Aplastic Anemia: Presence in Human Bone Marrow of Cells That Suppress Myelopoiesis* (Thymus-Derived Lymphocytes/Suppressor Cells/Differentiation) WALT A
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Proc. Natl. Acad. Sci. USA Vol. 73, No. 8, pp.2890-2894*, August 1976 Medical Sciences Aplastic anemia: Presence in human bone marrow of cells that suppress myelopoiesis* (thymus-derived lymphocytes/suppressor cells/differentiation) WALT A. KAGAN, JoAo A. ASCENSAO, RAJENDRA N. PAHWA, JOHN A. HANSEN, GIDEON GOLDSTEIN, ELISA B. VALERA, GENEVIEVE S. INCEFY, MALCOLM A. S. MOORE, AND ROBERT A. GOOD Memorial Sloan-Kettering Cancer Center, New York, N.Y. 10021 Contributed by Robert A. Good, May 24, 1976 ABSTRACT Bone marrow from a patient with aplastic all negative. There was no history of exposure to drugs or anemia was shown by multiple criteria to have a block in early chemical agents known to be capable of producing aplastic myeloid differentiation. This block was overcome in vitro by anemia. elimination of marrow lymphocytes. Furthermore, this differ- entiation block was transferred in vitro to normal marrow by coculturing with the patient's marrow. We suggest that some METHODS cases of aplastic anemia may be due to an immunologically Cell Separation. Heparinized bone marrow was obtained based suppression of marrow cell differentiation rather than from the posterior iliac crest of the patient and normal adult to a defect in stem cells or their necessary inductive environ- donors in 10 to 20 separate 0.5-ml aspirations. The cells were ment. separated according to density differences by centrifugation The hematopoietic system in man is thought to develop from on a Ficoll-hypaque gradient by the method of Boyum (5). Cells a common stem cell analogous to the spleen colony forming unit present at the plasma/Ficoll-hypaque interface were collected, in mice (CFU-s) (1), which then differentiates into committed and this heterogeneous mixture was then separated according progenitor cells of the granulocytic and monocytic series to size by velocity sedimentation at unit gravity by the method (CFU-c) (2), megakaryocytic, lymphoid, and erythroid lines, of Miller and Phillips (6). and then passes through several more differentiation steps into Surface Markers. Complement receptors on cells were as- mature effector cells. Aplastic anemia is a disease characterized sayed by rosette formation with sheep erythrocytes (SRBC) by a marked decrease in the production of erythrocytes, leu- coated with antibody and complement (7). SRBC (Flow Labs) kocytes, and platelets. The etiology of this kind of bone marrow were incubated for 30 min at 370 with an equal volume of failure is complex, and about 50% of all cases are idiopathic. rabbit IgM antibody against SRBC (Cordis) diluted by 1So. There has been considerable speculation about the possible role These cells (EA) were then incubated 30 min at 370 with 1A of an immunologic mechanism in the pathogenesis of some volume of Swiss mouse serum as a source of nonlytic comple- cases (3, 4), but this mechanism has not yet been fully docu- ment. These cells (EAC) were washed twice and resuspended mented. We present here evidence for such a mechanism. to 2 X 108 cells per ml, and 50 Ml of EAC were incubated with 50 Ml of bone marrow cells at 3 X 106 cells per ml for 30 min at CASE REPORT 370. Three fields of 100 cells were counted in a hemocytometer, and cells were scored as positive if they bound four or more The patient was a 26-year-old white woman diagnosed as EAC. having aplastic anemia by bone marrow biopsy in July 1973 The number of thymus-derived (T) lymphocytes was de- after recurrent episodes of bruising. Therapeutic trials with termined by spontaneous rosette formation with SRBC (8). Fifty prednisone, testosterone, oxymethalone, folic acid, and pyri- microliters of bone marrow cells at 3 X 106 cells per ml were doxine did not result in any significant improvement in her mixed with 50 Ml of washed SRBC at 2 X 108 cells per ml and condition. 25 ,l of heat-inactivated SRBC-absorbed fetal calf serum. This The patient was referred to the Memorial Sloan-Kettering mixture was incubated for 5 min at 37?, centrifuged at 200 X Cancer Center in April 1975 as a candidate for bone marrow g for 5 min at 24°, and then incubated for 3-6 hr at 4°. Four transplantation. At that time her white cell count was 1500/ fields of 100 cells were counted in a hemocytometer, and cells mm3, with 65% lymphocytes, 30% neutrophils, and 5% mono were scored as positive if they bound four or more SRBC. cytes, and her platelet count was 10,000/mm3. Repeated bone Surface Marker Induction. Separated bone marrow cell marrow aspirations and biopsies showed marked hypocellula- fractions were incubated at 1.5 X 106 cells per ml in RPMI-1640 rity with 80% lymphocytes, 6% neutrophils, 5% immature containing 5% fetal calf serum for 8 hr at 37° in humidified 5% myeloid cells, 3% erythroid precursors, 5% monocytes, and an CO2 and 95% air. These cultures contained either medium absence of megakaryocytes. All medications were discontinued, alone (control), or ubiquitin (0.5 ,g/ml). After incubation, the and the patient remained clinically stable supported by trans- cells were washed twice, resuspended in RPMI-1640 to 3 X 106 fusions of frozen red blood cells and HLA matched platelets cells per ml, and assayed for surface markers. Ubiquitin was every 10 days. Screening tests for possible causes of the aplastic prepared as described (9). anemia, including the Ham test, sugar-water test, antinuclear Spontaneous DNA Synthesis. In the course of studying the antibody test, Coomb's test, and chest x-rays for thymoma, were response of marrow cells in mixed leukocyte culture, the spontaneous DNA synthesis of cells was measured by substi- Abbreviations: CFU-s, colony forming unit (spleen); CFU-c, colony irradiated marrow cells for irradiated allo- forming unit (culture); SRBC, sheep erythrocytes; T cell, thymus- tuting autologous derived lymphocyte. genic peripheral blood lymphocytes. * Part of this paper was presented at the American Society for Clinical Cells were resuspended at 1 X 106 cells per ml in RPMI-1640 Investigation meetings on May 3, 1976. with penicillin (50 units/ml), streptomycin (50 Mug/ml) and 15% 2890 Downloaded by guest on September 28, 2021 Medical Sciences: Kagan et al. Proc. Nati. Acad. Sci. USA 73 (1976) 2891 Table 1. Cellular composition of aplastic bone marrow separated (a) by Ficoll-hypaque centrifugation alone or (b) by Ficoll-hypaque centrifugation followed by velocity sedimentation la (b) 0 S. Sedimentation velocity x .0 (mm/hr) So u 0 Cell type (a) 6.8-4.1 3.8-3.4 3.2-2.8 0'. Lymphocyte 72% 17% 90% 100% Mature granulocyte 10% 34% 0% 0% Metamyelocyte 12% 12% 0% 0% Myelocyte 3% 6% 0% 0% Promyelocyte 0% 1% 0% 0% Normoblast 1% 18% 4% 0% mm/hr Monocyte 1% 6% 6% 0% Plasma cell 1% 4% 0% 0% FIG. 1. Distribution of nucleated cells in human bone marrow after separation first by Ficoll-hypaque centrifugation and then by Results are expressed as percent of all cells present in that velocity sedimentation. fraction. heat-inactivated pooled human AB serum. An aliquot of these 6 to 9 mm/hr, mainly promyelocytes, myelocytes, and me- was irradiated in a Cesium Gammacel 40 at 125 rad/min (1.25 tamyelocytes with a few myeloblasts, and erythroblasts. J/kg min) for 10 min for use as stimulator cells. Fifty microliters Fig. 1 also shows the distribution of cells in the marrow of the of responder marrow cells, 50 .ul of stimulator cells, and 100 /A patient with aplastic anemia. As expected, there was a striking of medium were placed in triplicate in round-bottom micro- deficit of cells in the mature and immature granulocyte regions plate wells (Cooke no. 1-221-24-1) and incubated for 96 hr at of this distribution. However, this separation technique sig- 370 in 5% CO2, 95% humidified air. Cells were labeled with nificantly enriched for the few mature and immature granu- 0.03 juCi of [14C]thymidine (New England Nuclear) for 24 hr locytes present in this patient's marrow. Table 1 gives the cel- and harvested; radioactivity was measured in a scintillation lular composition of the different regions of this distribution counter (Packard). The mean cpm of three wells is reported. and demonstrates that while granulocytes comprised only 25% CFU-c Assay. The assay for granulocyte-macrophage pro- of the marrow cells separated on a Ficoll-hypaque gradient, genitor cells (CFU-c) was performed by the method of Pike and 55% of the cells sedimenting from 4.1 to 6.8 mm/hr were Robinson (10) using feeder layers of normal human leukocytes granulocytes. Furthermore, velocity sedimentation reduced as a source of colony stimulating factors. Marrow cultures were the proportion of lymphocytes from 72% in the original marrow established at concentrations of 2 X 105 cells per ml, incubated separated on a Ficoll-hypaque gradient to only 17% in the for 7 days at 37° in 10% CO2 humidified air, and scanned for fraction sedimenting from 4.1 to 6.8 mm/hr. Tetrachrome- the presence of colonies consisting of more than 40 cells and stained cytocentrifuge preparations of patient's cells in each clusters of 3-40 cells under a dissecting microscope. of these regions are shown in Fig. 2. Marrow Cocultures. Marrow cells from the patient and two normal donors, which had been separated on a Ficoll-hypaque Elevated number of T lymphocytes in aplastic marrow gradient, were suspended at 1 X 106 cells per ml and were The distribution of cells bearing certain surface markers was mixed in equal proportions. The mixtures and original sus- next investigated. In normal adults, 17% of the cells in marrow pensions were then assayed as described above in the CFU-c assay.