Proc. Nati. Acad. Sci. USA Vol. 75, No. 6, pp. 2933-2936, June 1978 Medical Sciences
Hemopoietic stem cell transplantation using mouse bone marrow and spleen cells fractionated by lectins (peanut agglutinin/soybean agglutinin/graft-versus-host reaction/spleen colony-forming units)
YAIR REISNER, LEA ITZICOVITCH, ASHER MESHORER, AND NATHAN SHARON Department of Biophysics and the Experimental Animal Center, Weizmann Institute of Science, Rehovoth, Israel
Communicated by Paul A. Marks, March 27,1978
ABSTRACT Mouse bone marrow and spleen cells were fractionated with the aid of soybean agglutinin and peanut agglutinin. A test for spleen colony-forming units in the isolated fractions showed that the hemopoietic stem cells are agglutinated by both of these lectins. The capacity of the agglutinated fractions to reconstitute lethally irradiat alogeneic mice was investigated. A sequential fractionation of'splenocy'tes from SWR donors by soybean agglutinin and peanut agglutinin, or a single fractionation by soybean agglutinin of splenocytes from BALB/c donors, afforded a cell fraction that successfully reconstituted lethally irradiated (BALB/c X C57BL/6)F1 mice, without complications due to graft-versus-host reaction.
Grafting allogeneic hemopoietic cell transplants to irradiated animals, or severely immune-deficient patients, has failed in most cases due to the presence in the transplant of cells that cause a graft-versus-host (GVH) reaction. In this communication we report on the isolation of a cell fraction from mouse bone marrow and spleen that is enriched with hemopoietic stem cells and depleted of GVH activity. We used this fraction to reconstitute lethally irradiated allogeneic mice. During the past few years numerous techniques for cell separation, based mainly on cell size, density, and charge, have been devised (1-3). Recently we have demonstrated that selective agglutination by lectins can be used for effective separation of mouse lymphocytes into biologically distinct subpopulations (4, 5). This method is simple, rapid, and reproducible and is based on differences in cell surface receptors rather than in size or density of cells. With this technique, immature and mature thymocytes were separated by the aid of peanut agglutinin (PNA, a lectin specific for D-galactosylI3(1- 3)-N-acetyl-D-galactosamnine) (4), and mouse splenocytes were fractionated into B and T cells by using soybean agglutinin (SBA, a lectin specific for terminal N-acetyl-D-galactosamine and D-galactose) (5); in both cases, the agglutinated cells were devoid of GVH activity. Studies of the binding of the lectins to those subpopulations, both before and after neuraminidase treatment, have led us to conclude that the receptors for both PNA and SBA are present on the surface of immature thymocytes; that in mature thymocytes only the SBA receptor is present (the PNA receptor being masked by sialic acid); and that in peripheral blood lymphocytes, as well as in most spleen T cells, both the PNA and the SBA receptors are masked by sialic acid. On the basis of the above findings, we have postulated that the hemopoietic stem cells may also possess PNA and SBA receptors on their surfaces (4). To test this assumption, we have now isolated a cell fraction from mouse bone marrow and spleen by agglutination with both SBA and PNA. We demonstrate by spleen colony assay and by reconstitution of irradiated The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
allogeneic animals that this fraction is depleted of GVH activity and enriched with hemopoietic stem cells. MATERIALS AND METHODS Animals. Female (BALB/c X C57BL/6)F1 hybrids and female SWR mice were used as recipients. Donor animals were BALB/c and SWR female mice. The recipients were 8-12 wk old and the donors' ages were 6-8 wk. All mice were raised at the Weizmann Institute Animal Center. Irradiation. Mice were exposed to a single dose of 900 R (1 R = 2.6 x 10-4 C/kg) from a y-beam 150A, 60Co source, produced by Atomic Energy of Canada, with focus-to-skin distance of 75 cm and 65 R/min dose rate. Cell Suspensions. Bone marrow was obtained from the femurs and suspended in phosphate-buffered saline (Pi/NaCI). Spleen suspension was prepared by straining the tissue in Pi/ NaCl solution through nylon gauze. The cells were then washed twice in Pi/NaCl and resuspended in the same buffer at the required final concentration. Nucleated cells were counted in Turk's solution. Spleen Colony-Forming Units (CFU-S) Assay. Details of the procedure have been previously described (6). Bone marrow and spleen suspensions were suspended in Pi/NaCl at various concentrations (see Results) and injected into the caudal vein of irradiated recipients. Each experimental group consisted of eight SWR mice' whose spleens were examined 8 days after injection. Radioprotection. Various fractions of splenocytes (see Results) suspended in Pi/NaCl (5 X 107 cells per ml) were injected intravenously into irradiated recipients (0.2 ml per animal) within 24 hr after the irradiation. Lectins. PNA and SBA were purified by affinity chromatography on a column of Sepharose-N-(e-aminocaproyl)-jlD-galactopyranosylamine (7, 8). Solutions. Lysing buffer: 155 mM ammonium chloride/10 mM potassium bicarbonate/0.01 mM EDTA in water; Pi/NaCl: 50 mM sodium phosphate buffer, pH 7.4, in saline. RESULTS AND DISCUSSION Separation of CFU-S-enriched fractions by agglutination with SBA and PNA Fractionation of splenocytes by SBA was carried out according to the method of Reisner et al. (5) with minor modifications. The splenocyte suspension (2 X 108 cells in 0.5 ml of Pi/NaCI) was incubated in polystyrene tubes (17 X 100 mm) with SBA (0.5 ml, 2 mg/ml of Pi/NaCI) for 5 min at room temperature. The cells were then gently layered with a pasteur pipette on top of a solution of bovine serum albumin (5% wt/vol in Pi/NaCl,
Abbreviations: CFU-S, spleen colony-forming units; GVH, graft-versus-host; Pi/NaCI, phosphate-buffered saline; PNA, peanut agglutinin; SBA, soybean agglutinin. 2933
2934
Medical Sciences: Reisner et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
Table 1. Average number of CFU-S in fractionated mouse splenocytes
Cell fraction
1 X 107
Average number of CFU-S* Number of cells injected 4 X 105 2 X 106
8 X 104
Relative enrichmentt
yieldt
0 0
2.8 1 0.5
50 + 10 50 10
>40 >40
Single fractionation 9.1 + 1.3 >40 3.2 d 0.8 19.8 A 2.1 16.8 i 2.1 >40 5.2 + 0.2 NCO
2.6 ± 0.25 0
>40
Sequential fractionation 17.5 1 1.2 >40
3.6 + 0.8
Agglutinated by SBA Unagglutinated by SBA
>40 >40
Agglutinated by PNA Unagglutinated by PNA
Agglutinated by SBA and PNA Agglutinated by SBA and unagglutinated by PNA
>40
7.3
>40
:
1.1
%
15 ± 5 85 :- 5 2.4 + 0.1
20 1 5
80 + 5
0
* Each experimental group consisted of eight mice and the average CFU-S value + SEM was calculated. The CFU-S could not be counted accurately when the value was above 40. t The relative enrichment value is the ratio between CFU-S in the agglutinated fraction and the unagglutinated fraction. Both fractions were subjected to identical treatment throughout the fractionation (see text) and injection procedure. The maximal differences in the values obtained at various cell concentrations were within the indicated limits. Percentage of each fraction out of the total number of cells recovered in both the agglutinated and unagglutinated fractions. § In two animals the CFU-S value was above 40 and the average could not be calculated.
50-ml conical glass tube. After 15 min at room of the agglutinated cells sedimented, whereas the unagglutinated cells remained on the surface of the bovine serum albumin solution. The bottom and top fractions were removed separately by pasteur pipettes and transferred to 15-ml conical plastic tubes. The cells were then suspended in D-galactose (10 ml, 0.2 M) in Pi/NaCl. After 10 min at room temperature, the cells were collected by centrifugation (200 X g, 5 min) and washed twice with the D-galactose solution. Finally the cells were washed twice with Pi/NaCl. About 6 X 107 cells were obtained from both the agglutinated and the unagglutinated fractions. It has been shown previously that the agglutinated fraction is highly enriched in B cells and depleted of T cells, whereas the unagglutinated fraction is comprised mainly of T cells and is depleted of B cells (5). We have now tested these fractions for CFU-S activity (6). As shown in Table 1, the agglutinated fraction is enriched 2.8-fold in hemopoietic activity relative to the unagglutinated fraction. A similar fractionation of bone marrow cells resulted in total agglutination (more than 90% of the cells were recovered in the bottom fraction), thus strengthening our assumption that the SBA receptor is present on hemopoietic cells as well as on B cells (data not shown). Staining of the bone marrow and spleen cells with PNA conjugated to fluorescein isothiocyanate revealed receptors for the lectin on only a small proportion of the cells (10-20%). Incubation of the cells (108/0.2 ml of Pi/NaCl) with PNA (0.25 ml, I mg/ml of Pi/NaCl) for 30 min at room temperature [conditions under which PNA-positive thymocytes were readily separated from the PNA-negative cells (4)], led to the formation of only a few small aggregates, which could not be separated efficiently from the single, unagglutinated cells.
40 ml) in
a
temperature, most
Cell fraction Agglutinated by PNA Unagglutinated by PNA
Fractionation of bone marrow or spleen cells by PNA could be achieved, however, when rabbit erythrocytes, which carry the PNA receptors (9), were added to the incubation mixture to form mixed aggregates of nucleated mouse spleen or bone marrow cells with rabbit erythrocytes. These aggregates were large enough to be separated from the unagglutinated fraction.
The spleen or bone marrow cells in PBS (3 X 108 cells/0.5 ml) incubated in polystyrene tubes (17 X 100 mm) with PNA (0.3 ml, 1 mg/ml) for 5 min at room temperature, and then rabbit erythrocytes (1.8 X 109 cells in 0.3 ml of Pi/NaCl) were added. After an additional 2 min, large aggregates comprised mainly of rabbit erythrocytes were formed and separated from the unagglutinated tells as described above for the SBA fractionation. The aggregates were dissociated with D-galactose and washed as above, and the two fractions were treated with ammonium chloride (10 ml) to lyse the rabbit erythrocytes. After four additional washings with 2 ml of PBS, 3.6-X 107 cells and 4.8 X 107 cells were recovered in the agglutinated fraction of spleen and bone marrow, respectively, whereas 2.3 X 108 cells and 2.1 X 108 cells were recovered from the corresponding unagglutinated fraction. Viability, as judged by the trypan blue exclusion test, was above 85% in all fractions. Analysis of the agglutinated cells showed that they were significantly enriched in CFU-S activity as compared to the unagglutinated ones (Tables 1 and 2), indicating that the PNA receptor is also exposed on the hemopoietic stem cells. In order to further test our assumption that both SBA and PNA receptors are present on hemopoietic stem cells, we attempted a sequential fractionation of splenocytes by both lec-
were
tins, starting with SBA. This fractionation was carried out under the same conditions as described for each fractionation alone, except that fractionation by PNA was performed on the sple-
Table 2. Average number of CFU-S in fractionated bone marrow cells* Average number of CFU-S Number of cells injected 1.6 X 104 8 X 104 4 X 105 2 X 106 >40 >40
>40 21 + 2.5
* More than 90% of the bone marrow cells were agglutinated by SBA.
18.4 i 0.6 3 4 0.5
2.0 + 0.3 0
Relative enrichment
% yield
5.0 + 1.1
20 + 5 80 ± 5
Medical Sciences: Reisner et al.
Proc. Natl. Acad. Scd. USA 75 (1978)
nocyte fraction agglutinated by SBA after dissociation of the aggregates to a single cell suspension by D-galactose. The total
yield of the twice-fractionated subpopulation was about 5%; this fraction is enriched in CFU-S relative to the SBA-agglutinated fraction (Table 1). Thus, additional support is provided for our suggestion that both SBA and PNA receptors are present on hemopoietic stem cells. Because the stem cells constitute only 0.1% of the nucleated cells in bone marrow, and 0.01% of nucleated cells in spleen (10), it is evident that most of the cells agglutinated by SBA and PNA do not possess the capacity to form spleen colonies. The agglutinated cells may represent various stages of differentiation of the lymphoid and myeloid lines, e.g., prethymocytes or pre-B cells, as indicated by the high incidence of PNA-positive cells in mouse embryonic liver (H. Rabinowich, T. Umiel, A. Globerson, Y. Reisner, and N. Sharon, unpublished data) and in various types of acute myeloid and lymphocytic leukemias in humans (Y. Reisner, M. Binyaminov, N. Sharon, and B. Ramot, unpublished data) and mice (A. Harris and Y. Reisner, unpublished data). Reconstitution of lethally irradiated mice by fractionated splenocytes from allogeneic mice The reconstitution of lethally irradiated mice was tested in a parent-to-F1 system [BALB/c (BALB/c X C57BL/6)F1] and in a complete allogeneic system [SWR (BALB/c X C57BL/6)F1]. In the former combination (a typical experiment is shown in Fig. 1), transplantation of the splenocyte fraction that is agglutinated by SBA resulted in complete survival (100%) of the irradiated recipients, whereas transplantation of the unagglutinated fraction or the unseparated splenocytes resulted in a high incidence of mortality. However, in the second system (in which there is no common parent of donor and recipient), we found that the fractionation by SBA was not sufficient to avoid a mortality due to the GVH reaction (mortality incidence in four experiments ranged between 50% and 70%). This GVH reaction may be caused by a small population of T cells that is still present in the SBAagglutinated fraction (5) [although the B cells that comprise the major subpopulation in this fraction (5) may possibly contribute to the overall mortality in this system]. Because PNA does not --
--
2935
bind to mature T or B cells (4, 5), we tried to reconstitute these mice with the twice-agglutinated fraction, namely the SBAagglutinated cells that were further fractionated by agglutination with PNA. The results of a typical experiment are shown in Fig. 2. Grafting lethally irradiated allogeneic mice with unfractionated splenocytes or with cells unagglutinated by SBA resulted in high mortality (13/15 and 15/15 respectively) within the first 30 days after irradiation. The two mice surviving the first 4 wk were suffering from wasting disease (delayed GVH reaction) and died within the second month after transplantation. In a parallel experiment, the few surviving mice were sacrificed 4 and 5 wk after transplantation. Spleen histology (Fig. 3) and bone marrow differential count revealed typical GVH symptoms (11, 12): damage to the white pulp of the spleen, a very high percentage of granulocytes (55-81%), and a low lymphocytic count (1-4%) in the bone marrow. Interestingly, the red pulp (myelo-, erythro-, and thrombopoiesis) in the spleen of these mice was normal or slightly hyperplastic (Fig. 3), indicating that complete hemopoiesis had occurred and that the high mortality rate among these mice was due to acute GVH reaction and not to deficiency of hemopoietic stem cells. Among the mice grafted with the twice-agglutinated fraction, only 1 out of 15 died (Fig. 2). The remaining mice have survived more than 6 mo. Spleen histology (Fig. 3) and bone marrow differential count (granulocytes, 19-35%; lymphocytes, 34-47%), when taken 4 and 5 wk after transplantation in a parallel experiment, revealed that in reconstituted mice both spleen and bone marrow were completely restored. These results are in line with our previous reports (4, 5), in which we tested the GVH activity of fractionated splenocytes and thermocytes in newborn mice. Mouse spleen cells can serve as a good model for allogeneic bone marrow transplantations in humans, because both mouse splenocytes and human bone marrow aspirates cause an early type of GVH reaction that is fatal in most cases, whereas mouse bone marrow cells may cause only a mild type of delayed GVH reaction (10).
_
I
W ~ I
{
15-
E 1
15
0
E
6
0
E
6
5-H
>101 0
E
0 ~~
~
~
~
~
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:
E
5
10
15
20
25
30
90
Time, days FIG. 1. Cumulative mortality of irradiated (BALB/c X C57BL/6)F1 mice after transplantation with splenocytes (107 cells per animal) from BALB/c mice, starting with 15 mice in each group. Grafts: A-A, unfractionated splenocytes; 0-0, splenocytes agglutinated by SBA; 0-0, splenocytes unagglutinated by SBA; *- -, control without graft. -
0
5
10
15 20 25 30 90 Time, days FIG. 2. Cumulative mortality of irradiated (BALB/c X C57BL/6)F1 mice after transplantation with splenocytes (107 cells per animal) from SWR mice, starting with 15 mice in each group. Grafts: A-a, unfractionated splenocytes; -0O, splenocytes sequentially agglutinated by SBA and PNA; 0-0, splenocytes unagglutinated by SBA; 0 ----@, control without graft.
2936
Medical Sciences: Reisner et al.
Proc. Natl. Acad. Sci. USA 75(1978)
Aft ,firr*1is5Xf W S~ ~ ~ ~ ~ ~ *
*
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FIG. 3. Typical spleen histology of (BALB/c X C57BL/6)Fl mice irradiated by 900 R and grafted with splenocytes (107 cells per animal) of SWR donors. The specimen was taken from mice sacrificed 4 wk after transplantation. Grafts. (A) unfractionated splenocytes; (B) splenocytes sequentially agglutinated by SBA and PNA. Scale bar, 0.1 mm. 2. Sabalovic, D. & Serrou, B., eds. (1976) Techniques of Separation Preliminary binding studies on the interaction of fluorescein and Characterization of Human Lymphocytes (Editions isothiocyanate-conjugated PNA with human lymphocytes reINSERM, Paris, France). vealed that, as in its interaction with mouse lymphocytes, PNA 3. Dicke, K. A. & van Bekkum, D. W. (1971) Transplant. Proc. 3, binds to a small subpopulation in the bone marrow (5-15%) and 666-668. does not bind to normal human peripheral blood lympho4. Reisner, Y., Linker-Israeli, M. & Sharon, N. (1976) Cell. Immucytes. nol. 25, 129-134. The approach described in this communication for the iso5. Reisner, Y., Ravid, A. & Sharon, N. (1976) Biochem. Biophys.
lation of cells capable of reconstituting lethally irradiated allogeneic mice may prove useful for bone marrow transplantations in humans. This study was supported in part by National Institutes of Health Contract NCI-CB-74163 and by a grant from the Leukemia Research Foundation. N.S. is an Established Investigator of the Chief Scientist's
6. 7.
8. 9.
Bureau, Israel Ministry of Health. 1. Shortman, K., van Boehmer, H., Lipp, J. & Hopper, K. (1975) Transplant. Rev. 25,163-210.
10. 11. 12.
Res. Commun. 72,1585-1591. Till, J. E. & McCulloch, E. A. (1961) Radiat. Res. 14,213-222. Gordon, J. A., Blumberg, S., Lis, H. & Sharon, N. (1972) FEBS Lett. 24, 193-196. Lotan, R., Skutelsky, E., Danon, D. & Sharon, N. (1975) J. Biol. Chem. 250,8518-8523. Reisner, Y., Gachelin, G., Dubois, P., Nicholas, J. F., Sharon, N. & Jacob, F. (1977) Develop. Biol. 61,20-27. van Bekkum, D. W. (1974) Semin. Hematol. 11, 325-340. Simonsen, M. (1962) Prog. Allergy 6,349-467. Meshorer, A. & Webb, P. (1976) Exp. Pathol. 12,259-268.
Hemopoietic stem cell transplantation using mouse bone marrow and spleen cells fractionated by lectins (peanut agglutinin/soybean agglutinin/graft-versus-host reaction/spleen colony-forming units)
YAIR REISNER, LEA ITZICOVITCH, ASHER MESHORER, AND NATHAN SHARON Department of Biophysics and the Experimental Animal Center, Weizmann Institute of Science, Rehovoth, Israel
Communicated by Paul A. Marks, March 27,1978
ABSTRACT Mouse bone marrow and spleen cells were fractionated with the aid of soybean agglutinin and peanut agglutinin. A test for spleen colony-forming units in the isolated fractions showed that the hemopoietic stem cells are agglutinated by both of these lectins. The capacity of the agglutinated fractions to reconstitute lethally irradiat alogeneic mice was investigated. A sequential fractionation of'splenocy'tes from SWR donors by soybean agglutinin and peanut agglutinin, or a single fractionation by soybean agglutinin of splenocytes from BALB/c donors, afforded a cell fraction that successfully reconstituted lethally irradiated (BALB/c X C57BL/6)F1 mice, without complications due to graft-versus-host reaction.
Grafting allogeneic hemopoietic cell transplants to irradiated animals, or severely immune-deficient patients, has failed in most cases due to the presence in the transplant of cells that cause a graft-versus-host (GVH) reaction. In this communication we report on the isolation of a cell fraction from mouse bone marrow and spleen that is enriched with hemopoietic stem cells and depleted of GVH activity. We used this fraction to reconstitute lethally irradiated allogeneic mice. During the past few years numerous techniques for cell separation, based mainly on cell size, density, and charge, have been devised (1-3). Recently we have demonstrated that selective agglutination by lectins can be used for effective separation of mouse lymphocytes into biologically distinct subpopulations (4, 5). This method is simple, rapid, and reproducible and is based on differences in cell surface receptors rather than in size or density of cells. With this technique, immature and mature thymocytes were separated by the aid of peanut agglutinin (PNA, a lectin specific for D-galactosylI3(1- 3)-N-acetyl-D-galactosamnine) (4), and mouse splenocytes were fractionated into B and T cells by using soybean agglutinin (SBA, a lectin specific for terminal N-acetyl-D-galactosamine and D-galactose) (5); in both cases, the agglutinated cells were devoid of GVH activity. Studies of the binding of the lectins to those subpopulations, both before and after neuraminidase treatment, have led us to conclude that the receptors for both PNA and SBA are present on the surface of immature thymocytes; that in mature thymocytes only the SBA receptor is present (the PNA receptor being masked by sialic acid); and that in peripheral blood lymphocytes, as well as in most spleen T cells, both the PNA and the SBA receptors are masked by sialic acid. On the basis of the above findings, we have postulated that the hemopoietic stem cells may also possess PNA and SBA receptors on their surfaces (4). To test this assumption, we have now isolated a cell fraction from mouse bone marrow and spleen by agglutination with both SBA and PNA. We demonstrate by spleen colony assay and by reconstitution of irradiated The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
allogeneic animals that this fraction is depleted of GVH activity and enriched with hemopoietic stem cells. MATERIALS AND METHODS Animals. Female (BALB/c X C57BL/6)F1 hybrids and female SWR mice were used as recipients. Donor animals were BALB/c and SWR female mice. The recipients were 8-12 wk old and the donors' ages were 6-8 wk. All mice were raised at the Weizmann Institute Animal Center. Irradiation. Mice were exposed to a single dose of 900 R (1 R = 2.6 x 10-4 C/kg) from a y-beam 150A, 60Co source, produced by Atomic Energy of Canada, with focus-to-skin distance of 75 cm and 65 R/min dose rate. Cell Suspensions. Bone marrow was obtained from the femurs and suspended in phosphate-buffered saline (Pi/NaCI). Spleen suspension was prepared by straining the tissue in Pi/ NaCl solution through nylon gauze. The cells were then washed twice in Pi/NaCl and resuspended in the same buffer at the required final concentration. Nucleated cells were counted in Turk's solution. Spleen Colony-Forming Units (CFU-S) Assay. Details of the procedure have been previously described (6). Bone marrow and spleen suspensions were suspended in Pi/NaCl at various concentrations (see Results) and injected into the caudal vein of irradiated recipients. Each experimental group consisted of eight SWR mice' whose spleens were examined 8 days after injection. Radioprotection. Various fractions of splenocytes (see Results) suspended in Pi/NaCl (5 X 107 cells per ml) were injected intravenously into irradiated recipients (0.2 ml per animal) within 24 hr after the irradiation. Lectins. PNA and SBA were purified by affinity chromatography on a column of Sepharose-N-(e-aminocaproyl)-jlD-galactopyranosylamine (7, 8). Solutions. Lysing buffer: 155 mM ammonium chloride/10 mM potassium bicarbonate/0.01 mM EDTA in water; Pi/NaCl: 50 mM sodium phosphate buffer, pH 7.4, in saline. RESULTS AND DISCUSSION Separation of CFU-S-enriched fractions by agglutination with SBA and PNA Fractionation of splenocytes by SBA was carried out according to the method of Reisner et al. (5) with minor modifications. The splenocyte suspension (2 X 108 cells in 0.5 ml of Pi/NaCI) was incubated in polystyrene tubes (17 X 100 mm) with SBA (0.5 ml, 2 mg/ml of Pi/NaCI) for 5 min at room temperature. The cells were then gently layered with a pasteur pipette on top of a solution of bovine serum albumin (5% wt/vol in Pi/NaCl,
Abbreviations: CFU-S, spleen colony-forming units; GVH, graft-versus-host; Pi/NaCI, phosphate-buffered saline; PNA, peanut agglutinin; SBA, soybean agglutinin. 2933
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Medical Sciences: Reisner et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
Table 1. Average number of CFU-S in fractionated mouse splenocytes
Cell fraction
1 X 107
Average number of CFU-S* Number of cells injected 4 X 105 2 X 106
8 X 104
Relative enrichmentt
yieldt
0 0
2.8 1 0.5
50 + 10 50 10
>40 >40
Single fractionation 9.1 + 1.3 >40 3.2 d 0.8 19.8 A 2.1 16.8 i 2.1 >40 5.2 + 0.2 NCO
2.6 ± 0.25 0
>40
Sequential fractionation 17.5 1 1.2 >40
3.6 + 0.8
Agglutinated by SBA Unagglutinated by SBA
>40 >40
Agglutinated by PNA Unagglutinated by PNA
Agglutinated by SBA and PNA Agglutinated by SBA and unagglutinated by PNA
>40
7.3
>40
:
1.1
%
15 ± 5 85 :- 5 2.4 + 0.1
20 1 5
80 + 5
0
* Each experimental group consisted of eight mice and the average CFU-S value + SEM was calculated. The CFU-S could not be counted accurately when the value was above 40. t The relative enrichment value is the ratio between CFU-S in the agglutinated fraction and the unagglutinated fraction. Both fractions were subjected to identical treatment throughout the fractionation (see text) and injection procedure. The maximal differences in the values obtained at various cell concentrations were within the indicated limits. Percentage of each fraction out of the total number of cells recovered in both the agglutinated and unagglutinated fractions. § In two animals the CFU-S value was above 40 and the average could not be calculated.
50-ml conical glass tube. After 15 min at room of the agglutinated cells sedimented, whereas the unagglutinated cells remained on the surface of the bovine serum albumin solution. The bottom and top fractions were removed separately by pasteur pipettes and transferred to 15-ml conical plastic tubes. The cells were then suspended in D-galactose (10 ml, 0.2 M) in Pi/NaCl. After 10 min at room temperature, the cells were collected by centrifugation (200 X g, 5 min) and washed twice with the D-galactose solution. Finally the cells were washed twice with Pi/NaCl. About 6 X 107 cells were obtained from both the agglutinated and the unagglutinated fractions. It has been shown previously that the agglutinated fraction is highly enriched in B cells and depleted of T cells, whereas the unagglutinated fraction is comprised mainly of T cells and is depleted of B cells (5). We have now tested these fractions for CFU-S activity (6). As shown in Table 1, the agglutinated fraction is enriched 2.8-fold in hemopoietic activity relative to the unagglutinated fraction. A similar fractionation of bone marrow cells resulted in total agglutination (more than 90% of the cells were recovered in the bottom fraction), thus strengthening our assumption that the SBA receptor is present on hemopoietic cells as well as on B cells (data not shown). Staining of the bone marrow and spleen cells with PNA conjugated to fluorescein isothiocyanate revealed receptors for the lectin on only a small proportion of the cells (10-20%). Incubation of the cells (108/0.2 ml of Pi/NaCl) with PNA (0.25 ml, I mg/ml of Pi/NaCl) for 30 min at room temperature [conditions under which PNA-positive thymocytes were readily separated from the PNA-negative cells (4)], led to the formation of only a few small aggregates, which could not be separated efficiently from the single, unagglutinated cells.
40 ml) in
a
temperature, most
Cell fraction Agglutinated by PNA Unagglutinated by PNA
Fractionation of bone marrow or spleen cells by PNA could be achieved, however, when rabbit erythrocytes, which carry the PNA receptors (9), were added to the incubation mixture to form mixed aggregates of nucleated mouse spleen or bone marrow cells with rabbit erythrocytes. These aggregates were large enough to be separated from the unagglutinated fraction.
The spleen or bone marrow cells in PBS (3 X 108 cells/0.5 ml) incubated in polystyrene tubes (17 X 100 mm) with PNA (0.3 ml, 1 mg/ml) for 5 min at room temperature, and then rabbit erythrocytes (1.8 X 109 cells in 0.3 ml of Pi/NaCl) were added. After an additional 2 min, large aggregates comprised mainly of rabbit erythrocytes were formed and separated from the unagglutinated tells as described above for the SBA fractionation. The aggregates were dissociated with D-galactose and washed as above, and the two fractions were treated with ammonium chloride (10 ml) to lyse the rabbit erythrocytes. After four additional washings with 2 ml of PBS, 3.6-X 107 cells and 4.8 X 107 cells were recovered in the agglutinated fraction of spleen and bone marrow, respectively, whereas 2.3 X 108 cells and 2.1 X 108 cells were recovered from the corresponding unagglutinated fraction. Viability, as judged by the trypan blue exclusion test, was above 85% in all fractions. Analysis of the agglutinated cells showed that they were significantly enriched in CFU-S activity as compared to the unagglutinated ones (Tables 1 and 2), indicating that the PNA receptor is also exposed on the hemopoietic stem cells. In order to further test our assumption that both SBA and PNA receptors are present on hemopoietic stem cells, we attempted a sequential fractionation of splenocytes by both lec-
were
tins, starting with SBA. This fractionation was carried out under the same conditions as described for each fractionation alone, except that fractionation by PNA was performed on the sple-
Table 2. Average number of CFU-S in fractionated bone marrow cells* Average number of CFU-S Number of cells injected 1.6 X 104 8 X 104 4 X 105 2 X 106 >40 >40
>40 21 + 2.5
* More than 90% of the bone marrow cells were agglutinated by SBA.
18.4 i 0.6 3 4 0.5
2.0 + 0.3 0
Relative enrichment
% yield
5.0 + 1.1
20 + 5 80 ± 5
Medical Sciences: Reisner et al.
Proc. Natl. Acad. Scd. USA 75 (1978)
nocyte fraction agglutinated by SBA after dissociation of the aggregates to a single cell suspension by D-galactose. The total
yield of the twice-fractionated subpopulation was about 5%; this fraction is enriched in CFU-S relative to the SBA-agglutinated fraction (Table 1). Thus, additional support is provided for our suggestion that both SBA and PNA receptors are present on hemopoietic stem cells. Because the stem cells constitute only 0.1% of the nucleated cells in bone marrow, and 0.01% of nucleated cells in spleen (10), it is evident that most of the cells agglutinated by SBA and PNA do not possess the capacity to form spleen colonies. The agglutinated cells may represent various stages of differentiation of the lymphoid and myeloid lines, e.g., prethymocytes or pre-B cells, as indicated by the high incidence of PNA-positive cells in mouse embryonic liver (H. Rabinowich, T. Umiel, A. Globerson, Y. Reisner, and N. Sharon, unpublished data) and in various types of acute myeloid and lymphocytic leukemias in humans (Y. Reisner, M. Binyaminov, N. Sharon, and B. Ramot, unpublished data) and mice (A. Harris and Y. Reisner, unpublished data). Reconstitution of lethally irradiated mice by fractionated splenocytes from allogeneic mice The reconstitution of lethally irradiated mice was tested in a parent-to-F1 system [BALB/c (BALB/c X C57BL/6)F1] and in a complete allogeneic system [SWR (BALB/c X C57BL/6)F1]. In the former combination (a typical experiment is shown in Fig. 1), transplantation of the splenocyte fraction that is agglutinated by SBA resulted in complete survival (100%) of the irradiated recipients, whereas transplantation of the unagglutinated fraction or the unseparated splenocytes resulted in a high incidence of mortality. However, in the second system (in which there is no common parent of donor and recipient), we found that the fractionation by SBA was not sufficient to avoid a mortality due to the GVH reaction (mortality incidence in four experiments ranged between 50% and 70%). This GVH reaction may be caused by a small population of T cells that is still present in the SBAagglutinated fraction (5) [although the B cells that comprise the major subpopulation in this fraction (5) may possibly contribute to the overall mortality in this system]. Because PNA does not --
--
2935
bind to mature T or B cells (4, 5), we tried to reconstitute these mice with the twice-agglutinated fraction, namely the SBAagglutinated cells that were further fractionated by agglutination with PNA. The results of a typical experiment are shown in Fig. 2. Grafting lethally irradiated allogeneic mice with unfractionated splenocytes or with cells unagglutinated by SBA resulted in high mortality (13/15 and 15/15 respectively) within the first 30 days after irradiation. The two mice surviving the first 4 wk were suffering from wasting disease (delayed GVH reaction) and died within the second month after transplantation. In a parallel experiment, the few surviving mice were sacrificed 4 and 5 wk after transplantation. Spleen histology (Fig. 3) and bone marrow differential count revealed typical GVH symptoms (11, 12): damage to the white pulp of the spleen, a very high percentage of granulocytes (55-81%), and a low lymphocytic count (1-4%) in the bone marrow. Interestingly, the red pulp (myelo-, erythro-, and thrombopoiesis) in the spleen of these mice was normal or slightly hyperplastic (Fig. 3), indicating that complete hemopoiesis had occurred and that the high mortality rate among these mice was due to acute GVH reaction and not to deficiency of hemopoietic stem cells. Among the mice grafted with the twice-agglutinated fraction, only 1 out of 15 died (Fig. 2). The remaining mice have survived more than 6 mo. Spleen histology (Fig. 3) and bone marrow differential count (granulocytes, 19-35%; lymphocytes, 34-47%), when taken 4 and 5 wk after transplantation in a parallel experiment, revealed that in reconstituted mice both spleen and bone marrow were completely restored. These results are in line with our previous reports (4, 5), in which we tested the GVH activity of fractionated splenocytes and thermocytes in newborn mice. Mouse spleen cells can serve as a good model for allogeneic bone marrow transplantations in humans, because both mouse splenocytes and human bone marrow aspirates cause an early type of GVH reaction that is fatal in most cases, whereas mouse bone marrow cells may cause only a mild type of delayed GVH reaction (10).
_
I
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{
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15
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>101 0
E
0 ~~
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~
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:
E
5
10
15
20
25
30
90
Time, days FIG. 1. Cumulative mortality of irradiated (BALB/c X C57BL/6)F1 mice after transplantation with splenocytes (107 cells per animal) from BALB/c mice, starting with 15 mice in each group. Grafts: A-A, unfractionated splenocytes; 0-0, splenocytes agglutinated by SBA; 0-0, splenocytes unagglutinated by SBA; *- -, control without graft. -
0
5
10
15 20 25 30 90 Time, days FIG. 2. Cumulative mortality of irradiated (BALB/c X C57BL/6)F1 mice after transplantation with splenocytes (107 cells per animal) from SWR mice, starting with 15 mice in each group. Grafts: A-a, unfractionated splenocytes; -0O, splenocytes sequentially agglutinated by SBA and PNA; 0-0, splenocytes unagglutinated by SBA; 0 ----@, control without graft.
2936
Medical Sciences: Reisner et al.
Proc. Natl. Acad. Sci. USA 75(1978)
Aft ,firr*1is5Xf W S~ ~ ~ ~ ~ ~ *
*
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FIG. 3. Typical spleen histology of (BALB/c X C57BL/6)Fl mice irradiated by 900 R and grafted with splenocytes (107 cells per animal) of SWR donors. The specimen was taken from mice sacrificed 4 wk after transplantation. Grafts. (A) unfractionated splenocytes; (B) splenocytes sequentially agglutinated by SBA and PNA. Scale bar, 0.1 mm. 2. Sabalovic, D. & Serrou, B., eds. (1976) Techniques of Separation Preliminary binding studies on the interaction of fluorescein and Characterization of Human Lymphocytes (Editions isothiocyanate-conjugated PNA with human lymphocytes reINSERM, Paris, France). vealed that, as in its interaction with mouse lymphocytes, PNA 3. Dicke, K. A. & van Bekkum, D. W. (1971) Transplant. Proc. 3, binds to a small subpopulation in the bone marrow (5-15%) and 666-668. does not bind to normal human peripheral blood lympho4. Reisner, Y., Linker-Israeli, M. & Sharon, N. (1976) Cell. Immucytes. nol. 25, 129-134. The approach described in this communication for the iso5. Reisner, Y., Ravid, A. & Sharon, N. (1976) Biochem. Biophys.
lation of cells capable of reconstituting lethally irradiated allogeneic mice may prove useful for bone marrow transplantations in humans. This study was supported in part by National Institutes of Health Contract NCI-CB-74163 and by a grant from the Leukemia Research Foundation. N.S. is an Established Investigator of the Chief Scientist's
6. 7.
8. 9.
Bureau, Israel Ministry of Health. 1. Shortman, K., van Boehmer, H., Lipp, J. & Hopper, K. (1975) Transplant. Rev. 25,163-210.
10. 11. 12.
Res. Commun. 72,1585-1591. Till, J. E. & McCulloch, E. A. (1961) Radiat. Res. 14,213-222. Gordon, J. A., Blumberg, S., Lis, H. & Sharon, N. (1972) FEBS Lett. 24, 193-196. Lotan, R., Skutelsky, E., Danon, D. & Sharon, N. (1975) J. Biol. Chem. 250,8518-8523. Reisner, Y., Gachelin, G., Dubois, P., Nicholas, J. F., Sharon, N. & Jacob, F. (1977) Develop. Biol. 61,20-27. van Bekkum, D. W. (1974) Semin. Hematol. 11, 325-340. Simonsen, M. (1962) Prog. Allergy 6,349-467. Meshorer, A. & Webb, P. (1976) Exp. Pathol. 12,259-268.
