PROCESS FOR THE FRACTIONATION OF CELLS

Abstract: A method of cell separation by agglutination using lectins or agglutinins which selectively bind to cells devoid of graft-versus-host-activity and suitable for transplantation without undesirable side effects. The method comprises preparing a mixed cell suspension in a suitable medium, contacting this mixed cell suspension with a lectin, which may be peanut agglutinin or soybean agglutinin, or agglutinin that selectively binds to terminal galactose residues on the cell surface and forms aggregates of cells, separating the cells from the unaggregated cells, and contacting the separated cell aggregates with D-galactose solution in order to obtain single cells suitable for injection.

Description: FIELD OF THE INVENTION A process for fractionating cells so as to obtain a cell type devoid of graft versus host activity, which can be used for cell transplants. STATE OF THE PRIOR ART Grafting allogenic hemopoietic cell transplants to irradiated animals or severly immune deficient patients, has failed in most cases due to the presence in the _ transplant of cells which cause a graft versus host reaction. During the last few years a number of techniques for cell separation, based mainly on cell size, density and charge, have been devised but none could be applied to the removal of GVH activity from human bone-marrow. SUMMARY OF THE INVENTION A method is provided for the fractionation of cells so as to obtain a fraction devoid of graft versus host activity, which can be used for transplantation without undesired side effects. The fractionation is effected by means of certain agglutinins adapted to bind selectively to terminal galactose residues on cell surfaces. The substances used are of the type of galactose and fucose binding lectins, and among preferred leetins there may be mentioned soybean agglutinin, peanut agglutinin. It has been found that when different cell types are present, only stem cells interact with lectins of this type, while no interaction takes place with βature T-cells. This brings about an aggregation of the stem cells, the aggregates are easily separated from the other cells and the aggregates can be subsequently broke up to yield cells which can be injected. Mouse bone marrow and spleen cells were fractionate by means of agglutinins of the type defined above. A test forspleen colony forming .units in the isolated fractions showed that the hemopoietic stem cells are agglutinated by such lectins. Specific substances used were soybean and peanut agglutinin. The capacity of the agglutininated fractions to reconstitute lethally irradiated allogeneic mice was investigated. It was found that a sequential fractionation of splenocytes from inbred strains of mice designated SWR donors by soybean agglutinin and by means of peanut agglutinin or a single fractionation by soybean agglutinin of splenocytes from Balb/c donors, afforded a cell fraction substantially devoid of graft-versus-host activity, which successfully reconstituted lethally irradiated (Balb/c x C5 7BL)F. mice. The cell fractions thus obtained are substantially depleted of graft-versus-host (GVH) reaction, and thus it is possible to overcome difficulties encountered hitherto when it was tried to graft allogeneic hemopoiet cell transplants to irradiated mammals or to severely immune deficient patients. The difficulties are due to the presence of cells which cause a GVH reaction, and these are substantially removed from the cell fractions obtained according to the process of the present invention. Various lectins can be used, and it seems that the criteria for the choice of these are their capability to bind specifically to galactose and fucose moieties. Peanut agglutinin (PNA) is a lectin specific for D- galacto__yl-(l-3)-N-acetyl-D-galactosamine while soybean agglutinin (SBA) is a lectin specific for terminal N-acetyl-D-galactosamine and D-galactose. It seems that receptors for such lectins, and specifically from PNA or SBA are present on the surface of henopoietic stem cells, whereas in aost cells of the T-type the PNA and SBA receptors are aasked. When a aiixed population of cells is subjected to lectin treatment, the agglutinated cells are the ones which can be used subsequently for the treatment, the resulting fraction being substantially devoid of GVH reaction. The following Example is intended to illustrate the invention and is not to be construed in a limitative manner. EXAMPLE Animals. Female (Balb/c x C57BL/6)F. hybrids and female S R mice were used as recipients. Donor animals were Balb/c and SWR female mice. The recipients were 8-12 weeks old and the donors* age was 6-8 weeks. All mice were raised at the Weizmann Institute Animal Center. Irradiation. Mice were exposed to a single dose of 900 R from a Gamma beam 150A, Co source, produced by Atomic Energy of Canada, with F.S.D. of 75 cm and 65 R min dose rate. Cell suspensions. Bone marrow was obtained from the femurs and suspended in phosphate buffered saline (PBS). Spleen suspension was prepared by straining the tissue in PBS solution through nylon gauze. The cells were then washed twice in PBS 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. Bone marrow and spleen suspensions were suspended in PBS at various concentrations (see Results) and injected into the caudal vein of irradiated recipients. Each experimental group consisted of 8 SWR mice. Radioprotection. Various fractions of splenocytes 7 (see Results) suspended m PBS (5 x 10 cells/ml) were injected intravenously into irradiated recipients (0.2 ml/animal) within 24 hours after the irradiation. Lectins. .PNA and SBA were purified by affinity chromatography on a column of Sepharose-N- ( f -amino- caproyl)-_^-D-galactopyranosylamine. Separation of CFU-S enriched fractions by agglutination with SBA and PNA: Fractionation of splenocytes by SBA was carried out as follows: 8 The splenocyte suspension (2 x 10 cells in 0.5 ml PBS) was incubate _d in polystyrene tubes (17 x 100 mm) with SBA (0.5 ml, 2 mg/ml) for 5 minutes at room temperature. The cells were then gently layered with Pasteur pipette on top of a solution of bovine serum albumin (5% w/v in PBS, 40 ml) in a 50 ml conical glass tube. After 15 minutes at room temperature, most of th agglutinated cells sedimented, whereas 1_he unagglutinat cells remained on the surface of the bovine serum album 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 (0.2 M) in PBS. After 10 minutes at roo 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 PBS. 7 About 6 x 10 cells were obtained from both agglutinate and unagglutinated fractions. The agglutinated fraction is enriched 2.8 fold in hemopoietic activity relative to the unagglutinated fraction. A similar fractionation of bone marrow cells resul 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 hemo poietic cells as well as on B cells. Staining of bone marrow and spleen cells with PNA conjugated to fluorescein isothiocyanate revealed receptors for the lectin on only a small portion of the cells (10-20%). Incubation of the cells (10 8 /0.2 ml PBS) with PNA (250 r g in 0.25 ml PBS) for 30 min at room temper¬ ature, led to the formation of only a few small aggregates, which could not be separated efficiently from the single, unagglutinated cells. Fractionation of bone marrow or spleen cells by PNA could be achieved, however, when rabbit erythrόcytes, which carry the PNA receptors 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) were 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 10 9 cells in 0.3 ml PBS) were added.. After an additional 2 min, large aggre¬ gates comprised mainly of rabbit erythrocytes were formed and separated (as described above for the SBA fractiona- tion) from the unagglutinated cells. The aggregates were dissociated with D-galactose, washed as above, and the two fractions were treated with ammonium chloride to lyse the rabbit erythrocytes. After four additional washings by PBS, 3.6 x 10 7' cells and 4.8 x 107 cells were recovered in the agglutinated fraction of spleen and bone marrow, respectively, whereas 2.3 x 10 8 cells and 2.1 x 10°f. 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 indicating that the PNA receptor is also exposed on the hemopoietic stem cell. In order to further test our assumption that both SBA and PNA receptors are present on the hemopoietic stem cell, we attempted a sequential fractionation of splenocytes - - by both lectins. This fractionation was carried out under the same conditions as described for each fractionation alone, except that the second fractionation by PNA was performed on the splenocyte fraction already agglutinated by SBA (after dissociation of the aggregates to a single cell suspension by the sugar inhibitor of the lectin) . The total yield of the twice fractionated subpopulation was about 5% and, as can be seen in Table 1, this fraction is enriched in CFU-S relative to the SBA-agglutinated fraction. Thus, additional support is provided for our suggestion that both SBA and PNA receptors are present on the hemopoietic stem cell. Reconstitution of allogeneic lethally irradiated mice by fractionated splenocytes The reconstitution of lethally irradiated mice was tested in a "parent to F," system [Balb/c ^-(Balb/c x C57BL/6) ι] and in a complete allogeneic system [SWR- ^ (Balb/c x C57BL/6)Fι_. In the former combination (a typical experiment is shown in Fig. 1), transplantation of the splenocyte fraction which 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. Fig. 1 shows the cumulative mortality of irradiated (BALB/c x C57BL/6)Fι mice after transplantation with splenocytes (10^ cells per animal) from BALB/c mice starting with 15 mice in each group. Grafts:Δ ___, unfractionated splenocytes;Q ■ Q , splenocytes agglutinated by SBA; o " o, splenocytes unagglutinated by SBA; • •, control without graft. 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 morta- lity d e to GVH reaction (mortality incidence in four experiments ranged between 50-70%) . This GVH reaction may TABLE 1 Average number of CFU-S in fractionated mouse splenocytes Average number of CFϋ-S* Cell fraction Number of cells injected Relative Percent 1x10 2x10' 4x10" 8xl0 4 enrichment b vield c Single fractionation Agglutinated by SBA >40 >40 9.1+1.3 50+10 2.8+0.5 Unagglutinated by SBA >40 19.8+2.1 3.2+0.8 50+10 Agglutinated by PNA >40 >40 16.8+2.1 2.6+0.25 3.2+0.7 15+5 Unagglutinated by PNA ?40 N.C.d 5.2+0.2 0 85+5 Sequential fractionation Agglutinated by SBA and PNA >40 >40 17.5+1.2 3.6+0.8 2.4+0.1 20+5 Agglutinated by SBA and unagglutinated by PNA >40 >40 7.3+1.1 «. 80+5 l Each experimental group consisted of 8 mice and the average CFU-S value + standard Table 1, continued 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. TABLE 2 Average number of CFU-S in fractionated bone marrow cells Average number of CFU-S Cell fraction Number of cells injected Relative Percent 2x10 4xl0 5 8xl0 4 l * 6xl0 4 enrichment yield Agglutinated by PNA >40 >40 18.4+0.6 2.0+0.3 Unagglutinated by PNA >40 21+2.5 3+0.5 0 be caused by a small population of T cells which is still present in the SBA agglutinated fraction (although the B cells which comprise the major subpopulation in this fraction may possibly contribute to the overall mortality in this system) . Since PNA does not bind to mature T or B cells, we tried to reconstitute these mice with the twice agglutinated fraction, namely the SBA agglutinated cells which are further purified by agglutination with PNA. The results of a typical experiment are illustrated in Fig. 2 which shows the cumulative mortality of irradiated (BALB/c x C57BL/6)F 1 mice after transplantation with splenocytes (10' cells per animal) from SWR mice, starting with 15 mice in each group. Grafts:^ & , unfractionated splenocytes;Q——Q, splenocytes sequentially agglutinated by SBA and PNA; o— o, splenocytes unagglutinated by SBA; • •, control without graft. 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. The two mice surviving the first four weeks were suffering from wasting disease (delayed GVH reaction) and died within the second month after transplantation. Spleen histology and bone marrow differential count revealed typical GVH symptoms damage to the white pulp of the (myelo-, erthro- and thro bopoiesis) in the spleen of these mice was normal or slightly hyperplastic, 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 one out of fifteen died (Fig. 2) . The remaining mice have already survived more than six months. Spleen histology (Figs. 3 and 4) and bone marrow differential count, when taken four and five weeks after transplantation in a parallel experiment, revealed that in reconstituted mice compared with intact animals, both spleen and bone marrow were completely restored. Typical spleen histology of {BALB/c x C57BL/6)Fι mice irradiated by 900 R and grafted with splenocytes (10 7 cells per animal) of SWR donors is shown in Figs. 3 and 4. The specimen was taken from mice sacrificed 4 weeks after transplantation. Grafts: (Fig. 3), unfractionated splenocytes; (Fig. 4), splenocytes sequentially agglutinated by SBA and PNA. Scale bar, 0.1 mm. Mouse spleen cells can serve as a good model for allogeneic bone marrow transplantations in humans, since both mouse splenocytes and human bone marrow aspirates cause an early type of GVH reaction which is fatal in most cases, whereas mouse bone marrow cells may cause only a mild type of delayed GVH reaction. Preliminary binding studies on the interaction of fluorescein isothiocyanate conjugated * PNA with human lymphocytes revealed that, similarly to its interaction with mouse lymphocytes, PNA binds to a small subpopulation in the bone marrow (5-15%) and does not bind to peripheral blood lymphocytes. The new process for the isolation of cells capable of the reconstitution of lethally irradiated allogeneic mice is indicative of the potential of this process for bone marrow transplantations in humans. OMPI Λ, W1F0 ,-^-V

Claims: - - The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 1. A process for fractionating cells in order to obtain a cell type devoid of graft versus host activity (GVH activity) which comprises preparing a cell suspension in a suitable medium, contacting this with a solution of a suitable lectin so as to form cell aggregates, separa¬ ting the aggregates from non-aggregated cells, suspending the aggregates in a suitable medium so as to dissolve the aggregates and to obtain a suspension of single cells which can be used for injection. 2. A process according to claim 1 wherein the cells used are bone marrow or spleen cells. 3. A process according to claim 1 or 2, wherein the lectin is soybean agglutinin or peanut agglutinin. 4. A process according to claim 1, wherein the cells are suspended in phosphate buffered saline (PBS) . 5. A process according to claim 1, wherein the aggregates are contacted with a D-galactose solution in order to obtain single cells. 6. A process according to claim 1, wherein the single cells are washed with PBS. 7. A process according to claim 1, wherein the cells separated by aggregation are stem cells. 8. Fractionated cells devoid of GVH activity, produced by the process of claim 1. s -

Inventors: REISNER Y, SHARON N
Applicants: US GOVERNMENT
Application year: 1979
Main classification: A61K35
IPC codes: A61K35, C12N5