Autoimmunity, 1990, Vol. 5 , pp. 257-264 Reprints available directly from the publisher Photocopying pqmitted by license only

0 1990 Harwood Academic Publishers GmbH Printed in the United Kingdom

NORMAL SUPPRESSIVE T CELL FUNCTION OF

ACTIVATION IN TYPE 1 (INSULIN DEPENDENT) DIABETES MELLITUS

EPSTEIN-BARR VIRUS INDUCED B CELL

ALICE KAHAN’, JEAN-PIERRE LeFLOCH2, and JEANNINE CHARREIRE’

‘INSERM U-283, Hdpital Cochin, 27 rue du Faubourg Saint Jacques, 75674 Paris Cedex 14 and ’Service d’Endocrinologie, Hdpital HMondor, CrCteil, France

(Received May 18. 1989; infinal form October 17, 1989)

Several studies have demonstrated abnormalities of T cell regulation of Epstein-Barr virus-induced B cell activation in systemic autoimmune diseases such as rheumatoid arthritis, systemic lupus erythemdtosus, and systemic sclerosis. However, a normal suppressive peripheral T cell function was observed in Graves’ disease. To investigate whether this abnormality is a common feature to other autoimmune diseases, we studied T cell regulation of Epstein-Bar virus induced B cell activation in I5 newly diagnosed type 1 (insulin dependent) diabetes mellitus patients and 10 normal control subjects. Peripheral B lymphocytes infected with Epstein-Barr virus were cultured for 20 days in the presence or absence of autologous T cells at different ratios (1 : 1 and 1 : 4). IgM and IgG secretions into the supernatants were determined using an enzyme-linked immunosorbent assay. The extent of suppression when T cells were added, as measured by a suppression ratio, was not significantly different in type I (insulin dependent) diabetes mellitus patients and normal subjects.

We conclude that in type 1 (insulin dependent) diabetes mellitus, the autoimmume reactivity is not dependent upon a generalized suppression defect. I t can by hypothesized, therefore, that in type I diabetes mellitus as well as in Graves’ disease, a local or organ specific suppressor deficit may induce the autoimmune phenomena.

KEY WORDS: Suppressive T cell function, B lymphocyte activation, Epstein-Barr virus, type I (insulin dependent) diabetes mellitus.

INTRODUCTION

Although the etiology of type 1 (insulin-dependent) diabetes mellitus is not clearly understood, a large body of evidence strongly suggests that the immune system plays an important role in its pathogenesis. The presence of a variety of organ and tissue specific autoantibodies, cell mediated immune response to pancreatic antigen, pancreatic lymphocyte infiltration during an early stage of the disease, suggest an aberration in immune regulation.

The study of T lymphocyte subpopulations has provided conflicting results. Studies of phenotypic T cell subsets from peripheral blood lymphocytes (PBL) have generally

Address for correspondence: Alice Kahan, MD, INSERM U-283, Hdpital Cochin, 27 rue du Faubourg Saint Jacques, 75674 Paris Cedex 14, France. Tel.: ( I ) 42 34 18 32.

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258 A. KAHAN, J . LeFLOCH A N D J. CHARREIRE

shown a normal percentage of CD3+ cells'-'; some authors, however, demonstrate a low percentage of CD3+ cells, due particulary to the decrease of CD4+ cells"'. The percentage of CD8+ cells was found decreased in 40% of patients and responsible for the high ratios of CD4/CD8'.'.' in newly diagnosed insulin dependent diabetus mellitus patients; this abnormality gradually reverts to normal 12 months after onset of clinically detectable disease. An increased number of activated T cells, assessed either by anti-DR2*3.9"o or anti-IL 2 receptor (TAC)" monoclonal antibodies, was demonstrated in recent-diagnosed type I diabetes. The activated T cells were either CD4+ or CD8+".

A deficient concanavalin A (ConA) induced suppressor cell activity against the proliferative response of autologous or allogeneic lymphocytes to phytohemaggluti- nin' or concanavalin A", as well as against autologous or allogeneic mixed lymphocyte culture13 was also found in newly diagnosed type 1 diabetus mellitus patients. However, a wide variation in suppressor cell function was seen among patients with diabetes and a substantial minority were entirely normalI3. PBL from type 1 diabetus mellitus patients also showed a loss of theophylline sensitivity, this subpopulation being considered to have a suppressive activity'.

In an attempt to resolve these conflicting data, we investigated T lymphocyte suppressive function assessed by the ability to these cells to modulate B lymphocyte differentiation induced by Epstein-Barr virus (EBV) in v i m . It is now widely accepted that EBV is a polyclonal B cell activator14, whose action does not require the par- ticipation of helper T cells, although T cells can negatively regulate this process in EBV-immune subjects''. Several reports described defective EBV-specific suppressor T cell function in patients with systemic autoimmune disease such as rheumatoid arthritisI6-", systemic sclerosis19, and systemic lupus erythematosus2'. In contrast, in Graves' disease the EBV-specific suppressor T cell function was similar to that found in healthy EBV-immune control subjects, suggesting that the auto-immune reactivity in Graves' disease is probably dependent upon a specific thyroid suppression defect, rather than upon a generalized defect2'.

In the present study we investigated the suppressive T cells function of EBV induced B cell activation in newly diagnosed type 1 (insulin dependent) diabetes mellitus, in order to determine whether the auto-immune phenomena are due to an organ specific immuno-regulatory defect as in Graves' disease or a more generalized im- munoregulatory defect as in systemic auto-immune diseases.

PATIENTS AND METHODS

Patients

Fifteen patients (9 men and 6 women, mean age f SD: 35 f 10 years, range: 16 to 53 years) with type 1 (insulin dependent) diabetes mellitus were studied. One patient had an association of type 1 diabetes mellitus and Hashimoto's thyroiditis. The onset of the disease had been recent, one month or less at the time of the study. These patients were ketosis prone and insulin depedent. The mean haemoglobin A,, at the start of the study was 10.55 f 1.14% (range 7.6 to 11.7%); the mean f SD in normal subjects 4.5 f 0.35%. Ten normal subjects, age and sex matched, were also studied. All were EBV-immune as determined by their antibody titers to viral capsid antigen (VCA), assessed by a standard immunofluorescent technique. All patients and control subject gave their informed consent.

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259 NORMAL SUPPRESSIVE T CELL FUNCTION

Virus The B 95-8 marmoset lymphoblastoid line served as the source of transforming EBV as previously described".

Cell preparation and lymphocyte cultures

The mononuclear cells obtained from heparinized blood by density gradient centrifu- gation with a mixture of Ficoll-Paque (density 1.076) (Pharmacia Laboratories, Uppsala, Sweden) were separated into B and T cells by rosetting with amino-ethyl- isothiouronium bromide treated sheep red blood cells (AET-SRBC)" at 4 "C for 2 hours as described". For each patient and normal control subject, 3 cultures were performed : a. B cells alone (0.5 x lo6 cells/ml); b. B cells (0.5 x lo6 cells/ml) + autologous T cells (0.5 x lo6 cells/ml); c. B cells (0.5 x lo6 cells/ml) + autologous T cells (2 x lo6 cells/ml). All the cultures were established in Falcon 3047 multiwell plates (Becton-Dickinson, Palo Alto, CA) in 2 ml RPMI 1640 (Dutch modification, Flow Laboratories) medium, supplemented with 15% fetal calf serum, 2 mM glutamine, 1 mM pyruvate, non-essential aminoacids (Flow Laboratories), 5 x lo-' M 2-mercaptoethanol and gentamicin. The cells were infected with 200~1 B 95-8 supernatant (EBV) and cultured at 37 OC in a humidified atmosphere containing 5% carbon dioxide. Half of the supernatant was removed from each culture on the 8th day and replaced with an equal volume of fresh complete medium. The same procedure was subsequently performed at 12, 16 and 20 days of culture. The super- natants were filtered through a 0.45pm Millipore filter and stored at -2OOC.

Ig determination by ELISA

IgM and IgG concentration in the culture supernatants were measured using a double antibody sandwich ELISA method as previously described*'.

A suppression ratio was calculated as follows: B -(B + T)/B, B representing Ig secretion in B cell culture alone and (B + T) Ig secretion into B and autologous T cells cultures. A suppression ratio > 0.50 was considered a normal suppression and a suppression ratio < 0.50 a deficient suppression, as previously determined in normal EBV-immune subjects and in systemic auto-immune d i s e a s e ~ l ~ ~ ' ~ .

Statistics The statistical difference between IgM and IgG secretions in B cells cultures and autologous B and T cell cocultures were determined with the two tailed Student's t-test. For the differences between the suppression ratio in normal and type I (insulin dependent) diabetes mellitus patients chi-square test was performed. P values < 0.05 were considered significant.

RESULTS

Autologous T cell Mediated Regulation of IgM and IgG Secretion by in vitro EBV- activated B Cells IgM secretion in supernatants of EBV-activated B cell cultures and cocultures of B and different ratios of autologous T cells are shown in Table IA.

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260

Table 1 at different ratios : I : l (b) and 4:l (c) A. IgM B. IgG

A . KAHAN, J. LeFLOCH A N D J. CHARREIRE

Ig secretion into the supernatant of B cell cultures (a) and cocultures of autologous T and B cells

Day 16 of culture a b C a b C

Day 20 of culture

A. IgM ng/ml Normal subjects

Mean f SEM n = 10

SR

n = 15 IDDM patients

Mean f SEM

SR

2037 f 308

675 f 244 **

I02 f 22 *** 0.93

867 f

312 ** 0.68

2428 + 270

612 101 f 192

f 29 *** *** 0.96

435 f I48 *** 0.86

0.77 0.65 2879 f 764

2077 +

844 **

3119 f

763

I732 f

669 ** 0.48 0.59

B. IgG ng/ml Normal subjects

n = 10 Mean SEM

147 f 29

71 53 - + 13

I24 - + 24

*

*

I83 - + 43

73 k 17 *

23 f 4

95 f 19

**

**

f 10 *

IDDM patients

Mean f SEM n = 15

174 f 29

138 f 26

182 - + 27

I24 f 22 *

Cocultures (b) and ( c ) were compared to B cell culture (a) with Student's two tailed 1 test :

SR : mean suppression d o *p < 0.05. "p < 0.01. ***p < 0.001.

In 10 control subjects, T cells induced a significant suppression of IgM secretion (p < 0.01, two tailed Student's t test) at all T to B ratios studied (1 : I and 4: 1) on day 16, and 20 of culture. These results were similar to those found in our previous studies in EBV-immune normal

In 15 newly diagnosed type 1 (insulin dependent) diabetes mellitus patients, autologous T cells induced a significant suppression of IgM secretion (p < 0.01, two tailed Student's t test) at days 16 and 20 of culture with all T to B ratios.

The mean suppression ratio was lower in type 1 (insulin dependent) diabetes mellitus patients than in control subjects at the days 16 and 20 of culture. However, if we consider as normal suppression, a suppression ratio higher than 0.50 as previously de~cribedl'~'~, the analysis of individual IgM secretory responses indicated that, at day 16 and with a T to B lymphocyte ratio of 1:1, 9 of 15 (60%) of type 1 (insulin dependent) diabetes mellitus patients and 7 of 10 (70%) of normal controls had a normal suppression ratio; at day 20, 10 of 15 (67%) type 1 (insulin dependent) diabetes mellitus patients and 9 of 10 (90Y0) control subjects had a normal suppres- sion ratio (chi square = 0.46 and 2.15 respectively, p = non significant) (Figure l a and b). Moreover, with a T to B lymphocyte ratio of 4: 1, which reflects the ratio found in peripheral blood, 13 of 15 (87%) at 16 days of culture and all type 1 (insulin dependent) diabetes mellitus patients at 20 days of culture, had a normal suppression ratio (Figure Id). As expected, all normal control subjects had a suppression ratio higher than 0.50 with this T:B ratio at 16 and 20 days of culture (Figure 1 c).

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NORMAL SUPPRESSIVE T CELL FUNCTION 26 I

1 0 0

0 8 0

A

' e s E 8

8

Figure 1 IgM secretion suppression ratios. Top panels T to B ratio of 1 : I ; botrom panels T to B ratio of 4: I ; a and c : normal control subjects (n = 10) ; b and d : Type 1 (insulin-dependent diabetes mellitus patients ( = 15). The mean suppression ratio is shown by horizontal lines and the brackets indicate the SEM.

IgG secretion into supernatants of EBV-activated B cell cultures was significantly lower than IgM secretion in type 1 (insulin dependent) diabetes mellitus patients as well as in control subjects (Table 1 B). IgG secretion by autologous cocultures of T and EBV-activated B cells was significantly decreased compared to EBV-activated B cultures alone at day 16 and 20 of culture (p<O.O5 and <0.01, respectively)

The mean suppression ratio was not significantly different in type 1 (insulin dependent) diabetes mellitus patients and in control subjects.

IgM and IgG Secretion by EBV-activated B cell Cultures from Type I (insulin dependent) Diabetes Mellitus Patients and Normal Subjects

We compared IgM and IgG secretion by EBV-activated B cells cultured alone in type 1 (insulin dependent) diabetes mellitus patients and control subjects (Table 1 Aa and Ba). No difference was found between these groups, suggesting that metabolic changes of type 1 (insulin-dependent) diabetes mellitus patients have no influence on activation of B cells by EBV in vitro.

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262

DISCUSSION

A. KAHAN, J . LeFLOCH A N D J . CHARREIRE

An organ specific autoimmune reaction probably participates in the development of insulin dependent diabetes mellitus. A very tempting hypothesis of the etio-path- ogenesis of this disease could be the following: in genetically susceptible individuals, the beta cells of Langerhans islets are stimulated to express HLA class I1 molecules in response to an environmental agent, possibly a viral infection. Lymphokines such as interferon gamma in combination with either tumor necrosis factor or lymphotoxin could be mediators for the inappropiate HLA class I1 expression2’. Once expressed, these molecules present their own surface antigens to autoreactive helper T lym- phocytes. Therefore a T cell regulation defect could be one of the possible mechanisms of autoimmunity in type 1 (insulin dependent) diabetes mellitus: either a helper cell hyperfunction or a suppressor cell hypofunction or both may be responsible for the induction of the diabetic state. Moreover the immunoregulatory defect could be organ or antigen specific or a more generalized one.

A defective T helper activity was demonstrated in a subset of type 1 (insulin dependent) diabetes mellitus patients by autologous mixed lymphocyte reaction24, very similar to that found in Graves’ disease2’. This deficiency is probably related to a decreased interleukin 2 production26. On the contrary, in allogeneic mixed lymphocyte reaction, T cells from type 1 diabetes mellitus patients respond normally, suggesting that in these two reactions different subsets of T cells are activated. It is supposed that in autologous mixed lymphocyte reaction, autoreative T cells contain precursors of Concanvalin A inducible suppressor cellsz6.

A deficit in suppressive cell activity has been demonstrated by migration inhibition test2’ or by direct cytotoxicity on isolated murine pancreatic islets either by a chromium release assay2’ or more specifically by an increased non-stimulatedZ9 or stimulated insulin release3’. This cytotoxic effect is a T cell phenomenon3’, CD8’ cells being involved in the damage of pancreatic beta cells as it has been shown morphologically32. Furthermore, a deficient concanvalin A induced suppressor cell activity was also demonstrated in newly diagnosed type 1 (insulin dependent) diabetes mellitus patients ’.’2”’.

To analyse the role of suppressive T cells in newly diagnosed type 1 (insulin dependent) diabetes mellitus patients, we chose as experimental system the activation of B cells by EBV. In this system, helper T cells, as well as macrophages, do not interfere with the suppressive T cell activity; thus it is possible to study only the suppressive function of T cells.

T cell regulation of EBV-induced B cell activation was assessed by comparing IgM and IgG secretions by B cells cultured alone or cocultured with autologous T cells. In newly diagnosed type 1 (insulin dependent) diabetes mellitus patients, a significant suppression of IgM secretion by cocultures of T and EBV-activated B cells compared to that of EBV-activated B cells alone was demonstrated on day 16 and 20 of culture with the two T to B ratios used. With the T to B lymphocyte ratio of 4 : I , reflecting the situation in peripheral blood, the suppression ratio was higher than with the ratio 1 : l .

The mean suppression ratios were lower in insulin dependent diabetes mellitus patients than those found in healthy EBV-immune control subjects. However, if we consider as normal suppression, a suppression ratio higher than 0.50 as previously described’s*’9, the analysis of individual IgM secretory responses indicated no signifi- cant difference in insulin dependent diabetes mellitus patients and control subjects,

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NORMAL SUPPRESSIVE T CELL FUNCTION 263

whatever T to B lymphocyte ratio. A similar normal suppression ratio was evident for IgG secretion on day 16 and 20 of culture.

The presence of multiple autoantibodies included those directed against cytoplas- mic antigens of beta islet cells suggested a B lymphocyte hyperactivity in insulin dependent diabetes mellitus patients. The percentage of B lymphocytes in peripheral blood was always found within the normal range; there was no evidence for polyclonal activation as measured by the number of spontaneous or pokeweed mitogen (PWM) induced immunoglobulin-secretion plaque forming cells or IgM and IgG secretions into the supernatants of PWM stimulated lymphocytes’. In our study the IgM and IgG secretions in EBV-activated B cell cultures was similar in type 1 (insulin dependent) diabetes mellitus patients and in control subject suggesting a normal B cell activation in this system also.

In conclusion, in type 1 (insulin dependent) diabetes mellitus patients as in Graves’ disease, the autoimmune reactivity is not dependent upon a generalized suppression defect as it was found in systemic autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus and systemic sclerosis). It can be hypothesized, therefore, that in type 1 diabetes mellitus as well as in Graves’ disease, a local or organ specific suppressor deficit may induce the autoimmune phenomena.

Acknowledgements We are indebted to Mrs Martine Nedelec and Nicole Bazely for excellent technical assistance.

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