Manipulation of autoimmune diseases with T-suppressor cells: lessons from experimental SLE and EAE

Immunology Letters, 36 (1993) 109-116

0165 - 2478 / 93 / $ 6.00 © 1993 ElsevierSciencePublishers B.V. All rights reserved IMLET 01964

Minireview Manipulation of autoimmune diseases with T-suppressor cells: lessons from experimental SLE and EAE Y e h u d a S h o e n f e l d a, M i r i B l a n k a, R i n a A h a r o n i b, D v o r a T e i t e l b a u m b a n d R u t h A r n o n b aResearch Unit of Autoimmune Diseases, Department of Medicine B'. Sheba Medical Center, Tel-Hashorner, Israel," and bDepartment of Chemical Immunology, The Weizmann Institute for Sciences, Rehovot, Israel

(Received 18 March 1993; revision received25 March 1993; accepted 26 March 1993)

1.

Introduction

In the past two decades a large body of experimental evidence has accumulated showing that normal B lymphocytes are capable of synthesizing and secreting autoantibodies [1] and that naturally occurring autoreactive T cells are present in healthy individuals [2,3]. These new discoveries raised the question of the normal control mechanisms preventing B and T lymphocytes from exerting their autoaggressive potential in vivo, a process which may lead to autoimmune diseases. Today, it is generally believed that one of the central immunoregulatory impairments leading to autoimmunity is defective T-suppressor (Ts) activity [4,5]. Ts-cell dysfunctions have been described in most autoimmune conditions. Despite abundant data on Ts cells, the concept of a unique subset of Ts cells is still a matter of debate. However, suppressor phenomena do occur in vivo and in vitro, and indirect evidence favoring the existence of a Ts-cell subset is accumulating. In the present review, the validity of the Tscell concept will be examined and its employment in manipulating autoimmune diseases will be discussed. Key words: Autoimmunity;Autoimmunedisease; T-suppressor cells; Autoantibody Correspondence to." Y. Shoenfeld, M.D., Department of Medicine B', Sheba Medical Center, Tel-Hashomer52621, Israel. Tel.: 972-3-5302652; Fax: 972-3-5352855.

2.

T-suppressor cell function in animal models and humans with autoimmune diseases

Recently, we summarized the extensive evidence for decreased number and function of Ts in various autoimmune conditions [4]. Those conditions in animal models entail: B/W F1 mice [6], experimental myasthenia gravis, experimental allergic encephalomyelitis, experimental autoimmune uveitis, spontaneous autoimmune thyroiditis in an obese strain of chickens and in experimental autoimmune thyroiditis. The data in human autoimmune diseases are much more detailed and include: SLE, autoimmune thyroid disease, multiple sclerosis, myasthenia gravis, primary biliary cirrhosis, chronic active hepatitis, rheumatoid arthritis, insulin-dependent diabetes mellitus, Sjogren's syndrome, Goodpasture's syndrome and autoimmune hemolytic anemia (for details, see [4,5]). Previously, several authors have reported on the employment of Ts cells in immune-mediated conditions. Pachner and Kantor [6] were able to establish and maintain acetylcholine receptor (AChR)-specific Ts cell lines by incubating spleen cells from C57B1/6 mice immunized with AChR with the same antigen (AChR): upon i.v. injection of these Ts cells into syngeneic mice with experimentally induced myasthenia gravis, they reported a suppression of anti-AChR antibody production followed by an improvement in neuromuscular dysfunction. Similar results were 109

achieved following the induction of guanosinespecific Ts cell lines that were shown to suppress in vitro the response to nucleosides other than guanosine [7]. In another study, Liebling et al. [8] reported on the generation of Ts cells specific for DNA. These cells suppressed the production of anti-DNA by both autologous peripheral mononuclear cells (PBMC) and allogeneic PBMC derived from SLE patients. A Ts line was also isolated from spleen cells of rats primed with the retinal soluble antigen in the anterior chamber of the eye. This line could inhibit in vitro the proliferation of uveoretinitic Th lymphocytes and downregulate experimental autoimmune uveoretinitis (EAU) [gal. In the following survey, we address the effect of Ts cells in two representative autoimmune situations in animal models: SLE and EAE. 2.1.

Ts cell function in S L E

Several workers have reported a decreased number and activity of Ts cells in B/W F1 mice [9,10]; however, there are also contradictory findings [11,12]. Soluble immune response suppressor (SIRS) activity was found to be diminished in cultures of B/W lymphocytes, and injecting SIRS into B/W mice prevented lupus nephritis [13]. It has been reported that the major component of anti-DNA antibodies in B/W F1 mice undergoes a switch from IgM to IgG upon ageing and that this switch causes the severe autoimmune signs seen in these mice [14]. Sekigawa et al. [15] have demonstrated that the autoantibody class switch results from an age-dependent change in Ts-cell function, with a loss of the ability to suppress IgG anti-DNA antibodies upon ageing. In another murine model of SLE - the MRL/1 mouse strain - spleen and lymph node Ts cells were found to have increased activity; however, these suppressor cells have shown an inability to suppress autoantibody production [16]. Unregulated autoantibody production in patients with systemic lupus erythematosus (SLE) has been attributed to inadequate suppression. Indeed, decreased numbers and activity of Ts cells in SLE has been commonly reported [17,18]. Others [19] tried to correlate the CD4/CD8 ratio with clinical subtypes of SLE. There are reports of de110

creased Ts function in disease flare-ups, the function returning to normal during remissions [20,21]. Moreover, Liebling et al. [22] have shown that SLE patients were unable to produce specific anti-DNA Ts cells, despite retaining their ability to generate Ts cells directed against different antigens. A decreased percentage of suppressor-inducer (CD4+2H +) cells was also reported in SLE [23], with the maximal reduction occurring during active disease, especially in patients with nephritis [24,25]. However, there are reports showing increased Ts activity in lupus patients [19,26], and some workers even found decreased IL-2 production as a consequence of increased suppressor activity [27,28]. These seemingly paradoxical findings can be explained if we assume the existence of two types of Ts cells: those that suppress IL-2 production by CD4 cells showing excessive activity in SLE patients and those that suppress immunoglobulin production and are IL-2 dependent showing reduced activity. 2.1.1.

Our experience with Ts and S L E In a series of studies [29-32] we were able to identify a pathogenic anti-DNA idiotype defined as 16/6 Id. With this common (cross-reactive) Id we were able to induce an experimental SLE in various strains of naive mice [33]. The induction was carried out via the immunization of mice in their footpads with 1 fLg of the Id emulsified in adjuvant. Four months following the boost injection, the mice developed serological (e.g. antidsDNA, anti-Sm), clinical (increased ESR, leukopenia, thrombocytopenia, proteinuria) and histopathological (immune complex deposition in the kidneys) changes of SLE. Since in a preliminary study we were able to detect a lower activity of Ts cells against the pathogenic 16/6 Id, we decided to generate Ts cells specific for the Id and to try to immunomanipulate the induction of experimental SLE with these specific Ts cells. We have demonstrated the ability to generate in vitro suppressor cells (CD8 +), specific for a pathogenic idiotype 16/6 (CI), from a population of normal T cells exposed to silica beads coated with 16/6 Id + mAb [34]. The specificity of these Ts cells (noted Ts SA-2) was confirmed by the following assays: (a) Specific suppression of proliferation of 16/

6-specific T helper cell lines (Th. CII) when exposed to the Id. (b) Elimination of the inhibitory effect by pretreatment of the suppressor cells with the idiotype but not when incubated with the anti-idiotype or normal human IgM. (c) Suppression of 16/6 T cell line helper activity in in vitro anti-16/6 production by 16/6 mAbprimed B cells. The fact that only 16/6 Id + Ts cells elicited the effect, and not SA-2 (a control MAb) Ts cells, provided additional evidence that the Ts cells are 16/6 Id-specific. However, the mechanism by which this idiotype-specific Ts cell acts is unclear. Both the receptors of the 16/6 helper T cell line and 16/6 Ts cells are 'anti-16/6', and it is hard to believe that they could work via direct T-T cell contact. Preliminary studies with 12-day Ts-cell culture fluids clearly point to the possible involvement of specific idiotype soluble factors which might play a central role in the specific suppression activity. The 16/6-specific Ts-cell culture fluid suppressed specifically both the 16/6 helper cell line [35] proliferation and the in vitro antibody production. The inhibitory effect of the Id-specific Ts cells on the in vitro mAb production assay can be explained in two ways: (a) mechanical absorption of Id molecules by the T cells (actually we have demonstrated that the Id could abrogate the Ts activity in the T cell line proliferation assays) and (b) through secretion of inhibitory factors specific for the Id. 2.1.2.

Manipulation of experimental SLE with Ts specific for a pathogenic anti-DNA Id (16/6) Weekly treatment of naive mice immunized with 16/6 Id with the Id-specific Ts cells induced a decline in the titers of autoantibodies in the sera, without affecting the titer of the total immunoglobulins. The treatment also had an effect on the clinical manifestations (decreased ESR, higher WBC counts, and smaller proteinuria). Furthermore, following the Ts cell infusions, no immunoglobulin deposits could be demonstrated in the mesangium. However, it should be emphasized that this treatment was successful only when employed early in the disease and failed once the disease was well established (5 months after immunization with the 16/6 Id). It seems

that in this experimental model of SLE it is important to continue to infuse the Ts cells, otherwise the effect of the treatment fades with time. 2.2.

Ts cells and EAE

In the EAE system various conditions of unresponsiveness, such as natural resistance in certain strains of mice [36], spontaneous recovery from the disease [37], or resistance to second induction of the disease following recovery from an acute episode of EAE [38], have been attributed to the presence of suppressor cells. Suppressor cells were also demonstrated to be involved in the induction of tolerance to EAE, by administration of antigen either subcutaneously [39,40] or orally [41], T-cell vaccination [42,43] or immunization with T-cell receptor peptides [44]. The suppressor cells induced under these conditions could be isolated from spleen cells or lymph node cells of sensitized rats and mice and transfer resistance to EAE to syngeneic normal recipients [37-44]. Several types of cells were characterized as suppressor cells such as CD4 + [45,46], CD8 + [42,44,47] and C D 4 - C D 8 - [48,49] T cells, as well as B cells [45] and macrophages [50]. The suppressor cells were either antigen (BP)-specific [37-41] or with anti-idiotypic activity [42,44,48]. Some of the suppressor cells had cytotoxic activity [42,48], others mediated their inhibitory effects through a mechanism involving the secretion of TGF-fl [51] or IL-4 [52]. Recently, several groups have described the isolation of suppressor T cell lines exhibiting in vitro and in vivo inhibitory activities [42,46,53,54], emphasizing the role of suppressor T cells in the regulation of EAE. EAE is regarded as the experimental model of multiple sclerosis (MS). There are several lines of evidence pointing to impaired immune regulation in MS and the role of suppressor cells in the disease process. Thus, Ts-cell activity is diminished in the peripheral blood and cerebrospinal fluid lymphocytes of patients with acute exacerbations of MS when compared with patients in remission or controls [55,56]. Moreover, patients in the progressive stage of MS show persistently decreased Ts-cell activity, which disappears during remissions [57,58]. The overreactivity of B cells in MS as reflected in the increased production of immu111

noglobulins in CSF may also be related to a defect in immune regulation [59].

2.2.1. Our experience with Ts in EAE Our work on the involvement of suppressor cells in EAE which started several years ago demonstrated the role of suppressor cells in both the natural resistance to disease induction and artificially antigen-induced unresponsiveness to EAE. In the case of genetic resistance to EAE we had shown that pretreatment with either low-dose cyclophosphamide or irradiation before disease induction may convert otherwise resistant strains, e.g. the congenic strains of BALB (c, d and k) and NZB mice into EAE-sensitive [36]. These treatments are known methods for the elimination of suppressor cells [60,61], and hence our results serve as a strong indication that non-susceptibility to EAE in these genetically resistant strains is mediated by suppressor cells. Suppressor cell activity was also demonstrated by us in the case of antigen-induced resistance to EAE in mice [62]. Protection against EAE was induced by pretreatment with either mouse spinal cord homogenate (MSCH), myelin basic protein (BP) or Cop 1 - , a synthetic suppressant copolymer which had been found to induce a therapeutic effect on EAE and MS (reviewed in [63]). The antigen-induced state of unresponsiveness could be adoptively transferred to normal recipients by a transfer of spleen cells from non-susceptible donors, as a result of which 80% of the recipients did not develop EAE after challenge. Heat-killed cells or cells treated with anti-Thy-1 serum and complement were incapable of transferring the unresponsiveness. Cyclophosphamide was also effective in abolishing the induced unresponsiveness and suppression transfer activity. We have further demonstrated that the suppressor cells which mediate unresponsiveness to EAE also regulate the cellular immune response to BP in a specific manner. Thus, the suppressor cells inhibited delayed-type hypersensitivity (DTH) responses to BP, induced both by active immunization or passive transfer by effector cells, while D T H responses to PPD were not affected by the suppressor cells [64]. Moreover, a soluble suppressor factor was extracted from these cells 112

which had the same biological activities as the suppressor cells from which it originated. It suppressed specifically D T H reactivity to BP and blocked effector lymphocytes in vitro; furthermore, it was also capable of interfering with the induction of clinical EAE [65,66]. Recently, we demonstrated that Ts cell lines and hybridomas could be generated from spleen cells of these antigen-induced unresponsive mice that were efficient in blocking the various manifestations of EAE in vitro as well as in vivo [67].

2.2.2. Suppressor T-hybridomas Spleen cells from SJL/J, BALB/K and their F1 mice which had been rendered unresponsive to EAE, either by MSCH or by Cop-l, were fused with BW 5147 A K R lymphosarcoma cells. A fraction of the hybridomas obtained (2.5%) exhibited a functional in vitro suppressive effect on the response to BP, as measured by testing the activity of the supernatants on either the direct proliferation or the IL-2 secretion of effector T-cell lines. This inhibition was specific, since the hybridomas did not affect non-relevant control cell lines. The cellular response to the encephalitogen (BP) is the primary pathogenic process in EAE; inhibition of this response therefore implies that these hybridomas might be relevant to the regulation of EAE. Interestingly, we have noticed that the hybridomas which blocked the specific response to BP were themselves blocked by the same antigen, but not by PPD. This specific growth arrest was found in all the hybridomas that had shown in vitro suppressive activity, except for a few hybridomas which were weak inhibitors of the BP effector lines. Thus, a high correlation was demonstrated between the two phenomena. The hybridoma lines that had an in vitro suppressive activity were tested for their ability to prevent EAE in vivo. Only a few hybridomas of SJL/J origin had some effect on the development of the disease, but none of them caused more than 50% inhibition in comparison to the BW control cells. On the other hand, four hybridomas originating in BALB/K mice exerted a significant in vivo suppressive effect: two of them completely prevented the disease. The BALB/K resistant strain had been shown to maintain a natural high level of suppressor cells, which prevented the

manifestation of the disease [36], while the SJL/J mice that are susceptible to EAE have some malfunction of the suppressor cell arm [68,69]. This may account for the differences observed between the activity of the suppressor hybridomas originating from these strains. All of the hybridoma lines were more effective in the inhibition of EAE when injected at the time of disease induction than when injected a week before. The immediate effect induced by these cells in vivo suggests that these hybridomas are effector-suppressor cells that actually mediate the suppression of the disease rather than activating an additional subset of suppressor cells. High correlations have been found between the suppressive effect exerted by the hybridoma supernatants in vitro and the efficiency of the hybridoma cells to inhibit the manifestation of the disease in vivo. The suppressive activity demonstrated by the hybridomas both in vitro and in vivo indicates that these hybridomas are indeed suppressor hybridomas that can regulate EAE. The disadvantage of using malignant lines as a therapeutic method have led us to use a more natural approach to obtain suppressor cells for EAE, namely, the establishment of an IL-2-dependent suppressor line.

2.2.3. Suppressor T-line An additional approach to obtain suppressor cells relevant to EAE was to grow an IL-2-dependent suppressor line. The line was generated from spleen cells of mice that had been rendered unresponsive to EAE by means of Cop-1. The Cop-1 specific line of the CD4 + phenotype suppressed the in vitro response of an encephalitogenic line specific to rat BP. This was demonstrated by the inhibition of both the proliferation and IL-2 secretion of the effector line, an effect which was proportional to the number of inhibiting cells. However, this was not a non-specific interference, such as competition for growth factors in the media, since a lysozyme control line did not exhibit such a suppressive effect when co-cultured under the same conditions. Injection of 10 7 cells of an IL-2-dependent cell line inhibited almost completely the development of EAE as reflected in both the incidence (85% inhibition) as well as the clinical score of the dis-

ease (83% inhibition). As demonstrated for the in vitro reactivity of the Ts line, the in vivo effect was dose-dependent and specific, since no inhibition was observed in mice treated with the lysozyme control line. Moreover, the lysozyme line even caused a non-specific stimulation of the disease. Like the T-suppressor hybridomas, the IL-2dependent line was effective when injected at the time of disease induction, i.e. immediate effect, suggesting that these are effector-suppressor cells that actually mediate the suppression of the disease. What is the target of the Ts cells? The Ts cells reacted with epitope(s) shared between BP and Cop-l, and no significant anti-idiotypic response to an encephalitogenic line could be demonstrated. Several modes for the suppressive activity of these antigen-specific cells could be considered. One possibility is that the Ts cells compete with the effector cells for the binding to BP when linked to MHC, thus blocking the activation of the encephalitogenic cells. Such a mechanism requires the suppressor cells to recognize the same determinant as the pathogenic cells, i.e. the 89101 encephalitogenic determinant of the SJL/J strain. However, no experimental confirmation of this model was found as we could not demonstrate cross-reactivity of this line with encephalitogenic epitopes. Another possibility is that Ts cells recognize an epitope present on Cop-1 and BP but different from the encephalitogenic determinant. Such epitopes on antigen molecules which selectively stimulate T-suppressor rather than Thelper lymphocytes are referred to as 'suppressive determinants'. The concept that T-helper and Tsuppressor repertoires differ is based on the observation that the determinants which induce suppressor or helper T-cells are distinct and not overlapping [70]. Such 'suppressive determinants' have also been demonstrated for BP [71]. The Ts cells stimulated by such 'suppressive determinants' could mediate the suppression of EAE. 3.

Conclusion

As emerges from this review and other publications [9,71], in the past decade Ts cells have been one of the focal points in immunological research. We believe that the abundant d a t a on Ts-cell 113

TABLE 1 Ts-cell manipulations as a treatment of autoimmune disease (modified from [4]). (A) Suppressor-cell manipulations in animal experimental models Drug/therapy Experimental model and animal Androgens Con-A

SLE-(B/W) Fl mice SLE-(B/W) F~ mice

PGE Thymic hormone Cy-A Soluble immune response suppression Ts specific for pathogenic Id Ts specific for MBP or encephalitogenic cells Activation of antigen-specific Ts cells Ts line specific to retinal soluble antigen

SLE-MRL-Ipr mice (B/W) Fi mice EAE, rat, guinea pig and primates SLE-(B/W) Fi mice SLE-BALB/c EAE-Lewis rats and mice EAMG, mice EAU, rats

Results

Reference

Prolonged survival Prolonged tolerance to foreign antigens Prolonged survival Enhanced Ts-cell function Prevention of EAE Decreased autoantibody levels increased survival Prevention of experimental SLE Prevention of EAE

[74] [13] [75] [76] [77] [13] [34] [42,46,54,59~7]

Inhibition of autoantibody production Inhibition of uveitogenic Th cell and down-regulation of EAU

[78] [8a]

(B) Suppressor-cell manipulations in human autoimmune diseases Treatment

Disease

Results

Reference

Cy-A

MS

[79]

Cy-A Thymic hormone

SLE, RA, polymyositis, chronic biliary cirrhosis Autoimmune hemolytic anemia

Modest slowing of progression of clinical disease Clinical improvement

TLI TLI

RA Chronic progressive MS

function favors the view that Ts cells do exist. It is currently believed that Ts cells are responsible for maintaining self-tolerance and preventing autoimmunity, either directly or via the secretion of soluble factors. A decreased number and function of Ts cells were reported under almost all autoimmune conditions including SLE-prone mice and mice with EAE, as well as in patients with SLE and MS [4]. Our work features the role Ts cells may play in the induction as well as modulation of autoimmune conditions such as SLE and EAE and the effect of two types of specific Ts in EAE. In the EAE system as discussed before, several other groups had also demonstrated the immunomodulation of EAE by Ts cells directly isolated from spleen and lymph nodes [37-40] or by in vitro maintained Ts lines [42,46,53,54]. 114

Increased Ts-cell activity and clinical improvement Clinical improvement Stabilization of the disease

[80] [81] [81] [79]

In view of the potentially pathogenic role of BP-specific T-cell responses in MS [72,73] and the role of the 16/6 idiotype in human SLE [32], the approaches presented here and summarized in Table 1 may be of relevance in experimental as well as in autoimmune diseases.

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