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Stem Cells, Vol. 17, No. 5, 242-252, September 1999
© 1999 AlphaMed Press

Human Hematopoietic Stem/Progenitor Cells Generate CD5+ B Lymphoid Cells in NOD/SCID Mice

Enrico M. Novellia, Manuel Ramíreza, Wing Leunga,b, Curt I. Civina,b

a Departments of Oncology and
b Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Key Words. CD34+ cells • NOD/SCID chimeras • Xenogeneic stem cell transplantation • Lymphopoiesis • CD5+ B cells • Immunophenotyping • Hematopoiesis

Dr. Curt I Civin, Oncology 3-109, Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, Maryland 21287-5001, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Financial Disclosure
 References
 
The nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenotransplantation model is increasingly utilized to study both human lymphohematopoietic stem/progenitor cells and committed cell types. Human B lymphoid cells develop and proliferate in this model. We found high numbers of CD19+CD5+ B lymphoid cells in the bone marrows and spleens of NOD/SCID mice transplanted with human CD34+ stem/progenitor cells. The CD5+ cells accounted for a particularly large percentage of the B lymphoid cells in the spleens of chimeras analyzed three months after transplantation. CD19+CD5+ cells from all the analyzed chimeras coexpressed HLA-DR, surface IgM, CD20, CD38, CD43, and CD45. However, CD19+CD5+ cells were negative for {kappa} light chain, CD10, CD11a, CD11b, CD15, CD21, CD22, CD23, CD25, CD34, CD35, CD44, CD62L, CD69, and CD71. Cell surface expression of the {lambda} light chain, surface IgD, CD9, and CD40 antigens was detected in some but not all chimeras. Thus, the CD19+CD5+ cell population detected in our study has the phenotype of previously described CD5+ B lymphoid cells in humans and other species. The origin and role of the B lymphoid cells which express CD5 cell surface glycoprotein are poorly understood. The malignant cells in B lymphoid chronic lymphocytic leukemia express CD5, and the numbers of CD5+ B lymphoid cells are elevated in several autoimmune conditions. The human-NOD/SCID chimera system may provide an in vivo model to investigate the maturation and development of this cryptic human CD5+ B lymphoid cell subpopulation.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Financial Disclosure
 References
 
After transplantation into sublethally irradiated immunodeficient mice, human lymphohematopoietic stem/progenitor cells (HSCs) generate sustained human multilineage hematopoiesis in this xenogeneic in vivo environment. Many groups have utilized these human-mouse (hu/mu) hematopoietic chimeras as model systems to assess HSC biology [1-9]. An additional application of these hu/mu chimera models is to investigate the in vivo biology of cell types of the individual human hematopoietic lineages [10-13]. Transplanted mature human B and T lymphocytes survive, proliferate, and are at least partially functional in the murine microenvironment. For example, transplanted mature human B cells secrete immunoglobulins [3, 14, 15], and transplanted mature human T cells generate a graft-versus-host-disease-like syndrome [3, 16-18].

Since in vitro assays for human lymphoid progenitor cells have not been widely available, it is of particular importance to be able to investigate human lymphoid cell development in hu/mu chimera models. In severe combined immunodeficient (SCID) mice, development of T cells from transplanted human CD34+ stem/progenitor cells requires cotransplantation of a human fetal thymus [10]. In the nonobese diabetic NOD/SCID hu/mu chimera model, T cells are not generated from transplanted CD34+ or T-cell-depleted hematopoietic human cell grafts [3, 19]. However, small numbers of T cells appear to develop from transplanted human CD34+ cells in bg/nu/xid mice [20], thus providing a model system for investigation of human T lymphopoiesis.

Although few human B lymphoid cells develop after transplantation of human CD34+ cells into bg/nu/xid mice, transplanted human CD34+ cells generate human CD19+IgM+ B lymphoid cells in NOD/SCID mice [4, 21, 22]. Recently, it has been shown that pre-B cells develop in the bone marrow (BM) of NOD/SCID mice transplanted with umbilical cord blood (CB) CD34+ cells, then mature in the spleen [23]. Thus, the NOD/SCID chimera model appears to provide a promising in vivo system for the study of B-cell development and maturation.

To further investigate B lymphopoiesis in the NOD/SCID model, we immunophenotyped the human B lymphoid cells in chimeras resulting from transplantation of human CD34+ cells into NOD/SCID mice. We were surprised to find a relatively large, persistent CD5+ B lymphoid cell population, a rare and poorly understood subset in normal human blood and lymphoid tissues.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Financial Disclosure
 References
 
Cell Sources
CB samples were from placentas of healthy newborns collected by Dr. Harris (University of Arizona Cord Blood Bank). Mobilized peripheral blood stem/progenitor cells (PBSCs) were obtained as small aliquots from autologous leukapheresis products from cancer patients undergoing chemotherapy at the Johns Hopkins Hospital under an Institutional Review Board-approved protocol. The PBSC mobilization regimen was cyclophosphamide (4 g/m2) followed by G-CSF (10 µg/kg/d) until the day of leukapheresis [2]. T-cell depletion and CD34+ cell purification were performed using immunomagnetic microspheres, as previously described [3, 24]. Because large numbers of cells were needed for each experiment, cells obtained from four to six donors were pooled to prepare transplant grafts.

Antibodies and FACS Analysis
Phycoerythrin (PE)-conjugated purified mouse anti-human CD5, CD19, and CD34; fluorescein isothiocyanate (FITC)-conjugated mouse anti-human HLA-DR, CD11a, CD20, CD22, CD34, CD44; and PE-conjugated and FITC-conjugated isotype control antibodies were purchased from Becton Dickinson (San Jose, CA). FITC-conjugated rat anti-mouse CD45 and PE-conjugated mouse anti-human CD45 were purchased from Sigma Chemical (St. Louis, MO). FITC-conjugated mouse anti-human CD13 was purchased from Dako Corporation (Carpinteria, CA). FITC-conjugated mouse anti-human surface IgM (sIgM), surface IgD (sIgD), {kappa}, {lambda}, CD9, CD10, CD15, CD21, CD23, CD25, CD35, CD38, CD40, CD43, CD62L, CD69, CD71; PE-Cychrome 5 (PE-CY5)-conjugated mouse anti-human CD45; and PE-CY5-conjugated isotype control were purchased from Pharmingen (San Diego, CA).

A FACSort (Becton Dickinson) flow cytometer equipped with an Argon laser tuned at 488 nm was used for fluorescence-activated cell sorter (FACS) analysis.

Transplantation of Human Cells
Cells were transplanted by tail vein injection into sublethally irradiated (350 cGy using a 137Cs gamma irradiator) six- to eight-week old NOD/LtSz-scid/scid (NOD/SCID) mice. In order to maximize levels of human cells in the engrafted hu/mu chimeras, mice received i.p. injections of the following growth factors every Monday, Wednesday, and Friday post-transplantation: interleukin 3 (IL-3), GM-CSF, and stem cell factor (SCF) (10 µg of each/dose) [2]. Mice were bred and maintained in pathogen-free conditions, as described [2, 3, 9]. Animal experiments were approved by the Animal Care and Use Committee of the Johns Hopkins Medical Institutions.

Analysis of Human Cell Engraftment
Mice were sacrificed 4-20 weeks after transplantation, as specified. Single-cell suspensions were prepared from the spleens, and BM cells were flushed from the removed femurs and tibiae [2, 3, 9]. Cells were counted and viability determined by trypan blue dye exclusion (88% ± 5%, in these experiments). Because these four bones contain ~25% of the total BM of a mouse [25], the total number of BM cells/mouse was estimated by multiplying the number of cells obtained by four. Human cells in the BM and spleens of the hu/mu chimeras were enumerated and cell lineages determined by three-color flow cytometry of cells immunostained using murine monoclonal antibodies, as described previously [2, 3, 9, 26].

Phenotypic Characterization of CD5+ B Cells
Highly engrafted hu/mu chimeras were subsequently analyzed by means of three-color flow cytometry to further characterize the CD5+ population. Immunostaining with CD5-FITC, CD19-PE, and CD3-PerCP was performed to exclude the possibility that the CD5+ population studied belonged to the T-cell lineage. In addition, BM and spleen mononuclear cells were stained with a combination of antibodies, including CD5-PE or CD5-FITC, CD19-PerCP or CD19-CY5, and a panel of FITC-conjugated antibodies (against human HLA-DR, sIgM, sIgD, {kappa}, {lambda}, CD9, CD10, CD11a, CD11b, CD15, CD20, CD21, CD22, CD23, CD25, CD34, CD35, CD38, CD40, CD43, CD44, CD62L, CD69, and CD71) and PE-conjugated antibodies (CD21 and CD34). Isotype controls were employed throughout.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Financial Disclosure
 References
 
CD5+ Human B Cells are Present in the BM and Spleen of hu/mu Chimeras
Sixteen NOD/SCID mice (#1-16) were evaluated for the presence of human hematopoiesis in their BM and spleens at one of three time points (30, 60, or 100 days) after transplantation of 5 x 105 human CB CD34+ cells. In a separate experiment, one mouse (#17) was evaluated at 140 days after transplantation of 5 x 105 human CB CD34+ cells. Every transplantation resulted in a hu/mu chimera with substantial levels of human hematopoiesis (Table 1Go, Fig. 1 Go). Other results from chimeras 1-16 were reported in a previous research article [2]. In chimeras analyzed one month after transplantation (chimeras #1-4), the BM contained an average of 30% human CD45+ cells (34 x 106 human cells per chimera, on average). At two months (#5-12), the BM contained 38% human CD45+ cells (38 x 106 human cells). At three months (#13-16), the BM contained 18% human CD45+ cells (22 x 106 human cells). In chimera #17 evaluated ~5 months after transplantation, the BM contained 55% human CD45+ cells (8.8 x 106 human cells).


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  		Table 1. Human CD19+CD5+ cells from hu/mu chimeras
 


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  		Figure 1. Flow cytometric analysis of a representative hu/mu chimera. (A, D) Isotype controls were used to assess nonspecific staining. (B) Cells from each chimera were immunostained and first analyzed by flow cytometry to assess the percentage of human cells in the marrow. Human cells were labeled with the anti-human CD45 but not anti-mouse CD45. (C) Analysis was then performed on gated human CD45+ cells to quantitate human CD45+CD19+ cells (E), and human CD45+CD19+CD5+ cells (F).

 
The BM from chimeras #1-9 and #17 were evaluated by multicolor flow cytometry for their numbers of human CD19+ and CD19+CD5+ B lymphoid cells. In BM from chimeras #1-4 analyzed one month after transplantation, an average of 56% of the human CD45+ cells coexpressed CD19, but no CD5+ cells were detected. In BM from chimeras #5-9 analyzed two months after transplantation, CD19+ cells comprised an average of 76% of the human CD45+ cells. An average of 8% of the CD19+ cells coexpressed CD5. In the BM from chimera #17 analyzed five months after transplantation, CD19+ cells comprised 81% of the human CD45+ cells, and 6% of the CD19+ cells coexpressed CD5.

The spleens of chimeras #1-17 were evaluated similarly, and with the exception of mouse #13, contained substantial numbers of human CD45+ cells at all time points. In spleens from chimeras #1-4, analyzed one month after transplantation, an average of 68% of the human CD45+ cells coexpressed CD19, and 12% of CD19+ cells coexpressed CD5. In spleens from chimeras #5-12 analyzed two months after transplantation, CD19+ cells comprised 49% of the human CD45+ cells, and 45% of CD19+ cells coexpressed CD5. In spleens from chimeras #14-16 analyzed three months after transplantation, CD19+ cells comprised 29% of the human CD45+ cells, and 63% of CD19+ cells coexpressed CD5 (chimera #13 was not analyzed for CD19 or CD5 expression, due to the low percentage of human CD45+ cells). In the spleen from chimera #17 analyzed five months after transplantation, CD19+ cells comprised 78% of the human CD45+ cells, and 49% of CD19+ cells coexpressed CD5.

The CD19+CD5+ cell populations from these chimeras had low forward and side light scattering properties (in the "lymphocyte region" of the forward versus side scatter flow cytogram) but were slightly higher in average side and forward scatter than the CD19+CD5- cells (Fig. 2Go). CD13+ myeloid cells accounted for most of the human CD45+CD19- cells recovered from the BM and spleens of all chimeras (data not shown). All samples were also immunophenotyped for the presence of T cells; no chimera had detectable CD3+CD5+CD19- cells (phenotypic T lymphoid cells), as assessed by flow cytometry (Fig. 3Go).



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  		Figure 2. CD19+CD5+ cells have higher average forward and side light scatter than CD19+CD5- cells. (A) Isotype controls were used to assess nonspecific staining. (B) Gating of CD19+CD5+ and CD19+CD5- cells. (C) Forward and side scatter dot plot of CD19+CD5- cells. (D) Forward and side scatter dot plot of CD19+CD5+ cells.

 


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  		Figure 3. No CD3+CD5+ cells are detected in hu/mu chimeras. (A) Isotype control for panel C. (B) Isotype control for panel D. (C and D) three-color flow cytometric analysis of the human CD5+ and CD19+ lymphocytes detected in a representative hu/mu chimera. The human lymphocytes were stained with CD5 FITC, CD19 PE, and CD3 PerCP.

 
CD5+ B cells were also detected in the BM and spleens of NOD/SCID chimeras which had been transplanted with mobilized blood CD34+ cells ("PBSC" data not shown), but the low levels of engraftment provided by this cell source in the chimeras prevented us from performing detailed phenotypic analyses.

Lymphoid Differentiation Antigens Expressed on CD5+ B cells
The BM of chimera #9 and the spleens of chimeras #5, 6, 10, 11, and 17 were further analyzed by three-color flow cytometry for coexpression of a panel of lymphoid differentiation antigens on CD19+CD5+ cells [27] (Table 2Go). Immature B cells which express neither surface immunoglobulin nor cytoplasmic µ chain have been operationally defined as pro-B cells, whereas pre-B cells are defined by the expression of cytoplasmic µ chain but not surface immunoglobulin [28]. Immature B cells express HLA-DR [29], surface {kappa} and {lambda} light chains, CD9 [30], CD10 (which is lost before cells acquire sIgM, but re-expressed on later B cells, upon their activation [31]), CD19 [32]), CD20 (expressed beginning at the pre-B cell stage [32]), CD34 (which is lost upon maturation to the pre-B cell stage [33]), CD38 (expressed on immature and terminally differentiated B cells [34]), and CD40 (a pan-B marker, except not expressed on plasma cells [35]). Other markers we employed are expressed on mature and/or activated B cells: sIgD [36], CD11a [37], CD21 [38], CD22 (expressed shortly after CD19 in the cytoplasm of early B cells, then on the surface of mature B cells [39]), CD23 (activation antigen expressed on cells which simultaneously express both membrane IgM and IgD [40]), CD25 (the IL-2 receptor [41]), CD35 (the complement receptor 1 [42]), CD43 (expressed on most lymphocytes except resting B cells [43]), CD44 [44], CD45 [45], CD62L (L-selectin [46]), CD69 (an early activation marker [47]), and CD71 (transferrin receptor [48]). Finally, we used the monocyte-associated markers CD11b (the C3bi receptor) expressed on normal and malignant CD5+ B cells [26, 49] and CD15 expressed on 25%-50% of the CD5+ B cells of patients with B-cell chronic lymphocytic leukemia and other chronic B cell disorders [49].


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  		Table 2. Human leukocyte differentiation antigens expressed on human CD5+ cells from hu/mu chimeras
 
The CD5+ cells present in the BM of hu/mu chimera #9 were negative for CD10, CD11a, CD22, CD23 (only 6% positive), CD34, and CD44. Most of the CD5+ cells present in the BM of chimera #9 were positive for HLA-DR, sIgM, CD9, CD20, CD38, CD40, CD43. Similar results were obtained for splenocytes of hu/mu chimeras #5, 6, 10, 11, and 17. The CD5+ cells in all chimeras tested were positive for HLA-DR (n = 5), sIgM (n = 5), CD20 (n = 5), CD38 (n = 3), CD43 (n = 5), and CD45 (n = 1). All chimeras analyzed were negative for {kappa} (cell surface), CD10, CD11a, CD11b, CD15, CD21, CD22, CD23, CD25, CD34, CD35, CD44, CD62L (one mouse showed only 6% positive cells), CD69, and CD71. In four of five chimeras analyzed, the CD5+ cells were sIgD+ (<=45% of the cells); in one of three chimeras analyzed, 10% of the CD5+ cells were positive for {lambda} (cell surface); in two of four chimeras analyzed, a subset of the CD5+ cells (<=35%) were CD9+; in three of five chimeras analyzed, the CD5+ cells were CD40+ (<15% of the cells). Figure 4Go shows the detailed results for a representative chimera (#17).



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  		Figure 4. Analysis of the leukocyte differentiation antigens expressed on human CD5+ cells detected in mouse #17. Human CD19+ cells were gated and analyzed for the coexpression of several differentiation antigens, with the respective percentages of CD19+CD5+ cells coexpressing the given antigen indicated. Data are not shown for several antigens which were not expressed on CD19+CD5+ cells (these negative results are stated in the text).

 

   Discussion
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 Introduction
 Materials and Methods
 Results
 Discussion
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In this study, we detected high numbers of human CD5+ B lymphoid cells in hu/mu chimeras generated by transplantation of human CB CD34+ stem/progenitor cells. The development and the role of B lymphoid cells expressing the surface antigen CD5 (also called B-1a) are enigmatic [50-64]. For example, it is not yet known whether CD5+ B cells derive from progenitors distinct from those which generate CD5- B cells or if they instead represent an activated B-cell subset [53]. Although CD5+ B cells have been described in several species [65], they have been characterized most fully in mice and humans. CD5+ B cells differ from CD5- B cells in their anatomical localization [66], gene usage [52], function and phenotype [26, 54].

Interestingly, the phenotype of the CD5+ B cells detected in the chimeras of the study herein is similar to that described for CD5+ B cells in humans and other species. By flow cytometry, CD5+ B cells appear larger and more granular than CD5- B cells, in both the hu/mu chimeras (Fig. 2Go) and in several species, including rats, sheep and humans [59]. This larger and more granular cytomorphology may be a sign of increased cell metabolism and activation [65]. Other features shared by the CD5+ B cells in the hu/mu chimeras and the CD5+ B cells described in the literature are coexpression of HLA-DR [54], sIgM and sIgD [54], CD9 [67], CD19 [59], CD20 [59], and CD43 [68, 69]. The low expression of {lambda} light chains in conjunction with sIgM observed in one chimera (Table 2Go) may be consistent with the observation that murine peritoneal CD5+ B cells are selectively enriched for cells expressing both sIgM and {lambda} light chains [70], but this requires more extensive investigation in larger numbers of hu/mu chimeras. CD11b was not detected in the single chimera whose CD5+ B cells we tested, whereas murine CD11b (Mac-1) is expressed on murine peritoneal but not splenic CD5+ B cells [54]. However, the percentage of CD5+ B cells which coexpressed CD11b was highly variable among animals in a study performed in sheep [65]. CD5+ B cells have been reported to express low levels of other myelomonocytic surface antigens, including CD15 [26]. In the hu/mu chimera we tested, CD15 was not expressed on the CD5+ B cells. The CD5+ B cells detected in the hu/mu chimera we tested were CD21-, CD22-, and CD44-. These results are in contrast with previous reports [26, 57, 67, 71, 72].

We were surprised to find that >60% of the human B lymphoid cells in the spleens of several of the hu/mu chimeras were CD5+. Interestingly, we observed that hu/mu chimeras sacrificed at late time points (60-140 days) showed higher percentages of CD19+CD5+ cells in both BM (6%-8% versus 0) and spleen (45%-63% versus 12%). It is possible that CD5+ B lymphoid progenitors preferentially undergo self-renewal in this hu/mu chimera model (a phenomenon also observed in the peritoneum of normal mice [26]). Alternatively, clonal expansion of CD5+ cells may occur in the chimeras. It is not possible to directly elucidate this possibility, since we did not perform a molecular analysis of immunoglobulin gene diversity in the chimeras. However, another group observed that human CD19+CD20+ cells sorted from the BM of hu/mu chimeras showed a polyclonal pattern of Ig gene expression [23]. The increase in the percentage of CD5+ B cells we observed in our study is paralleled by observations in human clinical studies. The CD5+ subset increased to high percentages up to one year post-transplant in patients transplanted with CD34+ sorted PBSC or BM [73-75].

In both mice and humans, the percentages of B cells expressing CD5 are high during fetal life and childhood; most murine fetal B lymphoid cells express CD5, as do most human IgM+ CB B lymphoid cells [73-75]. However, in young adult humans, CD5+ B cells are reduced in number, comprising 1%-30% of the B lymphocytes present in the spleen, lymph nodes, and peripheral blood. A further decrease in the numbers of CD5+ B cells is observed during late adulthood [76]; these cells are almost undetectable in human adult BM [61] and account for <5% of peripheral blood and peritoneal lymphocytes of humans older than 45 years [66]. Studies in the mouse suggest that CD5+ B cells are produced preferentially or exclusively early in the ontogeny and are still capable of self-replenishment in the adult [54, 58].

Although CD5+ B cells account for only a small percentage of B cells in normal adults, their numbers are highly increased in several neoplastic diseases, inflammatory states (autoimmune and alloimmune), and in the setting of BM transplantation (as mentioned above). The neoplastic B cells of most cases of chronic lymphoid leukemia and mantle cell lymphomas and some cases of diffuse, large B-cell lymphomas express CD5 [77, 78]. In patients with autoimmune diseases and in the early lymphoid recovery phase after BM transplantation, high numbers of nonmalignant CD5+ B lymphoid cells are present in the blood [79-83]. Intriguingly, it has been suggested that the hematopoietic reconstitution that takes place in the post-BM-transplantation period mimics the normal ontogeny of the hematopoietic and immune systems [72]. It is possible that similar phenomena occur in the xenogeneic human-NOD/SCID transplantation setting.

While the pathogenesis and the role of the increased numbers of CD5+ B cells observed in these conditions have not been clearly established, several factors have been shown to affect the size of the CD5+ B-cell pool. For instance, hormones (e.g., estrogens) and cytokines (e.g., IL-5) have been shown to cause an expansion of the CD5+ B-cell population [59, 84]. B-B-cell and B-T-cell interactions may also regulate the numbers of CD5+ B cells [59]. It is possible that the particular microenvironment of the NOD/SCID mice may favor expansion of the CD5+ B-cell population. In particular, since T cells do not develop from transplanted CD34+ cells in NOD/SCID mice, the absence of T cells may be pivotal in CD5+ B cell development in this model. Interestingly, in the clinical setting, T-cell suppression and lack of appropriate T-cell help are commonly observed in the recipient during the first year after BM transplantation [62]. As mentioned above, this is a situation in which increased numbers of CD5+ B cells have been detected in the recipient. Conversely, increases in the numbers of CD5+ B cells have not been detected in transplanted recipients who developed graft-versus-host disease, a T-cell-mediated disorder [26]. Finally, patients with SCID (in whom mature T cells are absent) also have high numbers of CD5+ B cells [62]. In order to explain the "independence" from T cells of CD5+ B cell development in the mouse, it has been proposed that CD5+ B cells in the adult are the remnant of a distinct fetal B-cell developmental pathway that is maintained by the presence of natural antigens without the requirement for T-cell help [57]. The CD5+ subset might be a source of low-affinity polyreactive natural antibodies that has been proposed to play a role in such disparate phenomena as T-cell-independent immunity against microorganisms, the "idiotype network," and immune reactivity against self-antigens [26, 69].

An alternative hypothesis for the expansion of the CD5+ B cell subset in the hu/mu chimeras is that CB CD34+ cells may have a higher potential for differentiating into CD5+ B cells. Even though we also observed CD5+ B cells in mice transplanted with PBSCs, it is possible that the high numbers of cells detected in mice transplanted with CB CD34+ cells may have been specifically related to the use of this immature source of stem cells. The hypothesis that CB may be particularly rich in CD5+ B-cell precursors is supported by the observation that in allogeneic transplantation studies conducted in mice, CD5+ B cells were exclusively obtained in the recipient mice when fetal or newborn tissues rather than adult BM were used as the stem cell sources for transplantation [85]; adult mouse BM may not contain cells that so heavily reconstitute the CD5+ B-cell population [26]. These results support the proposal that CD5+ B cells have a developmental origin distinct from CD5- B cells [85].

In summary, we have described a population of CD19+CD5+ cells which is highly represented in the spleens of NOD/SCID chimeras transplanted with human CB CD34+ cells. This population bears interesting similarities to CD5+ B cells observed in other species, including humans. Therefore, the human-NOD/SCID xenogeneic chimera assay may provide a useful in vivo tool to study the origin, development, and function of CD5+ B cells. For instance, future experiments may aim at exploring the role of CD5+ B cells in immune responses elicited in vivo in hu/mu chimeras.


   Financial Disclosure
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Financial Disclosure
References
 
The Johns Hopkins University holds patents on CD34 monoclonal antibodies and related inventions. CIC is entitled to a share of the sales royalty received by the University under licensing agreements between the University, Becton Dickinson Corporation, and Baxter HealthCare Corporation. This arrangement is being managed by the University in accordance with its conflict of interest policies.


   Acknowledgments
 
We thank Amgen, Inc.; Thousand Oaks, CA for providing human hematopoietic growth factors and Mrs. Laura Domina for her skilled secretarial assistance.

This work was supported in part by grant #PO1 CA70970 from the National Institute of Health, a grant from the National Foundation for Cancer Research, and an American Society of Clinical Oncology Young Investigator Award (WL).


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Financial Disclosure
 References
 

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accepted for publication July 31, 1999.



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