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Severe combined immunodeficiency with quantitatively normal but abnormally differentiated T lymphocytes
Volume 99 Number 2
intrauterine insults, it certainly reenforces the possibility of a single disease process having an effect upon multiple organs and organ systems. The etiology of this disease, including the bone marrow depression, remains obscure. There is no marked decrease in the marrow precursors and no apparent maturation arrest. Even the sideroblastic anemia seen is unusual because the red blood cells are macrocytic, rather than microcytic. The problem seems to be either an underlying metabolic or toxic process within the stromal microenvironment, or a defect in stem cell differentiation. The pathologic findings in the pancreas imply that the fibrosis may be a consequence of preceding inflammation. The paucity of iron deposition makes hemosiderosis as a cause of the pancreatic fibrosis unlikely. We are unable to implicate any known extrinsic cause of marrow suppression in our patient, either pre- or postnatally. She had a normal hver and spleen, as
B r i e f clinical and laboratory observations
26 1
contrasted with the hypoplasia of the spleen seen by Pearson et al. 1 Although our patient fits well into the syndrome described by Pearson, we believe that she may have a variant because of the very early age of onset, the severity of the bone marrow abnormalities, and the involvement of the thyroid gland. REFERENCES 1. Pearson HA, Lobel JS, Kocoshis SA, Naiman JL, Windmuller J, Lammi AT, Hoffman R, and Marsh JC: A new syndrome of refractory siderohlastic anemia with vacuolization of marrow precursors and exocrine pancreatic dysfunction, J PEDIATR95:976, 1979. 2. Schwachman H, Diamond LK, Oski FA, and Khan KT: The syndrome of pancreatic insufficiency and bone marrow dysfunction, J PEmAT~ 65:645, 1964. 3. Bodian M, Sheldon W, and Lightwood R: Congenital hypoplasia of the exocrine pancreas, Acta Pediatr 53:282, 1964.
Severe combined immunodeficiency with quantitatively normal but abnormally differentiated T lymphocytes A. Fischer,* A. Durandy, J. L. Virelizier, G. De Saint Basile, A. Lagrue, Paris, France,
E. Reinherz, S. Schlossman, Boston, Mass., and C. Griscelli, Paris France
THE SYNDROME of severe combined immunodeficiency has now been separated into various genetic, enzymologic, and immunologic subgroups? S C I D seems to result from an intrinsic T cell defect, ~ sometimes associated with membrane abnormalities$- ' or, more often, to be caused by a block in T cell differentiation.' However, the precise stages of human intrathymic differentiation of T lymphocytes remain largely ill-defined, preventing more specific studies of the cell or thymus defects in patients with SCID. We have identified previously unreported immunologic abnormalities in a patient with SCID, in w h o m peripheral T cells behaved like thymocytes blocked at a precise stage From Groupe d'Immunologie et Rhumatologie Pbdiatriques, INSERM U 132, Department of Pediatrics, Hbpital des Enfants Malades, and Division immunology, Sydney Farber Cancer Institute, and the Howard Medical School *Reprint address: Groupe de Recherches d'lmmunologie et de Rhumatologie Pediatriques, 1NSERM U 132, HOpital des Enfants Malades, 149, Rue de Sevres, 75015 Paris, France.
0022-3476/81/080261 +04500.40/0 9 1981 The C. V. Mosby Co.
of differentiation, as revealed by functional and cell surface characteristics. CASE REPORT
This girl was born under germ-~Yee conditions and was then maintained in a Trexler isolator at H6pital des Enfants-Malades. The patient was the second child of nonconsanguineous parents. Her sister died at age 22 months after repeated skin and pulmonary infections associated with severe chronic diarrhea. Postmortem histologic examination showed a profound hypoplasia of lymphoid organs including the thymus, in which, however, Abbreviations used SCID: severe combined immunodeficiency ADA: adenosine deaminase NP: nucleoside phosphorylase FTS: facteur thymique serique PHA: phytohemagglutinin Con A: concanavalin A PWM: pokeweed mitogen SIIl: polysaccharide antigen of pneumococcus MLR: mixed leukocyte reaction
262
Briefc#nical and laboratory observations
Table I. Cell surface characteristics of T and B lymphocytes Patient Lymphocytes/mm~ E rosettes (%) OKT1 ~ (%) OKT3* (%) OKT4' (%) OKT8 + (%) OKMI* (%) Surface Ig positive cells (%) Ia positive cells (%)
Normal values
1,500-2,500 35-62 52 52 52 52 17 20
2,000-3,000 71 _+ 7 60-80 60-80 35-50 25-35 5-20 5-15
15-30
5-15
Hassal corpuscles were seen. Immunologic data obtained four months before death showed a mild lymphopenia (1,000 to 1,200/mm~) with 25 to 35% T lymphocytes and 8 to 12% B lymphocytes. In vivo and in vitro lymphocyte functions were profoundly impaired. Serum concentrations of IgG, A, and M were within the normal range, and some antibodies against vaccinal antigens were detected. ADA and purine NP activities were normal in both the patient and her family. Physical examination of the younger sister was normal, but lymph nodes were not palpable and tonsils were not visible. A thymic shadow was seen on radiographs. Hemograms did not reveal lymphopenia but immunologic investigations demonstrated profound abnormalities. In the absence of any available HLA identical bone marrow donor, the patient was first treated with bovine thymosin fraction V (Goldstein et al,:' I mg/kg/day for two weeks), then with facteur thymique serique (Bach et al, '~ 10 ~,g/kg/day for two weeks) without any improvement of impaired immune functions. A transplantation of fetal liver (12 • 10~Vkg cells injected intravenously) and thymus (implanted in abdominal muscles) was performed at 6 months of life. No immunologic reconstitution or take has yet been observed ten months after transplantation. MATERIAL
AND METHODS
Isolation and enumeration of lymphocytes, sheep erythrocyte positive cells (E rosette+ or T cells), and membrane immunoglobulin-bearing lymphocytes (surface Ig + or B cells) were carried out as previously described. ~ Lymphocyte proliferation to mitogens (phytohaemagglutinin, concanavalin A, pokeweed mitogen), to antigens (tetanus toxoid), and to allogeneic cells, and cell-mediated cytotoxicity were performed in accordance with methods previously described. ~ The quinacrine fluorescence method was used to distinguish blasts from male and female donors in a mixed leukocyte reaction. In vitro maturation of patient B lymphocytes into i m m u n o g l o b u l i n containing cells and helper function of patient T lymphocytes were tested in seven-day PWM-induced cultures or cocultures, as previously described. ~ Suppressive assay was per-
The JournalqfPediat~es June 1981 formed using Con A activated T cells tested on PWMstimulated control leukocytes. I m m u n o g l o b u l i n serum concentrations were measured by immunodiffusion. Anticytomegalovirus antibodies (complement fixation and passive hemagglutination) and antibodies to tetanus toxold, to polio virus (neutralization), and to S I I I pneumococcal polysaccharide (complement fixation) were titrated after stimulation with corresponding antigens. Autoantibodies to mitochondria, red cells, nuclear antigens, smooth muscle, reticulin, and specific organs were determined by immunofluorescence. Peripheral blood T cells and monocytes were analyzed by indirect immunofluorescence using previously described mouse monoclonal antibodies to h u m a n peripheral T cells and thymocytes (OKT1, OKT3, OKT4, and OKT8 antisera) and monocytes (OKM1 antiserum). 7~ Anti-Ia (common public antigen of HLA-D borne by B lymphocytes and monocytes) monoclonal antiserum was also used? The percentage of positive cells was determined with a cytofluorograph. RESULTS Cell surface characteristics of T and B lymphocytes (Table l). The patient had a n o r m a l n u m b e r of lymphocytes with a slight decrease of the ratio of T/B lymphocytes as judged by E rosetting and m e m b r a n e i m m u n o globulin-bearing cells. H L A antigens were normally detected on the m e m b r a n e of total leukocytes (HLA-A and B) and of B lymphocytes and monocytes (HLA-D). Studies with monoclonal anti- T cell subset antibodies revealed an abnormal distribution, since all T lymphocytes were detected by the different monoclonal antibodies (OKTI, 3, 4, and 8) (Table I). T and B cell functions. In contrast with very low proliferative responses to mitogens or tetanus toxoid (whatever the dose of the stimulating agent, or the length-three to eight d a y s - o f the cultures), responses in MLR were repeatedly found to be positive (Table II). Study of cell surface markers at the sixth day of MLR, mixing leukocytes and irradiated leukocytes from a male donor, clearly showed that 85% of the blasts were T cells (E positive cells) and were q u i n a c r i n e negative. Various methods to reconstitute proliferative responses to mitogens or tetanus toxoid, including addition of FTS, thymosin fraction V, normal monocyte supernatant, desoxycytidine (10 ~M), red blood cell lysate, or lonophore R A 23 187 were attempted but all failed. Other T cell functions, including delayed skin test reactivity to PHA (3 /*g) and tetanus toxoid (after sensitization), in vitro helper function, Con A -induced suppressive function, and cellmediated cytotoxicity were absent. The serum i m m u n o globulin values were normal or transiently increased and
Volume 99 Number 2
Brief clinical and laboratory observations
263
Table II. T and B lymphocyte functions
Patient T cell proliferative responses* to Phytohemagglutin (Acpm) Concanavalin A (Acpm) Pokeweed (~cpm) Tetanus toxoid (dxcpm) Mixed leukocyte reaction Cell-mediated lympholysis (%)t Helper assay (%)~ Suppressive assay (%)w lgG (mg/ml) IgM (mg/ml) IgA (mg/ml) IgE (IU/ml) Antibodies to CMV Pneumococcal SIII Poliovirus Tetanus toxoid (IU) Autoantibodies to milochondria
5 5 5 5 36 0 0 0 1,000-2,000 80-250 10-25 40-200 1:40 0 1:8 0.1 1.10~
I
Normal values ++_] DS 150 _+ 20 200 _+ 30 50 _+ 10 25 _+ 8 42 _+_ 12 60 _+ 15 127 _+ 40 70 _+ 30 850-1,350 40-84 2-22 10-40 I: 16 0.1 _
*Results are expressed in Acpm X 10*a (minus background). J-Results are expressed as percent of total release of 51 chromium. The maximal specificrelease is given. SResults are the number of immunoglobulincontaining cells(M + G + A) at the seventh day of culture in presence of PWM for 1,000cells. Patient's or control E positive cells were added to control E negative cells (4:1). w are expressed by the percent of suppression of the number of immunoglobulin-containingcells in PWM-stimulated control culture by addition of Con A-activated T cells as compared to non-activated cells. The cells were added at the radio 1:1. monoclonal IgG and IgM were detected by ~lectrophoresis and immunoelectrophoresis of serum proteins. Antibody formation was dissociated (Table II). The patient's B lymphocytes were normally able to mature in vitro into immunoglobin (IgG, IgM, or IgA) containing cells with the help of normal T Lymphocytes. DISCUSSION The wide heterogeneity of SCID has been documented, ~ but little is known about the exact level(s) at which differentiation of T cell development can be blocked. We have identified a previously unreported type of SCID characterized by a normal n u m b e r of peripheral T lymphocytes with full expression of H L A antigens and sheep erythrocyte receptors. Such T cells were shown to proliferate in a one-way MLR, but were unable to exert any other immunologic functions, including mitogen-induced proliferation. The diagnosis of SCID has recently been extended to cases in which a subnormal number Of T cells can be found in blood, contrasting with functional impairment of cellular immunity. 4 This defect seems to be caused by a membrane abnormality, since in vitro addition of calcium ionophore A 23 187 resulted in a normal proliferative response of leukocytes. This type of defect can be excluded in our patient, whose T lymphocytes behaved as mature thymocytes unable to mature normally into functional peripheral T cells.
Indeed, there is a striking analogy between the in vitro pattern observed in our patient and the known properties of mature thymocytes (10% of thymus cells which are able to proliferate in M L R but not after stimulation with mitogens)2 The hypothesis of a blockade of T cell maturation at this stage was consistent with the T cell phenotype observed in our patient, Mature thymocytes bear the T1 antigens together with T3, T~, and T, antigens acquired during early intrathymic differentiation. ~ Normal peripheral T cells were shown to bear T1, T~, and either T4 and T, antigens, but not both T4 and T8 antigens. 7 An immaturity of the patient's T lymphocytes could explain the original immunologic abnormalities observed, including the presence of autoantibodies and the tendency to heterogeneity restriction of serum immunoglobulins as already observed in some S C I D patients? However, the exact level of the genetic lesion remains unsettled, SCID may be the result of either intrinsic dysfunction of precursor T lymphocytes 2 or of a thymus defect. TM The observations that thymic epithelial cells of the patient's sister could have been functional, since Hassal corpuscles were seen, and that treatment of our patient with thymosin or with facteur thymique serique was unable to enhance the i m m u n e responsiveness, are in favor of an intrinsic lymphocyte defect. Our observation is, to our knowledge, the first example of precise determination of the differentiation arrest in T cell development in SCID
264
Brief clinical and laboratory observations
confirmed by recent studies performed in other SCID patients." Cell surface characteristics together with analysis of cell functions show that the SCID syndrome may occur in the presence of a normal n u m b e r of circulating E rosette-forming cells. The demonstration of an abnormal accumulation in the blood of immature T cells unable to differentiate into functional T lymphOcytes illustrated the considerable interest of using monoclonal antibodies to T-cell differentiation antigens in immunodeficiencies. ADDENDUM Several months after a fetal thymus and liver transplantation a normal distribution of T cell subpopulations defined by immunofluorescence study with monoclonal antibodies was observed, whereas T cell functions were not modified. This was observed despite any evidence of prolonged take of fetal cells. Thus, the persistent T cell dysfunction cannot be anymore.correlated with an abnormal distribution of T cell subset markers. We thank M. Wyss, M.D., and M. Jeannet, M.D. (Geneva), who investigated the sister of our patient. REFERENCES 1. Fudenberg HH, Good RA, Goodman HC, Hitzig W, Kunkel H, Roitt IM, Rosen FS, Rowe DS, Seligman M, and Soothill JR: Primary immunodeficiencies, Bull WHO 45:125, 1971. 2. Griscelli C, Durandy A, Virelizier JL, Ballet J J, and Daguillard F: Selective defect of precursor T cells associated with apparently normal B lymphocytes in severe combined
The Journal q[ Pediatrics June 1981 immunodeficiency disease, J PEDIATR93:404, 1978. 3. Touraine JL, Betuel H, Souillet G, and Jeune M: Combined immunodeficiency disease associated with absence of ceil surface HLA-A and B antigens, J PEDIATR93:47, 1978. 4. Gehrz RC, McAulffe J, Linner KM, and Kersey JH: Defective membrane function in a patient with severe combined immunodeficiency disease, Clin Exp Immunol 39:344, I980. 5. Goldstein AL, Guha A, Zatz MA, and White A: Purification and biological activity of thymosin, hormone of the thymus gland, Proc Nail Acad Sci USA 69:1800, 1972. 6. Bach JF, Dardenne M, Pleau JM, and Roja J: Biochemical characterisation of a serum thymic factor, Cell Immunoi 266:55, 1977. 7. Reinherz EL, Kurg PC, Goldstein G, Levey RH, and Schlossman SF: Discrete stages of human intrathymic differentiation: analysis of normal thymocytes and leukemic iymphoblasts of T lineage, Proc Nail Acad Sci USA 77:1588, 1980. 8. Breard JM, Reinherz EL, Kung PC, Goldstein G, and Sehlossman SF: A monoclonal antibody reactive with human peripheral blood monocyte, J Immunol (in press). 9. Reinherz EL, Kung PC, Goldstein G, and Schlossman SF: A monoclonal antibody with selective reactivity with functionally mature human thymocytes and cell peripheral human T ceils, J Immnnol 123:1312, 1979. 10. Pike KW, Dosch HM, Ipp MM, and Gelfand EW: Demonstration of an intrathymic defect in a case of severe combined immunodeficiency disease, N Engl J Med 28:421, 1975. 11. Reinherz EL, Schlossman SF, and Rosen FS: Human immunodeficiency states resulting from disorders of T cell maturation and regulation, in Primary Immunoddficiencies INSERM Symposium No. 16, 1980, p 109.
Sustained depression of monocyte cytotoxicity in a boy with disseminated nontuberculous mycobacteriosis Troels Herlin,* Thomas Thelle, Knud Kragballe, Niels Borregaard, and Kristian Thestrup-Pedersen, Aarhus, Denmark
IN CHILDREN, infections with nontuberculous mycobacteria are usually localized, resulting in swelling of superficial lymph nodes? Disseminated infections caused by Mycobacterium intracellulare and the closely related Mycobacteriurn avium are very rare?, ~ M. intracellulare
occurs ubiquitously and is of low virulence. A widespread infection is, therefore, presumed to take place only in an immunodeficient host. This report describes a sustained
See related article, p. 268.
From the Department of Pediatrics, Aarhus Kommunehospital, and Departments of Dermatology and Medicine, Marselisborg Hospital, University of Aarhus. Supported by The Danish Medical Research Council (Grant No. 512-20089). *Reprint address: Departmentof Dermatology, Marselisborg Hospital, DK-8000Aarhus C, Denmark.
Abbreviations used PPD: purified protein derivative PHA: phytohemagglutinin ADCC: antibody-dependent cell-mediated cytotoxicity HMPS: hexose monophosphate shunt PMA: phorbol myristate acetate
0022-3476/81/080264 + 04500.40/0 9 1981 The C. V. Mosby Co.
intrauterine insults, it certainly reenforces the possibility of a single disease process having an effect upon multiple organs and organ systems. The etiology of this disease, including the bone marrow depression, remains obscure. There is no marked decrease in the marrow precursors and no apparent maturation arrest. Even the sideroblastic anemia seen is unusual because the red blood cells are macrocytic, rather than microcytic. The problem seems to be either an underlying metabolic or toxic process within the stromal microenvironment, or a defect in stem cell differentiation. The pathologic findings in the pancreas imply that the fibrosis may be a consequence of preceding inflammation. The paucity of iron deposition makes hemosiderosis as a cause of the pancreatic fibrosis unlikely. We are unable to implicate any known extrinsic cause of marrow suppression in our patient, either pre- or postnatally. She had a normal hver and spleen, as
B r i e f clinical and laboratory observations
26 1
contrasted with the hypoplasia of the spleen seen by Pearson et al. 1 Although our patient fits well into the syndrome described by Pearson, we believe that she may have a variant because of the very early age of onset, the severity of the bone marrow abnormalities, and the involvement of the thyroid gland. REFERENCES 1. Pearson HA, Lobel JS, Kocoshis SA, Naiman JL, Windmuller J, Lammi AT, Hoffman R, and Marsh JC: A new syndrome of refractory siderohlastic anemia with vacuolization of marrow precursors and exocrine pancreatic dysfunction, J PEDIATR95:976, 1979. 2. Schwachman H, Diamond LK, Oski FA, and Khan KT: The syndrome of pancreatic insufficiency and bone marrow dysfunction, J PEmAT~ 65:645, 1964. 3. Bodian M, Sheldon W, and Lightwood R: Congenital hypoplasia of the exocrine pancreas, Acta Pediatr 53:282, 1964.
Severe combined immunodeficiency with quantitatively normal but abnormally differentiated T lymphocytes A. Fischer,* A. Durandy, J. L. Virelizier, G. De Saint Basile, A. Lagrue, Paris, France,
E. Reinherz, S. Schlossman, Boston, Mass., and C. Griscelli, Paris France
THE SYNDROME of severe combined immunodeficiency has now been separated into various genetic, enzymologic, and immunologic subgroups? S C I D seems to result from an intrinsic T cell defect, ~ sometimes associated with membrane abnormalities$- ' or, more often, to be caused by a block in T cell differentiation.' However, the precise stages of human intrathymic differentiation of T lymphocytes remain largely ill-defined, preventing more specific studies of the cell or thymus defects in patients with SCID. We have identified previously unreported immunologic abnormalities in a patient with SCID, in w h o m peripheral T cells behaved like thymocytes blocked at a precise stage From Groupe d'Immunologie et Rhumatologie Pbdiatriques, INSERM U 132, Department of Pediatrics, Hbpital des Enfants Malades, and Division immunology, Sydney Farber Cancer Institute, and the Howard Medical School *Reprint address: Groupe de Recherches d'lmmunologie et de Rhumatologie Pediatriques, 1NSERM U 132, HOpital des Enfants Malades, 149, Rue de Sevres, 75015 Paris, France.
0022-3476/81/080261 +04500.40/0 9 1981 The C. V. Mosby Co.
of differentiation, as revealed by functional and cell surface characteristics. CASE REPORT
This girl was born under germ-~Yee conditions and was then maintained in a Trexler isolator at H6pital des Enfants-Malades. The patient was the second child of nonconsanguineous parents. Her sister died at age 22 months after repeated skin and pulmonary infections associated with severe chronic diarrhea. Postmortem histologic examination showed a profound hypoplasia of lymphoid organs including the thymus, in which, however, Abbreviations used SCID: severe combined immunodeficiency ADA: adenosine deaminase NP: nucleoside phosphorylase FTS: facteur thymique serique PHA: phytohemagglutinin Con A: concanavalin A PWM: pokeweed mitogen SIIl: polysaccharide antigen of pneumococcus MLR: mixed leukocyte reaction
262
Briefc#nical and laboratory observations
Table I. Cell surface characteristics of T and B lymphocytes Patient Lymphocytes/mm~ E rosettes (%) OKT1 ~ (%) OKT3* (%) OKT4' (%) OKT8 + (%) OKMI* (%) Surface Ig positive cells (%) Ia positive cells (%)
Normal values
1,500-2,500 35-62 52 52 52 52 17 20
2,000-3,000 71 _+ 7 60-80 60-80 35-50 25-35 5-20 5-15
15-30
5-15
Hassal corpuscles were seen. Immunologic data obtained four months before death showed a mild lymphopenia (1,000 to 1,200/mm~) with 25 to 35% T lymphocytes and 8 to 12% B lymphocytes. In vivo and in vitro lymphocyte functions were profoundly impaired. Serum concentrations of IgG, A, and M were within the normal range, and some antibodies against vaccinal antigens were detected. ADA and purine NP activities were normal in both the patient and her family. Physical examination of the younger sister was normal, but lymph nodes were not palpable and tonsils were not visible. A thymic shadow was seen on radiographs. Hemograms did not reveal lymphopenia but immunologic investigations demonstrated profound abnormalities. In the absence of any available HLA identical bone marrow donor, the patient was first treated with bovine thymosin fraction V (Goldstein et al,:' I mg/kg/day for two weeks), then with facteur thymique serique (Bach et al, '~ 10 ~,g/kg/day for two weeks) without any improvement of impaired immune functions. A transplantation of fetal liver (12 • 10~Vkg cells injected intravenously) and thymus (implanted in abdominal muscles) was performed at 6 months of life. No immunologic reconstitution or take has yet been observed ten months after transplantation. MATERIAL
AND METHODS
Isolation and enumeration of lymphocytes, sheep erythrocyte positive cells (E rosette+ or T cells), and membrane immunoglobulin-bearing lymphocytes (surface Ig + or B cells) were carried out as previously described. ~ Lymphocyte proliferation to mitogens (phytohaemagglutinin, concanavalin A, pokeweed mitogen), to antigens (tetanus toxoid), and to allogeneic cells, and cell-mediated cytotoxicity were performed in accordance with methods previously described. ~ The quinacrine fluorescence method was used to distinguish blasts from male and female donors in a mixed leukocyte reaction. In vitro maturation of patient B lymphocytes into i m m u n o g l o b u l i n containing cells and helper function of patient T lymphocytes were tested in seven-day PWM-induced cultures or cocultures, as previously described. ~ Suppressive assay was per-
The JournalqfPediat~es June 1981 formed using Con A activated T cells tested on PWMstimulated control leukocytes. I m m u n o g l o b u l i n serum concentrations were measured by immunodiffusion. Anticytomegalovirus antibodies (complement fixation and passive hemagglutination) and antibodies to tetanus toxold, to polio virus (neutralization), and to S I I I pneumococcal polysaccharide (complement fixation) were titrated after stimulation with corresponding antigens. Autoantibodies to mitochondria, red cells, nuclear antigens, smooth muscle, reticulin, and specific organs were determined by immunofluorescence. Peripheral blood T cells and monocytes were analyzed by indirect immunofluorescence using previously described mouse monoclonal antibodies to h u m a n peripheral T cells and thymocytes (OKT1, OKT3, OKT4, and OKT8 antisera) and monocytes (OKM1 antiserum). 7~ Anti-Ia (common public antigen of HLA-D borne by B lymphocytes and monocytes) monoclonal antiserum was also used? The percentage of positive cells was determined with a cytofluorograph. RESULTS Cell surface characteristics of T and B lymphocytes (Table l). The patient had a n o r m a l n u m b e r of lymphocytes with a slight decrease of the ratio of T/B lymphocytes as judged by E rosetting and m e m b r a n e i m m u n o globulin-bearing cells. H L A antigens were normally detected on the m e m b r a n e of total leukocytes (HLA-A and B) and of B lymphocytes and monocytes (HLA-D). Studies with monoclonal anti- T cell subset antibodies revealed an abnormal distribution, since all T lymphocytes were detected by the different monoclonal antibodies (OKTI, 3, 4, and 8) (Table I). T and B cell functions. In contrast with very low proliferative responses to mitogens or tetanus toxoid (whatever the dose of the stimulating agent, or the length-three to eight d a y s - o f the cultures), responses in MLR were repeatedly found to be positive (Table II). Study of cell surface markers at the sixth day of MLR, mixing leukocytes and irradiated leukocytes from a male donor, clearly showed that 85% of the blasts were T cells (E positive cells) and were q u i n a c r i n e negative. Various methods to reconstitute proliferative responses to mitogens or tetanus toxoid, including addition of FTS, thymosin fraction V, normal monocyte supernatant, desoxycytidine (10 ~M), red blood cell lysate, or lonophore R A 23 187 were attempted but all failed. Other T cell functions, including delayed skin test reactivity to PHA (3 /*g) and tetanus toxoid (after sensitization), in vitro helper function, Con A -induced suppressive function, and cellmediated cytotoxicity were absent. The serum i m m u n o globulin values were normal or transiently increased and
Volume 99 Number 2
Brief clinical and laboratory observations
263
Table II. T and B lymphocyte functions
Patient T cell proliferative responses* to Phytohemagglutin (Acpm) Concanavalin A (Acpm) Pokeweed (~cpm) Tetanus toxoid (dxcpm) Mixed leukocyte reaction Cell-mediated lympholysis (%)t Helper assay (%)~ Suppressive assay (%)w lgG (mg/ml) IgM (mg/ml) IgA (mg/ml) IgE (IU/ml) Antibodies to CMV Pneumococcal SIII Poliovirus Tetanus toxoid (IU) Autoantibodies to milochondria
5 5 5 5 36 0 0 0 1,000-2,000 80-250 10-25 40-200 1:40 0 1:8 0.1 1.10~
I
Normal values ++_] DS 150 _+ 20 200 _+ 30 50 _+ 10 25 _+ 8 42 _+_ 12 60 _+ 15 127 _+ 40 70 _+ 30 850-1,350 40-84 2-22 10-40 I: 16 0.1 _
*Results are expressed in Acpm X 10*a (minus background). J-Results are expressed as percent of total release of 51 chromium. The maximal specificrelease is given. SResults are the number of immunoglobulincontaining cells(M + G + A) at the seventh day of culture in presence of PWM for 1,000cells. Patient's or control E positive cells were added to control E negative cells (4:1). w are expressed by the percent of suppression of the number of immunoglobulin-containingcells in PWM-stimulated control culture by addition of Con A-activated T cells as compared to non-activated cells. The cells were added at the radio 1:1. monoclonal IgG and IgM were detected by ~lectrophoresis and immunoelectrophoresis of serum proteins. Antibody formation was dissociated (Table II). The patient's B lymphocytes were normally able to mature in vitro into immunoglobin (IgG, IgM, or IgA) containing cells with the help of normal T Lymphocytes. DISCUSSION The wide heterogeneity of SCID has been documented, ~ but little is known about the exact level(s) at which differentiation of T cell development can be blocked. We have identified a previously unreported type of SCID characterized by a normal n u m b e r of peripheral T lymphocytes with full expression of H L A antigens and sheep erythrocyte receptors. Such T cells were shown to proliferate in a one-way MLR, but were unable to exert any other immunologic functions, including mitogen-induced proliferation. The diagnosis of SCID has recently been extended to cases in which a subnormal number Of T cells can be found in blood, contrasting with functional impairment of cellular immunity. 4 This defect seems to be caused by a membrane abnormality, since in vitro addition of calcium ionophore A 23 187 resulted in a normal proliferative response of leukocytes. This type of defect can be excluded in our patient, whose T lymphocytes behaved as mature thymocytes unable to mature normally into functional peripheral T cells.
Indeed, there is a striking analogy between the in vitro pattern observed in our patient and the known properties of mature thymocytes (10% of thymus cells which are able to proliferate in M L R but not after stimulation with mitogens)2 The hypothesis of a blockade of T cell maturation at this stage was consistent with the T cell phenotype observed in our patient, Mature thymocytes bear the T1 antigens together with T3, T~, and T, antigens acquired during early intrathymic differentiation. ~ Normal peripheral T cells were shown to bear T1, T~, and either T4 and T, antigens, but not both T4 and T8 antigens. 7 An immaturity of the patient's T lymphocytes could explain the original immunologic abnormalities observed, including the presence of autoantibodies and the tendency to heterogeneity restriction of serum immunoglobulins as already observed in some S C I D patients? However, the exact level of the genetic lesion remains unsettled, SCID may be the result of either intrinsic dysfunction of precursor T lymphocytes 2 or of a thymus defect. TM The observations that thymic epithelial cells of the patient's sister could have been functional, since Hassal corpuscles were seen, and that treatment of our patient with thymosin or with facteur thymique serique was unable to enhance the i m m u n e responsiveness, are in favor of an intrinsic lymphocyte defect. Our observation is, to our knowledge, the first example of precise determination of the differentiation arrest in T cell development in SCID
264
Brief clinical and laboratory observations
confirmed by recent studies performed in other SCID patients." Cell surface characteristics together with analysis of cell functions show that the SCID syndrome may occur in the presence of a normal n u m b e r of circulating E rosette-forming cells. The demonstration of an abnormal accumulation in the blood of immature T cells unable to differentiate into functional T lymphOcytes illustrated the considerable interest of using monoclonal antibodies to T-cell differentiation antigens in immunodeficiencies. ADDENDUM Several months after a fetal thymus and liver transplantation a normal distribution of T cell subpopulations defined by immunofluorescence study with monoclonal antibodies was observed, whereas T cell functions were not modified. This was observed despite any evidence of prolonged take of fetal cells. Thus, the persistent T cell dysfunction cannot be anymore.correlated with an abnormal distribution of T cell subset markers. We thank M. Wyss, M.D., and M. Jeannet, M.D. (Geneva), who investigated the sister of our patient. REFERENCES 1. Fudenberg HH, Good RA, Goodman HC, Hitzig W, Kunkel H, Roitt IM, Rosen FS, Rowe DS, Seligman M, and Soothill JR: Primary immunodeficiencies, Bull WHO 45:125, 1971. 2. Griscelli C, Durandy A, Virelizier JL, Ballet J J, and Daguillard F: Selective defect of precursor T cells associated with apparently normal B lymphocytes in severe combined
The Journal q[ Pediatrics June 1981 immunodeficiency disease, J PEDIATR93:404, 1978. 3. Touraine JL, Betuel H, Souillet G, and Jeune M: Combined immunodeficiency disease associated with absence of ceil surface HLA-A and B antigens, J PEDIATR93:47, 1978. 4. Gehrz RC, McAulffe J, Linner KM, and Kersey JH: Defective membrane function in a patient with severe combined immunodeficiency disease, Clin Exp Immunol 39:344, I980. 5. Goldstein AL, Guha A, Zatz MA, and White A: Purification and biological activity of thymosin, hormone of the thymus gland, Proc Nail Acad Sci USA 69:1800, 1972. 6. Bach JF, Dardenne M, Pleau JM, and Roja J: Biochemical characterisation of a serum thymic factor, Cell Immunoi 266:55, 1977. 7. Reinherz EL, Kurg PC, Goldstein G, Levey RH, and Schlossman SF: Discrete stages of human intrathymic differentiation: analysis of normal thymocytes and leukemic iymphoblasts of T lineage, Proc Nail Acad Sci USA 77:1588, 1980. 8. Breard JM, Reinherz EL, Kung PC, Goldstein G, and Sehlossman SF: A monoclonal antibody reactive with human peripheral blood monocyte, J Immunol (in press). 9. Reinherz EL, Kung PC, Goldstein G, and Schlossman SF: A monoclonal antibody with selective reactivity with functionally mature human thymocytes and cell peripheral human T ceils, J Immnnol 123:1312, 1979. 10. Pike KW, Dosch HM, Ipp MM, and Gelfand EW: Demonstration of an intrathymic defect in a case of severe combined immunodeficiency disease, N Engl J Med 28:421, 1975. 11. Reinherz EL, Schlossman SF, and Rosen FS: Human immunodeficiency states resulting from disorders of T cell maturation and regulation, in Primary Immunoddficiencies INSERM Symposium No. 16, 1980, p 109.
Sustained depression of monocyte cytotoxicity in a boy with disseminated nontuberculous mycobacteriosis Troels Herlin,* Thomas Thelle, Knud Kragballe, Niels Borregaard, and Kristian Thestrup-Pedersen, Aarhus, Denmark
IN CHILDREN, infections with nontuberculous mycobacteria are usually localized, resulting in swelling of superficial lymph nodes? Disseminated infections caused by Mycobacterium intracellulare and the closely related Mycobacteriurn avium are very rare?, ~ M. intracellulare
occurs ubiquitously and is of low virulence. A widespread infection is, therefore, presumed to take place only in an immunodeficient host. This report describes a sustained
See related article, p. 268.
From the Department of Pediatrics, Aarhus Kommunehospital, and Departments of Dermatology and Medicine, Marselisborg Hospital, University of Aarhus. Supported by The Danish Medical Research Council (Grant No. 512-20089). *Reprint address: Departmentof Dermatology, Marselisborg Hospital, DK-8000Aarhus C, Denmark.
Abbreviations used PPD: purified protein derivative PHA: phytohemagglutinin ADCC: antibody-dependent cell-mediated cytotoxicity HMPS: hexose monophosphate shunt PMA: phorbol myristate acetate
0022-3476/81/080264 + 04500.40/0 9 1981 The C. V. Mosby Co.