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The Journal of Immunology, 1998, 161: 5391-5398.
Copyright © 1998 by The American Association of Immunologists

Characterization of a B Cell Progenitor Present in Neonatal Bone Marrow and Spleen But Not in Adult Bone Marrow and Spleen1

Kim M. Moscatello2, Kristen L. Biber2, Deborah C. Dempsey, Robert Chervenak and R. Michael Wolcott3

Department of Microbiology and Immunology, Louisiana State University Medical Center, Shreveport, LA 71130


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The neonatal period marks an important time in mammalian immunologic development, yet it is often ignored in studies of lymphocyte development. We identified a cell population with the phenotype heat stable Ag (HSA)low lin- CD43low that contained B cell progenitors at a high frequency in the neonatal bone marrow and spleen. Although cells with a similar phenotype can be identified in the bone marrow and spleen of adult animals, these populations showed a greatly reduced frequency of B cell progenitors. B lineage cells were detected after 7 days in culture at a frequency of 1:15 when HSAlow lin- CD43low cells from neonatal bone marrow were cultured on stromal cells and IL-7 under limiting dilution conditions. Under similar conditions, the equivalent population in adult bone marrow had a frequency of B cell progenitors that was less than 1:2000. The expression of terminal deoxynucleotidyl transferase in freshly sorted neonatal HSAlow lin- CD43low cells suggested that cells committed to the lymphocyte lineage were present in this population. These data suggested that the HSAlow lin- CD43low population of cells represents a pool of B lineage precursors that may be responsible for filling the immune compartment early in neonatal life.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The ontogeny of the adult immune system begins early in gestation when pluripotent hematopoietic stem cells (PHSCs)4, which collect in the aorta-gonad-mesonephros, simultaneously seed the fetal liver, thymus, spleen, and bone marrow (1, 2). Consequently, B and T lymphocytes develop in a temporal manner that is believed to be dependent on the maturation of the hematopoietic microenvironment (2). More specifically, committed B lymphocytes are detected in the bone marrow at day 17 of gestation, and this is the site where active B lymphopoiesis occurs throughout the subsequent lifetime of the animal (2).

Much of what is known about B cell development has been focused on the fetal liver and the adult bone marrow, while B cell development during neonatal life has received less attention (3, 4, 5, 6). The neonatal period represents a unique time in the life of the animal in that passive immunity from mother to infant is relatively short-lived and therefore the infant is left with the task of developing a mature and functional immune system. One hypothesis is that during neonatal life a period of rapid lymphocyte proliferation would be required until the immune system reaches a state of homeostasis, at which point rapid expansion of the immune system would no longer be needed. We are interested in learning the mechanisms that govern this phase of immune development.

Our studies of B cell ontogeny were focused on early lymphocyte progenitors because of their potential role in filling immune compartments early in life before steady state B lymphopoiesis is established in the animal. We have defined a unique cell population based on its differential expression of several lineage and functional markers known to be important in early hematopoiesis and B lymphopoiesis. This cell population was shown to be present only at a time during ontogeny when B lymphopoiesis and hematopoiesis are abundant, i.e., during neonatal development. This population of cells, which has the phenotype of heat stable antigen (HSA)low lin- CD43low (lin, lineage mixture against Ter-119, Gr-1, Mac-1, and B220) and contributes to the pool of B lineage precursors in the spleen and bone marrow of neonatal mice but not in adult bone marrow or spleen after lymphopoiesis has achieved homeostasis.


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

Six- to 8-wk-old male and female C57BL/6J mice were purchased from The Jackson Laboratories (Bar Harbor, ME). C57BL/6J mice were bred, and the appearance of a vaginal plug was scored as day 0 of gestation. Postcoital female mice were individually caged and maintained on a high protein breeding diet (Harlan Teklad, Bartonville, IL) during gestation.

Flow cytometry and cell sorting

Neonatal and adult mice were euthanized, and bone marrow and spleen were removed. Bone marrow was obtained by flushing the femurs and tibias with PBS supplemented with 1% BSA (PBS/BSA). Spleens were dispersed into single cell suspensions by grinding between the frosted ends of two glass slides. Cells were centrifuged at 200 x g for 10 min, resuspended in 1 ml of PBS/BSA, and counted with a hemacytometer. For flow cytometric analysis, staining reactions were conducted in 96-well U-bottom plates by resuspending 1 x 106 cells in 50 µl of the appropriately diluted Ab for 15 min on ice. Cells were washed with FACS buffer (PBS, 2% newborn calf serum, and 1 g/L sodium azide) and centrifuged at 200 x g for 3 min per wash. Samples were analyzed on an Epics Profile II and Elite work station software (Coulter, Hialeah, FL). Four-color analysis and cell sorting was done on a Vantage flow cytometer (Becton Dickinson, San Jose, CA), and analysis was accomplished with Cell-Quest software (Becton Dickinson, San Jose, CA). RBCs and debris were excluded on the basis of forward angle and 90° light scatter. Cell sorting and analysis were made available through the Louisiana State University Medical Center Core Facility for Flow Cytometry (Shreveport, LA).

For cell sorting, cells were stained in 15-ml conical centrifuge tubes using the appropriately diluted Ab at a concentration of 1 ml Ab/50 x 106 cells. Cells were incubated on a shaking platform in a cold room at 4°C for 30 min. Cells were washed with sterile FACS buffer, without sodium azide, and centrifuged for 10 min at 200 x g. Stained cells were resuspended in a final volume of 50 x 106 cells/ml. Deflected cells were collected in sterile FACS buffer and kept at 4°C.

Abs, unless otherwise indicated, were all purchased from PharMingen (San Diego, CA) and titrated before use to determine the optimal concentration for each Ab. mAbs included: anti-CD45R (B220, clone RA3-6B2) conjugated to FITC, biotinylated anti-CD24 (HSA, clone M1/69), anti-CD43 (leukosialin, clone S7) conjugated to phycoerythrin, anti-Gr-1 (myeloid differentiation Ag, clone RB6-8C5) conjugated to FITC, and anti-Ter-119 (clone Ter-119), which was purchased unlabeled and was conjugated to FITC by dialyzing Ter-119 mAb against FITC (Sigma, St. Louis, MO) at a final concentration of 1 µg/ml FITC in 500 ml carbonate-bicarbonate buffer (0.2 M solution of anhydrous sodium carbonate and 0.2 M solution of sodium bicarbonate). Streptavidin-RED670 was purchased from Life Technologies (Gaithersburg, MD). Anti-Mac-1, clone M1/70 (7), and anti-FcRII, clone 2.4G2 (8), were produced at Louisiana State University Medical Center from clones purchased from American Type Culture Collection (Manassas, VA) and purified using protein G columns. Anti-Mac-1 was conjugated to FITC as described above for anti-Ter-119.

Colony assays in semisolid medium

Sorted cells were placed in Iscove’s modified Dulbecco’s medium (IMDM) (Sigma), with no pH indicator, and were serially diluted. Three hundred microliters of various concentrations of sorted cells were placed in 3 ml MethoCult M3434 (Stem Cell Technologies, Vancouver, Canada), vortexed, and incubated on ice for 10 min. Cells were then dispensed using a 5-ml syringe with a 19-gauge needle into two, 35 x 10 mm petri dishes in 1.1 ml volumes and placed into a larger petri dish (100 x 15 mm). A third petri dish filled with sterile buffer was placed into the larger petri dish and left uncovered to maintain a humidified environment. Cultures were incubated at 37°C in 6.5% CO2 for 12–18 days. Microscopic colonies were read at various times over an 18-day period. MethoCult M3434 contains the cytokines and growth factors IL-3, IL-6, stem cell factor, and erythropoietin and is capable of detecting CFU-erythroid, burst-forming unit-erythroid, CFU-macrophage, CFU-granulocyte-macrophage, and CFU-granulocyte, erythroid, and monocyte/macrophage colonies. Each experiment was completed at least three times in duplicate.

In vitro culture and limiting dilution analysis

Sorted cells were cultured with irradiated (1800 rad via a 6-MeV linear accelerator) OP42 stromal cells (a generous gift from Dr. Paul Kincade, Oklahoma Research Foundation, Oklahoma City, Oklahoma) and 250 U/ml of recombinant human IL-7 (rHu IL-7; Genzyme, Cambridge, MA) in IMDM (supplemented with 10% FCS, glutamine, gentamicin, and 2-ME) under bulk culture or limiting dilution conditions. Supernatant from the J558 IL-7 murine IL-7 producing cell line was also used as a source of IL-7 for the in vitro cultures (a generous gift from Drs. Antonius Rolink and Fritz Melchers, Basel Institute for Immunology, Basel, Switzerland). Cells cultured in bulk were harvested and analyzed at various time points for expression of B220 and HSA as described earlier. Bulk cultures were maintained weekly by aspirating 50% of the spent media and replacing it with fresh media and IL-7. Cell proliferation was determined by counting cultured cells in the presence of eosin dye using a hemacytometer. Limiting dilution cultures were done in 96-well flat-bottom plates (Corning, Corning, NY) with 30 replicates of each fivefold dilution. Cultures were scored after 7 days for lymphocyte colony formation. Colonies consisting of eight or more connected cells were counted as positive. The frequency of responding cells was calculated when 37% of the wells were negative for growth (9).

Western blot analysis

Total cellular protein was extracted from either whole cell lysates of thymus, spleen, cultured cells, NIH3T3 fibroblasts (10), or freshly sorted cells as follows: cells were resuspended at a concentration of 107 cells/ml in PBS/BSA. The following protease inhibitors were added at a final concentration of 10 µg/ml: leupeptin, PMSF, and aprotonin (all from Sigma). Cells were incubated with the protease inhibitors for 30 min on ice. One milliliter of cells was transferred to a 1.5-ml microcentrifuge tube and pelleted at 12,000 rpm for 5 min, then the supernatant was discarded. Cell pellets were resuspended in 1 ml of SDS-sample buffer (0.0625 M Tris-HCl, 10% glycerol, 2% SDS, 5% 2-ME, and 2.5% saturated bromphenol blue), briefly vortexed, boiled for 4 min, and then centrifuged at 12,000 rpm for 5 min. The pellet, if any, was resuspended, and the lysate was boiled for another 4 min and then centrifuged as above. Protein determinations were made using a Bio-Rad protein assay (Bio-Rad, Hercules, CA), and equal amounts of protein of each sample were loaded onto a 7.5% SDS-PAGE gel and resolved. Proteins were transferred to nitrocellulose membrane by electroblotting. The nitrocellulose blots were blocked overnight at 4°C on a shaking platform in Tris-buffered saline (TBS) (0.02 M Tris, 0.5 M NaCl, pH 7.5) with 5% nonfat dry milk. Membranes were washed in TBS with 2% Tween (TBST) twice for 5 min and once for 20 min at room temperature. The primary Ab, anti-terminal deoxynucleotidyl transferase (TdT) (Supertechs, Bethesda, MD), was diluted in TBS/1% nonfat dry milk, added to the blot, and incubated overnight at 4°C on a shaking platform. Membranes were washed in TBST as described above and incubated for 2 h at room temperature with a biotinylated goat F(ab')2 anti-rabbit Ig (Southern Biotechnology, Birmingham, AL) appropriately diluted in TBS/1% nonfat dry milk. Membranes were washed in TBST as described above, then incubated with streptavidin-horseradish peroxidase (PharMingen) diluted in TBST and incubated for 30 min at room temperature. Detection of proteins was performed with the ECL kit from Amersham (Arlington Heights, IL), which was used as indicated by the manufacturer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HSAlow lin- CD43low cells are enriched for B lineage precursors in the bone marrow and spleen of neonatal mice

The initial characterization of B cell progenitor activity was determined in neonatal mice because this is a time of extensive hematopoiesis in the animal. Bone marrow cells were prepared from 2–3-wk-old mice and stained as indicated in Materials and Methods to permit the characterization of cells with the phenotypes of HSAlow lin- CD43low and HSAlow lin- CD43high. As can be seen in Fig. 1GoA, when neonatal bone marrow is gated on the lin- fraction, the two phenotypes fall into distinct populations that were well separated. Each of the populations was sorted using the gates indicated by the boxes in Fig. 1GoA. The HSAlowlin- CD43high cells were gated based on a clean single positive CD43high peak (Fig. 1GoA), and the HSAlow lin- CD43low population was gated slightly below that peak at approximately one decade in width. The lower panels of Fig. 1GoA show the reanalysis of the sorted cell populations. In initial experiments, cells were cultured under a variety of conditions; a) media only, b) IL-7 only, c) stromal cells only, and d) stromal cells and IL-7. The cells that were cultured in media or IL-7 only showed no colony formation. The cells cultured on stromal cells revealed a few small colonies; however, the addition of IL-7 produced several robust lymphoid colonies. Limiting dilution analysis, under conditions that propagate B cells, was used to determine the frequency of B cell progenitors in the purified cell populations. Cells were placed into culture on stromal cells with added IL-7 with 30 replicates of each dilution. The cells were cultured for 1 wk and then scored for the presence of lymphoid colonies. The frequency of B cell progenitors in both the HSAlow lin- CD43low and HSAlow lin- CD43high populations was approximately 1 in 15 (Fig. 1Go, B and C, respectively). The identification of the lymphoid colonies as B lineage cells was confirmed by staining representative cultures with FITC-anti-B220 Abs, followed by flow cytometric analysis and treatment of positive cultures with LPS to induce the cells to differentiate and secrete IgM, which was detected by ELISA (data not shown). One possible source of the B cell progenitors in the two sorted cell populations is the presence of multipotential stem cells. Two methods were used to determine the presence of multipotential stem cells. The first method was based on numerous reports (11, 12, 13, 14) that detection of cobblestone area forming cells (CAFC), when bone marrow cells are cultured on a stromal layer, can be equated with the presence of pluripotent or omnipotent stem cells. Both cell populations were cultured under limiting dilution conditions for more than 4 wk and were observed weekly for CAFC. Cultures of the HSAlow lin- CD43low cells produced only two colony types, one with lymphoid morphology as mentioned above and the other with monocyte/macrophage morphology, which was confirmed by staining representative cultures with Mac-l Abs (data not shown). The frequency of monocyte progenitors in the HSAlowlin- CD43low populations was 1 in 70. No CAFC were detected in the HSAlow lin- CD43low cell cultures through 4 wk of observation. By contrast, cultures of the HSAlow lin- CD43high population yielded multiple colony types, including a few CAFC that persisted for more that 4 wk, suggesting the presence of PHSC in this population of cells.



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  		FIGURE 1. Purification and limiting dilution analysis of HSAlow lin- CD43low/high cells from neonatal bone marrow. HSAlow lin- CD43low/high cells were sorted from neonatal bone marrow (A). Neonatal bone marrow was stained with Abs against HSA, CD43, and a lineage mixture (B220, Mac-1, Ter-119, and Gr-1). HSAlow CD43low/high cells were gated on the lin- fraction of neonatal bone marrow and sorted by FACS. The percentage of total events was 0.49% in the CD43low sort gate and 0.56% in the CD43high fraction. Limiting dilution analysis (30 replicates per dilution) was performed on either the HSAlow lin- CD43low cells (B) or the HSAlow lin- CD43high cells (C). Sorted HSAlow lin- CD43low/high cells were cultured on irradiated OP42 stromal cells and IL-7 and were scored for lymphocyte colony formation after 1 wk.

 
The second method used to detect multipotential stem cells in the sorted populations was culture in semisolid medium containing a mixture of cytokines that allow for the detection of hematopoietic progenitors. Various concentrations of HSAlow lin- CD43low/high cells were sorted from neonatal bone marrow and plated in MethoCult methylcellulose medium. The plates were cultured for 12–18 days at 37°C and scored for microscopic colonies. Nonlymphoid colonies were detected at a frequency of 1 in 75 within the HSAlow lin- CD43low population and at a frequency of 1 in 12 within the HSAlow lin- CD43high population (Table IGo). While the MethoCult system is capable of detecting monocyte/macrophage, granulocyte, and erythroid colonies, the HSAlow lin- CD43low population produced only colonies of the monocyte/macrophage lineage, whereas the HSAlow lin- CD43high population produced multipotent colonies consisting of monocyte/macrophages, granulocytes and erythroid cells and various combinations thereof. When whole bone marrow was used as a control, colonies of several different lineages were also detected. Together, these data suggest that the HSAlow lin- CD43high population contains primitive hematopoietic progenitors whereas the HSAlow lin- CD43low population contains cells that are developmentally more restricted. Because the HSAlow lin- CD43low population of cells appeared to be less primitive, we focused on this population as a candidate population for neonatal B cell progenitors.


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  		Table I. Hematopoietic colony assays in semi-solid medium

 
The frequency of B cell progenitors in the HSAlow lin- CD43low population of neonatal spleen was examined as well. The HSAlow lin- CD43low population was sorted from the spleens of 2–3-wk-old neonatal mice and placed into culture under limiting dilution conditions on stromal cells and IL-7 as described above for the neonatal bone marrow. Pre- and postsort analyses are shown in Fig. 2GoA. The frequency of B cell progenitors in the HSAlow lin- CD43low population of neonatal spleen was calculated to be approximately 1 in 285 (Fig. 2GoB). Thus, while the B cell progenitor activity within the HSAlow lin- CD43low population is less frequent in the neonatal spleen than in neonatal bone marrow, the splenic HSAlow lin- CD43low population present in neonates is capable of B cell genesis.



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  		FIGURE 2. Purification and limiting dilution analysis of HSAlow lin- CD43low cells from neonatal spleen. HSAlow lin- CD43low cells were sorted from neonatal spleen (A) and cultured (30 replicates) (B) on irradiated stromal cells and IL-7 for 1 wk under limiting dilution conditions as described in Materials and Methods. The percentage of cells within the sort gate was 0.3%.

 
In vitro differentiation and proliferation of HSAlow lin- CD43low cells

During differentiation of PHSCs along the B lineage pathway, the cells acquire B220 and up-regulate the expression of HSA (3, 15). Thus, the acquisition of HSAhigh B220+ was used to assess the rate of differentiation of the sorted HSAlow lin- CD43low cells. The HSAlow lin- CD43low population of cells was sorted from neonatal bone marrow and plated at 1 x 104 cells/well in 12-well plates on irradiated stromal cells with IL-7. Cells were analyzed daily by flow cytometry for the change in expression of HSA and B220. After 1 day in culture, there was little differentiation into a HSAhigh B220+ phenotype. However, by day 4 in culture, 13.1% of the cells had differentiated, and by day 6 of culture, 38.5% of the cells exhibited a differentiated phenotype (Fig. 3Go).



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  		FIGURE 3. Cell surface phenotype of HSAlow lin- CD43low cells at various time points during in vitro culture. HSAlow lin- CD43low cells were sorted from neonatal bone marrow and cultured in bulk on OP42 stromal cells and IL-7. Cells were harvested from the cultures daily and stained with anti-HSA and anti-B220 Abs. Differentiated cells were detected by using gates set on the HSAhigh B220+ cells from whole neonatal bone marrow.

 
Four-color flow cytometric analyses were used to determine the extent of differentiation of the HSAlow lin- CD43low cells after 4 wk in culture. The Hardy system of B cell nomenclature was used to determine the extent of differentiation of the HSAlow lin- CD43low cells in culture (3). This system of B cell nomenclature is based on the differential expression of HSA, B220, CD43, BP-1, IgM, and IgD and is used to define B cell subpopulations from the least mature B cell (fraction A) to the most mature B cell (fraction F) (3). HSAlow lin- CD43low cells were sorted from neonatal bone marrow as described above and cultured in bulk on irradiated stromal cells with exogenous IL-7 for 4 wk. Cells were removed and labeled with Abs specific for HSA, B220, CD43, and BP-1, or B220, CD43, IgM, and IgD. While cells within fraction A–D were detected, the majority of cells had the phenotype B220+ CD43+ HSA+ BP-1+, which places them in fraction C. Cells within the other fractions were as follows: B > D > A (Fig. 4Go). These data suggested that because fractions A–D develop from the HSAlow lin- CD43low cells, these progenitors either precede fraction A cells or parallel fraction A in the B cell developmental pathway.



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  		FIGURE 4. Four-color flow cytometric analysis of HSAlow lin- CD43low cells after 4 wk in culture. HSAlow lin- CD43low cells were sorted from neonatal bone marrow, cultured in bulk on stromal cells and IL-7, and harvested after 4 wk and stained with Abs against B220, CD43, HSA, and BP-1 or B220, CD43, IgM, and IgD. Fractions A–C were detected by gating on the B220+ CD43+ population and analyzing the gated population for expression of HSA and BP-1. Fraction A contained B220+, CD43+, HSA-, BP-1-; fraction B contained B220+, CD43+, HSA+, BP-1-; and fraction C contained B220+, CD43+, HSA+, BP-1+. Likewise, fractions D–F were detected by gating on B220+ CD43- cells and analyzing the gated population for expression of IgM and IgD. Fraction D contained B220+, CD43-, IgM-, IgD-; fraction E contained B220+, CD43-, IgM+, IgD-; and fraction F contained B220+, CD43-, IgM+, IgD+.

 
The proliferative capacity of the HSAlow lin- CD43low cells in vitro was determined by performing daily cell counts. After 2 days in culture, the cell number did not substantially increase. However, over the next 4 days of the experiment the cell number increased approximately fourfold (Fig. 5Go). While the expansion of the HSAlow lin- CD43low cultures coincided with the time point at which the cells differentiated (Fig. 3Go), it is impossible to conclude whether the HSAlow lin- CD43low cells are proliferating or if proliferation is the result of a subsequent phenotype, such as fraction C.



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  		FIGURE 5. Proliferation of the HSAlow lin- CD43low cells at various time points during in vitro culture. HSAlow lin- CD43low cells were sorted from neonatal bone marrow and cultured in bulk on stromal cells and IL-7. Cells were harvested every other day and live cell counts were performed using a hemacytometer. The data is representative of two separate experiments.

 
HSAlow lin- CD43low cells in adult bone marrow and spleen have a low frequency of B cell progenitors

Because the HSAlow lin- CD43low cells from bone marrow and spleen contained a high frequency of B lymphocyte progenitors during neonatal life, we questioned whether a similar population from adult bone marrow had B cell progenitor activity as well. Adult bone marrow (Fig. 6GoA) and spleen (Fig. 6GoB) were collected, stained, sorted, and cultured under limiting dilution conditions as described above. HSAlow lin- CD43low populations were detected in both the spleen and bone marrow of adult mice; however, when examined for lymphoid colonies in B cell limiting dilution assays, few if any lymphoid colonies were detected (frequency < 1:2000) even though the initial cell concentrations for the adult limiting dilution assays were three (spleen) and four (bone marrow) times the initial concentrations plated for the equivalent populations in neonatal mice (Table IIGo).



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  		FIGURE 6. Detection of HSAlow lin- CD43low cells from adult bone marrow and spleen. Adult bone marrow (A) or spleen (B) was stained with Abs against HSA, CD43, and a lineage mixture as described in Fig. 1Go. The percentage of total events within the sort gate was 1.2% in adult bone marrow and 0.2% in adult spleen.

 

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  		Table II. Frequency of B cell progenitors from HSAlow lin- CD43low fraction of adult bone marrow and spleen

 
HSAlow lin- CD43low cells express TdT in neonatal and adult mice

The HSAlow lin- CD43low cells from neonatal bone marrow were analyzed for the expression of TdT as an indication of lymphoid lineage commitment. TdT is a lymphocyte-specific protein whose expression is limited predominantly, if not exclusively, to B and T cells early in their developmental pathways (16, 17, 18, 19). TdT expression by freshly sorted and 1-wk-old cultures of HSAlow lin- CD43low cells was measured by Western blot analysis. Thymocytes served as a positive control and splenocytes as the negative control. As shown in Fig. 7Go, TdT was detected in whole cell lysates of thymus, cultured HSAlow lin- CD43low cells, and freshly sorted HSAlow lin- CD43low cells, but not in splenocytes. The expression of TdT by the HSAlow lin- CD43low cells is consistent with the hypothesis that lymphocyte-committed precursors reside within this population.



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  		FIGURE 7. TdT protein expression in the HSAlow lin- CD43low cells from neonatal bone marrow. A, Pre- and postsort analysis of the HSAlow lin- CD43low cells sorted from neonatal bone marrow. Whole cell lysates of HSAlow lin- CD43low cells sorted from neonatal bone marrow, HSAlow lin- CD43low cells after 1 wk in culture, thymocytes, and spleen were separated by SDS-PAGE and analyzed for the expression of TdT by Western blot analysis (B) as described in Materials and Methods. Equivalent amounts of protein were loaded for each sample.

 
TdT expression was also examined from the HSAlow lin- CD43low population in adult bone marrow to determine whether the HSAlow lin- CD43low cells from adult bone marrow have lost the capacity for B cell genesis via a suppressive mechanism in adult animals or because the adult HSAlow lin- CD43low population is comprised of significantly fewer B lineage progenitors. TdT expression was analyzed in HSAlow lin- CD43low cells isolated from adult bone marrow as described above. Thymocytes and 1-wk-old cultures from neonatal HSAlow lin- CD43low cells served as positive controls, while NIH3T3 cells served as a negative control. As shown in Fig. 8Go, TdT was detected in HSAlow lin- CD43low cells isolated from adult bone marrow. TdT expression was not detected in NIH3T3 cells. These data suggest that the HSAlow lin- CD43low population from adult bone marrow still contains precursors of the B cell lineage, but at a much lower frequency than the equivalent population in neonatal mice.



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  		FIGURE 8. TdT protein expression in the HSAlow lin- CD43low cells from adult bone marrow. A, Pre- and postsort analysis of the HSAlow lin- CD43low cell sorted from adult bone marrow. Whole cell lysates of HSAlow lin- CD43low cells sorted from adult bone marrow, cultured neonatal HSAlow lin- CD43low cells, NIH3T3 fibroblasts, and thymocytes were separated by SDS-PAGE and analyzed for TdT by Western blot analysis (B) as described in Materials and Methods. Equivalent amounts of protein were loaded for each sample.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we describe a novel population of B lineage precursors that expresses low levels of HSA and CD43 and is negative for the lineage markers B220, Mac-1, Ter-119, and Gr-1. The cell surface proteins by which we have characterized the HSAlow lin- CD43low population are not unique to the B cell lineage. In fact, HSA and CD43 are expressed on a wide variety of lymphohematopoietic cells during at least some part of their development, including T cells, macrophages, erythroid cells, and hematopoietic stem cells (3, 20, 21, 22, 23, 24). However, it is the coordinate and temporal regulation of HSA and CD43 in the absence of other lineage marker expression that distinguishes the HSAlow lin- CD43low population of B cell progenitors from other precursors of the B cell lineage. Furthermore, this is the first report of a B cell population that is temporally regulated, because B cell progenitor activity is detected at a high frequency from the HSAlow lin- CD43low population in the bone marrow and spleen of neonatal mice and not in the bone marrow and spleen of adult animals. Not only was the frequency of B cell progenitors less in the adult HSAlow lin- CD43low population when compared with the neonatal HSAlow lin- CD43low population, but the total number of B cell progenitors was also considerably less in the adult population. In experiments testing as many as 3000 adult HSAlow lin- CD43low cells/well (data not shown), limiting dilution of 1 cell/well was never reached. Therefore, the frequency was <1:3000. Based on a frequency of <1:3000, the total number of B cell progenitors in the HSAlow lin- CD43low population sorted from the femurs and tibias of adult mice was <1.0 x 104 compared with >1.0 x 106 B cell progenitors sorted from the equivalent bones in neonatal mice. Thus, neonatal animals contain at least 100-fold more HSAlow lin- CD43low B cell progenitors than adult animals.

Differentiation of the HSAlow lin- CD43low cells to the B cell lineage was determined to characterize the subsequent phenotypes of the HSAlow lin- CD43low cells after in vitro culture. The HSAlow lin- CD43low cells were determined to differentiate into cells expressing B220 and high levels of HSA within 4 days of culture on stromal cells with exogenously supplemented IL-7. Furthermore, the HSAlow lin- CD43low cells are capable of differentiating into fractions A–D when grown under conditions favoring B cell development. These data suggest that the HSAlow lin- CD43low cells developmentally precede fraction A cells as the HSAlow lin- CD43low cells give rise to fraction A cells in vitro. However, because the percentage of fraction A cells in culture was extremely low, it is possible that the HSAlow lin- CD43low cells parallel fraction A in the B cell developmental pathway.

Cell proliferation experiments revealed that cell division occurred from the HSAlow lin- CD43low population in vitro. It is impossible to extrapolate from these experiments whether the HSAlow lin- CD43low cells differentiate into B lineage precursors and then divide, possibly after having reached fraction C, or whether the HSAlow lin- CD43low cells undergo a few rounds of cell division and then differentiate along the B cell developmental pathway.

The multipotentiality of the HSAlow lin- CD43low cells was tested in vitro using hematopoietic colony assays. These studies showed that cells in the HSAlow lin- CD43low population of neonatal bone marrow not only had cells capable of development into B lineage cells, but also a low frequency of cells with the potential to develop into cells of the monocyte/macrophage lineage. Whether the HSAlow lin- CD43low cells represent a population of bi/multipotent progenitors capable of differentiation into multiple hematopoietic lineages or represent two populations each committed to a particular lineage cannot be resolved from these experiments. However, the HSAlow lin- CD43low population from neonatal bone marrow had at least a subpopulation of cells that expressed the lymphocyte-specific protein TdT. These data are consistent with the hypothesis that the HSAlow lin- CD43low cells are not multipotent for B cells and macrophages, but rather contain B220- precursors that are, at the very least, committed to the lymphoid lineage.

The HSAlow lin- CD43low population contains a high frequency of B cell progenitors, as demonstrated by limiting dilution analysis on stromal cells and IL-7. However, the fact that the HSAlow lin- CD43low population contributes to the generation of B cells does not constitute the uniqueness of this population, as several populations of primitive B cell progenitors have been previously described. For example, Cumano et al. characterized a bipotential precursor of B cells and macrophages from day 12 fetal livers that are similar to the HSAlow lin- CD43low B cell precursors that we have described in that the bipotential cells are negative for the expression of B220 and Mac-1 (25). Other similarities between the B cell/macrophage precursor and the HSAlow lin- CD43low population include the fact that we have isolated and cloned HSAlow lin- CD43low cells from the fetal liver (data not shown), and it appears that they have the potential to develop into both B cells and macrophages. However, the B cell/macrophage precursor has never been convincingly demonstrated in the bone marrow of adult or young mice. Furthermore, the frequency of B cell progenitors within the bipotential cell population was increased when isolated based on the expression of the cell surface protein AA4.1 (26), whereas the HSAlow lin- CD43low population was not increased in frequency by the inclusion of AA4.1 in the sorting strategy (data not shown). Thus, we initially entertained the possibility that the bipotent B cell/macrophage precursors and the HSAlow lin- CD43low cells were equivalent populations. However, based on the data presented here we believe that they represent two separate populations of cells.

Li et al. have described, as of yet, the earliest B lineage precursor in the adult bone marrow (27, 28). This population, designated A1, is a subdivision of fraction A cells initially described by Hardy several years earlier (3, 27, 29). The A1 subpopulation shares some common features of both the bipotential B cell/macrophage precursors and the HSAlow lin- CD43low population, yet bears some distinctive features as well. The A1 subpopulation and the HSAlow lin- CD43low cells are distinguishable in the expression of B220 and HSA, as A1 is positive for the common leukocyte Ag B220 and lacks the expression of the HSA glycoprotein, whereas the HSAlow lin- CD43low cells have not yet acquired B220 on the cell surface and have begun to express low levels of HSA (27). Regardless of the similarities between the A1 subpopulation of B cell progenitors and the HSAlow lin- CD43low population, the paramount difference is in the fact that we have not been able to isolate HSAlow lin- CD43low cells with B cell potential from the spleen or bone marrow of adult animals in amounts equivalent to neonatal bone marrow, the implications of which are discussed below.

Recently, Kondo et al. have published a report describing a cell population with the phenotype lin- IL-7R+ Thy-1- Sca-1low c-kitlow that is restricted to the lymphoid lineage (T, B, and NK cells) (30). This common lymphoid progenitor (CLP) also has the phenotype of HSAlow CD43low, which is similar to the HSAlow lin- CD43low population described in this report. The status of the IL-7R on the surface of the HSAlow lin- CD43low cells is currently unknown because the IL-7R Ab was unavailable at the time these studies were completed. However, the HSAlow lin- CD43low population is c-kit+ and Thy-1-, and a fraction of HSAlow lin- CD43low cells were positive for Sca-1 (data not shown). Furthermore, in preliminary studies (K.M.M. et al., manuscript in preparation) we have found that the HSAlow lin- CD43low population from neonates, but not from adults, was capable of reconstituting the B and T cell compartments of Rag1-/- mice, suggesting that CLPs may exist within this population in neonatal mice but not in the analogous population from adults. Taken together, the HSAlow lin- CD43low population and the CLP population described by Kondo et al. appear to be similar in phenotype and activity. However, the HSAlow lin- CD43low population we have characterized contains monocyte progenitors that have been demonstrated in vitro, whereas monocyte progenitor activity has not been documented in the CLP population. Finally, in comparing the CLP from Kondo’s group to the HSAlow lin- CD43low cells described in this paper, the CLP were isolated from adult murine bone marrow (between 4–10 wk of age), whereas the HSAlow lin- CD43low population appears to be active primarily in the neonatal period. Because the precise time that the HSAlow lin- CD43low population becomes diminished in its capacity to generate B cells is currently under investigation, it is not known whether the CLP and the HSAlow lin- CD43low population from neonatal mice are equivalent populations.

Although the HSAlow lin- CD43low population of cells appears to be an early intermediate in the B cell developmental pathway, it is interesting that the HSAlow lin- CD43low B lineage precursors were detected at a high frequency in the spleen and bone marrow of neonatal mice but not in the spleen and bone marrow of adult mice. However, the HSAlow lin- CD43low cells from adult bone marrow express the lymphocyte-specific protein TdT, suggesting that the adult HSAlow lin- CD43low population still contains cells with B lineage potential but at a low frequency. This is the first demonstration of a unique developmental intermediate that may be involved in the genesis of the immune system in neonatal mice. We hypothesize that the HSAlow lin- CD43low cells are present at an important point during ontogeny when the lymphocyte compartment is likely in a state of rapid expansion. The HSAlow lin- CD43low cells would serve the function of expanding the B lymphocyte compartment of the immune system until homeostasis is achieved. Once the lymphocyte compartment has reached steady state levels, the HSAlow lin- CD43low population would no longer be required and would thus decrease, but remain in the bone marrow and/or spleen at extremely low frequencies. In recent experiments (K.M.M. et al., manuscript in preparation), we observed that irradiation of adult animals resulted in an increase of B cell progenitor activity in the HSAlow lin- CD43low population of cells isolated from adult bone marrow. This is consistent with the hypothesis that the HSAlow lin- CD43low cells are up-regulated during periods where rapid lymphocyte expansion is necessary.

A second explanation for the "disappearance" of the HSAlow lin- CD43low cells in adult mice concerns the effects of age on the immune system. The effects of aging on B cell development (28, 31, 32, 33) and hematopoiesis (34, 35, 36) have been well documented. And, while the effects of aging on the capacity of hematopoietic stem cells to self-renew is somewhat controversial, there are several lines of evidence to suggest that the bone marrow in aged mice is diminished in its capacity to generate B cells (28, 31, 32). Recent reports have further demonstrated that the decline of B lymphopoiesis associated with aging reflects a diminished ability of the pre-B cell fraction to proliferate (31). Furthermore, the defect in the proliferative capacity of pre-B cells was determined to be related to the impaired ability of pro-B cells to respond to IL-7 (28). The HSAlow lin- CD43low cells described in this report represent a point in B cell development that is before the pro-B cell stage by several different criteria. Because there was no decline in the number of pro-B cells reported by Stephan et al., which might be indicative of a problem preceding the pro-B cell stage, we do not believe that the ontologic specificity observed in the HSAlow lin- CD43low population is due to the effects of aging on the immune system.

A third hypothesis for the failure of the HSAlow lin- CD43low cells from adult bone marrow to produce B cell progenitors is that the adult cells have different growth factor requirements for proliferation and differentiation. This hypothesis seems unlikely because the culture system that was used is capable of supporting PHSCs.

In summary, we have characterized a population of HSAlow lin- CD43low cells from neonatal bone marrow and spleen that contains a high frequency of B lineage progenitors. The most striking observation made is that the HSAlow lin- CD43low population is capable of yielding B lineage cells when isolated from neonatal mice but not when isolated from adults. While much of B cell development and lymphocyte development is, in general, focused on the adult murine bone marrow and the fetal liver, we have demonstrated that the bone marrow from neonatal mice is not analogous to the bone marrow from adult mice and thus the developmental events in one do not necessarily parallel the other.


   Footnotes
 
1 This work was supported in part by the National Institute on Alcohol Abuse and Alcoholism (Grant AA09876) and by funds provided through the Center for Excellence in Cancer Research Treatment and Education and the Center of Excellence in Arthritis and Rheumatology, Louisiana State University Medical Center, Shreveport, LA. Back

2 K.M.M. and K.L.B. were supported by National Research Service Award Fellowships (Grants AA05427 and AA05441, respectively) awarded by the National Institute on Alcohol Abuse and Alcoholism. Back

3 Address correspondence and reprint requests to Dr. R. Michael Wolcott, Department of Microbiology and Immunology, Louisiana State University Medical Center, P.O. Box 33932, Shreveport, LA 71130. Back

4 Abbreviations used in this paper: PHSC, pluripotent hematopoietic stem cell; HSA, heat stable Ag; lin, lineage mixture against Ter-119, Gr-1, Mac-1 and B220; TBS, Tris-buffered saline; TBST, Tris-buffered saline with 2% Tween; TdT, terminal deoxynucleotidyl transferase; CAFC, cobblestone area forming cells; CLP, common lymphoid progenitor. Back

Received for publication February 27, 1998. Accepted for publication July 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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