- Email: [email protected]
Selective defect of OKT4+ T-lymphocytes in severe immunodeficiency
588
Clinical and laboratory observations
EBV infection. That 100% of cultures of non-T cells repeatedly showed transformed cells emphasizes that these patients had chronic active EBV infection. The reason for the failure in these children to eradicate EBV infection is not known. Recently, it has been proposed that T cell-specific killing is important for the eradication of EBV infection. 6,'~ However, our results in these children indicate that the degree of EBV-specific cytotoxic T cell activity bears no direct relationship to the level of EBV virocytemia. Patient I had no detectable EBV-specific cytotoxic T cell activity, and patient 2 had a normal level of activity; nevertheless, patient 2 had 20 times more EBV virocytes. Equally surprising was the finding that despite a normal level of EBV-specific cytotoxic T cell activity, patient 2 continued to have a high level of EBV virocytemia more than 16 months after the onset of EBV infection. Our finding that active EBV infection can persist despite normal EBV-specific T cell cytotoxicity indicates that this cytotoxic mechanism is not sufficient for control of EBV infection. It is likely that multiple defects in T or B cell immunity are responsible for the failure in these children to eradicate EBV infection. Further studies are needed to sort out the essential mechanisms for clearing EBVinfected cells.
The Journal of Pediatrics October 1983
REFERENCES 1. Chang RS: Infectious mononucleosis. Boston, 1980, GK Hall Medical Publishers, pp 101-112. 2. Bar RS, DeLor C J, Clausen KP, Hurtubise P, Henle W, Hewetson JR: Fatal infectious mononucleosis in a family. N Engl J Med 290:363, 1974. 3. Virelier J-L, Lenoir G, Griscelli C: Persistent Epstein-Barr virus infection in a child with hypergammaglobulinaeimia and immunoblastic proliferation associated with a selective defect in immune interferon secretion. Lancet 2:231, 1978. 4, Tobi M, Ravid Z, Feldman-Weiss V, Ben-Chetrit E, Morag A, Chowers I, Michaeli Y, Shalit M, Knobler H: Prolonged atypical illness associated with serological evidence of persistent Epstein-Barr virus infection. Lancet 1:61, 1982. 5. Ballow M, Seeley J, Purtilo D, St Onge S, Sakamoto K, Rickles F: Familial chronic mononucleosis. 97:821, 1982. 6. Tsoukas CD, Fox RI, Slovin SF, Carson DA, Pellegrino M, Fong S, Pasquali JL, Ferrone S, Kung P, Vaughan J H : T lymphocyte-mediated cytotoxicity against autologous EBV genome-bearing B cells. J Immunol 126:1742, 1981. 7. Rocchi G, de Felici A, Ragona G, Heinz A: Quantitative evaluation of Epstein-Barr virus-infected mononuclear peripheral blood leukocytes in infectious mononucleosis. N Engl J Med 296:132, 1977. 8. Misko IS, Moss D J, Pope JH: HLA antigen-regulated restriction of T lymphocyte cytotoxicity to Epstein-Barr virus. Proc Natl Aead Sci USA 77:4247, 1980. 9. Ho M: The lymphocyte in infections with Epstein-Barr virus and cytomegalovirus. J Infect Dis 143:857, 1981.
We thank Ms. Carol Dahlstrom for typing and editorial help, and Ms. Nenita Arias for technological assistance.
Selective defect o f OKT4 + T-lymphocytes in severe immunodeficiency S. Tsuchiya, M.D., M. Minegishi, M.D., M. Imaizumi, M.D., S. Nakae, M.D., S. Tamura, M.D., T. Konno, M.D., and K. Tada, M.D. Sendal, Japan
RECENT ADVANCES in the characterization and identification of human T cell subsets by cell surface makers have made possible the further analysis of the cellular mechanisms of immunodeficiency caused by abnormality developed T cells. 1 In relation to the two major subsets of peripheral T cells, namely helper/inducer ( O K T 4 +) and
From the Department of Pediatrics and Division of Blood Transfusion, Tohoku University School ~f,~Medicine. Supported in part by a grant from the Ministry of Health and Welfare, Japan. Reprint requests: T. Konno, M.D., Department of Pediatrics, Tohoku University School of Medicine, 1-I Seiryo-machi, Sendai 980, Japan.
suppressor/cytotoxic (OKT8 +) T cells, an acquired deficiency of O K T 4 § T cells among adult homosexual men and a primary deficiency of O K T 8 § T cells of a 6-month-old boy and an 18-month-old girl have been reported. 24 CMV Con A PHA PWM
Cytomegalovirus Concanavalin A Phytohemagglutinin Pokeweed mitogen
We observed a 5-month-old boy with a deficiency of O K T 4 + T cells whose clinical manifestations of cellular immunodeficiency had been evident as early as his second month of age, probably because of primary deficiency of O K T 4 + T cells.
Volume 103 Number 4
Clinical and laboratory observations
T a b l e I. Cell surface characteristics of peripheral mononu-
Table II. T- and B-lymphocyte functions
clear cells
j Lymphocytes/mm E rosettes (%) OKT3 + (%) OKT4 + (%) OKT6 + (%) OKT8 + (%) Surface lg positive ceils (%) Peroxidase positive cells (%)
Patient |200 to 2800 56 to 65 52 1 0 53 6 25 to 35
Patient
Normal values 4000 60 60 30 0 25 5 20
to to to to
6000 75 75 50
to 35 to 10 to 30
CASE REPOR T This infant boy was born at 41 weeks gestation; his birth weight was 3370 gin, and the Apgar score at five minutes was 6. He was the first child of parents who were first cousins. Since 2 months of age, he had had intractable diarrhea containing blood, and been treated at Hachinohe City Hospital without any improvement. At age 5 months, he was admitted to Tohoku University Hospital. On admission, physical examination showed a malnourished pale infant weighing 5525 gin. He had neither skin rash, palpable lymph nodes, nor hepatosplenomegaly. Laboratory examinations showed the following values: hemoglobin 9.2 mg/dl, hematocrit 29%, and WBC 8500/ram 3, with 51% neutrophils, 34% lymphocytes, 3% eosinophils, 12% monocytes, and 1% basophils. Serum total protein was 8.2 gm/dl, with 43.5% albumin, 4.8% a~globulin, 13.2% az-globulin, 4.6% /3-globulin, and 33.9% "yglobulin. SGOT activity was 29 IU, SGPT t5 IU, and LDH 437 IU. Serum IgG concentration was 3800 mg/dl, containing monoclonal IgG with X light chain; IgM was 79 mg/dl; IgA was undetectable. A thymic shadow was not seen on chest radiograph. Endoscopic examination revealed a cobblestone-fike appearance in the descending colon, suggesting Crohn disease, but histologic studies did not show any noncaseating granulomas, but only inflammatory changes. Subpopulations of peripheral T- and B-lymphocytes were within normal limits (E rossette+ ceils 59%, surface Ig + cells 6%). At age 6 months, the patient had dyspnea with peripheral cyanosis. Interstitial pneumonia was identified on Chest radiograph, and a needle lung biopsy proved the presence of Pneumocystis carinii pneumonia. Immunologic investigation demonstrated distinct abnormalities in the T-lymphocytes, as described below;adenosine deaminase and purine nucleoside phosphorylase of red blood cells were normal. Cultured thymie epithelium was implanted intraperitoneally, resulting in no immunologic reconstitution. Despite trimethoprim-sulfamethoxazole and pentamidine therapy, respiratory distress progressed, and the patient required artificial respiration. He died at age 8 months. Autopsy examination showed a profound hypoplasia of the thymus without Hassal corpuscles and lymphoid organs, and interstitial pneumonia without cytoplasmic inclusions or giant cells.
589
T cell proliferative responses* Phytohemagglutinin (Acpm) Concanavalin A (Acpm) Pokeweed (Acpm) Mixed-leukocyte reaction (Acpm) Pokeweed-induced lg productionS" Bp Tp Tp "l" Bp Tp + BF TF + BF T~ + Bp BF TF Antibodies to CMV Complement fixation titer Early antigen Membrane antigen Anti-B isohemagglutination titer Natural killer activity~
2 6 0.2 3
to to to to
Normal values +- 1 SD 875 940 220 385
4.6 20 3 15
4 1 4 l0 370 68 2 1
_+ 100 -+ 150 _+ 60 _+ 80
---------
1 : 256 1:256 1 : 16 1 : 16
---1 : 16 to 1:64
4
5to 15
BF, Tv, Father's B and T ceils; Bp, Tv, patient's cells. *Results expressedin Acpm• 10 2 (minus background). "['Resultsexpressedin ng/ml x 10-~ lgG secretedin culture supernatantsat seventh day of culture in presenceof PWM. Patient's or father's E negative ceils (2 X 105)werecultured alone or with mitomycinC-treated patient's or father's E positiveceils (5 X 10s). ~Results expressedas spontaneousnatural killer activity in Iytic units/106 cells. were carried out as previously described? Blastogenic stimulation of lymphocytes by phytohemagglutinin, concanavalin A, pokeweed mitogen, and allogeneic cells were performed
in
accordance
with
methods
previously
described? In vitro induction of B cells into Ig-producing cells, and helper function of T cells were examined in seven-day cocultures stimulated by P W M as previously described. 6 Virus isolation and serologic tests for cytomegalovirus were performed as reported previously, 7 Natural killer activity against K562 target cells was performed as previously described? Peripheral blood T cells were analyzed by indirect immunofluorescence with monoclonal antibodies to h u m a n peripheral T cells and thymocytes (OKT3, OKT4, OKT6, and OKT8
antibodies; Ortho
Pharmaceutical, Raritan, N J)? C o m p l e m e n t - d e p e n d e n t lysis of T-lymphocytes with patient serum was carried out according to the method o f M o r i m o t o et al. 9 RESULTS
MATERIALS
AND
METHODS
Cell surface c h a r a c t e r i s t i c s o f peripheral l y m p h o c y t e s .
Isolation of peripheral blood lymphocytes and identifi-
The patient had a normal n u m b e r of lymphocytes, E
cation of sheep erythrocyte positive cells (E rosette + cells) and m e m b r a n e Ig bearing lymphocytes (surface Ig + Cells)
rosette + cells (T cells), and surface Ig + cells (B cells) (Table I). Monoclonal increase o f surface 3'-X positive B
590
Clinical and laboratory observations
cells was not demonstrated despite the presence of monoclonal IgG with X light chain in serum. Analysis of T cell subsets with monoclonal antibodies revealed an abnormal distribution with an absence of OKT4 + T cells; OKT3 and OKT8 antigens were expressed as 52% and 53%, respectively, on peripheral mononuclear cells from the patient. Cytotoxic assay showed that 20% to 26% of normal T cells examined were killed with the various dilutions of the patient's serum. However, the existence of antibodies that react selectively with the OKT4 + T cell subset in patient serum was not evident. Indirect immunofluorescence studies with monoclonal antibodies showed that after lysis of ,normal T cells (OKT4 + 63%, OKT8 + 35%) with the patient's serum and complement, 61% of the residual T cell population was OKT4 +, whereas with control serum it was 69%, with OKT4 antibody 27%, and with complement only 6O%. H L A typing revealed that the patient had A2w31, B5, the father A2w31, B5, and the mother A9w31, B5. T and B cell functions. Proliferative responses to PHA, Con A, and PWM of peripheral lymphocytes from the patient were very low, and mixed-lymphocyte reaction to allogeneic lymphocytes was also diminished (Table II). In PWM-induced Ig synthesis in cocultures, the patient's T cells induced Ig production in neither the patient's nor the father's B cells. The patient's T cells did not suppress Ig production in Cocultures of the father's T and B cells. The patient's B cells produced Ig in combination with the father's T cells, one fifth the amount of that from his father's B cells. Skin tests for dinitrochlorobenzene and Candida yielded negative results. Natural killer activity of the patient's lymphocytes was normal. A serum antibody titer of CMV and anti-B isohemagglutinin was demonstrated; CMV was isolated from urine. Karyotype analysis performed with PHA and crude culture supernatant of interleukin 2 showed 46,XY. The PHA-induced blasts were not examined for OKT4 expression. DISCUSSION The clinical and laboratory findings in this patient, namely, intractable diarrhea with failure to thrive starting as early as his second month of age, P. carinii pneumonia, and markedly diminished blastogeneic responses to mitogens and to allogeneic cells despite the presence of E rosette + cells, appear to be characteristic of primary ceil-mediated immunodeficiency. In view of the faulty cellular differentiation in this case, primary immunodeficiency was characterized by a normal number of periPheral T cells with expression of sheep erythrocyte receptors and HLA antigens but without OKT4 antigen detected by a monocional antibody The OKT4 + T cells are one of the two major T-lymphocyte subpopulations competent for
The Journal of Pediatrics October 1983
helper and inducer functions, in contrast to the OKT8 + T cells, which are for suppressor and eytotoxic functions. The deficiency of OKT4 + T cells could be substantiated by the absence of the helper function in the patient's T cells in PWM-induced in vitro Ig production studies. However, this deficiency could not explain hypergammaglobulinemia with monoclonal IgG production in the patient. In spite of the presence of OKT8 § ceils, the patient's T-lymphocytes (Tp), which were not activated by Con A, showed no suppressive effect on Ig production in cocultures with the father's T and B cells (TF, BF; 2280 ng/ml in TF + TB vs 2510 ng/ml in T r + BF + Tp). This may imply a functional defect of T cells phenotypically defined as cytotoxic/suppresso r cells. However, it remains t o be ascertained whether the suppressor cell activities were actually deficient in the patient. A similar case of immunologic dysfunction in severe combined immunodeficiency has been reported. ~~In that case, serum Ig values were elevated, with monoclonal IgG and IgM, but PWM-induced in vitro Ig production studies romaled no helper function of T cells despite the presence of OKT4 § T ceils. A deficiency of helper T cells has been recognized in patients with acute and chronic graft-versus-host disease after bone marrow transplantation. H In Our patient, maternal lymphocytes defined by H L A typing and karyotype analysis could not be identified. This finding would seem to exclude the possibility of graft-versus-host disease caused by intrauterine lymphoid engraftment. An acquired type of OKT4 + T cell deficiency has been found among homosexual men. Such a disease also is considered to be caused by severe CMV infection; T cell imbalance in favor of suppressor/cytotoxic T cells had been suggested as a general phenomenon in viral disease, including CMV infection J: Our patient shed C M V in urine and had antibodies against CMV, indicating that he had had CMV infection. However, no cerebral Or visceral involvement suggesting congenital CMV infection was found. No cytomegalic inclusions were revealed i n the biopsied lung tissue or colonic mucosa. Autopsy did not show any evidence of cytomegalic inclusion disease. I n view of such negative findings, the CMV infection seen in our patient seems to be an acquired infection commonly occurring in early infancy, and not a cause'of the primary digease: The parents of our patient were consanguineous, indicating a possible hereditary cause for the disease, although no other contributory family history was found, The majority of patients with severe combined immunodeficiency and thymic hypoplasia have persistent lymphopenia. ~3However, in our patient lymphocyte counts varied from 1200 to 2800. It is not clear how an almost normal
Volume 103 Number 4
number of lymphocytes was present in a patient with thymic hypoplasia and absence of Hassal corpuscles. The pathogenesis of this disease remains unknown, but our observation is, to our knowledge, the first example demonstrating a selective deficiency of O K T 4 + T cells (helper/inducer ceils) in primary immunodeficiency. Further analysis of cellular and biochemical mechanisms of immunodeficiency might enable us to determine the precise role of immunoregulatory T cells in vivo. We thank Dr. M. Kudo for the referral of the patient, and Dr. K. Kamagai for measurement of natural killer activity. REFERENCES
1. Reinherz EL, Schlossman SF: The differentiation and function of human T lymphocytes. Cell 19:821, 1980. 2. Gottlieb MS, Sehroff R, Sehanker HM, Weisman JD, Fan PT, Wolf RA, Saxon A: Pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men. N Engl J Med 305:1425, 1981. 3. Phan-dinh-tuy F, Durandy A, Grischelli C, Bach M: T-cell subset analysis by monoclonal antibodies in primary immunodeficiencies. Scand J Immunol 14:193, 1981. 4. Font~n G, De La Concha EG, Rodriguez MCG, Zabay JM, Carrasco S, Alba J, Pascual-Salcedo D, Ojeda JA: Severe combined immunodeficiency with disbalance and functional abnormalities in the T-lymphocyte subsets. Clin Immunol lmmunopathol 24:432, 1982. 5. Tsuchiya S, Konno T, Tada K, Ono Y: Epstein-Barr virusinduced lymphoblastoid ceil lines from patients with primary immunodeficiency diseases. Seand J Immunol 11:155, 1980.
Clinical and laboratory observations
59 1
6. Tsuchiya S, Nakae S, Konno T, Tada K, Ono Y: Estimation of B cells transformed by Epstein-Barr virus in patients with congenital agammaglobulinemia. Tohoku J Exp Med 135:379, 1981. 7. Numazaki Y, Oshima T, Tanaka A, Konno T, Tazawa Y, Karita M, Ishii A, Hirota K, Watabe N, lshida N: Demonstration of IgG EA (early antigen) and IgM MA (membrane antigen) antibodies in CMV infection of healthy infants and in those with liver disease. 3 PEt)~ATr~97:545, 1980. 8. Kumagai K, Itoh K, Suzuki R, Hinuma S, Saitoh F: Studies of murine large granular lymphocytes, I. Identification as effector cells in NK and K cytotoxicities. J Immunol 129:388, 1982. 9. Morimoto C, Reinherz EL, Abe T, Homma M, Schlossman SF: Characteristics of anti-T-cell antibodies in systemic lupus erythematosus: Evidence for selective reactivity with normal suppressor cells defined by monoclonal antibodies. Clin lmmunol lmmunopathol 16:474, 1980. 10. Fischer A, Durandy A, Virelizier JL, De Saint Basile G, Lagrue A, Reinherz E, Schlossman S, Grischelli C: Severe combined immunodeficiency with quantitatively normal but abnormally differentiated T lymphocytes. J PEDrATR99:261, 1981. 11. Freidrich W, O'Reilly R J, Koziner B, Gerhard DR Jr, Good RA, Evans FL: T-lymphocyte reconstitution in recipients of bone marrow transplants with and without GVHD: Imbalance of T-cell subpopulations having unique regulatory and cognitive functions. Blood 59:696, 1982. 12. Bach M-A, Bach J-F.: The use of monoclonal anti-T cell antibodies to study T cell imbalance in human disease. Clin Exp Immunol 45:499, 1981. 13. lmmun0deficiency. WHO Tech Rep Ser No. 630, 1978.
Neonatal transient thyrotoxicosis resulting from maternal TSH-binding inhibitor immunoglobulins Hideki Yagi, M.D., Masao Takeuchi, M.D., Kanji Nagashima, M.D., Shigeyoshi Suzuki, M.D., Takayoshi Kuroume, M.D., Yasuhiro Iida, M.D., Junji Konishi, M.D., and Keigo Endo, M.D. Maebashi Gunma and Sakyoku Kyoto, Japan
TRANSIENT THYROTOXICOSIS is a serious, life-threatening disease in neonates? Its pathogenesis has not been fully established, but the placental transfer of thyroid-
From the Departments of Pediatrics, Maebashi Red Cross Hospital and Gunma University School of Medicine, and the Department of Nuclear Medicine, Kyoto University School of Medicine. Reprint requests: Kanji Nagashima, M.D., Department of Pediatrics, Gunma University School of Medicine, 3-29-22. Showamachi, Maebashi-shi, Gunma-ken, Japan.
stimulating immunoglobulins such as L A T S , L A T S protector, and thyroid-stimulating antibody from the mother with Graves disease is considered the main cause. 2-4 Clinical signs usually subside with the disappearance of thyroid-stimulating IgG in the serum of the neonate. 2,3 LATS MCHA TBII TGHA TSAb
Long-acting thyroid stimulator Thyroid microsome hemagglutination antibody TSH-binding inhibitor immunoglobulins Thyroglobulin hemagglutination antibody Thyroid-stimulating antibody
Clinical and laboratory observations
EBV infection. That 100% of cultures of non-T cells repeatedly showed transformed cells emphasizes that these patients had chronic active EBV infection. The reason for the failure in these children to eradicate EBV infection is not known. Recently, it has been proposed that T cell-specific killing is important for the eradication of EBV infection. 6,'~ However, our results in these children indicate that the degree of EBV-specific cytotoxic T cell activity bears no direct relationship to the level of EBV virocytemia. Patient I had no detectable EBV-specific cytotoxic T cell activity, and patient 2 had a normal level of activity; nevertheless, patient 2 had 20 times more EBV virocytes. Equally surprising was the finding that despite a normal level of EBV-specific cytotoxic T cell activity, patient 2 continued to have a high level of EBV virocytemia more than 16 months after the onset of EBV infection. Our finding that active EBV infection can persist despite normal EBV-specific T cell cytotoxicity indicates that this cytotoxic mechanism is not sufficient for control of EBV infection. It is likely that multiple defects in T or B cell immunity are responsible for the failure in these children to eradicate EBV infection. Further studies are needed to sort out the essential mechanisms for clearing EBVinfected cells.
The Journal of Pediatrics October 1983
REFERENCES 1. Chang RS: Infectious mononucleosis. Boston, 1980, GK Hall Medical Publishers, pp 101-112. 2. Bar RS, DeLor C J, Clausen KP, Hurtubise P, Henle W, Hewetson JR: Fatal infectious mononucleosis in a family. N Engl J Med 290:363, 1974. 3. Virelier J-L, Lenoir G, Griscelli C: Persistent Epstein-Barr virus infection in a child with hypergammaglobulinaeimia and immunoblastic proliferation associated with a selective defect in immune interferon secretion. Lancet 2:231, 1978. 4, Tobi M, Ravid Z, Feldman-Weiss V, Ben-Chetrit E, Morag A, Chowers I, Michaeli Y, Shalit M, Knobler H: Prolonged atypical illness associated with serological evidence of persistent Epstein-Barr virus infection. Lancet 1:61, 1982. 5. Ballow M, Seeley J, Purtilo D, St Onge S, Sakamoto K, Rickles F: Familial chronic mononucleosis. 97:821, 1982. 6. Tsoukas CD, Fox RI, Slovin SF, Carson DA, Pellegrino M, Fong S, Pasquali JL, Ferrone S, Kung P, Vaughan J H : T lymphocyte-mediated cytotoxicity against autologous EBV genome-bearing B cells. J Immunol 126:1742, 1981. 7. Rocchi G, de Felici A, Ragona G, Heinz A: Quantitative evaluation of Epstein-Barr virus-infected mononuclear peripheral blood leukocytes in infectious mononucleosis. N Engl J Med 296:132, 1977. 8. Misko IS, Moss D J, Pope JH: HLA antigen-regulated restriction of T lymphocyte cytotoxicity to Epstein-Barr virus. Proc Natl Aead Sci USA 77:4247, 1980. 9. Ho M: The lymphocyte in infections with Epstein-Barr virus and cytomegalovirus. J Infect Dis 143:857, 1981.
We thank Ms. Carol Dahlstrom for typing and editorial help, and Ms. Nenita Arias for technological assistance.
Selective defect o f OKT4 + T-lymphocytes in severe immunodeficiency S. Tsuchiya, M.D., M. Minegishi, M.D., M. Imaizumi, M.D., S. Nakae, M.D., S. Tamura, M.D., T. Konno, M.D., and K. Tada, M.D. Sendal, Japan
RECENT ADVANCES in the characterization and identification of human T cell subsets by cell surface makers have made possible the further analysis of the cellular mechanisms of immunodeficiency caused by abnormality developed T cells. 1 In relation to the two major subsets of peripheral T cells, namely helper/inducer ( O K T 4 +) and
From the Department of Pediatrics and Division of Blood Transfusion, Tohoku University School ~f,~Medicine. Supported in part by a grant from the Ministry of Health and Welfare, Japan. Reprint requests: T. Konno, M.D., Department of Pediatrics, Tohoku University School of Medicine, 1-I Seiryo-machi, Sendai 980, Japan.
suppressor/cytotoxic (OKT8 +) T cells, an acquired deficiency of O K T 4 § T cells among adult homosexual men and a primary deficiency of O K T 8 § T cells of a 6-month-old boy and an 18-month-old girl have been reported. 24 CMV Con A PHA PWM
Cytomegalovirus Concanavalin A Phytohemagglutinin Pokeweed mitogen
We observed a 5-month-old boy with a deficiency of O K T 4 + T cells whose clinical manifestations of cellular immunodeficiency had been evident as early as his second month of age, probably because of primary deficiency of O K T 4 + T cells.
Volume 103 Number 4
Clinical and laboratory observations
T a b l e I. Cell surface characteristics of peripheral mononu-
Table II. T- and B-lymphocyte functions
clear cells
j Lymphocytes/mm E rosettes (%) OKT3 + (%) OKT4 + (%) OKT6 + (%) OKT8 + (%) Surface lg positive ceils (%) Peroxidase positive cells (%)
Patient |200 to 2800 56 to 65 52 1 0 53 6 25 to 35
Patient
Normal values 4000 60 60 30 0 25 5 20
to to to to
6000 75 75 50
to 35 to 10 to 30
CASE REPOR T This infant boy was born at 41 weeks gestation; his birth weight was 3370 gin, and the Apgar score at five minutes was 6. He was the first child of parents who were first cousins. Since 2 months of age, he had had intractable diarrhea containing blood, and been treated at Hachinohe City Hospital without any improvement. At age 5 months, he was admitted to Tohoku University Hospital. On admission, physical examination showed a malnourished pale infant weighing 5525 gin. He had neither skin rash, palpable lymph nodes, nor hepatosplenomegaly. Laboratory examinations showed the following values: hemoglobin 9.2 mg/dl, hematocrit 29%, and WBC 8500/ram 3, with 51% neutrophils, 34% lymphocytes, 3% eosinophils, 12% monocytes, and 1% basophils. Serum total protein was 8.2 gm/dl, with 43.5% albumin, 4.8% a~globulin, 13.2% az-globulin, 4.6% /3-globulin, and 33.9% "yglobulin. SGOT activity was 29 IU, SGPT t5 IU, and LDH 437 IU. Serum IgG concentration was 3800 mg/dl, containing monoclonal IgG with X light chain; IgM was 79 mg/dl; IgA was undetectable. A thymic shadow was not seen on chest radiograph. Endoscopic examination revealed a cobblestone-fike appearance in the descending colon, suggesting Crohn disease, but histologic studies did not show any noncaseating granulomas, but only inflammatory changes. Subpopulations of peripheral T- and B-lymphocytes were within normal limits (E rossette+ ceils 59%, surface Ig + cells 6%). At age 6 months, the patient had dyspnea with peripheral cyanosis. Interstitial pneumonia was identified on Chest radiograph, and a needle lung biopsy proved the presence of Pneumocystis carinii pneumonia. Immunologic investigation demonstrated distinct abnormalities in the T-lymphocytes, as described below;adenosine deaminase and purine nucleoside phosphorylase of red blood cells were normal. Cultured thymie epithelium was implanted intraperitoneally, resulting in no immunologic reconstitution. Despite trimethoprim-sulfamethoxazole and pentamidine therapy, respiratory distress progressed, and the patient required artificial respiration. He died at age 8 months. Autopsy examination showed a profound hypoplasia of the thymus without Hassal corpuscles and lymphoid organs, and interstitial pneumonia without cytoplasmic inclusions or giant cells.
589
T cell proliferative responses* Phytohemagglutinin (Acpm) Concanavalin A (Acpm) Pokeweed (Acpm) Mixed-leukocyte reaction (Acpm) Pokeweed-induced lg productionS" Bp Tp Tp "l" Bp Tp + BF TF + BF T~ + Bp BF TF Antibodies to CMV Complement fixation titer Early antigen Membrane antigen Anti-B isohemagglutination titer Natural killer activity~
2 6 0.2 3
to to to to
Normal values +- 1 SD 875 940 220 385
4.6 20 3 15
4 1 4 l0 370 68 2 1
_+ 100 -+ 150 _+ 60 _+ 80
---------
1 : 256 1:256 1 : 16 1 : 16
---1 : 16 to 1:64
4
5to 15
BF, Tv, Father's B and T ceils; Bp, Tv, patient's cells. *Results expressedin Acpm• 10 2 (minus background). "['Resultsexpressedin ng/ml x 10-~ lgG secretedin culture supernatantsat seventh day of culture in presenceof PWM. Patient's or father's E negative ceils (2 X 105)werecultured alone or with mitomycinC-treated patient's or father's E positiveceils (5 X 10s). ~Results expressedas spontaneousnatural killer activity in Iytic units/106 cells. were carried out as previously described? Blastogenic stimulation of lymphocytes by phytohemagglutinin, concanavalin A, pokeweed mitogen, and allogeneic cells were performed
in
accordance
with
methods
previously
described? In vitro induction of B cells into Ig-producing cells, and helper function of T cells were examined in seven-day cocultures stimulated by P W M as previously described. 6 Virus isolation and serologic tests for cytomegalovirus were performed as reported previously, 7 Natural killer activity against K562 target cells was performed as previously described? Peripheral blood T cells were analyzed by indirect immunofluorescence with monoclonal antibodies to h u m a n peripheral T cells and thymocytes (OKT3, OKT4, OKT6, and OKT8
antibodies; Ortho
Pharmaceutical, Raritan, N J)? C o m p l e m e n t - d e p e n d e n t lysis of T-lymphocytes with patient serum was carried out according to the method o f M o r i m o t o et al. 9 RESULTS
MATERIALS
AND
METHODS
Cell surface c h a r a c t e r i s t i c s o f peripheral l y m p h o c y t e s .
Isolation of peripheral blood lymphocytes and identifi-
The patient had a normal n u m b e r of lymphocytes, E
cation of sheep erythrocyte positive cells (E rosette + cells) and m e m b r a n e Ig bearing lymphocytes (surface Ig + Cells)
rosette + cells (T cells), and surface Ig + cells (B cells) (Table I). Monoclonal increase o f surface 3'-X positive B
590
Clinical and laboratory observations
cells was not demonstrated despite the presence of monoclonal IgG with X light chain in serum. Analysis of T cell subsets with monoclonal antibodies revealed an abnormal distribution with an absence of OKT4 + T cells; OKT3 and OKT8 antigens were expressed as 52% and 53%, respectively, on peripheral mononuclear cells from the patient. Cytotoxic assay showed that 20% to 26% of normal T cells examined were killed with the various dilutions of the patient's serum. However, the existence of antibodies that react selectively with the OKT4 + T cell subset in patient serum was not evident. Indirect immunofluorescence studies with monoclonal antibodies showed that after lysis of ,normal T cells (OKT4 + 63%, OKT8 + 35%) with the patient's serum and complement, 61% of the residual T cell population was OKT4 +, whereas with control serum it was 69%, with OKT4 antibody 27%, and with complement only 6O%. H L A typing revealed that the patient had A2w31, B5, the father A2w31, B5, and the mother A9w31, B5. T and B cell functions. Proliferative responses to PHA, Con A, and PWM of peripheral lymphocytes from the patient were very low, and mixed-lymphocyte reaction to allogeneic lymphocytes was also diminished (Table II). In PWM-induced Ig synthesis in cocultures, the patient's T cells induced Ig production in neither the patient's nor the father's B cells. The patient's T cells did not suppress Ig production in Cocultures of the father's T and B cells. The patient's B cells produced Ig in combination with the father's T cells, one fifth the amount of that from his father's B cells. Skin tests for dinitrochlorobenzene and Candida yielded negative results. Natural killer activity of the patient's lymphocytes was normal. A serum antibody titer of CMV and anti-B isohemagglutinin was demonstrated; CMV was isolated from urine. Karyotype analysis performed with PHA and crude culture supernatant of interleukin 2 showed 46,XY. The PHA-induced blasts were not examined for OKT4 expression. DISCUSSION The clinical and laboratory findings in this patient, namely, intractable diarrhea with failure to thrive starting as early as his second month of age, P. carinii pneumonia, and markedly diminished blastogeneic responses to mitogens and to allogeneic cells despite the presence of E rosette + cells, appear to be characteristic of primary ceil-mediated immunodeficiency. In view of the faulty cellular differentiation in this case, primary immunodeficiency was characterized by a normal number of periPheral T cells with expression of sheep erythrocyte receptors and HLA antigens but without OKT4 antigen detected by a monocional antibody The OKT4 + T cells are one of the two major T-lymphocyte subpopulations competent for
The Journal of Pediatrics October 1983
helper and inducer functions, in contrast to the OKT8 + T cells, which are for suppressor and eytotoxic functions. The deficiency of OKT4 + T cells could be substantiated by the absence of the helper function in the patient's T cells in PWM-induced in vitro Ig production studies. However, this deficiency could not explain hypergammaglobulinemia with monoclonal IgG production in the patient. In spite of the presence of OKT8 § ceils, the patient's T-lymphocytes (Tp), which were not activated by Con A, showed no suppressive effect on Ig production in cocultures with the father's T and B cells (TF, BF; 2280 ng/ml in TF + TB vs 2510 ng/ml in T r + BF + Tp). This may imply a functional defect of T cells phenotypically defined as cytotoxic/suppresso r cells. However, it remains t o be ascertained whether the suppressor cell activities were actually deficient in the patient. A similar case of immunologic dysfunction in severe combined immunodeficiency has been reported. ~~In that case, serum Ig values were elevated, with monoclonal IgG and IgM, but PWM-induced in vitro Ig production studies romaled no helper function of T cells despite the presence of OKT4 § T ceils. A deficiency of helper T cells has been recognized in patients with acute and chronic graft-versus-host disease after bone marrow transplantation. H In Our patient, maternal lymphocytes defined by H L A typing and karyotype analysis could not be identified. This finding would seem to exclude the possibility of graft-versus-host disease caused by intrauterine lymphoid engraftment. An acquired type of OKT4 + T cell deficiency has been found among homosexual men. Such a disease also is considered to be caused by severe CMV infection; T cell imbalance in favor of suppressor/cytotoxic T cells had been suggested as a general phenomenon in viral disease, including CMV infection J: Our patient shed C M V in urine and had antibodies against CMV, indicating that he had had CMV infection. However, no cerebral Or visceral involvement suggesting congenital CMV infection was found. No cytomegalic inclusions were revealed i n the biopsied lung tissue or colonic mucosa. Autopsy did not show any evidence of cytomegalic inclusion disease. I n view of such negative findings, the CMV infection seen in our patient seems to be an acquired infection commonly occurring in early infancy, and not a cause'of the primary digease: The parents of our patient were consanguineous, indicating a possible hereditary cause for the disease, although no other contributory family history was found, The majority of patients with severe combined immunodeficiency and thymic hypoplasia have persistent lymphopenia. ~3However, in our patient lymphocyte counts varied from 1200 to 2800. It is not clear how an almost normal
Volume 103 Number 4
number of lymphocytes was present in a patient with thymic hypoplasia and absence of Hassal corpuscles. The pathogenesis of this disease remains unknown, but our observation is, to our knowledge, the first example demonstrating a selective deficiency of O K T 4 + T cells (helper/inducer ceils) in primary immunodeficiency. Further analysis of cellular and biochemical mechanisms of immunodeficiency might enable us to determine the precise role of immunoregulatory T cells in vivo. We thank Dr. M. Kudo for the referral of the patient, and Dr. K. Kamagai for measurement of natural killer activity. REFERENCES
1. Reinherz EL, Schlossman SF: The differentiation and function of human T lymphocytes. Cell 19:821, 1980. 2. Gottlieb MS, Sehroff R, Sehanker HM, Weisman JD, Fan PT, Wolf RA, Saxon A: Pneumocystis carinii pneumonia and mucosal candidiasis in previously healthy homosexual men. N Engl J Med 305:1425, 1981. 3. Phan-dinh-tuy F, Durandy A, Grischelli C, Bach M: T-cell subset analysis by monoclonal antibodies in primary immunodeficiencies. Scand J Immunol 14:193, 1981. 4. Font~n G, De La Concha EG, Rodriguez MCG, Zabay JM, Carrasco S, Alba J, Pascual-Salcedo D, Ojeda JA: Severe combined immunodeficiency with disbalance and functional abnormalities in the T-lymphocyte subsets. Clin Immunol lmmunopathol 24:432, 1982. 5. Tsuchiya S, Konno T, Tada K, Ono Y: Epstein-Barr virusinduced lymphoblastoid ceil lines from patients with primary immunodeficiency diseases. Seand J Immunol 11:155, 1980.
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6. Tsuchiya S, Nakae S, Konno T, Tada K, Ono Y: Estimation of B cells transformed by Epstein-Barr virus in patients with congenital agammaglobulinemia. Tohoku J Exp Med 135:379, 1981. 7. Numazaki Y, Oshima T, Tanaka A, Konno T, Tazawa Y, Karita M, Ishii A, Hirota K, Watabe N, lshida N: Demonstration of IgG EA (early antigen) and IgM MA (membrane antigen) antibodies in CMV infection of healthy infants and in those with liver disease. 3 PEt)~ATr~97:545, 1980. 8. Kumagai K, Itoh K, Suzuki R, Hinuma S, Saitoh F: Studies of murine large granular lymphocytes, I. Identification as effector cells in NK and K cytotoxicities. J Immunol 129:388, 1982. 9. Morimoto C, Reinherz EL, Abe T, Homma M, Schlossman SF: Characteristics of anti-T-cell antibodies in systemic lupus erythematosus: Evidence for selective reactivity with normal suppressor cells defined by monoclonal antibodies. Clin lmmunol lmmunopathol 16:474, 1980. 10. Fischer A, Durandy A, Virelizier JL, De Saint Basile G, Lagrue A, Reinherz E, Schlossman S, Grischelli C: Severe combined immunodeficiency with quantitatively normal but abnormally differentiated T lymphocytes. J PEDrATR99:261, 1981. 11. Freidrich W, O'Reilly R J, Koziner B, Gerhard DR Jr, Good RA, Evans FL: T-lymphocyte reconstitution in recipients of bone marrow transplants with and without GVHD: Imbalance of T-cell subpopulations having unique regulatory and cognitive functions. Blood 59:696, 1982. 12. Bach M-A, Bach J-F.: The use of monoclonal anti-T cell antibodies to study T cell imbalance in human disease. Clin Exp Immunol 45:499, 1981. 13. lmmun0deficiency. WHO Tech Rep Ser No. 630, 1978.
Neonatal transient thyrotoxicosis resulting from maternal TSH-binding inhibitor immunoglobulins Hideki Yagi, M.D., Masao Takeuchi, M.D., Kanji Nagashima, M.D., Shigeyoshi Suzuki, M.D., Takayoshi Kuroume, M.D., Yasuhiro Iida, M.D., Junji Konishi, M.D., and Keigo Endo, M.D. Maebashi Gunma and Sakyoku Kyoto, Japan
TRANSIENT THYROTOXICOSIS is a serious, life-threatening disease in neonates? Its pathogenesis has not been fully established, but the placental transfer of thyroid-
From the Departments of Pediatrics, Maebashi Red Cross Hospital and Gunma University School of Medicine, and the Department of Nuclear Medicine, Kyoto University School of Medicine. Reprint requests: Kanji Nagashima, M.D., Department of Pediatrics, Gunma University School of Medicine, 3-29-22. Showamachi, Maebashi-shi, Gunma-ken, Japan.
stimulating immunoglobulins such as L A T S , L A T S protector, and thyroid-stimulating antibody from the mother with Graves disease is considered the main cause. 2-4 Clinical signs usually subside with the disappearance of thyroid-stimulating IgG in the serum of the neonate. 2,3 LATS MCHA TBII TGHA TSAb
Long-acting thyroid stimulator Thyroid microsome hemagglutination antibody TSH-binding inhibitor immunoglobulins Thyroglobulin hemagglutination antibody Thyroid-stimulating antibody