Further evaluation of autoreactive T cells in hydatid patients

Immunology Letters, 40 (1994) 59~3 0165 - 2478 / 94 / $ 7.00 © 1994 Elsevier Science B.V. All rights reserved IMLET 02100

Further evaluation of autoreactive T cells in hydatid patients F. Q u i n t i e r i a'*, R. R i g a n 6 a, O. Pugliese a, A. Teggi b a n d A. S i r a c u s a n o a aLaboratorio di Immunologia, Istituto Superiore di Sanitc~, Viale Regina Elena 299, 00161 Roma, Italy; and blF Cattedra di Malattie Infettive, Universitgt "La Sapienza', Viale Regina Elena 330, 00161 Roma, Italy (Received 29 November 1993; accepted 26 January 1994)

I. Summary

We evaluated the peripheral autoreactive response in patients with Echinococcus granulosus who showed a negative humoral response as compared to seropositive patients and healthy controls. For this purpose, a limiting dilution analysis (LDA) of autologous mixed lymphocyte cultures was established to both estimate frequency of autoreactive T cells and, by analysing the shape of the curves, to clarify the mechanisms that underlie the autoreactive response. Different LDA curves were observed between healthy controls and patients, suggesting that different cell interactions are involved in the two populations. More interestingly, all hydatid patients, independent of their humoral response, showed a higher number of autoreactive T cells than controls. Precisely, subjects with a negative humoral response showed a range of values for autoreactive T cells exactly between the value ranges observed in seropositive and normal subjects. The present data also show that the increase of autoreactive T cells in hydatid patients correlates with the production of specific antibodies.

2. Introduction

Autoreactive T cells proliferate in response to autologous MHC class II antigens, in the absence of known processed peptides. Such proliferation is detected by the autologous mixed lymphocyte reaction (AMLR) that provides an in vitro means for evaluating cell interactions. It has been studied in normal population and in different pathologies, especially Key words: Autoreactive T cell; Hydatid disease; Serodiagnosis; Limiting dilution analysis *Corresponding author: Dr. F. Quintieri, Laboratorio di Immunologia, Istituto Superiore di Sanitfi, Viale Regina Elena 299, 00161 Roma, Italy. SSD1 0 1 6 5 - 2 4 7 8 ( 9 4 ) 0 0 0 1 6 - K

autoimmune diseases where both an impairment and an increase were observed [1-3]. Recently, we studied the autoreactive phenomenon in hydatidosis, due to Echinococcus granulosus infection in man, showing a high increase in peripheral autoreactive T cells in patients compared to normal subjects [4]. Serodiagnosis of hydatid disease in man has been of great use to clinicians since no parasitological diagnosis is possible using non-invasive procedures and the clinical signs of the disease are non-specific. Although there has been an improvement in the sensitivity of techniques applied to immunodiagnosis, a high percentage of patients still test false-negative. The aim of this study was to analyze the proliferative response of T lymphocytes to autologous peripheral blood mononuclear cells (PBMC) in 14 hydatid patients and to compare it to the serological response. For this purpose, we performed a limiting dilution analysis (LDA) of AMLR cultures that provides both quantitative and qualitative information on the immune response under analysis. In this case, in addition to the number of autoreactive T cells that can be calculated in different ways depending on the curve obtained, the shape of the curve reflects the mechanism regulating the autoreactive response.

3. Materials and Methods

3.1. Blood samples Human blood samples were obtained from 14 patients with clinically diagnosed hydatidosis (12 with hepatic localization, 1 with multiple localization and 1 with pulmonary localization), and from 12 sex- and age-matched healthy blood donors. In the 4 patients who tested negative with all the immunodiagnostic techniques, the diagnosis of hydatidosis was confirmed by surgery.

3.2. Antigens

Sheep hydatid fluid (HF) was collected in the area of Sardinia from fertile cysts. The following antigen preparations were used: (1) a crude concentrated HF, prepared by the method of Bombardieri et al. [5] and (2) an HF fraction obtained by precipitation at low ionic strength (0.005 M acetate buffer, pH 5.0) according to Oriol et al. [6]. 3.3. Serological tests

Indirect hemagglutination (IHA) was carried out as described by Iacona et al. [7]. Double diffusion for antigen 5 (DD5) was performed using the pattern of double diffusion previously described [5]. Antigen 5 monoclonal antibody was employed as control antiserum to recognize arc-5-positive sera by a reaction of identity [8]. Immunoelectrophoresis (IEP) was performed with agarose at a concentration of 10 g/1 in 0.05 M barbital buffer, pH 8.6 [9]. Immunoblotting (IB) was performed as previously described [10]. 3.4. Cell preparation

Peripheral blood was taken by venopuncture into preservative-free heparin and PBMC were separated from plasma by density gradient centrifugation (Lymphoprep, Nyegaard, Oslo, Norway). Cells were washed twice and resuspended in RPMI-1640 (Flow Laboratories, Irvine, UK) containing 25 mM gluta-

mine, 100 U/ml penicillin, 100 #g/ml streptomycin and 10% fetal calf serum (FCS) inactivated at 56°C for 30 rain. 3.5. Autoreactive response

To prepare responder T cells, a nylon-wool column method was used [11]. Briefly, 5 x 106 PBMC in 0.5 ml of RPMI-1640 with 10% FCS (complete medium) was applied to a nylon-wool column (0.12 g of nylon wool) and the column was incubated at 37°C for 45 min in a CO2 incubator. After incubation, the non-adherent cells were gently eluted with 12 ml of prewarmed (37°C) complete medium. The effluent population contained between 84 and 90% T cells, as assessed by FACS analysis. Enriched T lymphocytes were cultured in fiat-bottomed 96-well microtiter plates at graded concentrations ranging from 390 to 50 000 cells/well with 50 000 autologous irradiated (6000 R) PBMC in each well. Cultures were set up in 24 replicate wells in a total volume of 200/A of complete medium. Recombinant IL-2 (rlL2, Janssen Chimica, Beerse, Belgium) was added at days 7 and 10 of culture. At day 12 of incubation, 20 h before termination, 0.5 #Ci/well of [3H]methylthymidine (specific activity: 5 mCi/mmol; Amersham Italia, Milan) was added. The proliferative activity of each well was considered positive when the counts per minute (cpm) exceeded the mean of the negative control (T cells alone) by at least 3 standard deviations. Analysis of the distribu-

TABLE 1 SEROLOGICAL RESULTS OF H E M A G G L U T I N A T I O N (IHA), I M M U N O B L O T T I N G (IB), I M M U N O E L E C T R O P H O R E S I S (IEP), DOUBLE D I F F U S I O N FOR A N T I G E N 5 (DD5) AND F R E Q U E N C Y OF AUTOREACTIVE CELLS (AUT) IN HYDATID PATIENTS Patient

Age (years)

Loc. of cysta

IHA b

L.B. A.F. P.S. A.T. G.M.

51 48 61 65 59

H H H P H

160 < 160 < 160 < 160 160

P.S. S.M. M.T. A.M. Z.L.

32 39 44 58 50

M H H H H

P.C. T.G. A.S. F.S.

31 80 53 20

H H H H

m

IEP

DD5

_

m

m

m

AUT 1/7500 1/9500 1/8000 1/7500 1/8000

(13.3x (10.5 x (12.5 × (13.3 x (12.5 x

(100× 10 -5 ) (83 x 10 5) (83 x 10 -5) (40 x 10 -5) (76.9 x 10 5) (100x 10 5) (200× 10 5) (55.5x 10 5) (50 x 10 -5)

_

m

_

m

÷

+

160 320 640 >40000 2560

-

÷

+ +

+ +

1/1000 1/1200 1/1200 1/2500 1/1300

1280 >40000 2560 640

+ + + +

+ + + +

1/1000 1/500 1/1800 1/2000

%ocalization of the cyst was hepatic (H), pulmonary (P) or multiple (M). bReciprocal of titre.

60

IB

m

10 5) 10 -5) 10 5) 10 5) 10 -5)

tion of precursor cells was performed by plotting the negative logarithm of the fraction of the non-responding cultures ( - l n F o ) on the y axis and the cell input on the x axis. The semilog plot was used to estimate the frequency of autoreactive precursor cells because, by interpolating the non-responding cultures at the level of 0.37, the size of the sample containing an average of one precursor cell can be estimated [12]. The precursor frequency of cultures exhibiting non-'single-hit' kinetics was roughly calculated as described by Lefkovits and Waldmann [13]. Briefly, F o = 0 . 3 7 was plotted from line 0.1 and AN cells were read directly on the titration curve; frequency = I/AN. The D R specificity of autoreactive T lymphocytes was assessed as reported in a previous paper [4]. The proliferative response of autoreactive T cell lines was blocked by anti-class II monoclonal antibodies, in agreement with results of restriction experiments.

TABLE 2 FREQUENCY OF PERIPHERAL AUTOREACTIVE T CELLS

Subjects (n)

Range

Healthy controls ( 1 2 ) Responder patients ( 1 0 ) Non-responder patients (4)

1/8000-1/35000 1/14000 1/500-1/2500 1/1250 1/7500-1/9500 1/7750

Wilcoxon rank sum test Healthy controls vs. responder patients Healthy controls vs. non-responder patients Responder patients vs. non-responder patients

P < 0.005 P < 0.05 P < 0.005

cells/well

A

I

I

3.6. Statistical analysis Linear portions of L D A curves were analyzed by a linear least-square regression analysis of the semilogarithmic plot of the number of cells per well versus the fraction of negative cultures. Correlation values ranged from 0.964 to 0.995. Differences between frequencies of autoreactive T cells were analyzed using the non-parametric Wilcoxon rank sum test. Correlation index between frequencies of autoreactive T cells and I H A antibody titers was evaluated by the Spearm a n rank test.

Median

Sill I

I

|

!

DNI I

I

GlIB I

I

I

|

IM I

!

ISIN I~_

0.37

01 0

",

B

•

!

m

I

I

N,w

n

o~

037

4. Results

Serological results and the frequency of autoreactive T cells obtained from 14 hydatid patients are shown in Table 1. Four patients testing negative with all the techniques used for immunodiagnosis (IEP, DD5, I H A , IB) were named non-responders; 10 patients testing positive in at least two of the serological tests used were named responders. Fig. 1 shows an example of the L D plots found in normal subjects (A), responders (B) and non-responders (C). Controls show a straight line passing through the origin ('single-hit' kinetics), meaning that only one population is limiting, in this case the peripheral autoreactive T cells. N o r m a l subjects show a range of autoreactive T cells between 1/8000 and 1/ 35000 (Table 2). On the contrary, all the hydatid patients show a curve with a linear portion at high cell concentrations (non-'single-hit' kinetics), mea-

.~

o,,I-01

0.1 4

\

~

o

Fig. 1. LDA of peripheral autoreactive T cell from a normal subject (A), a responder (B) and a non-responder (C) hydatid patient. Number of cells added per culture is plotted on the x axis. Fraction of negative cultures is plotted on the y axis. The data represent values from one representativeexperiment for each group studied. 61

ning that more than one population is limiting due to impairment of population(s) that normally cooperate in autoreactivity. Two ranges of autoreactive cells were defined: one from 1/7500 to 1/9500 in non-responder patients and the other from 1/500 to 1/2500 in responder patients. Table 2 also shows values of P obtained with the Wilcoxon rank sum test, carried on the different groups of numbers of autoreactive cells defined by LDA. Normal subjects, non-responder and responder patients have significantly different autoreactive cell numbers. Fig. 2 shows the distribution of the number of autoreactive cells in hydatid patients according to their serological reactivity. Responder and non-responder patients define two distinct groups of values; moreover, the number of autoreactive T cells and the serological reactivity correspond for most patients. The Spearman rank test confirms a correlation between specific anti-hydatid antibody titers (IHA) and the number of autoreactive T cells (r = 0.068; P < 0.05).

5. Discussion

In this study the proliferative response of T lymphocytes to autologous PBMC in 14 hydatid patients was evaluated and compared to their serological re-

N U t ~ E R OF AUTOREACTNE CELLS xIO "f

.

•

*o

oll

Fig. 2. Distribution of the frequency of peripheral autoreactive T cells in responder (R) and non-responder (NR) hydatid patients. Median value of autoreactive T cells in normal subjects is 6.7 x 10 -5"

62

sponses. We previously demonstrated a very high proliferative response of peripheral autoreactive T cells in hy, datid patients compared to normal subjects [4]. Such an increase could be due to the persistent presence of parasite antigens. These antigens probably induce an elevated production of INF-v and, consequently, increase the HLA class II antigen expression on stimulator cells and/or mimic 'self-molecules leading to an enhanced autoreactive response [14]. In addition, LD experiments indicated that different mechanisms lead to peripheral autoreactive T cell proliferation in hydatid patien~ts compared to healthy controls. Chronic activation of the immune system does not alone explain the increased AMLR frequencies, as in systemic sclerosis [15], and in Crohn's disease (data not published) an enhancement of autoreactive T cells has not been found. Our present study extends the evaluation of peripheral autoreactivity in hydatid patients, including seronegative subjects. Several reasons can cause the lack of detectable antibodies in patients' sera: (a) cyst localization (pulmonary localization gives a high rate of negativity); (b) time of infection; (c) patient's age (children are often seronegative); and (d) the presence of circulating antigen which binds the specific antibody to form circulating immune complexes, thus rendering the antibody unavailable for detection [16]. The non-responder patients used in this study behave similarly to responder patients, showing the same kinetics in LDA and also an elevated number of autoreactive T cells compared to normal subjects. Interestingly, their values for autoreactive T cells range between those observed in responder hydatid patients and normal subjects. A lower level of parasite antigen could explain this phenomenon. Finally, our data demonstrate a correlation (P<0.05) between serum antibody levels (IHA) and the peripheral autoreactive response in hydatid patients in contrast to their mitogen and antigen proliferative response [17]. In fact, we can identify responders and non-responders on the basis of their number of autoreactive cells in peripheral blood. Further investigation is requested to better define the mechanism that underlies autoreactivity and the role of hydatid antigens in enhancing this phenomenon. The significance of the autologous immune response in the protection or pathogenesis of hydatid disease has to be clarified.

Acknowledgements T h e a u t h o r s t h a n k Prof. G . V i c a r i for h e l p f u l c o m m e n t s o n the m a n u s c r i p t . T h e t e c h n i c a l a s s i s t a n c e o f M r . L. B e r n a r d i n i is v e r y m u c h a p p r e c i a t e d . T h i s p r o j e c t w a s p a r t i a l l y f i n a n c e d b y G r a n t 92.01290 f r o m the C o n s i g l i o N a z i o n a l e delle R i c e r c h e .

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