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 differentiation 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. q I~~~~~~~~~ RESULTS Abnormal cell distribution in aplastic marrow I w w I Bone marrow cells from a normal adult and a patient with aplastic anemia were separated first according to density by centrifugation on a Ficoll-hypaque gradient. This provides a suspension of cells with a density of less than 1.077 g/cm3, which includes all normal marrow cells except some mature *^s4 segmented granulocytes and erythrocytes. This mixture was * . then separated by velocity sedimentation, a method that sep- *0 i arates cells primarily according to size differences. We have a shown that this two-dimensional cell separation procedure separates normal bone marrow into several relatively homo- geneous cell fractions (11). Fig. 1 shows the distribution with a ___ _ respect to sedimentation velocity of cells present in normal bone FIG. 2. Bone marrow cells from a patient with aplastic anemia marrow. The region from 2 to 3 mm/hr contained only small after separation by Ficoll-hypaque centrifugation and velocity sedi- and medium lymphocytes, from 3 to 4 mm/hr, a mixture of mentation. Cytocentrifuge preparations were stained with tetra- monocytes, normoblasts, and large lymphocytes, from 5 to 6 chrome. (a) Cells sedimenting 2.8-3.2 mm/hr, (b) cells sedimenting mm/hr only band forms and segmented neutrophils, and from 3.4-3.8 mm/hr, andI'*(c) cells sedimenting 4.1-6.8 mm/hr. Downloaded by guest on September 28, 2021 2892 Medical Sciences: Kagan et al. Proc. Natl. Acad. Sci. USA 73 (1976) Table 2. Percentage of cells that form rosettes with untreated SRBC (E rosette) in human bone marrow 1.00 separated by only Ficoll-hypaque centrifugation or by Ficoll-hypaque centrifugation and velocity sedimentation

% E rosettes 0 .75 0 a 0 Marrow cells Normals Aplastic P < 0 CN I Ficoll-hypaque 17 ± 6 36 ± 2 0.020 0 2.5-3.5 mm/hr 21 ± 5 38 ± 1 0.025 x .501_ E For the patients' marrow, mean and standard error of triplicate 0 measurements of one sample is shown. For normal marrow separated by Ficoll-hypaque centrifugation, mean and standard error of mean of six marrows is shown. For normal marrow separated by Ficoll-hypaque centrifugation and velocity sedimentation, mean .25 and standard error of mean of three marrows is shown. Differences between normal and aplastic were analyzed by Student's t test.

separated by Ficoll-hypaque centrifugation and 21% in the 10-3.5 3.5-2.5 Ficoll- lymphocyte-enriched fraction (2.5-3.5 mm/hr of marrow mm/hr mm/hr hypaque separated by Ficoll-hypaque centrifugation and velocity sedimentation) have receptors for SRBC (E rosette). In this patient (Table 2), 36% of the cells in the marrow separated by Ficoll- hypaque centrifugation and 39% of the cells in the fraction sedimenting from 2.5 to 3.5 mm/hr had this T cell marker. Thus, the marrow of this patient had a much higher proportion of lymphocytes than normal, and a much higher proportion of b these lymphocytes bore the marker characteristic of mature T cells. 0 x Induction of complement receptors by ubiquitin E Q. We have previously shown (11) that 5-20% of cells in the fraction enriched for immature granulocytes that do not yet express the receptor for complement can be induced to express this marker after incubation for 8 hr with ubiquitin at 0.5 1 Ag/ml. As such, the induction of complement receptors can be used as an assay for the isolation and differentiative capacity of immature myeloid cells. Table 3 shows the percentage of cells induced to express this marker in the marrow of this patient 8 6 4 2 Ficoll- separated by Ficoll-hypaque centrifugation and that separated mm/hr hypaque by Ficoll-hypaque centrifugation and velocity sedimentation. FIG. 3. Spontaneous DNA synthesis measured by incorporation Only when slowly sedimenting small cells were removed from of [3H]thymidine by human bone marrow cells separated by only the patient's marrow could a small proportion of cells in the Ficoll-hypaque centrifugation or Ficoll-hypaque centrifugation and granulocyte-enriched fraction could be induced to express this velocity sedimentation. Cells were labeled for 24 hr after 4 days in marker. culture. (a) Marrow from a patient with aplastic anemia and (b) marrow from a normal donor. Mean and standard error of triplicate Spontaneous DNA synthesis measurements of one sample is reported. Aplastic velocity sedimentation fractions each contained 42% of total cells recovered. Normal The marrow separated by Ficoll-hypaque centrifugation and velocity sedimentation fractions each contained 17% of total cells that separated by Ficoll-hypaque centrifugation and velocity recovered. sedimentation were assayed for their spontaneous proliferation as measured by thymidine incorporation in autostimulated Table 3. Percentage of cells that express a complement mixed leukocyte culture. The results are shown in Fig. 3a. The receptor in the marrow of a patient with aplastic anemia spontaneous proliferation of cells in the faster sedimenting lymphocyte-depleted fraction was six times greater than in the % EAC rosettes marrow separated by Ficoll-hypaque centrifugation alone. Since the marrow was separated into two fractions, each con- Ubiquitin taining 42% of the total number of bone marrow cells, this Marrow cells Control (0.5 P < gg/ml) represents a marked enhancement of spontaneous cellular Ficoll-hypaque 13.1 ± 1.2 11.4 ± 1.1 0.40 proliferation rather than just selection of proliferating cells. For 2.5-3.5 mm/hr 9.4 ± 2.3 8.1 ± 2.3 0.80 comparison, the spontaneous proliferation of normal bone 3.5-10 mm/hr 7.4 ± 1.0 12.2 ± 1.1 0.02 marrow cells is shown in Fig. 3b. In this case, the proliferation of cells in the marrow separated by Ficoll-hypaque centrifu- Mean and standard error of triplicate measurements of one gation was equal to the sum of the proliferation in each velocity sample are reported. Differences between control and ubiquitin sedimentation fraction multiplied by the percentage of cells were analyzed by Student's t test. in that fraction. Downloaded by guest on September 28, 2021 Medical Sciences: Kagan et al. Proc. Natl. Acad. Sci. USA 73 (1976) 2893

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0 0 0 9 a 6.8-4.1 3.8-3.4 2.8-3.2 Ficoll- Apr. June Aug. Oct. Dec.. mm/hr mm/hr mm/hr hypaque FIG. 4. Sequential determinations of the number of colonies FIG. 5. Number of granulocyte-monocyte colonies (CFU-c) after marrow from a patient with aplastic anemia (CFU-c) formed by cells 7-day culture at 2 X 105 cells per ml in soft agar of marrow from a after 7-day culture at 2 X 105 cells per ml in soft agar. patient with aplastic anemia. Cells were separated by either Ficoll- hypaque centrifugation alone or by Ficoll-hypaque centrifugation and Isolation of CFU-c in aplastic marrow by velocity velocity sedimentation. sedimentation was then im- Fig. 4 demonstrates serial determinations of granulocyte colony shown in Table 4. One aliquot of the cells plated mediately in the CFU-c assay, and a second aliquot of cells was cells in the patient's marrow. Throughout the forming (CFU-c) incubated in suspension culture for 12 hr before plating. The period of study the number of CFU-c was subnormal or absent, number of colonies in mixtures of allogeneic normal marrow a characteristic of aplastic anemia. However, a brief rise in the was approximately the average of the number of colonies in the number of CFU-c could be demonstrated periodically. At a individual marrow cultures. However, the culture of a mixture time when no CFU-c could be demonstrated in the patient's of normal marrow with the patient's marrow showed 60% in- whole marrow, cell fractions separated by Ficoll-hypaque velocity sedimen- hibition of the normal colony formation with no preculturing centrifugation and by Ficoll-hypaque and and 82% inhibition after 12 hr of preculture. tation were assayed. As shown in Fig. 5, the marrow separated by Ficoll-hypaque centrifugation had no detectable CFU-c, but the faster sedimenting large cell fraction showed 14 CFU-c DISCUSSION per 105 cells. Aplastic anemia can be broadly viewed as involving a defect at one of three levels of hemopoietic differentiation, which we Suppression of CFU-c in normal marrow by aplastic have termed for convenience types I, II, and III. Type I is due marrow to an intrinsic stem cell defect. In type II, an inductive stimulus Further experiments demonstrated that cells present in the bone is absent from the differentiative environment. Alternatively, marrow of this patient actively suppress granulocyte prolifer- in the type III process, stem cells and the appropriate envi- ation and differentiation. Bone marrow was obtained from two ronment are both present and functional but differentiation is normal donors and the patient and separated on a Ficoll-hy- actively suppressed. Some immunodeficiency diseases have paque gradient. The cells were mixed in equal proportions as been classified according to this scheme. Severe combined

Table 4. Colony formation in soft agar (CFU-c assay) by marrow cells from normal donors and a patient with aplastic anemia Cells plated after 12-hr Cells plated immediately suspension culture (16) Marrow donor Colonies/105 cells Clusters/105 cells Colonies/105 cells Clusters/105 cells W.K. (normal) 12.5 ± 2.5 70.5 ± 14.5 11.0 ± 4.0 66.5 ± 13.5 J.A. (normal) 7.2 ± 7.5 31.5 ± 3.5 10.5 ± 5.5 52.5 ± 12.5 A.M. (aplastic) 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Observed W.K. + J.A. 12.7 ± 2.0 48.5 ± 11.0 13.5 ± 1.5 73.5 ± 21.5 Expected W.K. + J.A. 9.9 51.0 10.8 59.5 Observed/expected 128% 95% 125% 123% Observed W.K. + A.M. 2.5 ± 2.5 22.5 ± 7.5 1.0 ± 0.0 7.3 ± 0.3 Expected W.K. + A.M. 6.3 35.3 5.5 33.3 Observed/expected 40% 64% 18% 22% Cells were plated at 2 X 105 cells per ml in duplicate; mean and standard errors are reported. Downloaded by guest on September 28, 2021 2894 Medical Sciences: Kagan et al. Proc. Natl. Acad. Sci. USA 73 (1976)

immunodeficiency can be viewed as a type I lymphoid stem tests described in this paper may allow the diagnosis of different cell defect (12), the DiGeorge syndrome serves as an example kinds of aplastic anemia and lead to the development of ther- of a type II defect in the thymic inductive environment (13), apeutic protocols designed to deal with specific pathogenetic and some cases of hypogammaglobulinemia may involve a type mechanisms, resulting in a more successful treatment for this III suppression of B lymphocyte differentiation (14, 15). disease. By several criteria the patient reported here has a block in myeloid differentiation. Her marrow had a lower number of This investigation was supported in part by U.S. Public Health granulocytes, a lower percentage of complement receptor Service Research Grants CA-08748, CA-05826, and CA-17404; Con- bearing cells, and a lower level of spontaneous proliferation than tract CB-53868 from the National Cancer Institute; National Institute So- normal. In addition, her marrow could not respond to the in- of Health grants NS-11457 and AI-11843; the American Cancer ciety, the National Foundation-March of Dimes; the Zelda R. Wein- ductive influence of ubiquitin with the appearance of com- traub Cancer Fund; and by the Judith Harris Selig Memorial Fund. plement receptor bearing cells or to the inductive influence of J.A. is the recipient of a fellowship from the Instituto de Alta Cultura, colony stimulating factor with myeloid or monocytic colony Lisbon, Portugal, and from the J. M. Foundation. We are grateful to formation. Dr. Morton Coleman for referring this patient to us for analysis. We We suggest that this block is due to the active suppression of thank Olga Kucarova for her expert technical assistance, Judy Kempa myelopoiesis by a mononuclear cell. It is not due to a defective for the illustrations, and John Hlinka for the photomicrographs. We inductive environment since the CFU-c assay provides an en- also acknowledge the Upjohn Co., Kalamazoo, Mich., for providing vironment that can support normal myelopoiesis. Similarly, the the horse anti-human thymocyte globulin. We thank Dr. J. Abbott and block cannot be due to the absence of stem cells since we could Dr. F. P. Siegal for their constructive-criticism of the manuscript. establish myeloid differentiation, as measured by complement receptor induction, spontaneous proliferation, and colony 1. Till, J. E. & McCulloh, E. A. (1961) Radiat. Res. 14,213-222. formation after removal from the patient's marrow of a pop- 2. Bradley, T. R. & Metcalf, D. (1966) Aust. J. Exp. Biol. Med. Sci. 44,287-299. ulation of small cells by velocity sedimentation. Finally, cells 3. Krantz, S. B. (1972) Blood 39,347-360. present in the patient's marrow suppressed the myelopoietic 4. Boxer, L. A., Greenberg, M. S., Boxer, G. J. & Stossel, T. P. (1975) capacity of normal marrow cells. N. Engl. J. Med. 293,748-753. We have therefore demonstrated the presence of suppressor 5. Boyum, A. (1968) Scand. J. Clin. Lab. Invest. 21,77-89, suppl. cells in the marrow of a patient with aplastic anemia both by 97. achieving normal differentiation after their removal from the 6. Miller, R. G. & Phillips, R. A. (1969) J. Cell. Physiol. 73, 191- patient's marrow and by suppressing normal differentiation 202. after their addition to normal marrow. Additional evidence to 7. Ross, G. D., Rabellino, E. M., Polley, M. J. & Grey, H. M. (1973) J. Clin. Invest. 552,377-385. support this interpretation comes from studies of Ascensao et 8. Bentwich, Z., Douglas, S. D., Siegel, F. P. & Kunkel, H. G. (1973) al. (16), which demonstrate a 10-fold increase in colony for- Clin. Immunol. Immunopathol. 1, 511-522. mation in the CFU-c assay after treatment in vitro of this pa- 9. Goldstein, G., Scheid, M., Hammerling, U., Boyse, E. A., tient's marrow with horse anti-human thymocyte globulin and Schlesinger, D. H. & Niall, H. D. (1975) Proc. Natl. Acad. Sci. complement. From the present studies and those of Ascensio USA 72, 11-15. et al., we can define this suppressor cell as having a density of 10. Pike, B. L. & Robinson, W. A. (1970) J. Cell. Comp. Physiol. 76, less than 1.077 g/cm3, sedimenting from 2 to 4 mm/hr, and 77-84. being sensitive to the action of horse anti-human thymocyte 11. Kagan, W. A., Incefy, G. S., Gupta, S., Siegal, F., Goldstein, G. globulin. This suggests that the suppressor cell is a small T & Good, R. A. (1976) in Leucocyte Membrane Determinants lymphocyte, but further studies must be performed to establish Regulating Immune Reactivity; Proceedings of the 10th Leu- cocyte Culture Conference, eds. Eijsvoogel, V. P., Roos, D. & fully its identity. Zeijlemaker, W. (Academic Press, New York), pp. 719-725. The present studies have certain clinical implications. There 12. Incefy, G. S., Touraine, J. L., Touraine, F., L'Esperance, P., Siegal, are several reports of patients with aplastic anemia who have F. P. & Good, R. A. (1975) Trans. Assoc. Am. Phys. 87, 258- received bone marrow transplants after preconditioning with 262. cyclophosphamide, total body irradiation, or horse anti-human 13. DiGeorge, A. M. (1968) Immunologic Deficiency Diseases in thymocyte globulin. Some of these patients reject their allografts Man. Birth Defects Original Article Series 4: no. 1, eds. Bergsma, and then recover from their aplasia with complete regeneration D. & Good, R. A. (The National Foundation, New York). of their own marrow (17, 18). Common to all of these cases is 14. Waldman, T. A., Brodes, S., Blaese, R. M., Durm, M., Blackman, the preconditioning with immunosuppressive agents. We M. & Strober, W. (1974) Lancet 2,609-613. 15. F. P. & M. Clin. Res. 23,297A. suggest that these patients may be suffering from an autoim- Siegal, Siegal, (1975) into 16. Ascensio, J., Pahwa, R., Kagan, W. A., Hansen, J., Moore, M. & mune disorder. Thus aplastic anemia might be divided type Good, R. (1976) Lancet 1, 669-671. I stem cell defects, type II differentiative environment defects, 17. Jeannet, M., Rubenstein, A., Pelet, B. & Kummer, H. (1974) or type III suppression disorders. The case we have reported Transp. Proc. 6,359-363. here appears to involve a type III suppression disorder. We are 18. Thomas, E. D., Storb, R., Giblet, E. R., Longpre, B., Weiden, P. currently studying several other cases of aplastic anemia that L., Fefer, A., Witherspoon, R., Clift, R. A. & Buckner, C. D. do not appear to involve this mechanism. We hope the in vitro (1976) Exp. Hematol. 4,97-102. Downloaded by guest on September 28, 2021