T-cell responses to myelin basic protein in normal and MBP-deficient mice

Journal of Neuroimmunology 84 Ž1998. 131–138

T-cell responses to myelin basic protein in normal and MBP-deficient mice Izumi Yoshizawa a , Roderick Bronson b, Martin E. Dorf a , Sara Abromson-Leeman

a,)

a

b

Department of Pathology, HarÕard Medical School, Boston, MA 02115, USA Department of Pathology, Tufts UniÕersity Schools of Medicine and Veterinary Medicine, Boston, MA 02111, USA Received 18 June 1997; received in revised form 15 October 1997; accepted 15 October 1997

Abstract BALBrc mice are resistant to the development of experimental autoimmune encephalomyelitis ŽEAE. after immunization with myelin basic protein ŽMBP.. Previous studies of BALBrc mice suggest that MBP-specific T-cells can eventually be cloned from these mice, although they are either initially present in very low frequencies or are functionally anergic. To determine what role endogenous MBP expression plays in shaping the BALBrc T-cell repertoire, MBP-deficient BALBrc mice were constructed by breeding the shiÕerer Ž shirshi . mutation onto the BALBrc background. These mice lack all conventional isoforms of MBP due to a deletion of MBP exons 3–7. Studies of the MBP-directed response of these mice suggest that endogenous MBP expression is directly responsible for EAE resistance in BALBrc mice, by quantitatively affecting expression of the T-cell repertoire. In contrast to wild-type BALBrc T-cells, uncloned T-cells from BALBrc shirshi mice immunized with MBP proliferate in vitro to MBP and MBP peptides 59–76 and 89–101 and are able to induce severe EAE upon transfer to BALBrc recipients expressing MBP. q 1998 Elsevier Science B.V. Keywords: Experimental allergic encephalomyelitis; Myelin basic protein: BALBrc shiverer mice; T-cell repertoire

1. Introduction Several strains of BALBrc mice are resistant to induction of experimental autoimmune encephalomyelitis ŽEAE. when immunized with central nervous system ŽCNS.-derived myelin basic protein ŽMBP., or various peptide fragments of MBP ŽLevine and Sowinski, 1974; Bernard, 1976; Fallis et al., 1987.. Disease resistance is reflected in vitro by the absence of proliferative responses of MBPprimed lymphocytes to either native MBP or its peptides ŽFallis et al., 1987; Abromson-Leeman et al., 1993.. Continual in vitro restimulation of MBP-primed T-cells, however, reveals the presence of T-cells capable of recognizing MBP with high affinity and causing EAE upon transfer into syngeneic BALBrc recipients. Of sixteen disease-inducing clones previously studied in detail, all are CD4 q TH 1 cells, secreting IL-2 and IFN-g , but no IL-4, upon activation with MBP or appropriate peptides. Epitopes defined by these clones include residues 59–76 Žby 13 of )

Corresponding author. Tel.: q1 617 4320786; fax: q1 617 4322789.

0165-5728r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 7 . 0 0 2 0 5 - 1

16 clones., 151–168 Žby 2 of 16 clones. and 137–155 Žby one clone.. A minimum of six different T-cell receptors ŽTCR. are represented in this panel, as judged by TCR Vb phenotypes ŽAbromson-Leeman et al., 1993, 1995.. The low frequency with which MBP-reactive cells are isolated from BALBrc mice suggests that a selection process is involved in at least the quantitative development of the T-cell repertoire. Both central and peripheral tolerance induction mechanisms depend upon the presence of particular peptiderMHC complexes, either in the thymus at the time of T-cell development or in the periphery ŽSha et al., 1988; Rellahan et al., 1990; Ramsdell and Fowlkes, 1992; Ashton-Rickardt et al., 1994; Sebzda et al., 1994; Antonia et al., 1995.. Because embryonic forms of MBP have been found in spleen and thymus ŽCampagnoni et al., 1993; Mathisen et al., 1993; Fritz and Kalvakolanu, 1995., it is tempting to speculate that they may play a role in shaping both the qualitative and quantitative nature of the relevant T-cell repertoire. MBP is not expressed in shiverer Ž shirshi . mice, due to a recessive mutation resulting in deletion of MBP exons

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3–7 ŽRoach et al., 1983, 1985.. The shiverer mutation was bred onto BALBrc mice in order to determine what role, if any, endogenous MBP plays in determining MBP unresponsiveness and thus resistance to MBP-induced EAE.

2. Materials and methods

and the indicated antigens. Triplicate wells were pulsed with 1 m Ci 3 H-thymidine for the last 20 h of a 48 h culture period and radioactivity incorporated determined and expressed as mean cpm of triplicate culture wells. Stimulation index ŽS.I.. was calculated by dividing the mean cpm of cultures with antigen by the mean cpm of cultures in the absence of antigen.

2.1. Mice

2.5. Production of supernatants and cytokine assays

BALBrcHa female mice were purchased from Harlan Bioproducts ŽIndianapolis, IN. and used between the ages of 6–10 weeks. C3HrFe shirshi and shirq mice were purchased from Jackson Laboratories, Bar Harbor, ME. BALBrc shirshi mice were obtained by crossing C3H shirshi male mice with BALBrcHa females. At the F2 generation H-2 d homozygous shirshi males were identified phenotypically Žtremors are apparent by the third postnatal week. and backcrossed to BALBrc females. A cross-intercross mating scheme Ž10 generations. was used to transfer the shirshi mutation onto the BALBrc background, yielding the N5F2 generation used in this report. ShiÕerer mice were used at 4–12 weeks of age; their lifespan is generally 90–150 days ŽReadhead et al., 1987..

T-cell supernatants from secondary cultures were prepared in triplicate using 10 5 T-cells and 5 = 10 5 irradiated syngeneic spleen cells with the indicated antigens. Supernatants were harvested at either 20 h, for IL-2 and IL-4 determinations, or 48 h, for IFN-g measurements. Quantitation of units of IL-2 was determined by adding supernatants or recombinant standard IL-2 or IL-4 to HT-2 target cells in the presence and absence of antibodies specific for IL-2 or IL-4, as previously described ŽAbromson-Leeman et al., 1995.. Quantitation of IL-4 and IFN-g was performed using a capture ELISA system, according to the manufacturer’s protocol ŽPharmingen, San Diego, CA..

2.2. Antigens Purified mouse and guinea pig MBP were prepared as previously described ŽDeibler et al., 1972. and were generously provided by Dr. Al-Sabbagh and Dr. H. Weiner ŽAutoimmune, Lexington, MA and Center for Neurologic Diseases of the Brigham and Women’s Hospital, Boston, MA.. Overlapping peptides of MBP, generally 20-mers, were kindly provided by Dr. Ariel Miller ŽCenter for Neurologic Diseases, Brigham and Women’s Hospital, Boston, MA.. Mouse MBP peptides 59–76 ŽHTRTTHYGSLPQKSQHGR. and 89–101 ŽHFFKNIVTRRTPP. were synthesized by Dr. Chuck Dahl, Department of Biological Chemistry, Harvard Medical School, Boston, MA. 2.3. Immunization protocol Mice were primed by injection of 200 m g MBP or peptides emulsified in CFA, in three subcutaneous sites over the flanks. Draining lymph nodes were removed after 10–11 days. 2.4. Proliferation assay Lymph node cells were cultured for one week at 5 = 10 6 cells per well of 24 well plates with 10 m grml mouse MBP in DMEM supplemented with 0.8% normal mouse serum, sodium pyruvate, 2 mM L-glutamine and 50 m M 2-mercaptoethanol. Cells were then harvested, washed and restimulated in secondary cultures containing 5 = 10 4 cells per well, with 5 = 10 5 irradiated syngeneic spleen cells

2.6. In ÕiÕo adoptiÕe transfer of disease Guinea pig MBP- and peptide 89–101-primed lymphocytes were harvested after 4 days of primary culture with mouse MBP or peptide 89–101, respectively. Mouse MBP-primed lymphocytes were harvested on day 3 of secondary culture with mouse MBP. In vitro stimulated lymphocytes were washed three times and injected i.v. into lightly irradiated Ž390 R. BALBrc recipients, followed by i.v. injection on days 0 and 3 with 200 ng purified pertussis toxin ŽList Biological Laboratories, Campbell, CA.. Low dose irradiation and pertussis injections are reported to facilitate passive transfer of EAE ŽTrotter et al., 1985.. Mice were monitored daily for clinical signs of disease and scored as follows: 0, no clinical signs of disease; 1, complete tail limpness, incontinence and ruffling of fur; 2, all of the above, plus moderate hind limb weakness andror unsteady gait; 3, complete hind limb paralysis; 4, complete hind limb paralysis, with some forelimb weakness andror severe cachexia Ž25% loss of body weight. and 5, moribund state. 2.7. Histopathologic eÕaluation For histologic evaluation, mice were killed at the indicated times; nervous system tissues were fixed in Bouin’s solution, paraffin-embedded and sections stained and counter-stained with hematoxylin and eosin. 2.8. DeriÕation of MBP-specific T-cell clones Cell suspensions from draining lymph nodes removed 14 days after immunization of BALBrc shirshi mice

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with guinea pig MBP were stimulated with 25 m grml of mouse MBP as previously described ŽAbromson-Leeman et al., 1995.. On day 7, viable blasts were collected and restimulated with 10 m grml mMBP and irradiated BALBrc spleen cells. Cells were cloned by limiting dilution 14 days later, using irradiated BALBrc spleen and 10 m grml mMBP. Positive wells were screened for reactivity to MBP and overlapping MBP peptides.

3. Results 3.1. Comparison of proliferatiÕe responses to MBP and MBP peptides by secondary cultures of BALBr c and BALBr c shi r shi T-cells In order to obtain MBP-deficient BALBrc mice, the mutated gene was introduced by crossing BALBrc mice with C3HrFe shirshi mice. After ten generations of crossing and backcrossing, BALBrc shirshi mice of the N5F2 generation are ) 90% homozygous for alleles derived from the parental BALBrc strain. When BALBrc

Table 1 Comparison of secondary in vitro proliferative responses to MBP and MBP peptides by BALBrc shi r shi and BALBrc T-cells Antigen

BALBrc shi r shi CPM

a

BALBrc CPM

S.I.

Experiment 1: Guinea pig MBP-primed gpMBP 27,478"2,902 21.4 mMBP 24,139"1,437 18.7 MBP 11–30 3,101"755 2.4 MBP 59–76 21,464"620 16.7 MBP 89–101 20,288"1,488 15.8 MBP 137–155 2,701"298 2.1 MBP 151–168 3,730"1,955 2.9 Medium 1,284"80 —

5,306"833 3,924"357 3,801"708 4,344"579 2,993"368 3,043"441 3,127"273 2,246"15

2.4 1.7 1.7 1.9 1.3 1.4 1.1 —

Experiment 2: Mouse MBP-primed gpMBP 53,518"883 mMBP 55,428"3,113 MBP 11–30 10,443"2,021 MBP 59–76 45,331"940 MBP 89–101 50,274"544 MBP 137–155 13,845"3,083 MBP 151–168 12,497"2,075 Medium 10,181"735

NT 2,651"567 NT 2,649"64 2,539"294 NT NT 2,429"7

NT 1.1 NT 1.1 1.0 NT NT —

a

S.I.

b

5.3 5.4 1.0 4.5 4.9 1.4 1.2 —

BALBrc shi r shi and BALBrc mice were primed with 200 m g gpMBP ŽExp. 1. or mMBP ŽExp. 2. in CFA. 10–11 days after immunization, draining lymph node cells from at least 3 individual mice were pooled and cultured in 24-well plates with 10 m grml mMBP. After 7 days, cells were harvested, washed and 5=10 4 cellsrwell restimulated with the indicated antigens in triplicate wells, with 5=10 5 irradiated BALBrc spleen cells. MBP was used at 25 m grml, peptides at 10 m grml. 3 H-thymidine uptake was measured for the last 20 h of 48 h culture. Secondary proliferative responses to each stimulus are presented as mean cpm of triplicate wells"standard error. b Stimulation index; the ratio of mean cpm with antigen to mean cpm without antigen.

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shirshi mice are primed with either guinea pig MBP ŽTable 1, experiment 1. or mouse MBP ŽTable 1, experiment 2., significant secondary in vitro proliferative responses to autologous MBP are observed. Utilization of peptides of mouse MBP shows that epitopes recognized by BALBrc shirshi T-cells include residues 59–76, previously defined as the target of a substantial number of BALBrc-derived T-cell clones ŽAbromson-Leeman et al., 1995. and 89–101. In contrast, T-cell populations from wild-type BALBrc mice with normal expression of MBP do not proliferate in vitro in response to either mouse MBP or its peptides ŽTable 1, experiments 1 and 2.. 3.2. Cytokine production by BALB r c shi r shi and BALB r c mice To confirm the differences in MBP-responsiveness between BALBrc shirshi and normal BALBrc mice, supernatants from secondary in vitro cultures were harvested at either 20 or 48 h after stimulation, to test for the presence of IL-2, IL-4 and IFN-g produced in response to MBP or its peptides. Results of these measurements are given in Table 2. Secondary cultures of T-cells from MBP-deficient BALBrc shirshi mice secreted significant levels of IL-2, IL-4 and IFN-g in response to autologous MBP, whether in vivo priming was with guinea pig MBP ŽTable 2, experiment 1. or with mouse MBP ŽTable 2, experiment 2.. A tendency for peptide 59–76 to elicit more IL-2 than IL-4 and for peptide 89–101 to elicit more IL-4 than IL-2, was noted. In sharp contrast to the BALBrc shiÕerer mice, secondary cultures of either guinea pig MBP- or mouse MBP-primed normal BALBrc T-cells secreted no detectable IL-2, IL-4 or IFN-g after stimulation with either MBP or its peptides ŽTable 2, experiments 1 and 2.. 3.3. Encephalitogenicity of BALB r c shi r shi- and BALB r c-deriÕed MBP-primed T-cells Although BALBrc shirshi mice develop significant T-cell responses to MBP, the absence of endogenous MBP in these mice precludes the development of EAE, since MBP is a target antigen of inflammatory lesions of EAE. Development of encephalitogenic T-cells in these mice can be assessed, however, by in vivo transfer of MBP-primed and in vitro-activated T-cells to naive BALBrc recipients. BALBrc shirshi or wild-type BALBrc mice were primed with either guinea pig or mouse MBP, or peptide 89–101, and stimulated in culture with either mouse MBP Žthe first two groups. or peptide 89–101 Žthe third group.. Cultured cells were harvested and injected into BALBrc recipients, as described in the legend to Table 3. Recipients of MBPor peptide-primed T-cells from BALBrc shirshi mice developed moderate to severe EAE, as shown in Table 3,

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Table 2 Cytokine production from secondary lymph node cultures Antigen

BALBrc shirshi supernatants IL-2

Experiment 1: Guinea pig MBP-primed gpMBP 500 mMBP 380 MBP 11–30 - 100 MBP 59–76 210 MBP 89–101 170 MBP 137–155 - 100 MBP 151–168 - 100 Medium - 100

BALBrc supernatants

IL-4

IFN-g

IL-2

IL-4

IFN-g

367 322 - 20 72 254 - 20 - 20 - 20

16.2 17.4 -1 17.4 5.2 -1 -1 -1

- 100 - 100 - 100 - 100 - 100 - 100 - 100 - 100

- 20 - 20 - 20 - 20 - 20 - 20 - 20 - 20

-1 -1 -1 -1 -1 -1 -1 -1

1,276 1,371 - 20 90 1,187 - 20 - 20 - 20

16.8 17.2 -1 27.2 5.6 -1 -1 -1

NT - 100 NT NT NT NT NT - 100

NT - 20 NT NT NT NT NT - 20

NT -1 NT NT NT NT NT -1

a

Experiment 2: Mouse MBP-primed gpMBP 900 mMBP 940 MBP 11–30 - 100 MBP 59–76 1,100 MBP 89–101 140 MBP 137–155 - 100 MBP 151–168 - 100 Medium - 100

a BALBrc shirshi and BALBrc mice were primed with 200 m g gpMBP or mMBP in CFA. 10–11 days after immunization, lymph node cells pooled from at least three mice were cultured with 10 m grml mMBP for 7 days. After washing, 10 5 cellsrwell were restimulated with the indicated antigen and 5 = 10 5rwell irradiated BALBrc spleen cells. MBP’s were used at 25 m grml, peptides at 10 m grml. Supernatants were harvested at 20 or 48 h for quantitative analysis of IL-2rIL-4 secretion and IFN-g production, respectively. Units of IL-2 were determined after blocking proliferation of the HT-2 cell line with specific antibody. Levels of IL-4 and IFN-g were assayed by ELISA, as described in Section 2.

while recipients of MBP- or peptide-primed T-cells from normal BALBrc mice failed to develop any clinical signs of EAE. Sections from tissues taken on days 15–21 post-transfer from two BALBrc recipients of mMBP-primed and stimulated BALBrc shirshi T-cells are shown in Fig. 1. In brain, cerebellar white matter is replaced by densely packed macrophages and perivascular cuffs of lymphocytes are visible. Mixed inflammatory infiltrates including neutrophils and lymphocytes contribute to the focal hypercellularity of spinal roots. Inflammatory cells infiltrate the meninges and extend deeply into the parenchyma, where

several areas of white matter are replaced by dense infiltrates of macrophages, lymphocytes and neutrophils, with characteristic perivascular cuffs of cells. In contrast, Fig. 2 shows the absence on day 22 of any signs of CNS inflammatory disease in BALBrc recipients of mMBP-primed and stimulated BALBrc T-cells. As expected, BALBrc shirshi recipients of encephalitogenic clones derived from either BALBrc shirshi or BALBrc mice fail to show any clinical or histological signs of inflammatory disease Ždata not shown.. Mouse MBP-reactive T-cells were cloned from a BALBrc shirshi mouse immunized with gpMBP and

Table 3 Adoptive transfer of BALBrc shirshi- and BALBrc-derived T-cell lines into BALBrc recipients Antigena in vivo

in vitro

gpMBP

mMBP

mMBP

mMBP

89–101

89–101

a

Donor strain

Incidence of EAE

Mean day of onset

Mean maximal clinical score

BALBrc shirshi BALBrc BALBrc shirshi BALBrc BALBrc shirshi BALBrc

2r3 0r2 3r3 0r2 5r6 0r2

16.0 — 13.6 — 10.5 —

2 — 4 — 3.2 —

Lymphocytes from either BALBrc shirshi or wild-type BALBrc mice were collected after in vivo priming and in vitro activation with 10 m grml of the indicated antigens and irradiated BALBrc spleen cells. 2.5 = 10 7 mouse MBP-primed lymphocytes were injected after secondary in vitro activation; 3 = 10 7 gpMBP- and 89–101-primed cells were injected after primary in vitro stimulation. Recipient mice were scored daily for clinical signs of EAE as described in Section 2.

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Fig. 1. ŽA. Brain section of BALBrc recipient of mMBP-primed and stimulated BALBrc shirshi T-cells. Cerebellar white matter is replaced by densely packed macrophages. A perivascular cuff of lymphocytes is present around a blood vessel. H and E, 50 = . ŽB. Same section, 100 = . ŽC. Sacral spinal cord of same recipient as in ŽA., with meningeal infiltrates; large spinal root on left is hypercellular due to presence of mixed inflammatory infiltrate. 50 = . ŽD. Same section; mixed inflammatory cells include lymphocytes and a few neutrophils Žarrows.. ŽE. Dorsal aspect of thoracic spinal cord, from another recipient of BALBrc shirshi T-cells. There is perivascular cuffing of blood vessels, extending from meninges deeply into parenchyma and several areas of white matter have been replaced by dense infiltrates of macrophages, lymphocytes and neutrophils. Meningeal areas are also infiltrated by mixed cells; 50 = . ŽF. Same section, 100 = . Neutrophils and macrophages are present.

restimulated in vitro with mMBP. Epitopes within mMBP recognized by cloned cells were defined using overlapping MBP peptides. Clone 19F is a BALBrc shirshi-derived Vb 14q T-cell clone recognizing peptide 59–76 and I-Ad ; its pattern of cytokine secretion, shown in Table 4, is representative of the TH 1-type predominating after stimulation of MBP-primed lines with this peptide. When trans-

ferred to BALBrc recipients, clone 19F induces severe ascending paralysis, characteristic of EAE ŽTable 4.. Clone 810B is a BALBrc shirshi-derived Vb 6q TH1-type cell recognizing peptide 89–101 and I-E d . ŽOf four 89–101-recognizing T-cells cloned from BALBrc shirshi mice, two were TH1-type and two were TH2-type cells, based on analysis of their cytokines, data not shown.. Clone 810B

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Fig. 2. ŽA and B. 50 = and 100 = views of brain from recipient of mMBP-primed wild-type BALBrc T-cells. There is no evidence of cellular infiltrates. ŽC and D. 50 = and 100 = views of thoracic spinal cord from recipient of mMBP-primed wild-type BALBrc T-cells. Inflammatory cells are absent. H and E staining.

Table 4 Encephalitogenicity of BALBrc shi r shi-derived TH 1-type clones recognizing epitopes 59–76 and 89–101

can induce characteristic EAE upon adoptive transfer to BALBrc recipients ŽTable 4..

BALBrc shi r shi-derived T-cell clonesa

MBP epitope MHC restriction TCR Vb Cytokine production IL-2 ŽUrml. IL-4 Žpgrml. IFNg Žngrml. Induction of EAE b Incidence Day of onset Max. score c Inflammation a

19F

810B

59–76 I-Ad 14

89–101 I-E d 6

30 - 20 503

3.9 - 20 17

4r6 16 4 CNS

1r4 18 4 CNS

Clones 19F and 810B were established from lymph node cells of gpMBP immunized BABLrc shi r shi mice. T-cell clones were obtained by limiting dilution using mMBP after secondary culture with mouse MBP and were maintained by serial restimulation with irradiated BALBrc spleen cells and relevant peptide. b 1=10 7 T-cells were injected i.v. into recipient BALBrc mice three days after activation with BALBrc spleen cells and specific peptide. c Mice with a score of 4 accompanied by severe cachexia were sacrificed.

4. Discussion Susceptibility to development of EAE following immunization with myelin basic protein is genetically determined ŽLevine and Sowinski, 1974; Bernard, 1976; Fritz et al., 1985; Fallis et al., 1987.. Because multiple genes appear to control susceptibility, multiple mechanisms are probably involved ŽShaw et al., 1992, Duong et al., 1994; Segal and Shevach, 1996.. BALBrc mice are resistant to development of EAE following immunization with MBP. Resistance is reflected in the absence of an in vitro T-cell proliferative response. We have previously shown that despite the initial unresponsiveness of MBP-primed BALBrc lymphocytes to MBP in culture, MBP-specific T-cells can eventually be cloned from BALBrc mice, after multiple rounds of antigen stimulation in vitro, combined with the use of growth factor conditioned medium ŽAbromson-Leeman et al., 1993, 1995.. All MBP-reactive T-cells cloned from BALBrc mice to date have been T H 1

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cells; deliberate attempts to skew the repertoire toward T H 2 cell development have been negative Žunpublished data., suggesting that resistance in the BALBrc strains is not due to a predominating MBP-specific TH 2 response. The low frequency with which MBP-reactive cells are obtained from BALBrc mice suggests that either most MBP-reactive cells are deleted, or that they are present but are functionally tolerized or anergic. Experiments reported here attempt to examine whether resistance, as reflected in a low frequency of antigenspecific cells, is a result of tolerance Žeither clonal deletion or functional inactivation. which is dependent upon the presence of particular peptiderMHC complexes. We have taken advantage of the shirshi neurological mutant, functionally a knockout of MBP ŽRoach et al., 1983, 1985., to study whether expression of endogenous MBP plays a role in shaping the repertoire of MBP-reactive cells in the BALBrc mouse that is ultimately responsible for its resistance to EAE. Results presented in this report suggest that EAE resistance in BALBrc mice is a consequence of a tolerance mechanism in which expression of endogenous MBP is essential. Thus, unlike wild-type BALBrc mice, T-cells from BALBrc shirshi mice respond by proliferation and cytokine production in response to autologous MBP in secondary in vitro cultures. That these cells are readily able to cause EAE is shown upon adoptive transfer of activated cells to naive BALBrc recipients. In agreement with previous data ŽAbromson-Leeman et al., 1993., T-cells from BALBrc mice neither proliferate nor produce cytokines upon stimulation with MBP in culture, nor are encephalitogenic T-cells apparent after secondary in vitro stimulation. MBP-primed T-cells from BALBrc shirshi mice recognize two dominant epitopes. One, defined by peptide 59–76, was previously shown to be recognized by a majority of encephalitogenic clones derived from BALBrc mice ŽAbromson-Leeman et al., 1995.. A second epitope, defined by peptide 89–101, is recognized by T-cells from every BALBrc shirshi mouse tested Ž8 of 8 mice tested individually. and primes a highly encephalitogenic population of T-cells, as demonstrated by adoptive transfer of 89–101-primed cells into BALBrc recipients. The importance of this epitope in induction of disease in BALBrc mice has previously been shown ŽDuong et al., 1994.. Duong et al. reported that immunization of BALBrc mice with MBP peptide 91–107 in CFA can result in severe EAE if mice are treated daily with anti-IFN-g monoclonal antibody, but no disease is observed in immunized BALBrc mice not treated with anti-IFN-g . Despite these results, only two 89–101-recognizing T-cell clones have been derived from wild-type BALBrc mice from over 30 analyzed Žunpublished data.. The disparity in frequencies raises the possibility that T-cells recognizing the 89–101 epitope are regulated in either a qualitatively or quantitatively different manner than T-cells recognizing MBP 59– 76. Interestingly, populations of BALBrc shirshi T-cells

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stimulated with peptide 89–101 include both T H 0-type Žsecreting IL-4, as well as low levels of IL-2 and IFN-g . and TH1-type cells. In summary, studies of the MBP-specific T-cell response in BALBrc mice lacking MBP show that expression of endogenous MBP is directly responsible for MBPspecific tolerance and therefore EAE-resistance in BALBrc mice. In the absence of endogenous MBP expression, T-cells from BALBrc mice primed with MBP are able to proliferate and produce cytokines in vitro and are highly encephalitogenic in vivo. Recently, transcripts of developmentally-regulated isoforms of MBP have been identified in thymus and other non-nervous system tissues ŽCampagnoni et al., 1993; Mathisen et al., 1993; Fritz et al., 1985.. Studies reported here provide evidence for the role played by endogenous MBP expression in shaping the functional repertoire of BALBrc mice and thus the potential threat of autoimmune encephalomyelitis. The regulatory mechanisms involved are currently being studied. Acknowledgements This research was supported by grants from the National Multiple Sclerosis Society ŽRG 2620-A-1., the Multiple Sclerosis Foundation, and the National Institutes of Health ŽNS31152.. References Abromson-Leeman, S., Hayashi, M., Martin, C., Sobel, R., Al-Sabbagh, A., Weiner, H., Dorf, M.E., 1993. T-cell responses to myelin basic protein in experimental autoimmune encephalomyelitis-resistant BALBrc mice. J. Neuroimmunol. 45, 89–101. Abromson-Leeman, S., Alexander, J., Bronson, R., Carroll, J., Southwood, S., Dorf, M., 1995. Experimental autoimmune encephalomyelitis-resistant mice have highly encephalitogenic myelin basic protein ŽMBP.-specific T-cell clones that recognize a MBP peptide with high affinity for MHC Class II. J. Immunol. 154, 388–398. Antonia, S.J., Geiger, T., Miller, J., Flavell, R.A., 1995. Mechanisms of immune tolerance induction through thymic expression of a peripheral tissue-specific protein. Int. Immunol. 7, 715–725. Ashton-Rickardt, P.G., Bandeira, A., Delaney, J.R., Van Kaer, L., Pircher, H.-P., Zinkernagel, R.M., Tonegawa, S., 1994. Evidence for a differential avidity model of T-cell selection in the thymus. Cell 76, 651–663. Bernard, C.C.A., 1976. Experimental autoimmune encephalomyelitis in mice: Genetic controls of susceptibility. J. Immunogenet. 3, 263–274. Campagnoni, A.T., Pribyl, T.M., Campagnoni, C.W., Kampf, K., AmurUmarjee, S., Landry, C.F., Handley, V.W., Newman, S.L., Garbay, B., Kitamura, K., 1993. Structure and developmental regulation of Golli-mbp, a 105-kb gene that encompasses the myelin basic protein gene and is expressed in cells in the oligodendrocyte lineage in the brain. J. Biol. Chem. 268, 4930–4938. Deibler, G.E., Martense, R.E., Kies, M.W., 1972. Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Prep. Biochem. 2, 139–165. Duong, T.T., Finkelman, F.D., Singh, B., Strejan, G.H., 1994. Effect of anti-interferon-g monoclonal antibody treatment on the development of experimental allergic encephalomyelitis in resistant mouse strains. J. Neuroimmunol. 53, 101–107.

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