Impaired T-cell-independent B-cell maturation in systemic lupus erythematosus: Coculture experiments in monozygotic twins concordant for Klinefelter's syndrome but discordant for systemic lupus erythematosus

CLINICAL

IMMUNOLOGY

Impaired

AND

40.

IMMUNOPATHOLOGY

365-370

(19861

T-Cell-independent B-Cell Maturation Lupus Erythematosus:

in Systemic

Coculture Experiments in Monozygotic Twins Concordant for Klinefelter’s Syndrome but Discordant for Systemic Lupus Erythematosusl TILMANSCHLEGELBERGER~J~RNKEKOW. l~c~purtr~ettt In a case coculture formed. B-cell points

of‘ItIter-ml

Mdicitte.

of monozygotic

experiments Adequate

Chistitrrl

twins

with T-helper-cell

discordant

Alhwcht for

and maturation

1 ‘tril,cjr.\itv.

systemic

isoiogous (i.e.. genetically function in the SLE

responsiveness to T-cell-dependent to a T-cell-independent B-cell

L. GROSS

ANDWOLFGANG

lupus

identical) twin was

T-cell-independent defect in the

A’iel. G‘c,rmut~

erythematosuh lymphocytes demonstrated.

SLE

polyctonal twin.

tSLE). were perLack of activator\ 14% A~:IJCIM

Prurk. Inc.

INTRODUCTION

The systematic lupus erythematosus (SLE) is an autoimmune disease with complex immunoregulatory abnormalities. Clinically a B-cell hyperreactivity appears to be the precursor of the major effector mechanisms of disease. However. a variety of T-cell defects exist either primarily or secondarily, if not independently from, the B-cell abnormality (for review see (I )). In order to analyze B- and T-cell function in SLE. we have taken the opportunity to perform in vitro stimulation and coculture experiments in a case of identical twins, both with Klinefelter’s syndrome (KS), but discordant for SLE. The purpose of our study was first, to look for inappropriate immune response and regulation in the SLE patient and his clinically healthy brother: and second. to study cell cooperation of SLE lymphocytes with isologous (i.e.. genetically identical control) lymphocytes. The results obtained delineate that there is a T-cellindependent B-cell maturation defect in the SLE patient, whereas the healthy twin did not reveal any pathological reactions. CASE REPORT

The clinical features of this case with the rare coincidence of KS and SLE in monozygotic twins have been reported in detail elsewhere (2). Our SLE patient was hospitalized at 18 years of age. He presented with clinical symptoms of pericarditis. arthritis, and Raynaud’s phenomenon, and with serological findings of hypergammaglobulinemia, antinuclear antibodies (ANA; titer 1:5260), antibodies I Supported

by

the

Deutsche

Forschungsgemeinschaft.

Grant

Gr 609/J.

365 0090-1129/X6

$ I .50

Cup>nght ‘c IYBf, b, Academic Pre\\. Inc. A,, r,ght\ of rrproducnon I” any form reaensd

.X6

BKIEF C‘OMMUNI(‘A'fIOihS

against extractable nuclear antigens (anti-ENA: anti-SS-A). and cardiolipin atillbodies. Delayed-type hypersensitivity to recall antigens was absent. This constcllation allows the diagnosis of SLE in accordance with the rev ised criteria of‘ the American Rheumatism Association (3). The patient‘s brother did not show clinical or serological manifestations of an autoimmune or any other disease. He prcsented normal delayed-type hypersensitivity to recall antigens. These brothers are monozygotic twins; both have Klinefeltcr’a syndrome with a caryotype 47, XXY and a hypergonadotropic hypogonadism. METHODS The methods of the immunological studies reported here have been previously described extensively (4). In brief. peripheral blood mononuclear cells (MNC’) were isolated from freshly drawn heparinized blood by density gradient technique. In addition to unfractionated MNC. we studied lymphocyte subpopulations. Phenotypical differentiation of MNC was performed by immunofluorescence with monoclonal antibodies (OKT I I. Leu IO) and with alpha-naphthylesterase staining. MNC were depleted from T cells by rosette formation with 2-aminoethylisothiouronium-bromide-treated sheep red blood cells (E,,,). The remaining cells consisted mainly of B cells (60-90%) with a varying amount of lo-40%’ monocytes, and less than I c/f T cells. Such preparations are called “B cells” in this article. The E.,,-rosette-forming cells were treated with bidistilled water for osmotic lysis of the sheep red blood cells. The remaining lymphocytes were over 9.5% OKT I l-positive and are called “T cells” in this article. They were used for reconstitution by B cells with 5% T cells in coculture experiments. Lymphocyte stimulation was performed in microtiter plates with a final volume of 0.3 ml and a final cell density of IOh cells/ml. Cells were suspended in RPM1 1640 medium, supplemented with L-glutamine. antibiotics, and 10% heat-inactivated fetal calf serum. Cell proliferation was measured after a culture period of 5 days by incorporation of tritiated thymidine during a 4-hr pulse with 0.5 l.LCiiculture. Immunoglobulin secreted into the culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA) technique after 7 days of culture. As stimulants we used phytohemagglutinin (PHA: 5 pg/ml), and pokeweed mitogen (PWM: 5 pgiml) as phytomitogens. and Klrhsirll~r ,~,rrrrnlorrirle K43 membrane preparations (KlebsM: 100 pgiml) for T-cell-independent B-cell stimulation (4). RESULTS The in l’itr’o analysis of lymphocyte function was performed about 3 months after diagnosis of SLE. At this time the SLE patient did not show any clinical features of active disease anymore. but was under a medication of 4 mg prednisonelday. The proliferation assay showed normal spontaneous proliferation rates for both probands. The value for the SLE twin (96 cpm) was slightly lower than that of his brother (345 cpm). Stimulation of T cells with PHA gave normal proliferative responses for both when compared with healthy controls: the stimulation index

BRIEF

SLE twin MNC

FIG. 1. IgM synthesis of unfractionated pokeweed mitogen (PWM).

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mononuclear

healthy twin 1MNC

cells

tMNCt

without

toI

and with

(m)

(i.e., cpm with PHA: cpm without PHA) was 102 for the SLE twin and 63 for the brother. There was no statistically significant difference. The ELISA analysis of immunoglobulin concentrations in the culture supernatants demonstrated normal spontaneous B-cell differentiation for both twin brothers. T-Cell-dependent stimulation of B cells with PWM was first studied in cultures of unfractionated MNC. The SLE twin exhibited an almost complete nonresponse; in contrast, B cells of the healthy twin were stimulated to a considerable immunoglobulin production (Fig. 1). In subsequent coculture experiments, B cells of the SLE twin responded to PWM neither with autologous T cells nor with T cells of his healthy brother. In contrast, B ceils of the healthy twin, reconstituted with either autologous T cells or with T cells of the SLE twin, consistently gave rise to a considerable immunoglobulin production after PWM stimulation (Fig. 3). Stimulation with Klebsiella ptzeutnonirre membrane preparations was finally

+ SLE twin 1 cells SLE

FIG. 2. IgM of the identical

synthesis in cocultures twins. without (0)

+ healthy twin T cdts I twin

+ healthy twin T cells I

B cells

of B cells. reconstituted and with (m) pokeweed

healthy

+ SLE twin T cells twin

1

B cells

with 5% autologous mitogen (PWM).

or isologous

T cells

36X

BRIEF

C‘OMMUNI(‘ATIONS

SLE twin 0 cells FIG.

B-cell

3. IgM activator

synthesis Kleh.tic~//tr

of B cell\ without (i?) puc~rr~~~orric~c~ membrane

healthy twin 0 cells

and with Ia I KlehsM 1.

I the

T-cell-mdependent

polyclonal

performed for T-cell-independent B-ceil activation. Again. B cells of the healthy twin responded well with enhanced immunoglobulin secretion, whereas B cells of the SLE twin failed to respond significantly (Fig. 3). DISCUSSION Identical twins discordant for SLE represent an interesting model for the analysis of functional alterations of lymphocytes by in ~‘itrc> stimulation and coculture experiments, devoid of many problems related to genetic differences. In our case. one twin had presented with clinical and serological symptoms of active SLE. His clinically healthy brother did not show any abnormalities in B- and T-cell responsiveness to polyclonal activators and exhibited normal T/B-cell cooperation. This is of special interest with regard to the high concordance of monozygotic twins for autoantibodies and functional defects of lymphocyte subpopulations (5. 6). and with regard to a high incidence of various immune phenomena in KS (7. 8). We can postulate that the clinically healthy twin behaves like a normal healthy control in in llitw assays. This enables us to study the lymphocyte function of the SLE twin in cell-mixing experiments with genetically identical but “normal” lymphocytes. Such coculture experiments with lymphocytes of monozygotic twins discordant for SLE have not been done previously. The SLE twin exhibited normal spontaneous lymphocyte proliferation and normal T-cell stimulation by PHA. T-Cell-dependent stimulation of B cells with PWM, studied in cultures of unfractionated MNC, showed an almost complete nonresponse in the SLE patient. This lack of responsiveness to PWM is regularly found in SLE (9, IO). It is commonly explained by an inappropriate T-helper function ( 10, I1 1. On the other hand, a B-cell abnormality is a principal feature of SLE, but not only of active disease. It can also be found independently from disease activity (IO). For further differentiation. we stimulated isolated B cells (containing a certain amount of monocytes and non-B-non-T cells), reconstituted with 5% of either

BRIEF

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369

autologous or isologous T cells. The SLE twin’s B cells responded neither with autologous nor with perceivably normal isologous T cells. A normal T-helper function of the healthy twin can be assumed from the good response observed after stimulating MNC with PWM, as well as stimulating B cells, reconstituted with autologous T cells. PWM stimulation of the healthy twin’s B cells, reconstituted with SLE twin’s T cells, induced marked immunoglobulin secretion. It even significantly exceeded the response of the same B cells with autologous T cells, pointing to a predominance of helper over suppressor influences in the SLE twin. This demonstrates that SLE T cells do exert adequate helper properties in this PWM system. The functional defect is therefore localized to another cell population. This is in accordance with results of Fauci et 01. (9). who demonstrated normal T-helper activity and defective B-cell response in coculture experiments of irradiated and nonirradiated unfractionated lymphocytes from SLE patients and normal controls. Other investigators were able to restore responsiveness to mitogen (PWM) or antigen (e.g., trinitrophenyl (TNP), SRBC) by coculturing SLEMNC with normal control’s T cells (I I - 13). Schroeder and coworkers (1 I) performed coculture studies with allogeneic cells of normal controls in a case of identical twins discordant for SLE. They also found decreased responsiveness to PWM. Yet. the SLE twin’s T cells, cocultured with a normal control’s B cells, failed to support a PWM response. whereas control T cells were able to restore activation of SLE B cells, hence indicating a T-helper defect. In order to give further evidence for a T-cell-independent B-cell maturation defect in our case, we stimulated B cells with Klrhsiclla pnetrmotziac membranes, a true (i.e., T-cell-independent) polyclonal B-cell activator (4). The B-cell defect detected in the PWM system was also found after stimulation with KlebsM. After exclusion of a T-cell defect responsible for the lack of B-cell response in our case, an intrinsic B-cell maturation defect can be discussed. In \,itro hyporeactivity of B cells may be a result of in \,i\‘o hyperreactivity (9. 14). A preceding polyclonal B-cell activation itt l,il-o may abolish the responsiveness to a second polyclonal stimulus in ,sitro (15). Interestingly. at the time of study there were no features of disease activity anymore, and we were not able to find spontaneous immunoglobulin production increased in \~itro, as commonly observed in active SLE ( 14. 16). The B-cell hyporeactivity therefore seems to be a more persistent defect that exceeds the phase of in l-i\w hyperreactivity. Besides an intrinsic B-cell defect, an altered immunoregulation by defective monocyte function cannot be ruled out in our experimental setting. The presence of adherent mononuclear cells is necessary for the B-cell response to both PWM and KlebsM. Furthermore, suppressive effects diminishing the B-cell response are possible. Defective monocyte function in patients with SLE has been described (17). Further coculture experiments (e.g., mixing T-cell-depleted cell fractions of both twins, or stimulating with the monocyte-independent polyclonal B-cell activator Epstein-Barr virus) shall be carried out. Summarizing our findings, we demonstrated (a) the presence of T-helper activity in the SLE twin, possibly with predominance over T-suppressor influences, and (b) a T-cell-independent B-cell maturation defect in the SLE patient, possibly

370

BRlEF

C‘OMMUNIC‘ATIONS

due to an intrinsic B-cell defect. These findings arc in accordance with xomc previously published results. although coculture experiments with isologous lymphocytes of monozygotic twins have not heen done previously. On the other hand. OLK results arc in contrast to other investigations. The great variety of rcsuits obtained in similar experimental settings with different SLE patients i\ an interesting and well-known phenomenon. The varying occurrence and dcgrcc of immunoregulatory abnormalities among SLE patients emphasizes the heterogcneity of this disease a> well as the complexity of possible defect\. REFERENCES I.

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