Adoptively Transferred EAE in γδ T Cell-knockout Mice

Journal of Autoimmunity (1998) 11, 105–110

Adoptively Transferred EAE in ãä T Cell-knockout Mice Robert B. Clark and Elizabeth G. Lingenheld Department of Medicine, Division of Rheumatic Diseases, University of Connecticut Medical School, Connecticut, USA

Received 2 September 1997 Accepted 31 October 1997 Key words: EAE, ãä T cells, knockout mice

Recently there has been evidence suggesting that ãä receptor-bearing T cells may play a role in both multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). We have recently described approaches for the generation of encephalitogenic T-cell populations from EAE-resistant strains of mice. Using encephalitogenic T-cell lines and clones generated from wild-type C57BL/6 mice we have studied adoptively transferred EAE in C57BL/6-TCR ä-knockout mice. We now report that the adoptive transfer of encephalitogenic T cells into TCR ä T-knockout mice leads to clinical EAE that is not significantly different in severity or time course than that seen after transfer into wild-type C57BL/6 mice. We conclude that ãä T cells do not play an integral role in the mediation or regulation of the effector-phase mechanisms in EAE. © 1998 Academic Press Limited

Introduction

Material and Methods

Experimental allergic encephalomyelitis (EAE) is an animal model for the human disease multiple sclerosis (MS). EAE has classically been thought to be mediated by áâ receptor-bearing type 1 T-helper cells that are reactive to central nervous system (CNS)-derived autoantigens. However, recently, there has been much evidence suggesting that ãä T cells may also play a role in both EAE and MS. In this regard, there has been a demonstration, not confirmed in all studies, of an accumulation of ãä T cells in active plaque, cerebrospinal fluid (CSF) and peripheral blood (PB) of MS patients and in the CNS in EAE [1–13]. The in vivo function of ãä T cells is not as yet known. In models of autoimmunity, anti-tumor responses, models of allergic responses and in infections there have been suggestions that such T cells may subserve either a direct effector role and/or a regulatory function [14–18]. However, in murine EAE, in vivo depletion of ãä T cells using monoclonal antibodies has led to conflicting clinical outcomes, and the role of ãä T cells in the pathogenesis of EAE remains unclear [10, 19]. In the present study, we have taken advantage of our recently described ability to generate encephalitogenic áâ T-cell populations from EAE-resistant strains of mice [20], and have investigated adoptively transferred EAE in TCR ä-knockout mice.

Mice Female wild-type C57BL/6J mice and female TCR ä-knockout mice, homozygotes on a C57BL/6 background (C57BL/6J-Tcrdtm/Mom; JR2120), were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were 4–10 weeks of age when studied. The TCR ä-knockout mice were generated as previously described [21].

Antigens Porcine myelin basic protein (PMBP) was obtained as a gift from Eli Lilly Co, Indianapolis, IN. Murine myelin basic protein (MMBP) was prepared in our laboratory as previously described [22].

Generation of MBP-specific T-cell lines and clones Myelin basic protein-specific áâ T-cell lines and clones were generated using methods described previously [20]. Briefly, C57BL/6 mice were immunized in both hind footpads using a total of 100 ìg of PMBP emulsified in complete Freund’s adjuvant. The CFA contained 2.5 mg/ml of desiccated Mycobacterium tuberculosis H37RA (Difco, Detroit, MI). Eight to 10 days after this immunization, draining lymph nodes were removed and single cell suspensions prepared. These lymph node suspensions were cultured in ‘mouse media’ (MM) without IL-2, along with 25 ìg/ml of PMBP and in the presence of irradiated syngeneic

Correspondence to: Dr Robert B. Clark, Room L3056, Department of Medicine, University of Connecticut Medical School, 263 Farmington Avenue, Farmington, CT 06032, Fax: 860 679 1287. 105 0896-8411/98/010105+06 $25.00/0/au970180

© 1998 Academic Press Limited

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splenocytes as a source of additional antigenpresenting cells (APC). Mouse media is RPMI 1640 with 10% fetal calf serum and 5×10 −5 M 2-mercaptoethanol. Three days after initiation, these cells were centrifuged through ficoll-hypaque to remove dead cells, and then restimulated. This restimulation was accomplished by replating the cells at 0.5–1.0×106/well in IL-2 (recombinant human IL-2 [16–20 units/ml], Genzyme, Inc, Cambridge, MA) together with APC (5×106/well) and PMBP (25 ìg/ ml). T-cell lines were then generated by in vitro expansion from these cultures using a 2-week growth cycle. The 2-week growth cycle involved continuing the cultures in IL-2 for another 4 days (subsequent cycles involved 7 days total in IL-2). Cultures were then ‘rested’ by replacing the media with MM in the absence of IL-2 for the next 7 days. At the end of this week, the 2-week cycle was then restarted by stimulating the cultures with PMBP (25 ìg/ml), irradiated (2,600 rad) syngeneic splenocytes (3.1×106/24-well culture) and IL-2 (16–20 units/ml). Two encephalitogenic, wild-type C57BL/6-derived, áâ T-cell lines, ‘C-Apop’ and ‘Apop C-2’, were generated in this fashion as previously described [20]. In the present study, the áâ T-cell line ‘C-A-46’ was derived from the C-Apop line by limiting dilution cultures plated initially at five cells per well. In these limiting dilution cultures fewer than 60% of wells yielded growing lines. Also in the present study, the áâ T-cell clone ‘A-C-2-4’ was derived from the Apop C-2 line through limiting dilution cultures plated initially at one cell per well. In these cultures fewer than 60% of wells yielded growing clones.

R. B. Clark and E. G. Lingenheld

T cells were assayed only after they had been rested in the absence of IL-2 for 1 week. T cells were plated in 96-well plates in the absence of IL-2 and cocultured either with irradiated splenocytes alone (i.e. APC alone) or irradiated splenocytes and PMBP or MMBP (25 ìg/ml). After 48 h in culture, tritiated thymidine (New England Nuclear, Boston, MA; 2 ìCi/well) was added to the cultures and the wells were harvested with the aid of a semiautomated cell harvester 18 h later. The incorporation of tritium into cellular DNA was assayed using a beta-scintillation counter and the results expressed as mean and standard error of the mean of triplicate wells.

Assay for cytokine secretion T-cell lines and clones were assayed for cytokine secretion after they had been rested in the absence of IL-2 for 1 week. T cells (0.5×106/ml) were plated in MM in the absence of IL-2 in 24-well plates that had been coated with 10 ìg/ml anti-murine CD3 monoclonal antibody (Pharmingen, Inc, San Diego, CA). After 3 days, supernatants from these cultures were harvested, filtered, and frozen until assayed. Assays for murine gamma-interferon and murine TNF-á were performed using cytokine-specific ELISA kits (Genzyme, Inc, Cambridge, MA). Results are expressed as ng of cytokine per ml of supernatant generated by 0.5×106 cells plated per ml of MM.

Results Adoptive transfer of EAE The C-Apop and C-A-46 T-cell lines and the A-C-2-4 clone were used to transfer EAE adoptively using the approach described previously [20, 23, 24]. Unirradiated, naive, wild-type C57BL/6 or TCR ä-knockout mice, homozygotes on a C57BL/6 background, were the recipients. T cells were injected via intracardiac puncture. The T cells were injected at 4 days after the last stimulation with APC, PMBP, and IL-2, and 1 day after an additional feed with fresh IL-2. The number of T cells injected was 30×106 for the C-Apop line, 16×106 cells for the C-A-46 line, and 13×106 for the A-C-2-4 T-cell clone. Mice received one injection of T cells and then received no other treatments or interventions. All mice were followed for signs of EAE for a minimum of 3 months after the single injection of T cells. EAE was graded as previously described: Grade 1, flaccid tail; Grade 2, leg weakness and gait abnormality; Grade 3, hind leg dragging; Grade 4, hind leg dragging with front leg weakness; Grade 5, death [20, 23, 24].

Antigen-specific proliferative assays T-cell lines and clones were assayed for antigenspecific proliferation as previously described [23].

Characterization of the encephalitogenic T-cell lines and clones As previously described, we have generated MBPreactive, encephalitogenic, áâ T-cell lines from the ‘EAE-resistant’ C57BL/6 strain of mice [20]. In the present studies we used either one T-cell line (C-Apop), one subline (C-A-46), or one clone (A-C2-4) to mediate the adoptive transfer of EAE. C-Apop, C-A-46 and A-C-2-4, derived from the draining lymph notes of a wild-type C57BL/6 mouse immunized with porcine MBP, are characterized in Table 1. The C-Apop and C-A-46 T-cell lines and the A-C-2-4 T-cell clone are all CD4-positive, áâ-receptor positive T cells (data not shown). These lines and the clone have all been propagated in vitro using PMBP as the stimulating antigen. Nevertheless, all three populations proliferate both to porcine MBP and murine MBP (MMBP) presented in the context of irradiated C57BL/6 splenocytes as APC (Table 1). This is consistent with previously reported findings that encephalitogenic T cells immunized with a xenogeneic MBP respond to the self-antigen MMBP [22]. Both the lines and the clone secrete TNF-á and gamma interferon, making them type-1 helper T cells (Table 1). All three of these T-cell populations mediate EAE after a single intracardiac injection of 13–30×106

EAE in TCR ä-knockout mice

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Table 1. C57BL/6-derived encephalitogenic T cells 3

H cpm*

T cell

Line/ clone

APC†

APC/PMBP**

APC/MMBP**

C-Apop C-A-46 A-C-2-4

Line Line Clone

945 401 743

85,959 104,441 36,449

78,847 48,246 13,713

TNF‡

50 59 61

Gamma INF‡ 1,600 852 628

*Antigen specific proliferation assay: 3H cpm=Mean counts per minute of 3H of triplicate wells of 5×104 T cells stimulated by: †5×105 irradiated C57BL/6 splenocytes (APC) alone, and **APC together with 25 ìg/ml of PMBP or MMBP. Standard errors of the means were all less than 15%. ‡Concentration (ng/ml) of TNF-á or gamma interferon (gamma INF) secreted after stimulation of 0.5×106/ml T cells with immobilized anti-CD3 antibody.

cells, and do not require irradiation or other treatment of the naive recipient mouse.

Adoptive EAE

5

Adoptively transferred EAE EAE grade

4

The TCR ä-knockout mice were generated by disruption of the mouse Cä gene of the ãä TCR using homologous recombination as previously described [21}. These mice have no thymic or peripheral T cells bearing the ãä TCR, but in contrast, they have normal numbers of áâ TCR-bearing T cells in the thymus and the periphery, and these appear to be structurally and functionally normal. The TCR ä-knockout mice used in these studies were homozygotes on a C57BL/6 background. We compared the mediation of EAE in wild-type C57BL/6 recipient mice versus C57BL/6-TCR ä-knockout recipient mice after adoptive transfer of the encephalitogenic lines and clone. As can be seen in Figure 1, the intracardiac injection of either of the T-cell lines or the T-cell clone led to consistent and significant clinical signs of EAE in the wild-type mice. These clinical signs ranged from tail weakness (grade 1 EAE) to death (grade 5 EAE). The mean EAE clinical score of the wild-type mice in these studies was 2.36 (SEM=0.548; Figure 1). Significantly, the T-cell lines and clone also mediated severe EAE in the TCR ä-knockout mice. The clinical signs in the TCR ä-knockout mice ranged from tail weakness with a mild gait abnormality (grade 1.5 EAE) to death (grade 5 EAE). As can be seen in Figure 1, the adoptive transfer of the T-cell lines or clone into the TCR ä-knockout mice led to signs of EAE that were, on average, slightly (though not significantly) more severe than in the wild-type mice (Mann-Whitney rank sum test: P=0.264). The mean EAE clinical score of the TCR ä-knockout mice was 3.11 (SEM=0.466; Figure 1). The encephalitogenic T-cell lines and clone utilized in this study did not differ significantly among themselves in the severity of clinical EAE they mediated. Therefore, in Figure 1, we have not noted which T-cell population was used for any given mouse. However, the C-Apop T-cell line was injected into four pairs of mice (i.e. each pair consists of one wild-type and one TCR ä-knockout mouse), the C-A-46 T-cell line was injected into three such pairs of

3 2 1 0 Wild-type mice

γδ-knockout mice

Figure 1. Wild-type C57BL/6 mice (wild-type mice) or C57BL/6-ä T-cell receptor-knockout mice (ãä-knockout mice) received a single intracardiac injection of encephalitogenic T cells. One of three different encephalitogenic T-cell populations were used: C-Apop, 30×106; C-A-46, 16×106; or A-C-2-4, 13×106. Each T-cell population, when used, was injected into equal numbers of wild-type and TCR ä-knockout mice. Mice were observed for 4 months after the injection and the clinical signs of EAE were graded as previously described [23]. Each point represents the maximal clinical grade of EAE for one mouse.

mice, and the A-C-2-4 T-cell clone was injected into four pairs of wild-type and TCR ä-knockout mice (data not shown). The EAE in the TCR ä-knockout mice manifested as clinical signs that were identical to those seen in the wild-type mice in terms of kinetics of onset and clinical progression. In wild-type mice the day of onset of clinical signs was, on average, day 5 (mean=day 5.8, SEM=0.6) after the intracardiac injection of the encephalitogenic T cells. In TCR ä-knockout mice the day of onset of clinical signs was also, on average, day 5 (mean=day 5.6, SEM=0.85) after the intracardiac injection of the encephalitogenic T cells. In both wild-type and TCR ä-knockout mice the signs began with tail weakness and progressed (depending on the individual mouse) through hindleg weakness, hind-leg paralysis, front-leg weakness,

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and death. After injection of the T cells, clinical signs reached their maximum level in wild-type mice at a mean of day 8 (SEM=0.47). In TCR ä-knockout mice, clinical signs reached their maximum level at a mean of day 7.2 (SEM=0.85). In both wild-type and TCR ä-knockout mice this clinical level was either maintained or improved after this time point. Mice have been observed for up to 4 months and we have not as yet seen a second exacerbation of EAE in any of our wild-type or TCR ä-knockout mice.

Discussion The in vivo function of ãä T cells remains unclear. However, in models of anti-tumor responses, allergic responses and infections, there have been suggestions that such T cells may subserve either a direct effector role and/or a regulatory function vis-a`-vis áâ T cells [15–18]. In autoimmune diseases and models of autoimmunity there also have been reports of the relevance of ãä T cells. Vincent et al. demonstrated that synovial ãä T-cell clones from patients with Lyme arthritis express elevated levels of FasL and induce Fas–FasL-mediated apoptosis of CD4 + synovial lymphocytes, suggesting an immunoregulatory function of ãä T cells via a Fas-dependent process [25]. Peng et al., studying lupus-prone mice with a concomitant knockout of either áâ or ãä T cells, found evidence for both an effector and a regulatory function of ãä T cells [14]. ãä T cells have been implicated in the pathogenesis of MS, although the data remain somewhat conflicting and controversial. Selmaj et al. identified heat-shock protein (HSP) 65 expression in reactive immature oligodendrocytes in chronic MS plaques, and ãä T cells were found to colocalize to this site [2]. Given that ãä T cells have been reported to be stimulated by HSP, and that human ãä T cells have been found to be able to lyse cultured oligodendrocytes, these findings suggested a possible new and disease-relevant antigenic reaction in MS mediated by ãä T cells [5, 26]. However, using peripheral blood (PB) from MS patients in vitro, some investigators have found more evidence of an HSP 70 than an HSP 65 response [8, 27]. After in vitro PHA stimulation, Stinissen et al. found a significantly increased frequency of ãä T cells derived from the PB and CSF of MS patients compared to patients with other neurological diseases, although this finding also has not been confirmed by all groups [4, 5, 27]. In addition, there have been some conflicting results concerning the frequency of ãä T cells in the CNS in regard to the clinical phase of MS studied [7]. Regarding the in vivo effector or regulatory function of ãä T cells in MS, Burns et al. studied T-cell–T cell interactions in the PB of patients with MS. They found that, in vitro, MS PB responded to activated MBP-reactive T cells predominantly through an expansion of ãä T cells [28]. They postulated that the accumulation of ãä T cells in CNS lesions in MS may be a result of a recruitment by activated áâ T cells in the lesions.

R. B. Clark and E. G. Lingenheld

A number of investigators have examined the T-cell receptor structure utilized by MS ãä T cells. A predominant usage of Vä1, Vä2, or both, in MS plaque tissue and MS CSF has been described by various investigators [3, 5–8, 29]. The issue of oligoclonality of ãä T-cell receptors as discerned by sequencing of the receptors also remains unclear. Overall, the frequency, clinical correlation, antigenic-specificity, T-cell receptor structure, and in vivo function of ãä T cells in MS all remain controversial issues. EAE has classically been thought to be mediated by áâ type-1 T-helper cells that are reactive to CNSderived autoantigens. However, as in MS, a number of recent reports have suggested that ãä T cells may also play a role in EAE. Sobel and Kuchroo found small numbers of ãä T cells in early CNS lesions in both primary and áâ T-cell induced adoptively transferred EAE [13]. Gao et al. reported that in chronic EAE, HSP 60 was predominantly expressed on oligodendrocytes and astrocytes and that ãä T cells co-localized with this HSP 60 expression [9]. Rajan et al. found that in all stages of relapsing-remitting EAE, ãä T cells were increased in the CNS [10]. In contrast to these reports, Zeine and Owens found that in the recently remitted phase of murine EAE, there were no ãä T cells present in the CNS [12]. Olive studied the ãä T-cell receptor variable region repertoires of T cells infiltrating the brains of mice during the development of EAE and found that in early EAE a relatively restricted repertoire was used (biased towards Vã6) but that as the disease progressed, most Vã and Vä TCR regions were found [11]. Subsequently, this investigator immunized mice with T-cell receptor peptide specific for Vã6 chains and found that while this did not result in protection from primary EAE, it did result in a minor delay in onset of EAE and a minor reduction in disease severity [30]. Rajan et al. depleted the ãä T-cell population in vivo with a monoclonal antibody and found a significant reduction in the severity of the clinical signs of murine EAE together with a decrease in the percentage of CD3 + /ãä T cells in the CNS [10]. In contrast, Kobayashi et al. depleted ãä T cells in vivo (using a different monoclonal antibody from that used by Rajan et al.) and found that there was an earlier onset and increase in severity of clinical signs of EAE [19]. In addition, this group noted the onset of an exacerbation of the disease that was not seen in the nondepleted mice. Thus, the studies of Rajan et al. and Kobayashi et al., using in vivo depletion of ãä T cells, yielded directly conflicting results. It was suggested that the different outcomes might be a result of studying actively induced versus adoptively transferred EAE, using different strains of mice, and/or using different antibodies for in vivo-depletion of ãä T cells [19]. Thus, as in MS, significant controversy remains as to the role of ãä T cells in EAE. In the present study we have examined the role of ãä T cells in EAE using TCR ä-knockout mice. We have studied adoptively transferred EAE in wildtype and TCR ä-knockout mice using two áâ T-cell lines and one áâ T-cell clone derived from a wild-type C57BL/6 mouse. These encephalitogenic,

EAE in TCR ä-knockout mice

MBP-reactive T-cell populations were generated using recently described approaches for the study of EAE in resistant strains of mice [20]. We now report that the adoptive transfer of encephalitogenic, MBP-reactive, áâ T cells into TCR ä-knockout mice results in EAE that is not significantly different from that seen in wild-type recipient mice. The clinical EAE in TCR ä-knockout mice was only slightly more severe and did not differ in the kinetics of onset or progression from that seen in the wild-type mice. Adoptively transferred EAE represents a model for studying the effector phase of EAE. As such, our results suggest that ãä T cells do not play a significant role in the mediation or regulation of the homing to the CNS, the crossing of the blood–brain barrier, the entry into the CNS, the persistence in the CNS, or the mediation of tissue damage by our transferred encephalitogenic áâ T cells. As has recently been pointed out, results obtained using knockout mice to study disease models such as EAE may need to be interpreted cautiously [31]. This is because of the many compensatory mechanisms that can arise in knockout mice and this may be especially true when knockouts involving cytokines are involved. However, studies of knockout mice may still allow for some insight into the significance of the specific moiety missing, as well as concomitantly allowing for an identification of the range of compensatory approaches available. In knockout mice involving depletion of an entire cell population, such as ãä T cells, it may be that compensatory mechanisms for effector and/or regulatory functions normally subserved by the cellular population are less likely to arise. To the best of our knowledge, this is the first report of EAE in TCR ä-knockout mice. Our present study does not allow us to draw any conclusions concerning the relevance of ãä T cells in actively induced EAE, which includes the steps involved in the initial in vivo activation and expansion of encephalitogenic T cells. Thus, it remains possible that ãä T cells play an important role in the regulation of the initial priming and subsequent expansion of encephalitogenic áâ T cells in vivo. In addition, the present study does not allow us to draw definitive conclusions concerning the relevance of ãä T cells in exacerbating-remitting EAE. However, our findings do suggest that in the effector-phase mechanisms represented by the model of adoptively transferred EAE, ãä T cells do not play a crucial role in the mediation or regulation of the disease.

Acknowledgements This work was supported by a University of Connecticut School of Medicine Faculty Research Grant. The authors would like to thank Dr Steven J. Padula and Dr M. Tawfik Omran, Jr for their reading of the manuscript and their helpful suggestions. The authors would also like to thank Mr John Kane for his assistance in the preparation of the manuscript.

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