Severe combined immunodeficiency with T lymphocytes retaining functional activity

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

46,432-441

Severe Combined lmmunodeficiency Retaining Functional

t 1988)

with T Lymphocytes Activity

G. FONTAN,~ M. C. GARCfA RODRIGUEZ, S. CARRASCO,” J. M. ZABAY,~ AND E. G. DE LA CONCHAS Immunology fservice

Service

and *Department Hospital Hospital Chico

of Immunology,

of Pediatrics, Ciudad Sanitaria Provincial, Madrid; and #Service de San Carlos, Madrid, Spain

“La

Paz”,

Madrid;

of Immunology,

Two cases of severe combined immunodeficiency @CID) with normal numbers of T cells are reported. Studies of T-cell subsets showed an absence of TQT lymphocytes and a very low percentage of CD; cells in Patient 2. Functional studies of T cells from this patient showed a normal suppressor activity. Patient 1 had normal percentages of T-cell subsets and his lymphocytes showed helper and suppressor activities but to a lesser degree than normal controls. Both cases stressed the heterogeneity of SCID in which T cells could be present and retain some of their funCtiOnal activities. 0 1988 Academic Press. Inc.

INTRODUCTION The severe combined immunodeficiency syndrome (SCID) comprises a variety of diseases characterized by profound defects in T- and B-cell functions. The syndrome is due to a lack of stem cells (I), block of T-cell maturation (2), or membrane abnormalities (3). Lymphopenia affecting both T and B cells is usual, but normal or elevated numbers of circulating B lymphocytes can also be found (4). The finding of normal numbers of circulating T lymphocytes in SCID is infrequent, representing less than 10% of the total cases of the syndrome. Albeit proliferative responses to mitogens and alloantigens are always severely depressed, little is known about functional activities of these T cells. The engrafting of maternal T cells is a frequent complication of SCID, and some cases of SCID with normal T-cell numbers have been attributed to engraftment of maternal lymphocytes (5). Usually these engrafted T cells have been nonfunctional and showed expression of T-cell activation antigens (6,7). In some patients, maternal cells are identifiable only after amplification techniques (8), but in SCID cases with normal T-cell numbers due to maternal grafts, HLA typing revealed the maternal origin of these cells (6). We have studied two cases of SCID with normal T-lymphocyte numbers in which the patients’ T cells did not show “extra” reactions when typed for HLA antigens and whose lymphocytes retained T-cell regulatory activities. CASE REPORTS Case 1, An 1l-month-old with a history of vomiting,

male was admitted to “La Paz” Children’s Hospital diarrhea, and recurrent bronchitis and laryngitis since

’ TO whom correspondence should be addressed at Servicio de Inmunologia, Departamento de Laboratorio, Editicio de Anatomia Patol6gica. Ciudad Sanitaria “La Paz,” Paseo de la Castellana, 261, 28046 Madrid, Spain. 432 0090-1229188 $1 SO Copyright All rights

0 1988 by Academic Press. Inc. of reproduction in any form reserved

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the age of 4 months. One month before admission he developed ulcers in the soft palate, in which Candida albicans was grown. The patient had a persistent neutropenia (400-900 polymorphonuclear leukocytes/mm3). Case 2. A 5-month-old male was referred to our Hospital with a history of purulent rinorrhea and serous otitis when he was 1% months old. One month later he suffered from recurrent diarrhea caused by Salmonella enteriditis, as well as several episodes of respiratory tract infection. WBC was normal. A brother died when 8 months old from intractable diarrhea and bronchopneumonia. The immunologic criteria upon which the diagnosis of SCID was made in both patients are shown in Table 1. Erythrocytes from both children contained adenosine deaminase and nucleoside phosphorylase. None of the patients showed extra specifities when typed for HLA antigens, expressing HLA antigens on one parental and one maternal haplotype of the respective parents. Both children were treated with intravenous gammaglobulin (Endobulin Immuno Laboratories, Austria) and thymopoyetin (TP- 1, Serono Laboratories, Italy) and showed a significant improvement in their recurrent infections, but a chronic cough leading to bronchiectasias persisted in Patient 2, and Patient 1 died unexpectedly from an endotoxic shock, having shown a clear improvement in his proliferative responses to the mitogen PHA and to a lesser degree to concanavalin A (Con A) and pokeweed mitogen (PWM). No analytical changes were recorded in Patient 2. The postmortem examination of Patient I showed a rudimentary epithelial thymus devoid of Hassall’s corpuscles. The lymphocytes accumulated in the paracortical region of the lymph nodes and the postcapillary venules were permeated by lymphocytes. MATERIAL

AND METHODS

Lymphocyte isolation. Mononuclear cells were isolated from peripheral blood (PBM) or from tonsils obtained from subjects undergoing routine tonsillectomy, by centrifugation on a Ficoll-Hypaque gradient. Tonsillar cells and patients’ PBM were separated into T-cell- and B-cell (non-T)-enriched populations by rosetting with neuraminidase-treated sheep red blood cells (SRBC) (9). The T-cell fraction was contaminated by ~2% surface Ig-positive cells and <2% monocytes (OKMl + or nonspecific sterase+), and when cultured with PWM always failed to produce detectable Ig. Cell membrane marker analysis. B lymphocytes were identified as surfacebearing immunoglobulin cells (Slg) which were stained by direct immunofluorescence using fluorescein-conjugated F(ab’), fragments of specific antisera to human immunoglobulins (Meloy Laboratories, Springfield, VA) (10). T lymphocytes were identified by rosetting with neuraminidase-treated SRBC (11). PBM were analyzed by indirect immunofluorescence using previously described monoclonal antibodies (mab): OKT,, OKT,, OKT,, OKT,, OKT,,, and OKM, from Ortho Pharmaceuticals (Raritan, NJ); Leu 3a, Leu 11 (recognizing a human NK cell and neutrophil antigen) (12) and HLA-DR from Becton-Dickinson (Sunnyvale, CA); TQ, (recognizing a subpopulation of suppressor-inducer T cells) (13) from Coulter Electronics, Ltd. (Luton, UK); and Tat (recognizing the interleukin-2 receptor) (14) from Immunotech S.A. (Marseille, France).

G

6.8 1.5 20-80

A

M

340 25 IO-50

Serum Ig (mg/dl)

92 72 300-7.50

~-. 2.700 3.500 1.500

Lymphocytes (cells/mm3)

Note. Results are expressed in cpm x 103. U Antibodies against tetanus toxoid, E. cob, and heterophile b Patient versus control cells.

Normal range

1 2

Patient No.

IMMUNOLOGIC

antigens

l/128 Absent >1/16

Isoagglutinins

1 WITH

Absent Absent -

Other antibodies”

IN PATIENTS

TABLE FINDINGS

SCID

0.32 0.55 0.15-0.83

Spontaneous

0.31 0.14 18-60

PHA

Proliferative

0.29 0.33 12-50

Con A

0.14 0.34 8-37

PWM

responses MLRh ~ 0.72 1.7 8.5-65

_

2 >

z z

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435

Cell cultures. All cells were cultured in triplicates (0.25 ml per well) in flatbottomed microplates using RPM1 1640 medium supplemented with 100 p&ml penicillin, 200 mM L-glutamine, and 10% fetal calf serum. PBM were cultured at a cell density of 2.5 x lO’/ml. Lymphoproliferative responses to the mitogens phytohemagglutinin (PHA), Con A, PWM, and unidirectional mixed-lymphocyte reactions (MLR) were assessed by microculture techniques as described elsewhere (15, 16). In vitro immunoglobulin synthesis. Unfractionated PBM were cultured in supplemented RPM1 1640 at a cell density of 1 x 106/ml. Whenever it was possible to obtain T-cell- and B-cell-depleted populations from the patients, cocultures were performed. In these experiments, 1 x 106/ml tonsillar B cells were recombined with graded amounts (1 to 100 x 104/ml) of patients’ or controls’ T cells. Patients’ B cells were cocultured (1 plus 1 x l@/ml) with autologous or control T cells. In addition, unfractionated (T + B) tonsillar lymphocytes (1 x lO?ml) were cocultured with graded amounts of allogeneic T cells from patients or controls. For Ig production, 4 @ml of PWM was added. After 7 days the concentrations of IgG, IgA, and IgM in the supernatant were measured by an ELISA technique (17, 18). Other studies. IL-2 production was induced by incubating 5 x lo6 PBM in 0.2 ml of complete medium in the presence of PHA at a final concentration of 1% w/v for 24 hr in microculture plates. Supernatants were harvested and frozen at - 70°C until analysis. Measurement of IL-2 in supernatants were performed using the IL-Zdependent T-cell line CTL-2 (19). Results are expressed as units per milliliter after comparison with a standard curve made with purified IL-2 (Cellular Products, Inc., Buffalo, NY). Proliferative responses of patients’ cells supplemented with exogenous IL-2 and tymopoyetin, as well as lymphocyte capping of fluoresceinated Con A were performed as previously described (20, 21). RESULTS

Humorul immunity. Serum Ig levels and the results of specific antibody determinations are shown in Table 1. With the exception of increased levels of IgM and isoagglutinin anti-B titer in Patient 1, both patients had severely impaired humoral as showed by low Ig levels and lack of specific antibody formation. Both patients showed normal percentages of circulating B lymphocytes. When patients’ non-T cells (1 x 106/ml) were incubated with graded amounts of control T cells (0.002 to 1 x 106/ml), cultured for 7 days with PWM and the concentrations of Ig measured in the supernatant, none of the children’s cells produced significant amounts of IgG, IgA, or IgM. Lymphocyte markers. A summary of leukocyte marker analysis is given in Table 2. Patient 1 showed a normal number and percentage of lymphocytes as defined by the markers used. In contrast, Patient 2 showed a low percentage of OKT,-positive cells, values oscillating between 2 and 10%. Staining with the mab Leu 3a that recognizes a different epitope of the CD, structure showed similar results to those obtained with the OKT,. OKT,’ cells were persistently increased percentually and in absolute numbers. Lymphocytes from this patient were repeatedly nonreactive with the TQ, mab that recognizes a structure present in subsets from CD: and CD: cells. Autoantibodies masking the TQ, determinant

436

FONTAN TABLE

PHENOTYPIC

CHARACTERIZATION

Surface marker (%)

OF THE

PATIENTS’

Patient:

E rosette9 OKT3 OKT4 Leu 3a OKT8 OKT6

TQI Sk3 DR OKMl Leull a Cells forming rosettes with neuraminidase-treated

ET AL. 2 PERIPHERAL

BLOOD

MONONUCLEAR

CELLS

I

2

Normal range

57

53 58 6 8 52 I I I I5 23 I

45-80 50430 35-55 35-55 15-35 o-l 4&60 4-12 5-20 S-20 4-18

66 50 47 16 2 41 13 10 18 6

sheep red blood cells.

were discarded, since incubation of normal T cells with the serum of Patient 2 diluted 1/4caused no change in the percentage of TQC cells. PBM from Patient 2 when incubated for 24 hr with PHA did not express IL-2 receptors as assessed by staining with the Tat mab. Proliferative responses. Prior to treatment, DNA synthesis induced by the tested mitogens was repeatedly negative in both patients. Patient 2 showed a minimal proliferative response to unrelated PBM, being the stimulation index (patient versus control-patient versus patient/patient versus patient) of 1.75, well below the normal index (>12). PBM from both children were able to stimulate lymphoid cells from unrelated donors as well as from the respective parents (data not shown). Incubation of patients’ PBM with TP-1 (1 kg/ml), as well as supplementing of the PBM cultures with purified IL-2 (15 U/l x lo6 cells), did not restore the proliferative responses to PHA. In vitro Zg production. In several different probes patients’ PBM when incubated with PWM for 7 days did not produce Ig’s. When non-T cells from the patients were cocultured with normal T cells and stimulated with PWM, none of the patients were able to produce in vitro detectable amounts of Ig’s. Figure 1 shows the regulatory activity from patients T cells when cocultured with control B cells and stimulated with PWM. Patient 1 showed lower helper activity for Ig production than the day’s control. In fact a tenth of the number of T cells from the control was sufficient for reaching the maximal IgM production attained by patient T cells. Similar dose-response curves were obtained for IgG and IgA. In spite of partial reconstitution of the proliferative responses to mitogens after TP-1 treatment, no modification of T-cell-helper activity could be found in Patient 1. Patient 2 T cells were devoid of helper activity in this system, but his lymphocytes were able to suppress Ig production. Moreover, when graded amounts of Patient 2 T cells were cocultured with mononuclear tonsillar cells (T + B), suppressor activity, similar to that produced by T cells from the day’s control, was demonstrated again (Table 3). Monocyte-mediated suppression in these experiments seems unlikely since following Gmelig-Meyling and Waldmann data (22) mono-

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FIG. 1. IgM production in vitro. Graded numbers of normal T cells (O), Patient 1 T cells (m, or Patient 2 T cells (A) were added in cultures containing a constant amount of tonsillar B cells (1 x lo6 cells/culture). Cultures were stimulated with PWM (4 pi/ml) during 7 days and the IgM in the supernatant was determined by ELISA.

cytes are suppressive in concentrations starting at 0.3 x lO%nl. This concentration was never reached in our experiment. Moreover, previous work in 10 controls showed that further purification by adherence of the T-cell population did not modify the suppressive effect of the T cells in our system. Other studies. Spontaneous and colchicine-induced capping of Con A receptors were similar to those recorded in controls. When incubated for 24 hr and stimulated with PHA, PBM from both patients did not produce detectable amounts of IL-2. DISCUSSION

The patients described herein had a total and percentage count of T and B lymphocytes within the normal range, although they had laboratory evidence of SCID, including absence of proliferative responses to mitogens and alloantigens, inability of IL-2 production in vitro, and impaired Ig production in vivo and in vitro. Patient 1 showed high IgM serum levels and retained some antibody TABLE IgG AND IgM IO?CULTURE)

3

PRODUCTION IN PWM-STIMULATED COCULTURES OF MONONUCLEAR CELLS (1 x FROM A CONTROL AND GRADED AMOUNTS OF T CELLS FROM PATIENT 2 OR A NORMAL CONTROL

Ig in the supematant (&ml) T cells added (X 1w 0

0.5 2.5 10 50 250

Patient G 475 525 525 400 320 20

Control M

G

M

1075

475 775 480 310 290 45

1075 loo0 740 540 435 290

1000

750 625 450 300

438

FONTAN

ET AL.

formation. On this basis he could be diagnosed as having Nezeloff syndrome (23), but following the recommendations of the WHO group on primary immunodeticiencies (24), the diagnosis was SCID. Terms such as Nezeloff syndrome are no longer recommended for use by the aforementioned group. SCID is a very heterogeneous entity: normal or high serum Ig levels have been described in this syndrome (25-27). In the report on SCID and bone marrow transplantation made by Bortin and Rimm (28), 11 of 55 SCID cases showed IgM serum values between or above the normal range. Neutropenia is one of the several hematologic abnormalities found in SCID patients (29, 30). Recently Bagby ef al. included two cases of SCID with severe neutropenia in a series of neutropenic patients treated with prednisone (3 1). Several causes of SCID with normal numbers of T cells, such as maternal graft (S), lack of HLA expression (32), and adenosine deaminase deficiency (33), have been discarded. Membrane cytoskeleton abnormalities (3) producing spontaneous capping of Con A receptors (34) could also be ruled out. Patient 2 has in common with the few HIV-related illnesses with hypogammaglobulinemia (35) a low CD: cell count, but the familial, serological, and epidemiological data could reasonably rule out this etiology. In fact the patient was born in 1981 in one of the most isolated places of the Canary Islands where no HIV infection has been recorded to present. Both parents are HIV negative and the patient did not receive blood products prior to the diagnosis of the illness. Moreover an older brother born in 1979 probably died from a similar syndrome. The practical absence of OKTZ cells in our patient is not due to a lack of the epitope recognized by this mab (36), since the Leu 3a mab that defines a different epitope of the CD, structure also stained a low proportion of his lymphocytes. The absence of cell reactivity with the TQ, mab that stains subsets of CD: and CD: cells deserves special interest. The CD: TQ: lymphocytes are supposed to be the inducers of suppression (37). In our patient and at least in the system studied, suppressor activity was found in spite of the absence of this subset. Despite the presence of mature T cell markers, the lymphocytes in both patients did not proliferate in response to stimulation with mitogens or alloantigens. The proliferative defect is in agreement with the profound deficiency found in IL-2 production, but the proliferative defect could not fully account for a lack of IL-2 production: IL-2 unresponsiveness is also contributory, since supplementing stimulated patients’ PBM cultures with purified IL-2 did not restore the proliferative response. In Patient 2 there was a lack of expression of IL-2 receptors when his cells were stimulated with PHA. Nevertheless such deficiency in IL-2 production and responsiveness did not impede T-cell regulatory activities on Ig production. The T cells of Patient 1 were capable, albeit in a subnormal level, of helper and suppressor activities when cocultured with policlonally activated normal allogeneic B cells; the T cells from Patient 2 similarly treated showed only a suppressor activity in accordance with the phenotype of his circulating lymphocytes (38). In addition to the T-cell defects, the patients’ B cells when cocultured with normal T cells and stimulated with PWM were unable to produce in vitro Ig’s. In Patient 1, there was a lack of correlation between the in vitro assessment of Ig production and the in vitro finding of increased IgM in his serum. Differences

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between B cells from peripheral blood and B cells from lymph nodes or spleen in susceptibility to helper factor induction or to the polyclonal activator could be accounted for by the discrepancy (39). Recent findings (40) suggest extreme caution in postulating inherent B-cell lineage defects based on these data, since abnormal T-B cell interactions in ontogeny could account for the B-cell abnormality (41).

Only a few casesof selective deficiency of CD: cells leading to SCID have been described (42-45), and T-cell suppressor activity on allogeneic B cells by the remaining CD: cells could be demonstrated in none of the tested patients. Recently Jung et al. (40) described a case of SCID with CD7 deficiency in which the proliferative responses to mitogens were abnormal, but the T cells were found to provide help for the differentiation of normal B cells to Ig-secreting cells. In other instances as in the deficiency of OKT: ceils described by us (17) or in a case of SCID with abnormally differentiated T lymphocytes (46), the patients T cells did not show regulatory activities. To our knowledge Patient 1 is the first case of “Nezeloff syndrome” in which T-cell regulatory activities could be demonstrated. The regulatory activities were detected before the treatment with TP-I was started. A few cases of SCID and Nezeloff syndrome treated with thymic hormones showed clinical and analytical improvement (47). Some of the clusters of differentiation of T lymphocytes as well as some of the functional activities displayed by the mature T cells are believed to be acquired during the passage through the thymus of the T-cell precursors (48). Interestingly enough, the thymus of Patient 1, in which the partial response to the TP-1 suggests some grade of involvement of the thymus in the pathogenesis of the syndrome, showed the classical morphology of SCID. This morphology is similar to the embryonic epithelial thymus before the lymphoid colonization (49). Nevertheless we have to keep in mind that the thymus morphology obtained during autopsy showed the specimens to be different from those obtained by thymic biopsies and may not reflect the status of the organ during life (50). Moreover loss of Hassall’s corpuscles could be the result of “acquired” loss of thymic epithelium as shown in patients with biliary atresia or AIDS (51,52). In the casesof SCID with mature circulating T cells, two possibilities exist: either the thymus has been functioning, homing, and differentiating T cells and involutioned afterwards, or the differentiation of T cells has been acquired outside the thymus. The present cases as well as those mentioned in the literature underline the heterogeneity of the SCID syndrome in which normal numbers of T cells, absence of T cells, and presence or absence of mab-defined T cell subsets have been described. Furthermore, when present, T cells could retain functional activities or not. Such a heterogeneity could be caused by multiple primitive defects or could be only the reflection of environmental factors modulating the phenotypic and functional expression of a few primitive defects of the lymphoid stem cells or the microenvironment where they differentiate. REFERENCES 1. Incefy, Good,

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R., and

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ET AL.

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44. Duse, M., Maccario, R., Nespoli, L., Plebani, A., and Ugazio, A. G., In “Primary Immunodeliciency Diseases: Birth Defects, Original Article Series 19” (R. J. Wedgwood, F. S. Rosen, and N. W. Paul, Eds.), pp. 105-106, A. R. Liss, New York, 1983. 45. Edwards, K. M., Cooper, M. D., Lawton, A. R., Sanders, D. S., and Wright, P. F., J. Pediatr. 71, 70, 1984. 46. Fischer, A., Durandy, A., Virelizier, J. L., De Saint Basile. G., Lagrue, A., Reinherz, E., Schlossman, S., and Griscelli, C., J. Pediutr. 99, 261, 1981. 47. Sculof, R. S., and Goldstein, A. L., In “Recent Advances in Clinical Immunology” (R. A. Thompson and N. R. Rose, Eds.), pp. 243-286, Churchill Livingstone, London, 1983. 48. Stutman, O., Clin. Immunol. Allergy 5, 191, 1985. 49. Good, R. A., Peterson, R. D. A., Perey, D. Y., Finstad, J., and Cooper, M. D., In “Immunological Deficiency Diseases in Man: Birth Defects, Original Article Series 6” (D. Bergsma and R. A. Good, Eds.), pp. 17-34, National Foundation March of Dimes, New York, 1%8. 50. Borzy, M. S., Schulte-Wissermann, H., Gilbert, E., Horowitz, S. D., Pellett, J., and Hong, R., Clin. Immunol. Immunopathol. 12, 31, 1979. 51. Landing, B. H., Yutuc, I. L., and Swanson, V. L., In “Immunodeficiency: Its Nature and Etiological Significance in Human Disease” (N. Kobayashi, Ed.), pp. 3-35, University Park Press, Baltimore, 1978. 52. Elie, R., Laroche, A. C., Amoux, E., Guerin, J. M., Pierre, G., Malebranche, R., Seemayer, T. A., Dupuy, J. M., Russo, P., and Lapp, W. J., N. Engl. J. Med. 308, 841, 1983. Received August 13, 1987; accepted September 3, 1987