CLINICAL
IMML’NOLOGY
AND
IMMUNOPATHOLOGY
10,
344- 349 (1978)
Hypogammaglobulinemia followed by Aplastic Anemia with Suppressor Lymphocytes: A Case Report’ BRUCE H. LITTMAN,~‘~
CHARLES L. COOKE,
AND RONALD
HOFFMAN
Division of Immunology and Connective Tissue Diseases, Medical College of Virginia, School of Medicine. Virginia Commnnn~ealth University, Richmond. Virginia 23298. and the Department of Internal Medicine, Yale University School Medicine, Ne\z, Haven, Connecticut 06520
of
Received January 17. 1978 A patient with hypogammaglobulinemia was found 8 years later to have aplastic anemia. At this time immunoglobulin levels had returned to normal. Her peripheral blood lymphocytes suppressed erythroid colony formation by normal bone marrow cells. The association of these two conditions with suppressor T lymphocytes and the occurrence of these diseases in the same patient imply a common defect in T-lymphocyte control of cellular differentiation.
INTRODUCTION Recently much has been learned about control of the immune response. In particular, T-lymphocyte subpopulations have been found which are responsible for both help and suppression of antibody production (1). The suppressor function of T lymphocytes is important in the phenomenon of tolerance (2) because it prevents, in the normal state, the development of autoimmunity. Loss of this normal suppressor function has been implicated in disease states such as systemic lupus erythematosus (3). Of equal importance are disease states characterized by the presence of suppressor lymphocytes capable of inhibiting normal cellular functions. Some patients with common variable hypogammaglobulinemia (CVH) have suppressor T lymphocytes capable of inhibiting immunoglobulin synthesis (4). Some patients with aplastic anemia have been found to have lymphocytes which inhibit colony formation by normal bone marrow cells (5). The mechanism of suppression in either disease is unknown. In some patients with CVH, this suppression results in inhibition of cellular differentiation from the precursor to the functional state (6). The case here is the first report of a patient with suppressor lymphocytes who first developed hypogammaglobulinemia and then asplastic anemia associated with a return of immunoglobulin levels to normal.
’ This work was supported in part by an A.D. Williams small grant from the Medical College of Virginia, by the Anna Fuller Fund, New Haven, Connecticut, and by NIH Grants HE-10880 and AM-20317. This is publication No. 106 from the Charles W. Thomas Fund, Medical College of Virginia. * Address reprint requests to Dr. Littman, Box 263, MCV Station, Richmond, Virginia 23298. 3 Recipient of NIH Research Career Development Award No. AMOO336-01. 344 0090-1229/78/0103-0344$01.00/O Copyright All
right?
0 of
1978 reproduction
by
Academic in
any
Press.
Inc.
form
reserved
HYPOGAMMAGLOBULINEMIA
FOLLOWED
TABLE IMMUNOCLOBULIN
LEVELS
kG
is Adult
APLASTIC
345
A’NEMIA
1
BY RADIAL
Immunoglobulin
kA W
BY
IMMUNODIFFUSION
level (mg%)
1969
1912
1973
1974
1975
1977
Normal range”
577 131 128
540 1.50 56
570 135 56
730 170 74
670 200 108
1200 132 194
600- 1600 20-500 60-200
values
taken
from
Ref. (19).
CASE REPORT In 1969, a 56-year-old white female (R.P.) presented with complaints of a purulent nasal discharge, fatigue, and low y-globulin level. She had tested herself while employed as a laboratory technician and discovered that she was hypogammaglobulinemic. She had recurrent sinus infections and bronchitis, was a heavy smoker, and was allergic to sulfa drugs. She had normal vital signs and tenderness over the right maxillary sinus and rhonchi throughout the chest. CBC, differential white count, urinalysis, chest X ray, and hepatic enzymes were normal. An antinuclear antibody test (ANA) was negative. Serum protein electrophoresis revealed decreased y-globulin. The immunoglobulin levels (as determined by radial immunodiffusion) are given in Table 1. She had an IgG level approximately onehalf normal and normal levels of IgA and IgM. Radiographs of the paranasal sinuses showed changes due to chronic right maxillary sinusitis. She was treated with antibiotics and given injections of y-globulin, 10 ml every 2 weeks. The latter was continued for 2 years. Between 1972 and 1976, she underwent surgery for two herniated intervertebral discs, an abdominal hysterectomy, and an aortic endarterectomy with Dacron graft replacement. Prior to the hysterectomy she developed severe osteoporosis. spontaneous fractures of two lumbar vertebrae, and a fractured pelvis. In January 1977, she experienced epistaxis and, in February 1977, she noted rectal bleeding associated with a petechial skin eruption. For 8 months prior to this she took only acetaminophen and chloralhydrate. There was no splenomegaly. The platelet count was 13,00O/mm 3, the hemoglobin was 14.4 g/dl, and the leukocyte count was 6000/mm3 with a normal differential. Immunoglobulin values were now normal and are given in Table 1. The ANA test4 was now positive (1:200) with a nucleolar pattern. This ANA was not further characterized. Several lupus erythematosus cell tests were negative. There were reduced megakaryocytes and platelets in the bone marrow. Despite this, an initial diagnosis of idiopathic thrombocytopenic purpura with possible systemic lupus erythematosus (SLE) was considered. She was treated with prednisone, 80 mg/day, without a hematologic response. Over the next month her leukocyte count fell to 2000/mm3 (95% lymphocytes) and her hemoglobin concentration plummeted to 8.5 g/dl. A bone marrow 4 Indirect immunofluorescence
of mouse liver.
344
LITTMAN,
COOKE,
AND
HOFFMAN
biopsy on March 24, 1977, revealed hypoplasia of all elements. Oxymetholone was given without response. Febrile episodes began, but no infectious cause could be demonstrated. At this time studies were conducted which demonstrated lymphocytes capable of suppressing bone marrow erythroid colony formation. Cyclophosphamide was begun but resulted in a high fever and was discontinued. On May 1, 1977, she developed a transient right hemiparesis and became lethargic. She required frequent platelet transfusions. At her request no further complications of her illness were treated. She became hypotensive and died on May 27, 1977. An autopsy was not performed. MATERIALS
AND METHODS
Inhibition of bone marrow’ colony formation. Peripheral blood lymphocytes were separated from freshly drawn heparinized venous blood by Ficoll-Hypaque density-gradient sedimentation (7). The cells were then suspended in NCTC-109 medium and used within 30 min of collection. Bone marrow cells were obtained by aspiration from the posterior iliac crest of a hematologically normal donor. Informed consent was obtained from both the patient and the normal donors. The in vitro plasma-clot culture technique for measuring growth of erythroid colonies from human bone marrow has been described by Tepperman et al. (8). Dispersed bone marrow cells, in a final concentration of 6 x lo5 cells/l. 1 ml, were cultured in duplicate in the presence of 2 IU of erythropoietin. In the lymphocyte studies, 0.5 to 4 x lo5 lymphocytes in 0.1 ml of NCTC-109 were cocultured with normal bone marrow cells. Cultures were maintained in a humidified atmosphere of 4% carbon dioxide in air at 37°C. At 5 to 7 days of incubation, the clots were removed and transferred to glass slides, fixed in gluteraldehyde and stained with benzidine and hematoxylin. Under 100x magnification, each clot was examined, and erythroid colonies consisting of eight or more benzidine-positive cells were counted. Lymphocytes from the patient, a normal control, and an individual with thalessemia requiring multiple transfusions were studied simutaneously . T-Lymphocyte quantitation. T-Lymphocyte quantitation was accomplished by rosetting Ficoll-Hypaque-separated lymphocytes with sheep erythrocytes previously treated with neuraminidase. The method for this has been previously described (9). Lymphocytes rosetted with one or two erythrocytes were counted separately from those rosetted with three or more. Lymphocyte transformation. Lymphocyte transformation in response to specific antigens [streptokinase - streptodornase (SK-SD), Varidase, Lederle Laboratories, Pearl River, N.Y.; candida (Dermatophytin O), Hollister-Stier, Spokane, Wash.; and purified protein derivative (PPD), supplied by the U.S.Japan Cooperative Study, NIH, Bethesda, Md.] was performed as previously described (9). Briefly, lo5 Ficoll-Hypaque-separated lymphocytes were cultured for 6 days in quadruplicate microtiter plate wells with an without antigen using 0.2 ml of 15% AB serum in TC-199. One microcurie of [3H]thymidine was added 18 hr prior to harvesting with a multiple automated sample harvestor. Results for unstimulated and antigen-stimulated cultures are expressed as the mean counts per minute t SEM.
HYPOGAMMAGLOBULINEMIA
FOLLOWED
TABLE EFFFCI
BY
APLASTIC
347
ANEMIA
2
OF PERIPHERAL BLOOD LYMPHOCYWZS FROM PATIENT R. P., ONE MULTIPLY TRANSFUSEI) THALESSEMIC PATIENT, AND ONE NORMAL CONTROL ON ERYTHROID COLONY FORMATION BY NORMAL HUMAN BONE MARROW CELLS IN RESPONSE ‘1‘0 2 IU OF ERYTHROPOIETIN AFTER 7 DAYS OF CULTURE
Erythroid colonies/6 x 10’ bone marrow cells at lymphocyte concentration indicated Subject -
Normal Thalessemic (multiple transfusions) R. P.
0
0.5 x 10’
1.0 x 105
2.0 x 10s
4.0 x 105
1475260 -
144 + 34
188 t 81
296 t 60
127 x 44
-
124 2 49 54 k 24
144 75
163 56
139 5 24 51 k 14
i i-
36 42
t t
36 17
” Mean i SEM for duplicates.
RESULTS
inhibition of Erythroid Colony Formation The patient’s peripheral blood lymphocytes suppressed erythroid colony formation by normal bone marrow cells (Table 2). As few as 50,000 peripheral lymphocytes reduced bone marrow colony formation from 147 ZF26 to 54 + 24 erythroid colonies per 6 x 10’ bone marrow cells. Peripheral lymphocytes from a normal donor and from a multiply transfused patient with thalessemia had no such effect. T-Lymphocyte Number and Function In April 1977, the patient’s white blood cell count was 1000/mm3, including 87% lymphocytes and 13% monocytes. No polymorphonuclear leukocytes were observed. Seventy-five percent of her lymphocytes were sheep erythrocyte rosette positive (72% with three RBC/per lymphocyte and 3% with one or two RBC/per lymphocyte), a value within the normal range of 76 i- 12 (mean of 40 people + 2 SD). Lymphocyte transformation in response to stimulation with specific antigens resulted in a significant response although the amount of 13H]thymidine incorporation was less than that of a simultaneously tested normal individual and less than is usually observed for normal blood donors. These results are given in Table 3.
TABLE LYY~PHOCYTE
TRANSFORMATIOK
3
IN RESPONSE TO
[3H]Thymidine Antigen None SK-SD Candida PPD ‘I Mean + SEM.
Patient 253 1626 2092 6659
+ t k t
3Cr' 955 215 512
AN?.rorNrc S-rrMurArro~ incorporation
(cpm)
Normal control 1020 18223 13884 8005
t t k t
200 1424 990 490
348
LITTMAN,
COOKE,
AND
HOFFMAN
DISCUSSION
Waldmann et al. demonstrated that patients with CVH usually have T lymphocytes present in their blood capable of inhibiting immunoglobulin production by pokeweed mitogen-stimulated lymphocytes (4). Siegal er al. demonstrated that this phenomenon was associated with inhibition of B-lymphocyte differentiation into plasmacytoid, immunoglobulin-producing cells (6). These findings have been confirmed by other investigators (10). Some patients with CVH may have intrinsic B-lymphocyte defects (11). While some disorders of blood components are associated with autoantibodies, as is the case with idiopathic thrombocytopenic purpura (anti-platelet antibody) (12), autoimmune hemolytic anemia (anti-erythrocyte antibody) (13), and pure red cell aplasia (anti-erythroblast antibody) (14), no such autoantibody has been found in idiopathic aplastic anemia. On the other hand, this disorder and the Diamond-Blackfan syndrome (congenital hypoplastic anemia) have both recently been found to be associated with peripheral blood lymphocytes capable of suppressing erythroid colony formation by normal bone marrow cells (5. 15). In aplastic anemia, treatment of patients’ lymphocytes with anti-thymocyte globulin and complement prevents this in vitro suppression of bone marrow colony formation (16). This implies that the suppressor cell is a T lymphocyte. In addition, the association of variable hypogammaglobulinemia with thymoma is well documented (4, 6). In one of two patients with thymoma and hypogammaglobulinemia reported by Litwin and Zanjani (17), episodic red cell agenesis also occurred. Both of these patients had lymphocytes capable of suppressing immunoglobulin production and erythroid colony formation. The case reported here is interesting because of the initial onset of mild hypogammaglobulinemia, the resolution of this problem, and then the appearance of aplastic anemia associated with suppressor lymphocytes. It is tempting to postulate that suppressor lymphocytes were responsible for both diseases in this patient. This patient also developed a positive test for anti-nuclear antibody which may also represent a defect in suppressor T-lymphocyte function (3). There was no antemortem evidence for a thymoma but without an autopsy this possibility cannot be excluded. Laboratory studies confirmed the presence of suppressor lymphocytes capable of inhibiting erythroid colony formation by normal bone marrow, but did not include studies of suppression by lymphocyte subpopulations. Maximal suppression was observed even at the lowest concentration of patient lymphocytes. Normal numbers of T lymphocytes as determined by sheep erythrocyte rosette formation were found. While some of these cells functioned normally in response to antigenic stimulation, others had abnormal suppressor activity. Perhaps the quantitatively small response to antigen was due to fewer T lymphocytes belonging to the subclass responsible for delayed-type hypersensitivity and a greater proportion belonging to a suppressor subclass. This hypothesis is strengthened by results of studies in mice indicating that separate T-lymphocyte subpopulations are responsible for these functions (18). ACKNOWLEDGMENTS Human urinary ment of Physiology.
erythropoietin University
used in these studies was collected of Northeast. Corrientes. Argentina.
and concentrated and was further
by the Departprocessed by the
HYPOGAMMAGLOBULINEMIA
FOLLOWED
BY
APLASTIC
ANEMIA
349
Hematologic Research Laboratories. Children‘s Hospital, Los Angeles, Calif. We would also like to acknowledge the excellent secretarial assistance of Ms. Kay Jenkins.
REFERENCES 1. Gershon, R. K., In “Contemporary Topics in Immunology.“ Vol. 3. p. I. 1969. 2. Benjamin, D. C.. J. E~\-p. Mud. 141. 635. 1975. 3. Waldmann, T. A.. and Broder, S., In “Progress in Clinical Immunology” (R. Schwartz, Ed.). Vol. 3. p. 155. Grune and Stratton. New York. 1977. 4. Waldmann, T. A.. Broder, S.. Blaese. R. M.. c’t ~(1..Luncet 2, 609. 1974. 5. Hoffman, R.. Zanjani. E. D., Lutton. J. D., et al.. N. E~pl. J. Mud. 296. IO. 1977. 6. Siegal, F. P.. Siegal, M.. and Good, R. A., J. Cli,~. Inresr. 58, 109, 1976. 7. Boyam, A., Sccrnd. J. Clirl. Lab. In\vst. 21 (Suppl. 97). 77. 1968. 8. Tepperman. A. D.. Curtis. J. E.. and McCulloch, E. A., Blood 44, 659. 1974. 9. Littman, B. H.. David. J. R.. and Rocklin. R. E.. Cc//. Immctnol. 24, 241. 1976. 10. Broom, B. C.. de la Concha. E. G.. Webster, A. D. B.. et ol.. C/;rl. E.up. Itrmruno/. 23, 73. 1976. 1 I. de la Concha. E. G., Oldham, G.. Webster, A. D. B., et al.. C/in. E.rp. Immune/. 27, 208, 1977. 12. Harrington. W. J.. Minnich. V.. Hollingsworth. J. W.. et 01.. J. Luh. C/in. Med. 38. I. 1951. 13. Eyster. M. E.. Jenkins, D. E.. Jr.. Amer. J. Med. 46, 360. 1969. 14. Krantz. S. B.. Nrn, EngI. J. Mud. 291, 345, 1974. 15. Hoffman. R.. Zanjani, E. D.. Zalusky. R., et ul.. Sc,irncr 193. 899, 1976. 16. Ascensao. J.. Kagan, W.. Moore, M.. et (I/.. Lanc,rt 1, 669. 1976. 17. Litwin, S., and Zanjani. E.. Nature (London) 266, 57, 1977. 18. Huber, B.. Devinsky. O., Gershon, R. K.. and Cantor. H.. J. Erp. Med. 143. 1534. 1976. 19. Freedman, S. O., and Gold. P. (Eds.) “Clinical Immunology,” p. 606. Harper and Row. Hagerstown. Md.. 1976.
IMML’NOLOGY
AND
IMMUNOPATHOLOGY
10,
344- 349 (1978)
Hypogammaglobulinemia followed by Aplastic Anemia with Suppressor Lymphocytes: A Case Report’ BRUCE H. LITTMAN,~‘~
CHARLES L. COOKE,
AND RONALD
HOFFMAN
Division of Immunology and Connective Tissue Diseases, Medical College of Virginia, School of Medicine. Virginia Commnnn~ealth University, Richmond. Virginia 23298. and the Department of Internal Medicine, Yale University School Medicine, Ne\z, Haven, Connecticut 06520
of
Received January 17. 1978 A patient with hypogammaglobulinemia was found 8 years later to have aplastic anemia. At this time immunoglobulin levels had returned to normal. Her peripheral blood lymphocytes suppressed erythroid colony formation by normal bone marrow cells. The association of these two conditions with suppressor T lymphocytes and the occurrence of these diseases in the same patient imply a common defect in T-lymphocyte control of cellular differentiation.
INTRODUCTION Recently much has been learned about control of the immune response. In particular, T-lymphocyte subpopulations have been found which are responsible for both help and suppression of antibody production (1). The suppressor function of T lymphocytes is important in the phenomenon of tolerance (2) because it prevents, in the normal state, the development of autoimmunity. Loss of this normal suppressor function has been implicated in disease states such as systemic lupus erythematosus (3). Of equal importance are disease states characterized by the presence of suppressor lymphocytes capable of inhibiting normal cellular functions. Some patients with common variable hypogammaglobulinemia (CVH) have suppressor T lymphocytes capable of inhibiting immunoglobulin synthesis (4). Some patients with aplastic anemia have been found to have lymphocytes which inhibit colony formation by normal bone marrow cells (5). The mechanism of suppression in either disease is unknown. In some patients with CVH, this suppression results in inhibition of cellular differentiation from the precursor to the functional state (6). The case here is the first report of a patient with suppressor lymphocytes who first developed hypogammaglobulinemia and then asplastic anemia associated with a return of immunoglobulin levels to normal.
’ This work was supported in part by an A.D. Williams small grant from the Medical College of Virginia, by the Anna Fuller Fund, New Haven, Connecticut, and by NIH Grants HE-10880 and AM-20317. This is publication No. 106 from the Charles W. Thomas Fund, Medical College of Virginia. * Address reprint requests to Dr. Littman, Box 263, MCV Station, Richmond, Virginia 23298. 3 Recipient of NIH Research Career Development Award No. AMOO336-01. 344 0090-1229/78/0103-0344$01.00/O Copyright All
right?
0 of
1978 reproduction
by
Academic in
any
Press.
Inc.
form
reserved
HYPOGAMMAGLOBULINEMIA
FOLLOWED
TABLE IMMUNOCLOBULIN
LEVELS
kG
is Adult
APLASTIC
345
A’NEMIA
1
BY RADIAL
Immunoglobulin
kA W
BY
IMMUNODIFFUSION
level (mg%)
1969
1912
1973
1974
1975
1977
Normal range”
577 131 128
540 1.50 56
570 135 56
730 170 74
670 200 108
1200 132 194
600- 1600 20-500 60-200
values
taken
from
Ref. (19).
CASE REPORT In 1969, a 56-year-old white female (R.P.) presented with complaints of a purulent nasal discharge, fatigue, and low y-globulin level. She had tested herself while employed as a laboratory technician and discovered that she was hypogammaglobulinemic. She had recurrent sinus infections and bronchitis, was a heavy smoker, and was allergic to sulfa drugs. She had normal vital signs and tenderness over the right maxillary sinus and rhonchi throughout the chest. CBC, differential white count, urinalysis, chest X ray, and hepatic enzymes were normal. An antinuclear antibody test (ANA) was negative. Serum protein electrophoresis revealed decreased y-globulin. The immunoglobulin levels (as determined by radial immunodiffusion) are given in Table 1. She had an IgG level approximately onehalf normal and normal levels of IgA and IgM. Radiographs of the paranasal sinuses showed changes due to chronic right maxillary sinusitis. She was treated with antibiotics and given injections of y-globulin, 10 ml every 2 weeks. The latter was continued for 2 years. Between 1972 and 1976, she underwent surgery for two herniated intervertebral discs, an abdominal hysterectomy, and an aortic endarterectomy with Dacron graft replacement. Prior to the hysterectomy she developed severe osteoporosis. spontaneous fractures of two lumbar vertebrae, and a fractured pelvis. In January 1977, she experienced epistaxis and, in February 1977, she noted rectal bleeding associated with a petechial skin eruption. For 8 months prior to this she took only acetaminophen and chloralhydrate. There was no splenomegaly. The platelet count was 13,00O/mm 3, the hemoglobin was 14.4 g/dl, and the leukocyte count was 6000/mm3 with a normal differential. Immunoglobulin values were now normal and are given in Table 1. The ANA test4 was now positive (1:200) with a nucleolar pattern. This ANA was not further characterized. Several lupus erythematosus cell tests were negative. There were reduced megakaryocytes and platelets in the bone marrow. Despite this, an initial diagnosis of idiopathic thrombocytopenic purpura with possible systemic lupus erythematosus (SLE) was considered. She was treated with prednisone, 80 mg/day, without a hematologic response. Over the next month her leukocyte count fell to 2000/mm3 (95% lymphocytes) and her hemoglobin concentration plummeted to 8.5 g/dl. A bone marrow 4 Indirect immunofluorescence
of mouse liver.
344
LITTMAN,
COOKE,
AND
HOFFMAN
biopsy on March 24, 1977, revealed hypoplasia of all elements. Oxymetholone was given without response. Febrile episodes began, but no infectious cause could be demonstrated. At this time studies were conducted which demonstrated lymphocytes capable of suppressing bone marrow erythroid colony formation. Cyclophosphamide was begun but resulted in a high fever and was discontinued. On May 1, 1977, she developed a transient right hemiparesis and became lethargic. She required frequent platelet transfusions. At her request no further complications of her illness were treated. She became hypotensive and died on May 27, 1977. An autopsy was not performed. MATERIALS
AND METHODS
Inhibition of bone marrow’ colony formation. Peripheral blood lymphocytes were separated from freshly drawn heparinized venous blood by Ficoll-Hypaque density-gradient sedimentation (7). The cells were then suspended in NCTC-109 medium and used within 30 min of collection. Bone marrow cells were obtained by aspiration from the posterior iliac crest of a hematologically normal donor. Informed consent was obtained from both the patient and the normal donors. The in vitro plasma-clot culture technique for measuring growth of erythroid colonies from human bone marrow has been described by Tepperman et al. (8). Dispersed bone marrow cells, in a final concentration of 6 x lo5 cells/l. 1 ml, were cultured in duplicate in the presence of 2 IU of erythropoietin. In the lymphocyte studies, 0.5 to 4 x lo5 lymphocytes in 0.1 ml of NCTC-109 were cocultured with normal bone marrow cells. Cultures were maintained in a humidified atmosphere of 4% carbon dioxide in air at 37°C. At 5 to 7 days of incubation, the clots were removed and transferred to glass slides, fixed in gluteraldehyde and stained with benzidine and hematoxylin. Under 100x magnification, each clot was examined, and erythroid colonies consisting of eight or more benzidine-positive cells were counted. Lymphocytes from the patient, a normal control, and an individual with thalessemia requiring multiple transfusions were studied simutaneously . T-Lymphocyte quantitation. T-Lymphocyte quantitation was accomplished by rosetting Ficoll-Hypaque-separated lymphocytes with sheep erythrocytes previously treated with neuraminidase. The method for this has been previously described (9). Lymphocytes rosetted with one or two erythrocytes were counted separately from those rosetted with three or more. Lymphocyte transformation. Lymphocyte transformation in response to specific antigens [streptokinase - streptodornase (SK-SD), Varidase, Lederle Laboratories, Pearl River, N.Y.; candida (Dermatophytin O), Hollister-Stier, Spokane, Wash.; and purified protein derivative (PPD), supplied by the U.S.Japan Cooperative Study, NIH, Bethesda, Md.] was performed as previously described (9). Briefly, lo5 Ficoll-Hypaque-separated lymphocytes were cultured for 6 days in quadruplicate microtiter plate wells with an without antigen using 0.2 ml of 15% AB serum in TC-199. One microcurie of [3H]thymidine was added 18 hr prior to harvesting with a multiple automated sample harvestor. Results for unstimulated and antigen-stimulated cultures are expressed as the mean counts per minute t SEM.
HYPOGAMMAGLOBULINEMIA
FOLLOWED
TABLE EFFFCI
BY
APLASTIC
347
ANEMIA
2
OF PERIPHERAL BLOOD LYMPHOCYWZS FROM PATIENT R. P., ONE MULTIPLY TRANSFUSEI) THALESSEMIC PATIENT, AND ONE NORMAL CONTROL ON ERYTHROID COLONY FORMATION BY NORMAL HUMAN BONE MARROW CELLS IN RESPONSE ‘1‘0 2 IU OF ERYTHROPOIETIN AFTER 7 DAYS OF CULTURE
Erythroid colonies/6 x 10’ bone marrow cells at lymphocyte concentration indicated Subject -
Normal Thalessemic (multiple transfusions) R. P.
0
0.5 x 10’
1.0 x 105
2.0 x 10s
4.0 x 105
1475260 -
144 + 34
188 t 81
296 t 60
127 x 44
-
124 2 49 54 k 24
144 75
163 56
139 5 24 51 k 14
i i-
36 42
t t
36 17
” Mean i SEM for duplicates.
RESULTS
inhibition of Erythroid Colony Formation The patient’s peripheral blood lymphocytes suppressed erythroid colony formation by normal bone marrow cells (Table 2). As few as 50,000 peripheral lymphocytes reduced bone marrow colony formation from 147 ZF26 to 54 + 24 erythroid colonies per 6 x 10’ bone marrow cells. Peripheral lymphocytes from a normal donor and from a multiply transfused patient with thalessemia had no such effect. T-Lymphocyte Number and Function In April 1977, the patient’s white blood cell count was 1000/mm3, including 87% lymphocytes and 13% monocytes. No polymorphonuclear leukocytes were observed. Seventy-five percent of her lymphocytes were sheep erythrocyte rosette positive (72% with three RBC/per lymphocyte and 3% with one or two RBC/per lymphocyte), a value within the normal range of 76 i- 12 (mean of 40 people + 2 SD). Lymphocyte transformation in response to stimulation with specific antigens resulted in a significant response although the amount of 13H]thymidine incorporation was less than that of a simultaneously tested normal individual and less than is usually observed for normal blood donors. These results are given in Table 3.
TABLE LYY~PHOCYTE
TRANSFORMATIOK
3
IN RESPONSE TO
[3H]Thymidine Antigen None SK-SD Candida PPD ‘I Mean + SEM.
Patient 253 1626 2092 6659
+ t k t
3Cr' 955 215 512
AN?.rorNrc S-rrMurArro~ incorporation
(cpm)
Normal control 1020 18223 13884 8005
t t k t
200 1424 990 490
348
LITTMAN,
COOKE,
AND
HOFFMAN
DISCUSSION
Waldmann et al. demonstrated that patients with CVH usually have T lymphocytes present in their blood capable of inhibiting immunoglobulin production by pokeweed mitogen-stimulated lymphocytes (4). Siegal er al. demonstrated that this phenomenon was associated with inhibition of B-lymphocyte differentiation into plasmacytoid, immunoglobulin-producing cells (6). These findings have been confirmed by other investigators (10). Some patients with CVH may have intrinsic B-lymphocyte defects (11). While some disorders of blood components are associated with autoantibodies, as is the case with idiopathic thrombocytopenic purpura (anti-platelet antibody) (12), autoimmune hemolytic anemia (anti-erythrocyte antibody) (13), and pure red cell aplasia (anti-erythroblast antibody) (14), no such autoantibody has been found in idiopathic aplastic anemia. On the other hand, this disorder and the Diamond-Blackfan syndrome (congenital hypoplastic anemia) have both recently been found to be associated with peripheral blood lymphocytes capable of suppressing erythroid colony formation by normal bone marrow cells (5. 15). In aplastic anemia, treatment of patients’ lymphocytes with anti-thymocyte globulin and complement prevents this in vitro suppression of bone marrow colony formation (16). This implies that the suppressor cell is a T lymphocyte. In addition, the association of variable hypogammaglobulinemia with thymoma is well documented (4, 6). In one of two patients with thymoma and hypogammaglobulinemia reported by Litwin and Zanjani (17), episodic red cell agenesis also occurred. Both of these patients had lymphocytes capable of suppressing immunoglobulin production and erythroid colony formation. The case reported here is interesting because of the initial onset of mild hypogammaglobulinemia, the resolution of this problem, and then the appearance of aplastic anemia associated with suppressor lymphocytes. It is tempting to postulate that suppressor lymphocytes were responsible for both diseases in this patient. This patient also developed a positive test for anti-nuclear antibody which may also represent a defect in suppressor T-lymphocyte function (3). There was no antemortem evidence for a thymoma but without an autopsy this possibility cannot be excluded. Laboratory studies confirmed the presence of suppressor lymphocytes capable of inhibiting erythroid colony formation by normal bone marrow, but did not include studies of suppression by lymphocyte subpopulations. Maximal suppression was observed even at the lowest concentration of patient lymphocytes. Normal numbers of T lymphocytes as determined by sheep erythrocyte rosette formation were found. While some of these cells functioned normally in response to antigenic stimulation, others had abnormal suppressor activity. Perhaps the quantitatively small response to antigen was due to fewer T lymphocytes belonging to the subclass responsible for delayed-type hypersensitivity and a greater proportion belonging to a suppressor subclass. This hypothesis is strengthened by results of studies in mice indicating that separate T-lymphocyte subpopulations are responsible for these functions (18). ACKNOWLEDGMENTS Human urinary ment of Physiology.
erythropoietin University
used in these studies was collected of Northeast. Corrientes. Argentina.
and concentrated and was further
by the Departprocessed by the
HYPOGAMMAGLOBULINEMIA
FOLLOWED
BY
APLASTIC
ANEMIA
349
Hematologic Research Laboratories. Children‘s Hospital, Los Angeles, Calif. We would also like to acknowledge the excellent secretarial assistance of Ms. Kay Jenkins.
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