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Stem Cells, Vol. 16, No. 2, 99-106, March 1998
© 1998 AlphaMed Press

Characterization of Natural Suppressor Cells in Human Bone Marrow

Kikuya Sugiuraa, Savita Pahwab, Yoshihisa Yamamotoc, Konstantin Borisovd, Rajendra Pahwae, Robert P. Nelson, Jr.d, Junji Ishikawaf, Tomoko Iguchic, Naoki Oyaizub, Robert A. Goodd, Susumu Ikeharaa

a First Department of Pathology, Kansai Medical University, Moriguchi-City, Osaka, Japan;
b Department of Pediatrics, North Shore University Hospital-New York University School of Medicine, Manhasset, New York, USA;
c First Department of Internal Medicine, Kansai Medical University, Moriguchi-City, Osaka, Japan;
d Department of Pediatrics, All Children's Hospital, University of South Florida, St. Petersburg, Florida, USA;
e Department of Pediatrics, Schneider Children's Hospital, Long Island Jewish Medical Center, New Hyde Park, New York, USA;
f Novartis Pharma K.K. Takarazuka-City, Hyogo, Japan

Key Words. Natural suppressor • CD34+33+ cells • Myeloid progenitors

Correspondence: Dr. Susumu Ikehara, First Department of Pathology, Kansai Medical University, 10-15 Fumizono, Moriguchi-City, Osaka 570, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Natural suppressor (NS) cells, which exert nonspecific suppressive activity in an unprimed manner, have been found in mouse, rabbit and monkey bone marrow (BM). In the present study, we characterize NS cells in human BM. NS activity was found in a fraction of low density (1.055-1.065 g/ml) BM cells that had been depleted of T cells, B cells, and monocytes. The NS activity was significantly decreased by the depletion of CD34+ or CD33+ cells but not CD56+ cells. The NS activity was indeed detected in isolated CD34+ cells and further enriched in CD34+CD33+ cells. Hematopoietic progenitor cells committed to the myeloid lineage were also enriched in the CD34+CD33+ cells, which significantly correlated to the NS activity. From these findings, it is strongly suggested that NS activity in human BM is exerted by the myeloid hematopoietic progenitors. Since cell-to-cell contact was not necessary for the action, NS cells seemed to secrete soluble mediator(s). Transforming growth factor-ß1 and leukemia inhibitory factor were, however, not the candidates, based on experiments using neutralizing antibodies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural suppressor (NS) activity is defined as the ability of unprimed cells to exert nonspecific suppression in various immunological responses [1, 2]. NS cells show "null" surface phenotype and have been identified at the site of active hematopoiesis, including bone marrow (BM) [3-7], neonatal spleen [8, 9] and the spleen of mice after total lymphoid irradiation [1, 10, 11] or administration of Strontium 89 [12, 13]. These findings suggest that hematopoietic progenitor cells exert NS activity in BM. Indeed, we have previously found that NS activity is enriched in the fraction of the wheat germ agglutinin-high affinity [14, 15] and interleukin 3 receptor-positive [16] hematopoietic progenitor cells of mouse and monkey BM. These findings have been confirmed by other investigators [17, 18].

Graft-versus-host disease (GVHD) is one of the main obstacles in allogeneic BM transplantation, which is now a powerful strategy for the treatment of congenital immunodeficiencies, hematological neoplasias [19, 20] and aplastic anemia [21] in humans. However, the elimination of T cells from the donor BM (to prevent GVHD) is often associated with relapse of the malignancy or rejection of the BM graft [22]. In the murine model, it has been reported that NS cells prevent GVHD [23]. Furthermore, we have found that murine BM NS cells inhibit the proliferation of murine hematological neoplasias [24]. In this study, we attempt to isolate and characterize NS cells in human BM, and show that CD34+ CD33+ cells have potent NS activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
Specimens of human BM were obtained from normal donors after informed consent. All samples were collected using heparin.

Fractionation of BM Cells
BM samples were diluted 1:20 with calcium and magnesium-free Dulbecco's phosphate-buffered salt solution and placed on a Ficoll-Hypaque gradient (Lymphoprep; density 1.077 g/ml; Nycomed As; Oslo, Norway). After centrifugation at 500 x g for 30 min, whole mononuclear cells (WMNCs) were collected from the interphase, washed and suspended in RPMI 1640 medium (GIBCO; Grand Island, NY) containing 10% fetal bovine serum ([FBS]; Hyclone Laboratories; Logan, UT). T cells, B cells and monocytes in WMNCs were purged using monoclonal antibodies (mAbs) and immunomagnetic beads. In brief, WMNCs were incubated with mAbs to CD2 (clone OKT11; American Type Culture Collection: ATCC; Rockville, MD), CD14 (clone 3C10; ATCC), CD11b (clone OKM1; ATCC) and B lymphocyte (clone Lym-1; ATCC) for 30 min at 4°C. The effective concentration of these mAbs was determined in preliminary experiments. After the incubation, the cells were washed twice with RPMI 1640 medium and incubated with immunomagnetic beads coated with sheep antimouse IgG (Dynabeads M450; Dynal; Oslo, Norway; the target cells/beads ratio, 1:10) for 40 min at 4°C. After the beads (together with binding cells) were retained along the tube wall with a magnet for a few minutes, the supernatant containing negative cells was recovered. The procedure using the antimouse IgG beads was repeated once. By these procedures, more than 97% of CD14+, 98% of CD2+ cells, and 94% of CD19+ cells had been removed from the WMNCs (non-T B Mo).

The non-T B Mo population was then fractionated by equilibrium density centrifugation on a discontinuous gradient of Percoll (Pharmacia Biotech; Uppsala, Sweden) solution as previously described [25]. For the density separation, Percoll solution was prepared at densities of 1.075, 1.065, and 1.055 g/ml. The pH and osmolarity of these solutions were adjusted to pH 7.2 to 7.4 and 290 m Osm/Kg. After centrifugation at 1,400 x g for 30 min, cells were collected from each fraction: (Fr. 1) < 1.055 g/ml, (Fr. 2) 1.055 to 1.065 g/ml, (Fr. 3) 1.065 to 1.074 g/ml, > (Fr. 4) 1.075 g/ml. Cells in each fraction were used for the natural suppressor cell assay and in vitro hematopoietic colony formation assay.

Isolation of CD34+, CD34+CD33+ and CD34+CD33– Cells
Isolation of CD34+, CD34+CD33+ and CD34+CD33– cells was performed using the magnetic cell separation system ([MACS], Miltenyi Biotec GmbH; Bergisch-Gladbach, Germany) and CD34 Multisort Kit (Miltenyi Biotec) described by Miltenyi et al. [26]. In brief, non-T, B, Mo BM cells were incubated with anti-CD34 mAb (clone QBEND/10)-conjugated ferritdextran (microbeads) at 6°C for 30 min after the incubation with the Fc receptor-blocking reagent in the multisort kit. Labeled cells and unlabeled cells were separated in a high gradient magnetic field generated in a steelwool matrix in a separation column (MiniMACS Separation Column; Miltenyi Biotec) and inserted into the field of a permanent magnet. The positive cells were eluted from the column outside of the magnet. After repeating the separation procedure, the purity of the resultant CD34+ cells was 98% (Fig. 1). Microbeads were then separated from anti-CD34 mAbs using the release reagent in the multisort kit. The isolated CD34+ cells were labeled with fluorescein isothiocyanate-conjugated anti-CD33 mAb (clone MY9; Coulter Cytometry; Hialeah, FL), then further labeled with anti-FITC Ab-conjugated microbeads (Miltenyi Biotec). The CD34+ CD33+ cells were retained on the steelwool matrix of the separation column in a magnetic field, whereas the CD34+CD33– cells were eluted out. The CD34+CD33+ cells were eluted outside of the magnetic field. The purity of the CD34+CD33+ cell and CD34+CD33– cell populations was evaluated on a FACScan (Becton Dickinson Immunocytometry System; Mountain View, CA) after staining with phycoerythrin-conjugated anti-CD34 mAb (HPCA-2; Becton Dickinson) (Fig. 1). The isolated cell populations were cultured in Iscove's modified Dulbecco's medium (IMDM) with 10% FBS at the concentration of 1 x 106/ml. The culture supernatants were used for the assay of NS activity and measurement of nitrate and nitrite.



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  		Figure 1. FACS analysis of isolated CD34+, CD34+CD33–, CD34+CD33+ and CD34– cells. Cells in each isolated fraction were stained with phycoerythrin-conjugated anti-CD34 mAb and fluorescein isothiocyanate-conjugated anti-CD33 mAb. The fluorescent intensity, forward scatter (FSC) intensity, and side scatter (SSC) intensity were evaluated on a FACScan.

 
Assay for NS Activity
NS activity of BM cells was evaluated according to the ability to inhibit the mitogen responses of human peripheral blood lymphocytes (PBLs). In some experiments, the assay for suppressor activity was performed using a 0.4 mm double diffusion chamber ([DDC]; MILLICELL; Millipore; Bedford, MA), which prevents the cell-to-cell contact between BM cells (as suppressor cells) and PBLs or tumor cells. In brief, DDCs were placed into wells of 24-well plates (Costor; Cambridge, MA). PBLs (3 x 105 in 500 µl) along with phytohemagglutinin (PHA)-P (Difco Laboratories; Detroit, MI; 0.25% final) were placed in DDCs and various concentrations of BM cells in 500 µl of IMDM 10% FBS were placed in the wells. The cultures were incubated for 60 h at 37°C in a humidified atmosphere of 5% CO2 in air. After incubation, three aliquots (100 µl) were collected from the DDCs and the proliferation of PBLs was determined by measuring incorporation of [3H]-thymidine or [14C]-thymidine presented in the cultures for six h. In the other experiments, the supernatant of the BM cell culture was serially diluted and added to the cultures of PBLs (1 x 105/well) in the presence of PHA-P (0.25% final) in 96-well culture plates with flat bottom (Corning Glass Works; Corning, NY). The cultures were incubated for 60 h. The proliferation of PBLs was determined by measuring the incorporation of [3H]-thymidine presented in the cultures for six h. The percentage of suppression was calculated using the following formula:


(1)

Assay for Colony Formation
Triplicate cultures were set up using 35 mm Petri dishes (FALCON 1008, Becton-Dickinson Labware; Lincoln Park, NJ). An appropriate number of BM cells was incubated in one milliliter of culture in each dish containing 0.9% methylcellulose, 30% FBS, 1% bovine serum albumin, 3 units erythropoietin, 10–4 M 2-mercaptoethanol, 2 mM L-glutamine, 50 ng recombinant human stem cell factor, 10 ng recombinant human GM-CSF, and recombinant human interleukin 3 in IMDM (Methoccult GF H4434; Stem Cell Technologies, Inc.; Vancouver, BC, Canada). The dishes were incubated at 37°C in a humidified atmosphere with 5% CO2 in air. After 14 days in culture, numbers of colonies containing a minimum of 40 cells were counted. Representative colonies were picked up and stained with Wright-Giemsa for morphologic analyses.

Neutralization Assay of NS Activity
The supernatant (90 µl) collected from the culture of CD34+ cells was incubated with neutralizing Abs against transforming growth factor-ß1 ([TGF-ß1]; R&D Systems, Inc.; Minneapolis, MN; 100 µg), leukemia inhibitory factor ([LIF]; R&D Systems; 100 µg) or macrophage inflammatory factor-1{alpha} (R&D Systems; 100 µg) for 4 h at room temperature. To estimate neutralization, resultant solutions were added to assays for NS activity.

Statistics
All experiments were independently carried out two or more times. The sample number in each experiment is described in the legends of figures or footnote of tables. The significance of difference was calculated using Tukey-Kramer multiple comparison test after the confirmation that populations had equal standard deviations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enrichment of NS Activity in CD34+CD33+ BM Cell Fraction
The NS activity of the BM cells was evaluated according to their ability to inhibit the mitogen responses of human PBLs. Since we have found that NS cells in mouse BM produce soluble mediator(s) [14, 15], we used DDCs which prevent the cell-to-cell contact between effector (BM cells) and target (PBLs) in the assays for NS activity. We have also found that cells exerting NS activity in mouse and monkey BM are non-T B Mo. Therefore, we first attempted to confirm that human BM cells without markers of T cells, B cells and monocytes have suppressor activity. As shown in Table 1, the non-T B Mo BM cell fraction exerted suppressor activity. No suppressor activity was found in the isolated CD2+, CD19+ or aplastic adherent cells (data not shown). Also, no suppressor activity was detected in the fraction of nonerythroid BM cells with the density (<1,077 g/ml); 85% of these cells were neutrophils. After the equilibrium density separation on Percoll density gradients, the suppressor activity was enriched in cells with relatively low density (Fr. 2; 1.055-1.065 g/ml). The suppressor activity in the BM Fr. 2 was not affected by the depletion of cells bearing CD56, which composed 6.5% BM Fr. 2 cells. However, the suppressor activity was depleted or significantly reduced when cells in BM Fr. 2 were purged of cells bearing CD33 and CD34, which composed 19%-28% and 6%-19% of BM Fr. 2, respectively (Table 1). From these results, it is suggested that cells expressing both CD34 and CD33 (CD34+CD33+ cells) are candidates for NS cells in human BM, and that human NS cells may produce soluble mediator(s) for suppressive activity. To confirm this, we isolated CD34+, CD34+CD33+ and CD34+CD33– cells, then examined the suppressive activity in culture supernatants of the isolated cell populations. As shown in Figure 1, the CD34+ and CD34– cell populations were clearly separated using a MACS. We collected the supernatants daily for seven days from cultures of CD34+ and CD34– cells (1 x 106/ml), and examined the suppressive activity. Suppressive activity in the culture supernatants of CD34+ cells increased by day 4, reached a plateau on day 5 and then decreased from day 7; whereas the supernatants from the culture of CD34– cells had little suppressive activity at any period of culture (Fig. 2). The CD34+ cells were further separated into the CD34+CD33+ and CD34+CD33– subpopulations, as shown in Figure 1, and suppressive activity in the culture supernatants collected on day 4 was evaluated. As shown in Figure 3, the supernatants from the CD34+CD33+ cell culture had the most potent suppressive activity, whereas the supernatants from the CD34+CD33– cell cultures had little suppressive activity. After four days culture, the CD34–CD33+ cell population had indeed differentiated from CD34+CD33+ cells. However, more than 65% of the cultured cells still remained CD34+. Furthermore, we did not find any suppressive activity in the culture of isolated CD34–CD33+ cells (data not shown). Therefore, we conclude that the suppressive activity in non-T B Mo BMCs is exerted by the CD34+CD33+ cells.


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  		Table 1. Decrease in NS activity of human bone marrow after depletion of CD34+ or CD33+ cells
 


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  		Figure 2. Time course of suppressive activity in the culture supernatants of CD34+ cells and CD34– cells. Supernatants were collected daily for seven days from the culture of CD34+ or CD34– cells (1 x 106/ml). One hundred microliters of the supernatants were added to 100 microliters of the culture containing PBLs (1 x 105) and PHA (0.5%). The cultures were incubated for 60 h. The percentages of suppression were calculated as described in Materials and Methods . Experiments were carried out two times. Results are expressed as the mean of two experiments.

 


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  		Figure 3. Suppressive activity of the supernatants collected from the cultures of CD34+, CD34+CD33–, CD34+CD33+ and CD34– cells. Each isolated cell population (1 x 106/ml) was cultured for four days. The culture supernatants were serially diluted and added to the culture using PBLs along with PHA. The percentages of suppression were calculated, as described in Materials and Methods . Results are expressed as mean (symbol) and SD (bar). The sample size of CD34+ cell culture supernatants is eight; CD34– cell culture supernatants, eight; CD34+CD33+ cell culture supernatants, five; CD34+CD33– cell culture supernatants, five. At the concentration of 2–2 (25%), p < 0.001, "CD34+CD33+" versus "CD34+CD33–," "CD34+CD33+" versus "CD34–" and "CD34+" versus "CD34–"; p < 0.01, "CD34+CD33+" versus "CD34+" and "CD34+" versus "CD34+CD33–" by the Tukey-Kramer multiple comparison test.

 
Hematopoietic Activity of Fractionated BM Cells
The hematopoietic activity of WMNCs or fractionated BM cells was evaluated using an in vitro colony forming assay. BM Fr. 2 showed greater numbers of colony forming unit-granulocyte/macrophage ([CFU-GM]; myeloid precursor), BFU-E (erythroid precursor) and CFU-granulocyte/erythroid/macrophage/megakaryocyte ([CFU-GEMM]; multipotent precursor) than other density fractions or unseparated non-T B Mo fraction (data not shown). These three hematopoietic precursors were further enriched in the isolated CD34+ cell fraction, whereas a few progenitors were found in the CD34– fractions. When CD34+ cells were further separated into CD34+CD33+ cells and CD34+CD33– cells, both CFU-GM and CFU-GEMM activities were further enriched in the CD34+CD33+ cell population, whereas BFU-E activity was further enriched in the CD34+CD33– cell population (Fig. 4). Statistical analyses to estimate the correlation between suppressor activity and hematopoietic progenitor activity suggest that the suppressive activity of supernatants significantly correlates with the numbers of CFU-GM (r2 = 0.995) but not with those of BFU-E (r2 = 0.016). The numbers of CFU-GEMM also correlated to the suppressive activity. However, the correlation (r2 = 0.874) was less significant than that of CFU-GM.



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  		Figure 4. Colony forming activities of isolated CD34+, CD34+ CD33+ and CD34+CD33– cells. CFU-GEMM, BFU-E and CFU-GM activities in 1,000 cells of isolated populations were examined. Results are expressed as mean and SD (bar). Statistical differences were evaluated using the Tukey-Kramer multiple comparison test. The sample size of CD34+ cells is five; CD34+CD33– cells, five; CD34+CD33+ cells, five.

 
Roles of TGF-ß1 and LIF in NS Activity
We next attempted to clarify the soluble mediator(s) produced by the human BM. Moore et al. proposed TGF-ß1 as a candidate of the mediators for NS activity in the mouse BM [27]. In addition, Hatzfeld et al. recently found that CD34+ human hematopoietic progenitors secrete TGF-ß1 for the autocrine negative regulation of the cell cycle [28]. Therefore, we examined whether TGF-ß1 contributes to the suppressive activity in the culture supernatants of CD34+ cells. As shown in Figure 5 , 100 µg of anti-TGF-ß1 antibody completely neutralized the suppressive activity of TGF-ß1 (24 µg), which inhibited the PHA response of PBLs. However, the same dose of anti-TGF-ß1 antibody did not affect the suppressive activity of the CD34+ cell-culture supernatants. We also tested the effects of LIF on the suppressive activity of the CD34+ cell-culture supernatants, since it has been reported that LIF is produced by both rodent and human hematopoietic progenitor cell lines [29]. However, as shown in Figure 5, the neutralizing antibody against LIF did not affect the suppression of the CD34+ cell-culture supernatants.



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  		Figure 5. Neutralization assay of suppressive activity in the supernatants of CD34+ cell cultures. Supernatants collected from the cultures of CD34+ cells were incubated with neutralizing Ab against TGF-ß 1 (100 µg) or LIF (100 µg) for four h at room temperature. To estimate neutralization, resultant solutions were added to the PHA-responding cultures using PBLs. Results are expressed as mean ± SD of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously characterized the cells exerting NS activity in mouse BM. We have found that NS cells in mouse and monkey BM have low density, a high affinity (more cell surface receptor) to wheat germ agglutinin [14, 24], and the receptor for interleukin 3 [16]. From these results, it is anticipated that NS cells are hematopoietic progenitors committed to myeloid lineage [30, 31]. In the present study, of cells that lack lineage markers, those with relatively low density (BM Fr. 2: 1.055-1.065 g/ml) exerted greater NS activity and hematopoietic colony forming activity than the cells in other density fractions. The NS activity of BM Fr. 2 cells was completely eliminated by the depletion of CD34+ cells, whereas the depletion of CD56+ cells did not affect the NS activity. NS activity was also found in isolated CD34+ cells but not CD34– cells. NS activity was further enriched in CD34+CD33+ cells. Cells expressing the CD34 molecule are thought to be multipotent and erythroid progenitors [32, 33], whereas CD33-expressing cells are thought to be myeloid progenitors [33, 34]. Indeed, isolated CD34+CD33+ cells showed a strong CFU-GM activity. These findings strongly suggest that NS activity in human BM, as well as mouse BM, is exerted by myeloid hematopoietic progenitors. Although Schmidt-Wolf et al. have recently found that CD4–CD8–{alpha}ß+T cells in human BM exert suppressor activity in mixed lymphocyte reaction using autologous responder PBLs, they have found that the CD4–CD8-{alpha}ß– cells in a low density cell fraction, in which CFU-GM and BFU-E activities are enriched, also have the same suppressor activity [35]. More recently, Young et al. have reported data indirectly suggesting that CD34+ cells have NS activity [36]. They have found that increased numbers of CD34+ cells significantly correlate to the increased recurrence or metastasis of the human head and neck squamous cell carcinoma, which produces GM-CSF, and they concluded that the recurrence or metastasis may be due to the inhibition of the intra-tumoral T cell activity by the CD34+ suppressor cells. It has also recently been reported that increasing the doses of hematopoietic stem cells in BM transplantation has overcome the major histocompatibility complex barriers in the therapy for leukemia patients [37, 38]. Besides the increase in the ability of hematopoietic reconstitution, the suppressive activity exerted by the CD34+CD33+ myeloid progenitor cells may play a role in the prevention of either graft rejection by the residual host T cells or GVHD by the donor T cells. On the other hand, our findings that myeloid progenitors suppress cell proliferation indicate that the progenitors may play a role in the control of hematopoiesis under physiological conditions. Lemischka et al., using a technique for transferring foreign genes to hematopoietic stem cells, have proposed that normal hematopoiesis results from sequential activation of different stem cell clones rather than from an average contribution of the entire stem cell pool [39]. Our hypothesis seems to explain the dynamic behavior of stem cells in differentiation.

The suppressive activity in CD34+ cell culture supernatants was not affected by neutralizing Abs against TGF-ß1 and LIF. The neutralizing antibody to macrophage inflammatory protein-1{alpha} (a negative regulator of hematopoietic stem cells) had also no effect on the suppressive activity of the supernatant (data not shown). In mouse experiments, we have recently observed that the molecular weight of the suppressive mediator(s) in the supernatants of myeloid progenitors is less than 10,000. Recently, nitric oxide (NO–) has been reported to be a mediator of suppressive activity exerted by mouse low density BM cells [40] or by mouse granulocyte-macrophage progenitor cells [41]. In addition, Maciejewski et al. reported that NO synthase is induced in human CD34+ cells [42]. However, since the half life of NO– is three to six sec and it immediately changes to inactive form (nitrite or nitrate in solution), NO– cannot be a candidate for the suppressive mediator in the CD34+ cell culture supernatants, which are collected and stocked (for at least 24 h) before use for suppressive activity assay. In the studies described above [41, 42], NS cells were cultured together with target (spleen) cells in their assay.

We are now in the process of purifying NS factor(s) produced by human and mouse BM cells.


   Acknowledgments
 
The authors thank Suzan Lay, Roberta Hill, Maria Maecki and Yuki Matsui for their expert technical assistance, and Keiko Ando for her help in the preparation of the manuscript. This study was partially supported by National Institutes of Health grant AI28281, DA05161, AG05628 and AI22360, an American Cancer Society Institutional Grant 179-08, the Research Aid of Inoue Foundation for Science and a grant-in-aid from the Osaka Cancer Research Association, Grant-in-Aid for Scientific Research on Priority Areas, Grant-in-Aid for Japan Private School Promotion Foundation, the Ministry of Education, Science and Culture, Japan.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication on December 3, 1997.




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K. Sugiura, H. Hisha, J. Ishikawa, Y. Adachi, S. Taketani, S. Lee, T. Nagahama, and S. Ikehara
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