not shown). The highest yield of human cells in the spleen was achieved when the recipient SCID mice were not only pretreated with TM{beta}1 but also sublethally irradiated before Hu-PBL injection (2.1 ± 0.2 x 107 CD45+ cells). Under these conditions, 85–90% of the leukocytes residing in the murine spleen were of human origin (data not shown). Total body irradiation reduced the total number of mouse spleen cells 10-fold when compared with that of nonirradiated SCID mice. This explains the high percentage of intrasplenic human CD45+ cells in irradiated mice. Equally successful Hu-PBL transplantations were achieved in TM{beta}1-treated and irradiated NOD/SCID mice. In both SCID and NOD/SCID strains the extent of human cell engraftment varied very slightly from donor to donor or from mouse to mouse using PBL from a single donor. Irradiated and TM{beta}1-treated Hu-PBL-SCID or NOD/SCID mice died of xenoreactive graft-vs-host disease within 3–4 wk after Hu-PBL transfer. This acute graft-vs-host disease was easily diagnosed by the presence of a hunched back, ruffled fur, emaciation, and diarrhea.

Human B cells transiently predominate and are functional

One week after the inoculation of human PBL in the mouse spleens, B cells were strikingly predominant within the human intrasplenic cell population (Fig. 1GoA). B cell predominance was independent of the conditioning regimen of the animal host or of the mouse strain used. Human monocytes and NK cells, originally present in the inoculum, were not detected (data not shown), and human T cells constituted only a minor fraction.

A substantial drop of T and B cells occurred during the first days following intrasplenic injection of the Hu-PBL (Fig. 1GoB). In this period, Hu-PBL were never found in extrasplenic sites (blood, liver, lung; data not shown), suggesting that the loss of human cells in the spleen was due to cell death rather than to cell migration. From day 3 on, a vigorous B cell growth became apparent. Absolute numbers of intrasplenic human B cells reached a plateau at 1 wk posttransplantation and slowly declined afterward. The predominance of B lymphocytes within the human leukocyte population was transient and only apparent between days 7 and 10. This change of subset distributions was caused by the rapid intrasplenic expansion of the human T cell population (Fig. 1GoB), the emigration of human B cells from the spleen to other tissues (e.g., bone marrow, liver; data not shown), and by cell death. This transient B cell predominance was also observed when Hu-PBL derived from an EBV-negative blood donor were used, excluding the early expansion of EBV+ cells in these severely immune suppressed mice.

When human leukocytes were transferred in the peritoneal cavity of optimally conditioned SCID mice, T cells constituted the majority of the surviving human cell population at all times, whereas B cells only occurred in low numbers (Fig. 1GoC). The absolute number of B cells found in the spleen after intrasplenic transfer was 10-fold higher than that found in the peritoneal cavity following i.p. injection both at 1 and 2 wk after cell transfer. Total IgG and IgM production were also significantly higher in comparison with i.p. reconstitution (data not shown).

The functional integrity of the engrafted human B cells was demonstrated by an early and vigorous production of human IgG and IgM that appeared in the mouse blood without any added antigenic stimulus (Fig. 1GoD). Seven days after the transfer of the Hu-PBL, human Ig levels were significantly higher in NOD/SCID mice than in SCID mice although the number of surviving human B cells (Fig. 1GoA) and the kinetics of human B cell expansion (data not shown) were similar in both strains. The Ig repertoire was polyclonal as indicated by normal {kappa}{lambda} light chain ratios (data not shown).

Human B lymphocytes differentiate into plasmacytoid cells

During their stay in the murine spleen, the CD19+ B lymphocytes differentiated into lymphoblastoid and plasmacytoid cells. The blastoid phenotype was evidenced by the fact that the majority of the human CD45+ cells, isolated from the SCID spleen 7 days after Hu-PBL transfer, were enlarged (Fig. 2GoB) and expressed the transferrin receptor CD71 (Fig. 2GoD). CD71 is a marker for proliferation (21) that was totally absent on the cells at the time of inoculation (data not shown). Two populations of CD19+ cells could be distinguished: a small cluster with high CD19 expression levels (CD19high) and a major cluster characterized by low expression of CD19 (CD19low) (Fig. 2GoE). The CD19high cluster still expressed CD20 and low levels of CD38 (Fig. 2Go, F and G) as observed on B cells at the moment of inoculation (data not shown). These CD19high cells were representative of an activated lymphoblastoid B cell population. CD19low cells discontinued the expression of CD20 (CD20neg) and became strongly CD38 positive (CD38high) (Fig. 2Go, F and G). It is known that CD19 and CD20 are down-regulated and CD38 becomes highly expressed upon terminal differentiation of mature B cells into Ig-secreting plasma cells (22, 23, 24). Thus the CD19lowCD20negCD38high cells represented B lymphocytes with a plasmacytoid differentiation status. Both lymphoblastoid and plasmacytoid B cells were characterized by the appearance of the costimulatory molecule CD86 and the disappearance of CD21 and CD23 (Fig. 2Go, H–J). The latter markers both are known to be gradually lost upon stimulation and terminal differentiation (25). Furthermore, the plasmacytoid cells lost the expression of surface IgD and CD40 (Fig. 2Go, K and L) and the MHC class II protein HLA-DR (data not shown). The absence of CD40 is also a marker of terminally differentiated plasma cells (26). CD5, exposed at high density in some human autoimmune and B cell-derived lymphoproliferative disorders (27), was totally absent on the CD19+ cells present in the murine spleen (data not shown). The described CD19+ phenotypes could be observed both in untreated as well as in anti-asialo or TM{beta}1-pretreated and/or irradiated SCID or NOD/SCID mice (data not shown).



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  		FIGURE 2. Intrasplenically transplanted human B lymphocytes undergo lymphoblastoid and plasmacytoid differentiation. TM{beta}1-treated and irradiated SCID mice were inoculated in the spleen with 2 x 107 Hu-PBL. Human cells present in the spleen 7 days after inoculation were phenotyped by FACS analysis. Analysis was gated on the viable (propidium iodide-negative) human leukocyte subpopulation (region outlined in dot plot B). Murine lymphoid cells were gated out by staining with CyChrome-conjugated anti-mouse CD45 Ab. (Dot plots A and B show total spleen leukocytes without and with anti-mouse CD45 Ab, respectively.) Results represent engraftment with Hu-PBL from one donor. Identical human cell phenotypes and percentages were obtained using PBL from four other blood donors.

 
Kinetics of the appearance of different B cell phenotypes was studied in mice reconstituted with Hu-PBL derived from different donors (data not shown). After 3 days of in vivo incubation, the CD38, CD71, and CD86 activation markers appeared on the B lymphocytes concomitant with the vigorous B cell growth described above. At day 5, lymphoblastoid CD19high and plasmacytoid CD19low B cells could be distinguished and were equally represented. As time progressed, the plasmacytoid cell population became increasingly dominant and represented over 80% of the B cells in the spleen 14 days after transplantation.

The intrasplenic engraftment of B cells was T cell dependent. Human CD19+ B cell survival and human Ig production were barely detectable in optimally conditioned SCID mice reconstituted with T cell-depleted Hu-PBL (data not shown). CD4+ T helper cells rather than CD8+ CTLs played an important role as the absence or presence of CD8+ cells in the inoculum did not influence B cell survival, Ig production, or B cell differentiation (data not shown). Over 90% of the CD4+ T cell population isolated from the spleen at 1 week after engraftment of Hu-PBL were characterized by the membrane expression of the activation markers CD38 and HLA-DR, the proliferation markers CD25 (IL-2 receptor {alpha}) and CD71, and the memory cell marker CD45RO. This CD4+ T cell phenotype was observed in all mice engrafted with Hu-PBL derived from different donors.

Secondary Ag-specific Ig production in and human monoclonal hybridoma development from Hu-PBL-SCID mice reconstituted by PBL transfer in the spleen

Human Ag-specific Ig production was studied in optimally conditioned SCID mice engrafted with Hu-PBL isolated from a donor that was immune to the T cell-dependent HBsAg. The anti-HBs titer of this subject at the time of blood donation was 6981 IU/L. Without in vivo HBsAg immunization, only low levels of anti-HBs were detected in the mouse plasma (Table IGo). However, in the plasma of animals that had received a HBsAg boost in vivo, a vigorous secondary immune response was discernible within 1 week after Hu-PBL transfer. Thus, human peripheral blood-derived B cells could be reactivated in an Ag-specific manner during SCID engraftment. Similarly, cells isolated from the spleens of the immunized Hu-PBL-SCID mice continued to secrete considerable amounts of anti-HBs when transferred to a culture dish and maintained in vitro. No anti-HBs were produced upon in vitro culture of freshly isolated Hu-PBL in the absence or in the presence of HBsAg (data not shown). This observation indicates the very low frequency of HBsAg-specific plasma cells in the peripheral blood of a vaccinated individual.


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  		Table I. In vivo exposure to a recall Ag activates Ag-specific B cell clones

 
Spleen leukocytes isolated from in vivo immunized Hu-PBL-SCID mice, 7 days postinoculation, were chemically fused with the heteromyeloma cell line K6H6/B5. Fused cells were seeded in culture medium supplemented with hybridoma growth factors and the selection drugs hypoxanthine-aminopterin-thymidine and ouabain. Ouabain rapidly killed nonfused human B cells and thus prevented their anti-HBsAg Ab production. After 10 days, all microcultures contained clusters of viable cells; significant anti-HBsAg production was measured in 4% of these. Anti-HBs-positive cultures were sequentially cloned, and several monoclonal hybridoma cell lines were isolated that stably produced 42 ± 8 µg human IgG1 with an anti-HBsAg activity of 758 ± 71 IU/106 cells/day. The above-described specific in vivo and in vitro anti-HBsAg production and hybridoma generation was also achieved with Hu-PBL from other HBsAg-immune donors.

HumAbs specifically recognizing HCV Ags have also been developed using this strategy of intrasplenic cell transfer in SCID mice. Hu-PBL were isolated from a chronically infected patient with circulating Abs against core, E1, E2, NS3, and NS4 as shown by a Line Immuno Assay (LIA; Innogenetics) (Fig. 3GoA). Cells (107 per mouse) were injected in three optimally conditioned NOD/SCID mice without further in vivo immunization. On day 7 these animals were sacrificed and the spleen cells were isolated for fusion with the heteromyeloma line. At that time, plasma of these mice was harvested and examined for the presence of anti-HCV Abs. As shown in Fig. 3GoB, the INNO-LIA strips demonstrated the presence of circulating Abs toward core and E1 (in all three mice), to E2 (in mouse 3 only), to NS3A and NS3B (in all three mice), to NS4B1 (in mice 1 and 3), and to NS4B2 (in all three mice). In the serum of mouse 2 a faint reactivity with NS5A was visible. This suggests that the cell donor (chronic HCV patient) has circulating memory B cells recognizing NS5A, despite the absence of visible NS5A reactivity in the serum of this person. Furthermore, this experiment demonstrates that the recipients of the Hu-PBL produce approximately the same spectrum of Abs as the original cell donor even without an in vivo boost with HCV Ags. Following the cell fusion, hybridoma selection, and cloning, hybridoma have been generated that produce Abs directed against NS3A and NS4B1 (Fig. 3GoC).



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  		FIGURE 3. Analysis of serum from a chronically HCV-infected PBL donor (A), sera obtained at day 7 from three mice that were inoculated (on day 1) with 107 PBL from donor (B) and supernatants from four individual hybridomas (C) using LIAs. Two different assay formats have been used: the strip in panel A contained Ig controls, Core1, Core2, E1, E2, NS3, NS4A, NS4B, and NS5 lines, whereas the strips used in panels B and C were coated with Ig controls, Core, E1, E2, NS3A, NS3B, NS4A, NS4B1, NS4B2, and NS5A Ags.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hu-PBL-SCID constitute a unique model for fundamental research on human humoral and cellular immune responses. Most of the studied chimeras are constructed by injection of human blood-derived leukocytes in the peritoneum of SCID mice. Here, we describe an alternative route of engraftment through direct intrasplenic transfer of Hu-PBL. This more invasive procedure caused no mortality, and the rate of successful engraftment was over 90% equaling that described for i.p. graft deposition (28). No or very limited interdonor variability was observed. The major advantage of this transplantation route is the striking expansion of the human B cell population, which has never been documented upon i.p. engraftment of Hu-PBL. Thus, our intrasplenic chimeras may serve as a model to further investigate human B cell activation and may facilitate the production of human mAbs.

EBV, known to induce lymphomas in chimeras constructed with Hu-PBL derived from EBV-infected donors (29), was ruled out as the polyclonal stimulator of B cells in our experiments because prominent intrasplenic B cell proliferation was observed irrespective of the EBV-serostatus of the Hu-PBL donor. Furthermore, B cells did not express CD23, a marker for EBV-transformed lymphocytes, nor was there evidence for EBV-induced lymphoproliferative disorders in any of the examined chimeras.

T cells and, more specifically, the membrane expression of CD40 ligand on activated T cells have been shown to be responsible for the induction of spontaneous human Ig production in i.p. human-mouse constructs (18, 30, 31). Also in this study, CD4+ T helper cells were proven to play a major role in the activation and differentiation of human B cells. One week after engraftment, the majority of the T cells present in the spleen expressed a mature, activated memory phenotype. Tary-Lehmann and coworkers were the first to report on identical human T cell phenotypes present in the organs of i.p. constructed Hu-PBL mice (17). Thus, although activated T cells are present in both Hu-PBL-SCID models, major differences in B cell proliferation between i.p. and intrasplenic chimeras could be observed. Probably the splenic environment may provide a closer contact and improved cellular interaction between B and T lymphocytes resulting in a more prominent B cell proliferation. The vascularized murine spleen may also provide additional costimulatory molecules, cytokines, and growth stimuli, which might be absent in the peritoneum.

Monoclonal Abs can be applied for the treatment of infectious diseases and cancer (32, 33). Although mAbs from mice are relatively easy to produce and can be generated to bind to a number of Ags, their therapeutic utility is restricted by their immunogenicity in humans. Humans rapidly develop an Ab response to mouse Abs. These human anti-mouse Abs not only block the actions of the mouse Abs but may lead to allergic reactions. To reduce this problem, mouse mAbs are humanized by grafting the complementary determining regions (CDRs) of a mouse mAb, which form the Ag-binding loops, onto a framework of a human Ig molecule. Although these recombinant Abs are far less immunogenic in humans than the parent mouse mAb, their production is usually expensive, time consuming, and may result in a final Ab product with decreased specificity and affinity.

Several technologies have been developed to produce Abs with fully human protein sequences. One approach is called phage display technology, which involves the cloning of human Ab genes into bacteriophages to display Ab fragments on their surface for the selection of Ag specificity (34). However, isolated fragments are frequently of moderate affinity, and further genetic engineering is required to convert them into fully assembled Abs. Another approach is the transplantation of human B and T cells in immune deficient mice. Hu-PBL are generally transplanted i.p. followed by an in vivo stimulation with the appropriate recall Ag. Under these conditions, B cells constitute only a minor fraction of the surviving graft; they are scattered all over the host organism and are, therefore, hard to isolate for subsequent immortalization procedures. Nevertheless, HumAbs have been generated from cells isolated from i.p. reconstituted Hu-PBL-SCID through combinatorial gene library (35). This technique remains extremely laborious because a large number of clones has to be tested, resulting in Abs that are cloned as fragments whose biological activity is inferior to that of complete Ig proteins. Others have established cloned cell lines from visibly EBV-transformed tumors that were growing in the Hu-PBL-SCID and produced Abs specific for the immunizing Ag (36, 37). However, these tumors only express monoclonal or oligoclonal B cell repertoires (38), whereas the cell lines derived from them have rather low Ab production capacities and are frequently unstable.

The intrasplenic Hu-PBL-SCID model described here provides a direct and easy access to the rich memory compartment of human B cells that can be exploited for the production of a wide variety of stable and highly productive hybridomas. Within the first week of engraftment, B cells proliferate and remain abundantly present in the spleen. They can easily be recovered for immortalization procedures. Immunization of the Hu-PBL-SCID mouse with recall Ag resulted in enrichment of Ag-specific B cell clones. HumAbs could not only be developed from Hu-PBL derived from vaccine-induced immune donors (e.g., HBsAg) but also from Hu-PBL derived from a donor carrying the infectious agent (e.g., HCV). In the latter case, in vivo immunization with recall Ag was not even necessary to generate expanded repertoires of Abs to specific epitopes of the infectious agent. Our method is currently still limited in generating Abs to Ags to which the human B cell donor has already responded in vivo. Human autoantibodies, potentially useful for immunosuppressive and immunomodulatory function, could be obtained using appropriate autoimmune donors in the model. Similarly, Abs against specific MHC determinants for treating graft rejection could be derived from Hu-PBL from selected multiparous women.

Our method may be complementary to the most recently developed transgenic HumAb mouse model. These mice comprise fragments of the unrearranged human heavy and light chain Ig loci in addition to targeted disruption of the endogenous mouse Ig genes (39, 40). Upon immunization with an Ag of interest, the introduced transgenes undergo gene rearrangements, somatic mutations, and class switching to generate human Abs that can be accessed by standard hybridoma technology. In contrast with our Hu-PBL-SCID model, this model is well suited for primary immunization experiments and is not dependent on immune donors.


   Acknowledgments
 
We thank Dr. H. Spits and Dr. K. Weijer (Nederlands Kanker Instituut, Amsterdam, The Netherlands) for providing us with a NOD/SCID mouse breeding pair, Dr. K. Thielemans (Department of Physiology, Free University Brussels, Brussels, Belgium) for supplying the K6H6/B5 cell line, and Dr. T. Boterberg (Department of Radiotherapy, Nuclear Medicine and Experimental Cancerology, Ghent University Hospital, Ghent, Belgium) for assistance in irradiation of the animals.


   Footnotes
 
1 Part of this study was supported by a grant from the Flemish Institute for the Promotion of Scientific-Technological Research in Industry (97259447/HCV-3) and a grant from the Flemish Fund for Scientific Research (FWO-Vlaanderen G.0022.00). Back

2 Address correspondence and reprint requests to Dr. Stany Depraetere and Dr. Geert Leroux-Roels, Innogenetics N.V., Industriepark 7, B-9052, Ghent-Zwijnaarde, Belgium. Back

3 G.L. is a senior research assistant of the Flemish Fund for Scientific Research (FWO-Vlaanderen). Back

4 Abbreviations used in this paper: Hu, human; HBsAg, hepatitis B surface Ag; anti-HBs, Abs against HBsAg; HumAb, human mAb; NOD, nonobese diabetic; LIA, Line Immuno Assay; HCV, hepatitis C virus. Back

Received for publication November 3, 1999. Accepted for publication December 11, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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