Regulation of self-major histocompatibility complex reactive human T-cell clones

Int. J. lmmunopharmac., Vol. 12, No. 3, pp. 2 5 5 - 2 6 0 , 1990. Printed in Great Britain.

0 1 9 2 - 0 5 6 1 / 9 0 $3.00 + .00 International Society for Immunopharmacology.

REGULATION OF SELF-MAJOR HISTOCOMPATIBILITY COMPLEX REACTIVE H U M A N T-CELL CLONES M. S. GILARDINIMONTANI*'t F. DEL GALLO,* M. GOBBI,* G. LOMBARDI,* E. PICCOLELLA,* O. PUGLIESE* and V. COL~ZZI~ *Department of Cellular and Developmental Biology, I University of Rome; tlnstitute of Health, Rome; ~Department of Biology, II University of Rome, Italy (Received 27 July 1989 and in final form 31 October 1989)

Abstract -- The proliferative response of human T-lymphocyte clones, (TLC) specific for self-major

histocompatibility complex (MHC) products either alone or associated with PPD epitopes are inhibited in vitro by dexamethasone (DEX) and by a non-specific inhibitory factor(s) (nslNH) produced by PPD-activated T-cells. The inhibiting effect has been investigated by preincubating autoreactive and PPDspecific TLC with nslNH or DEX. Results obtained indicate that T-lymphocytes are the target of these two immunoregulatory molecules. Moreover, the addition of exogenous recombinant interleukin 2 (rlL-2) substantially reverses the inhibition observed in both nslNH- or DEX-treated cultures. Recognition of foreign antigens in the context of self major histocompatibility complex (MHC) products appears to be an essential feature for the activation of T-lymphocytes. It has been found, however, that T-lymphocyte stimulation either in vivo or in vitro frequently results in the induction of T-cells with specificity not only for nominal antigen but also for self-MHC determinants (Kotani, Mitsuya, Benson, James, & Strober, 1988; Faherty, Johnson, & Zauderer, 1985; Muller, Maier, Brinkmann, & Kaufmann, 1986). Whilst the in vivo significance of such cells is uncertain, it is none the less conceivable that the generation of autoreactive T-cells is actively regulated in vivo to avoid the expansion of such cells (Finnegan & Hodes, 1986). The induction and regulation of human T-cell clones specific for self-MHC products has been investigated in this paper. During the in vitro stimulation of peripheral blood mononuclear cells (PBMC) with mycobacterial antigens, either PPDspecific or self-MHC reactive T-cell clones have been generated. The regulation of such T-cell clones has been analyzed comparing their sensitivity to the synthetic glucocorticoid dexamethasone (DEX), and to a natural inhibitory factor (nslNH) obtained in vitro by antigen stimulation of PBMC with microbial antigens. This factor, that is acid (pH 2.5)

and temperature (56°C) stable and appears to possess two molecular weights of 30-35,000 and 60-65,000 (Lombardi, Di Massimo, Del Gallo, Vismara, Piccolella, Pugliese, & Colizzi, 1986) is released spontaneously by CD8 + T-cells separated by microbial antigen activated PBMC. Furthermore, the nsINH displays an antigen nonspecific and genetically unrestricted inhibitory activity on cell proliferation (Lombardi, Vismara, Piccolella, Colizzi, & Asherson, 1985b), as well as on IL-2 and immune interferon synthesis and NK cell induction (Lombardi et al., 1986). Likewise, DEX inhibits PBMC proliferation (Piccolella, Vismara, Lombardi, Guerritore, Piantelli & Ranelletti, 1986a) and lymphokine induced NK activity (Piccolella, Lombardi, Vismara, Del Gallo, Colizzi, Dolei & Dianzani, 1986b). Results reported in this paper suggest that nsINH and DEX regulate both antigenspecific and self MHC-reactive T-cell proliferation.

EXPERIMENTAL PROCEDURES

Cell preparation

PBMC from healthy subjects were isolated on a gradient of Lymphoprep (Nyeegaard and Co. A/S,

tAuthor to whom correspondence should be addressed at: Department of Cellular Development Biology, Via degli Apuli 1, 00185 Rome, Italy. 255

256

M.S. GILARDINIMONTANIet al.

Oslo, Norway). The cells were suspended at 106/ml in RPMI-1640 medium (Flow Laboratories, McLean, VA) supplemented with 10°70 heat inactivated human serum, 2 mM glntamine (Eurobio, Paris), and 100 I U / m l penicillin/streptomycin (culture medium) (Lombardi, Del Gallo, Vismara, Piccolella, De Martino, Garzelli, Puglisi & Colizzi, 1987).

Microbial antigens Purified protein derivative (PPD) from Mycobacterium tuberculosis (Statens Seruminstitut, Copenhagen, Denmark) and expressate fractions of the Mycobacteria strains H37Rv of M. tuberculosis, BCG of M. bovis, M. gordonae, and M. avium (Hewitt, Coates, Mitchison & Ivanyi, 1982) have been used for studying the specificity of T-cell clones. Tetanus toxoid (TT) and a polysaccharde fraction from C. albicans (MPPS; Piccolella, Lombardi & Morelli, 1980) were used as non-mycobacterial control antigens. Preparation o f EB V-B lymphocytes B-cells were enriched by rosetting (Piccolella et al., 1980) and the E cells, resuspended in medium with I0% heat inactivated serum at 10 6 cell/ml, were transformed with E p s t e i n - B a r r virus (EBV) obtained from the marmoset lymphoblastoid line B95-8 as previously described (Lombardi et al., 1987). Preparation o f T-lymphocyte clones" (TLC) PBMC from healthy PPD skin test positive donors were precultured in 24-well microtiter plates (Flow Labs) for 7 days in the presence of PPD. The blast cells were cloned by limiting dilution (0.3 cells/well) in Terasaki plates in the presence of irradiated autologous PBMC (5 x 10"/ml), rIL-2 (20 units/ml, Biogen SA, Geneva) and antigen (10/ag/ml) as previously described (Lombardi et al., 1987). Growing clones at day 7 were transferred to 96-well microtiter plates (Falcon) and then to 24-well trays (Flow Labs). At each transfer, the TLC received specific antigen, irradiated (2500 rads) autologous PBMC or irradiated (5000 rads) autologous EBV-B cells and rIL-2 (20 U/ml). Proliferation assay T-cell clones were cultured at 3 × 104 cells/well in microtiter trays in culture medium in the presence of antigen presenting cells (APC) which were either autologous irradiated PBMC (105 cells/well) or

autologous irradiated EVB-B cells (3 × 104 cells/ well) and antigenic preparations (10 ~g/ml, determined as optimal in preliminary experiments). Following 3 days of incubation, the cultures were pulsed with 0.5 ~Ci of tritiated methyl thymidine (3H-TdR, specific activity: 5 Ci/mmole, Radiochemicals Inc., Amersham) for 16 h and harvested onto glass fibre filters. 3H-TdR incorporation was measured by liquid scintillation spectroscopy. The results are expressed as mean counts per minute _+ standard deviation (S.D.) of the mean for ['riplicate cultures. The inhibition of cell proliferation mediated by nslNH or DEX was calculated according to the formula: (counts/rnin of control cultures counts/min of nslNH treated cultures)/ (counts/rain of control culture) x 100.

Dexamethasone treatment Dexamethasone (DEX, Sigma, Munich) was used at the 2 x 10-TM concentration chosen on the basis of a dose-response curve for the inhibition of lymphocyte proliferation assay (Piccolella et al., 1986a). TLC were preincubated with DEX for 3 h at 37°C. At the end of incubation period, TLC were resuspended in medium and maintained at 37°C for 3 h to allow the release of unbound DEX. Thereafter, the cells were washed three times and added to the cultures containing EBV-B cells.

Production o f n s I N H PBMC (5 x 105cell/ml) were precultured in flasks (Nunc Inter Med) for 5 days in presence of PPD (20/~g/ml) in a CO2 incubator. The cells were then harvested, washed, resuspended in fresh medium and cultured for 2 days at 2.5 x 106 cell/ml in the absence of antigens in new flasks. The 48 h filtered supernatant was routinely used as source of nsINH (Lombardi, Piccolella, Vismara, Colizzi & Zembala, 1985a). Aliquot samples stored at - 2 0 ° C maintained their inhibitory activity. Determination o f n s l N H activity The nsINH-containing supernatants, diluted 1:2 with culture medium, were tested for their ability to inhibit cell proliferation by their addition at the beginning of TLC cultures or by preincubating TLC for 3 h. In the latter case, cells were then collected, washed once, and added to the untreated autologous and irradiated APC.

257

Regulation of Self-Major Histocompatibility Complex Reactive H u m a n T-Cell Clones Table

1.

Proliferative

P/1 Medium A P C alone + IL-2 + PPD + H37Rv + M. bovis + M. gordonae

+ MPPS + TT

response

of mycobacterial reactive-TLC

PPD-specific T L C P/2 P/3 P/4

2 2 23 89 93 100 4 2 2

2 2 2 62 76 57 2 2 2

I 1 9 157 190 57 1 1 1

specific- and

self-MHC

Self-MHC reactive T L C A/1 A/2 A/3 A/4

2 2 59 185 285 150 3 2 2

1 389 ND 336 ND ND ND 314 ND

3 128 1386 152 ND ND ND 119 ND

1 121 198 104 ND ND ND 101 ND

1 457 718 421 ND ND ND 426 ND

T-cell clones (3 x 104 cells/well) were cultured for 3 days with irradiated autologous EBV-B cells (3 x 104 cells/well). The antigen preparations were added at the beginning of the cultures, whereas radioactive thymidine (0.5 MCi/well) was added 16 h before the end of cultures. The D N A synthesis was expressed as counts per minute ( x 10 -2) and the S.D. was always less than 10%. Autoreactive T-cell clones were tested also with autologous P BMC (105 cells/well) and the proliferation values were comparable with those obtained with EBV-B cells.

Table 2. Regulation of PPD-reactive (DG2/2) and of selfM H C reactive (CA2) T cell clones by distinct baches o f n s l N H and by D E X

Table 3. Effect of n s I N H or DEX preincubation on PPDspecific (DG2/2) and autoreactive (CA2) T cell clone proliferation

Cell culture treatment

T L C preincubation with

3H-TdR incorporation DG2/2 CA2

Experiment 1 Medium 19702 ± 2284 50% 4739 ± 912 (75) t n s I N H 5 25% 12247 + 1277 (37)* 12070 17597 ± 3554 50% n s I N H 6 25% 12%

4073 ± 851 8282 + 363 14359 ± 402

_+ 975 ± 330 (68)* +_ 349 (55)* + 650

(79) t 2835 ± 158 (69) + (57)* 4567 _+ 188 (50)* 5532 ± 220 (40)*

50% 14569 _+ 1487 (26) n s I N H 7 25°70 20793 + 2963 12% 23148 _+ 2489 Experiment 2 Medium DEX

9253 2906 4102 6471

3614 ± 197 (60)* 5656 ± 610 (38) 8372 _+ 60

29318 + 1445 6128 _+ 483 2110 ± 441 (92) + 980 ± 10 (91) +

Cell cultures containing T L C and PPD-pulsed EBV-B cells were treated with n s I N H and D E X (2 x 10 7M) and cultured for 3 days. The results, referred to two representative experiments, are reported as c o u n t s / min ± S.D. The n u m b e r s in parentheses indicate the percentage of inhibition. *P<0.01; tp<0.001 (Student t-test). RESULTS Several TLC were developed from human PBMC of healthy individuals cultured with PPD. The antigenic specificity of each TLC was determined by co-culture with APC and with a variety of antigenic

Experiment 1 Medium nsINH 6 Medium DEX Experiment 2 Medium nsINH 7 Medium DEX

3H-TdR incorporation DG2/2 CA2 16090 ± 1452 5869 ± 642 8508 ± 1109 (47) + 3863 ± 323 (34)* 5956 ± 441 2337 ± 7 3 1

42838 ± 2580 ( 6 1 ) t 2 6 0 0 9 ± 181 (39)*

7015 + 941 3124 _+ 215 (55)*

ND ND

31503 ± 1269 56342 ± 4203 18480 + 726 (37)*29986 ± 1132(47)*

T L C D G 2 / 2 and CA2 were preincubated at 106 cells/ml in the presence of D E X (2 x 10-TM) or n s l N H (50o70) for 3 h. At the end of incubation, the cells were spun down, washed and cultured for 3 days with the unpreincubated autologous irradiated EBV-B cells. The results, referred to two representative experiments, are reported as c o u n t s / min ± S.D. The n u m b e r s in parentheses indicate the percentage of inhibition. *P<0.05; tp<0.01 (Student t-test).

preparations. PPD-specific TLC and autoreactiveT L C w e r e c o n s i d e r e d , r e s p e c t i v e l y , t h o s e cells r e s p o n d i n g to A P C in t h e p r e s e n c e o f t h e mycobacterial antigen, PPD, a n d t h o s e cells responding to MHC glycoprotein expressed on the surface of APC without addition of nominal antigen. Table 1 shows that PPD-specific TLC also

M.S. GILARDIN1MONTANI et al.

258

Table 4. Effect of exogenous IL-2 on nsINH- or Dextreated cultures of PPD-specific (DG2/2) and autoreactive (CA2) TLC Cell culture treatment Experiment 1 -+ IL-2 + nslNH 6 + nsINH 6 + IL-2

3H-TdR incorporation DG2/2 CA2 15317 _+ 3086 73127 _+ 6100 3821 _+ 391 67553+ 3565

5833 _+ 465 55661 _+ 6714 1397 + 118 50993_+ 1597

5956 _+441 27884 _ 1168 728 _+445 28198+ 999

18695 + 1794 22546_+ 1938 3882 + 33 14354 + 1231

either nsINH or DEX resulted in a reduction of subsequent proliferation. The effect of exogenous IL-2 on nsINH- or DEXtreated cultures was then analyzed. Table 4 shows that rIL-2 was capable of reversing the inhibitory activity exerted by nsINH or DEX. However, the levels of thymidine incorporation of lL-2-treated cultures were always higher than those obtained in absence of exogenous IL-2.

Experiment 2 --

+ IL-2 + DEX +

DEX

+

IL-2

DG2/2 and CA2 TLC were cultured for 3 days with PPDpulsed or unpulsed EBV-B cells and nsINH (50%), DEX (2 x 10 7M) and rIL-2 (20 units/ml) in different combinations. The results, referred to two representative experiments, are reported as counts/min _+ S.D.

responded to M . tuberculosis strain H37Rv and M . bovis preparations. In contrast, they failed to respond to other mycobacterial antigen preparations such as M . gordonae, or to unrelated antigenic controls (MPPS and TT). However, autoreactiveTLC proliferated in the presence of only A P C and antigen had no additional effect. Autoreactive T-cell clones were also characterized for their HLArestriction with a variety of allogeneic A P C of known HLA, and membrane cluster differentiation antigens such as CD4 and CD8 (data not shown). Thereafter, we investigated the regulation of PPDspecific and of self-MHC reactive T-cell clone proliferations by nsINH and DEX. Data shown in Table 2 indicate that nsINH and DEX exert a statistically significant inhibitory activity on TLC proliferation. In fact, experiment 1 shows that nsINH produced by PBMC cultured for 5 days in the presence of P P D was able to similarly inhibit the proliferative response of both PPD-specific and self MHC-reactive TLC. This inhibition was dosedependent as evidenced in Table 2 for the 50°70 and 25% nsINH concentrations and was present in different batches of nsINH although at different levels of activity. The experiment 2 of the same table shows that both PPD-specific and self-MHC reactive T-cell clones were inhibited in their proliferation by the presence of DEX in culture. The target of these inhibitory activities was then investigated by preincubating PPD-specific and MHC-reactive TLC with either nsINH or DEX. It was found (Table 3) that preincubation of TLC with

DISCUSSION In this paper we describe that PBMC stimulation with mycobacterial antigens, such as PPD, results in the development either of T-cell clones reactive to PPD or of T-cell clones reactive to self-MHC molecules. Although this result is obtained using cloned T-cells and it may represent an artefact in vitro, a high frequency of autoreactive T-cells has been evidenced in PBMC cultures stimulated with PPD (Del Gallo, Lombardi, Piccolella, Gilardini Montani, Del Porto, Pugliese, Antonelli & Colizzi, 1990). In the course of mycobacterial infections, the immune system is exposed to modified autologous tissue antigens caused by the increased protein degradation and turnover, thus self antigens and mycobacterial antigens may cross-react in humoral and cellular responses (Shoenfeld & Isenberg, 1988). For example, the 65,000 mol. wt mycobacterial protein, which recent studies have shown to be an immune target in tuberculosis (Young, Lathriga, Hendrix, Sweetser & Young, 1 9 8 8 ) and in autoimmune arthritis (van Eden, Thole, van der Zee, Noordzij, van Embden, Hensen & Cohen, 1988), shares a high homology with eukariotic heat shock proteins (Shinnick, Vodkin & Williams, 1988). Hence a classical mechanism of molecular mimicry between mycobacteria and host antigens may be responsible for the induction of autoreactive T-cells (Raulet, 1988). The fact that T-cells with two distinct recognition patterns are induced by mycobacterial stimulation opens the question whether both cells are regulated by similar mechanisms. Recent studies have demonstrated that autocytotoxic T-cells are generated during stimulation with autologous peripheral blood lymphocytes and IL-2 in vitro and are physiologically down regulated by autologous suppressor T-cells (Rosenkrantz, Dupont & Flomenberg, 1985). It is unclear whether autoreactive T-cells are inhibited through direct cell-

Regulation of Self-Major Histocompatibility Complex Reactive Human T-Cell Clones to-cell contact or through the release of soluble factors by the regulatory population. Inhibitory molecules capable of down regulating cell proliferation and cell cytotoxicity have been reported to be produced by leukemic cells (Chiao, Heil, Arlin, Lutton, Choi & Leung, 1986), glioblastoma cells (Wrann, Bodmer, de Martin, Siepl, Hofer-Warbinek, Frei, Hofer & Fontana, 1987) and normal T-lymphocytes activated either in vitro or in vivo by antigens (Asherson, Colizzi & Zembala, 1986) and by microbial infections (Colizzi, Asherson, Malkovsky, Garrea & Ferluga, 1984; Teh, Ho & Williams, 1988). We have described a nsINH factor that is released in vitro by microbial antigen activated T-cells and which controls cell proliferation by interfering with the lymphokine cascade. The involvement of nsINH in the control of lymphokine pathways was formally demonstrated by showing that PBMC cultured in the presence of nsINH and PPD failed to release IFN and IL-2 and to express IL-2 receptor (Tac antigen) (Lombardi et al., 1985a; Lombardi et al., 1985b; Lombardi et al., 1986). However, recent experiments show that the incubation of T-cell clones with nsINH in the presence of exogenous IL-2, did not impair the expression of Tac antigens (unpublished results), confirming the observations that IL-2 itself regulates the IL-2 receptors (Smith, 1988). A number of other inhibitory factors which share similar physicochemical and functional properties with nsINH have been described in human systems. Con A-activated human mononuclear cells elaborate, within 8 to 24 h of culture, two distinct inhibitory factors of 3 0 - 45,000 mol. wt (Greene, Fleisher & Waldmann, 1981) and 60-90,000 mol.wt (Fleisher, Greene, Blaese & Waldmann, 1981), acid (pH 2.5) stable and temperature (56°C) unstable that inhibit mitogenand antigen-stimulated T-cell proliferation and B-cell immunoglobulin production respectively. Human myeloid leukemic cells (Chiao et al., 1986) produce constitutively an inhibitory factor with a molecular weight in the range of 40-60,000 which

259

suppresses mitogen- and alloantigen-stimulated proliferative responses of normal lymphocytes by drastically reducing IL-2 production. All these factors produced in different ways, present molecular weight ranges similar to nsINH but further analysis at molecular level will be needed to clarify the relationships between them. In this paper, we have compared the sensitivity of self MHC-reactive T-cell clones with that of PPDspecific T-cell clones to nsINH. Moreover, as inhibitory molecule control, we have used the synthetic corticosteroid DEX that previous studies have shown to inhibit T-cell proliferation (Piccolella et al., 1986a) and cytokine (IL-1, IL-2, IL-3, IL-4) transcription (for a review see Dupont, 1988). Results presented show that both PPD-specific and self MHC-reactive T-cell clones were similarly regulated by nsINH and DEX and that T-cells are the target of their inhibitory activities. The involvement of nsINH in the counterbalance of positive effect of the lymphokine cascade, as previously demonstrated (Lombardi et al., 1985b; Lombardi et al., 1986), was here confirmed by the finding that the inhibition of the TLC proliferation could be reversed by the addition of exogenous IL-2. Taken together these results suggest that a possible mechanism of action of nsINH may involve decreased synthesis of cytokines through molecular mechanisms that at present are unresolved. In conclusion, the fact that a natural product (nsINH) is capable of down regulating autoreactive T-cells in a manner similar to that of DEX, could be of practical interest in the identification of new molecules of potential usefulness in the immunopharmacological treatment of autoimmune diseases. Acknowledgments - - This study was supported by the

Italian Research Council grants No 88.00472.04 and No 88.00704.44 and by the Immunology of Tuberculosis (IMMTUB) component of the WHO special programme on Vaccine Development.

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