Adoptive transfer of experimental allergic encephalomyelitis and localization of the encephalitogenic epitope in the SWR mouse

Journal of Neuroimmunology, 31 (1991) 59-66 Elsevier

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JNI 01038

Adoptive transfer of experimental allergic encephalomyelitis and localization of the encephalitogenic epitope in the SWR mouse Anne H. Cross 1,2, George A. Hashim 4,5 and Cedric S. Raine 1.3 Departments of 1 Pathology (Neuropathology), 2 Neurology, and -~Neuroscience, and the Rose F. Kennedy Center for Research in Human Development and Mental Retardation, Albert Einstein College of Medicine, The Bronx, N Y, U.S.A,, and Departments of 4 Surgery and s Microbiology, St. Luke's-Roosevelt Hospital Center and Columbia University, New York, NY, (~S.A.

(Received 14 June 1990) (Accepted 24 August 1990)

Key words: Experimental allergic encephalomyelitis; Autoimmunity; Myelin basic protein; Encephalitogenic epitope; Multiple sclerosis

Summary Adoptively-transferred experimental allergic encephalomyelitis (EAE) was induced in the SWR (H-2 q) strain of mouse using lymph node cells, spleen cells, or cell lines sensitized to whole myelin basic protein (MBP). With the aid of synthetic peptides, the minimal encephalitogenic epitope of MBP for the SWR strain was localized to amino acids 87-99 of the MBP molecule. The 87-99 sequence is also encephalitogenic for the SJL strain of mouse and the Lewis rat. EAE was induced with a protocol similar to that for the induction of EAE in the SJL strain with the exception that sublethal irradiation of recipients was necessary. Mice developed typical clinical and pathological EAE 6-14 days post-transfer of cells sensitized to either whole MBP or peptide 87-99, after which they remitted. No relapses were observed. Thus, adoptively transferred EAE can be induced in irradiated H-2 q mice for which the encephalitogenic epitope is one of the nested encephalitogenic epitopes for SJL (H-2 ~) mice, namely residues 87-99.

Introduction Murine experimental allergic encephalomyelitis (EAE), a T cell-mediated autoimmune disease induced by sensitization to central nervous system (CNS) white matter or its major encephalitogenic component, myelin basic protein (MBP), is widely

Address for correspondence: Anne H. Cross, M.D., Department of Neurology, Albert Einstein College of Medicine, K436, 1300 Morris Park Avenue, Bronx, NY 10461, U.S.A.

used as a model for studies on the human CNS disease, multiple sclerosis (MS). Using CNS antigens in adjuvant, acute EAE can be actively induced by immunization of mice with certain major histocompatibility complex (MHC) backgrounds, e.g. H-2 s, H-2 u, H-2 q (Levine and Sowinski, 1973; Raine et al., 1980; Linthicum and Frelinger, 1982; Fritz et al., 1985). Similarly, susceptibility to MS appears to be linked to selected MHC types (McFarlin and McFarland, 1982). EAE can also be induced by the adoptive transfer by MBP-immune T cells. In the SJL (H-2 ~) and PL (H-2 °) strains, adoptively transferred EAE

0165-5728/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

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is a disease with a clinical course (relapses and remissions) and demyelinating pathology which resemble MS (Pettinelli et al,, 1981; Cross et al., 1987). Different regions of the MBP molecule are known to be encephalitogenic in the two strains: the encephalitogenic epitope for P L / J mice lies in the N-terminal first nine amino acids (Zamvil et al., 1986), and for the SJL/J strain, there are at least two overlapping or 'nested' epitopes encompassing amino acids 87-101 (Kono et al., 1988; Sakai et al., 1988; Fallis et al., 1989). The present investigation was undertaken to determine if EAE could be adoptively transferred in the SWR/J (H-2 q) strain and to define the encephalitogenic epitope(s) involved. The SWR and SJL strains share the lack of expression of MHC class II I-E molecules and a deletion of approximately 50% of the mouse genome encoding the variable region of the T cell receptor (TCR) beta chain (Behlke et al., 1986), although the two strains are not genetically related (Potter, 1978; personal communication, Jackson Laboratories). Since prevailing dogma states that specific T cell responses are governed by the interaction of the TCR with an antigen-MHC complex on the surface of an antigen-presenting cell, both MHC and TCR expression are crucial for antigen recognition. Thus, these deletions might be expected to limit antigen recognition in both strains. The present findings showed that EAE could be adoptively transferred in the SWR strain and the encephalitogenic epitope localized to the same region of MBP as for the SJL strain, but encompassed only one of the nested encephalitogenic epitopes.

Materials and methods

Animals Female SWR/J mice and female SJL/J mice (Jackson Laboratories, Bar Harbor, ME, U.S.A.) were used between 6 and 12 weeks of age and were housed and cared for in an NIH-approved facility, Antigens Guinea pig myelin basic protein (gpMBP) was prepared from guinea pig spinal cords (Rockland, Gilbertsville, PA, U.S.A.) according to the proce-

dure of Deibler et al. (1972). Its size and purity were verified by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Purified protein derivative (PPD) of tuberculin was obtained from Connaught Laboratories, PA, U.S.A. The MBP peptides V-H-F-F-K-N-I-V-T-P-R-T-P (residues 87-99) and H-F-F-K-N-I-V-T-P-R-T-P (8899) were synthesized by the Macromolecular Analysis Laboratory at the Albert Einstein College of Medicine using an Applied Biosystems 430a peptide synthesizer with N-methyl pyrrilodone/1-hydroxy benzotriazole t-Boc methodology. Other batches of peptide 87 99 and F-K-N1-V-T-P-R-T-P (90-99) were synthesized by a minor modification (Hashim et al., 1986) of the Merrifield solid-phase method (Merrifield, 1963). Synthetic MBP peptide F-K-N-I-V-T-P-R-T-P-P-P (90-101) (Peninsula Labs, Belmont, CA, U.S.A.) was a gift from Drs. Robert Fallis and Dale McFarlin, NIH, Bethesda, MD, U.S.A. Peptide 21 166 was obtained by cyanogen bromide cleavage of whole gpMBP using a modification of the method of Chou et al. (1984).

Immunization and transfer The method of immunization was similar to that published previously for SJL/J mice (Pettinelli et al., 1981). S W R / J mice were immunized subcutaneously in four sites over the flanks with 400 /zg of whole gpMBP or 400 /xg of purified peptide and 30 /~g of Mycobacterium tuberculosis, H37RA (Difco Laboratories, Detroit, MI, U.S.A.) emulsified in Freund's incomplete adjuvant (Difco). Ten days later, draining axial, brachial and inguinal lymph nodes were removed and a single-cell suspension prepared by pressing whole lymph nodes or spleens through sterile wire mesh screens, Spleen cell (SC) suspensions were depleted of erythrocytes using sterile ammonium chloride. Cell number and viability were assessed by trypan blue exclusion and cells were plated in 24-well Linbro plates (2 ml/well) at 4 x 106/ml in RPMI 1640 supplemented with 10% fetal calf serum (FCS, Gibco, Grand Island, NY, U.S.A.), penicillin G (100 units/ml, Sigma, St. Louis, MO, U.S.A.), streptomycin (100 /~g/ml, Gibco), glutamine (2 mM, Sigma) and whole gpMBP (50 /~g/ml) or MBP peptides (10 /~g/ml). Cultures were incubated at 37 °C in 7% CO 2 for 3-4 days.

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Clinical grading

F o r adoptive transfer, the cells were washed twice and injected via a tail vein into naive recipients. Except for the earliest experiments, all recipients were irradiated with 500 rad using a cesium-137 irradiator (Gammacell 40, A t o m i c Energy of Canada, Ottawa, Canada). 2 x 107 to 1 x l 0 s cells in 0.2-0.3 ml H a n k s ' balanced salt solution (Sigma) were necessary for the induction of EAE.

Mice were evaluated every 1 - 3 days and graded according to the following scale: grade 1, limp tail; grade 2, hindlimb weakness with difficulty fighting; grade 3, one limb plegic; grade 4, two limbs plegic; grade 5, tetra- or quadriplegic or moribund. Relapses were defined as worsening by at least one grade lasting 48 h or longer.

Pathology Proliferation assays

Mice were anesthetized by ether inhalation and perfused t h r o u g h the left cardiac ventricle with 40 ml of cold 2.5% glutaraldehyde, The C N S was removed and thin slices of brain, spinal cord and sacral roots were post-fixed in cold 1% osmium tetroxide for 60 min, after which sections were deh),drated through a graded series of ethanol, cleared in propylene oxide and embedded in Epon 812. Slides were stained with toluidine blue prior to light microscope examination.

To quantify the specific proliferative response to various antigens, cells were plated in triplicate or quadruplicate in 96-well flat-bottomed microtiter plates at 8 × l 0 s cells/well in complete m e d i u m supplemented with 1% normal mouse serum. M B P (25-100 /~g/ml), synthetic peptides ( 5 - 1 0 t~g/ml) or m e d i u m alone, were added to a final volume of 0.2 M / w e l l . F o r the final 8 h of culture, 1 /~Ci/well of [methyl-3H]thymidine, 25 C i / m m o l (Amersham Corp., Arlington Heights, VA, U.S.A.) was added to each well. The plates were harvested using a semi-automated multichannel harvesting device (Skatron, Sterling, CA, U.S.A.) onto glass fiber filters. Counts per minute were determined by liquid scintillation using standard technique. The stimulation index (SI) was calculated as follows:

Results

Irradiation is required for adoptive transfer of EAE Five naive recipients of 1 × 108 cells did not b e c o m e clinically ill and did not show signs of pathology. One recipient of a T cell line to M B P also remained well. However, after 500 rad whole b o d y irradiation, 15 recipients of 2 × 10 7 to 1 × l0 s l y m p h n o d e cells ( L N C ) or SC became acutely

mean cpm of cells cultured with antigen mean cpm of cells cultured without antigen

TABLE 1 SUMMARY OF EAE INDUCED IN THE SWR/J STRAIN

Non-irradiated recipients of MBP-sensifized cells * Recipients of MBP-sensitized cells * Recipients of 87-99-sensitized cells c * Recipients of 88-99-sensitized cells * Recipients of 90-101-sensitized cells * Recipients of 90-99-sensitized cells

Clinical EAE

Mean maximum clilaicalscore

SI a of cells used in transfer

0/5 16/16 6/6 0/3 0/6 0/5

0 3.2 2.6 0 0 0

LNC: 3.4, 3.4 LNC: 2.1, 4.1; SC: 2.3, 3.0, 3.7 LNC: 1.0, 1.8; SC: 1.4, 1.5 SC: < 1.0 LNC: 1.0; SC: 1.0 LNC: 1.1; SC: 1.3

Pathology b

7/7 4/4 0/2 0/2 0/1

a SI = stimulation index as defined in Materials and Methods. b Inflammatory cell infiltration and demyelination. Four animals received cells sensitized to 87-99 which was synthesized by the Merrifield method, two received cells sensitized to 87-99 synthesized by t-Boc methodology. * Irradiated with 500 rad prior to cell transfer.

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ill. One irradiated recipient of serially transferred SC derived from LNC recipients also became acutely ill. SC and LNC used in the experiments employing irradiated and non-irradiated recipients proliferated equally well to MBP (Table 1).

LNC and SC yielded no significant stimulation. Similarly, no proliferation above background to peptide 88-99 was detected using 88-99-sensitized cells. Clinical course

Peptide studies

When the 13 amino acid SJL encephalitogenic MBP peptide 87-99 was used as the immunogen and for stimulation in vitro, EAE resulted in 6 / 6 irradiated recipients beginning as early as day 6 post-transfer. Peptide 87-99 worked equally well when synthesized by either Merrifield or t-Boc methodology. Moreover, proliferation to both 8799 and whole MBP was demonstrated by these cells (Table 2). When the 12 amino acid S J L / J encephalitogenic MBP peptide 90-101 was used as the antigenic stimulus, no disease occurred in irradiated recipients. Likewise, when peptide 90-101 was used as both the immunogen and for stimulation in vitro, no disease resulted. Significant proliferation above background to peptide 90-101 was not seen in either situation (Table 2). Truncated peptides of 87-99 were examined next in an attempt to determine the minimal encephalitogenic epitope. None of five irradiated recipients of peptide 90-99-sensitized cells or of three recipients of peptide 88-99-sensitized cells developed EAE. Proliferation to 90-99 in vitro by

Irradiated recipients first showed signs of illness between days 6 and 14 post-transfer. Unlike the SJL or PL strains, overnight loss of approximately 20% of body weight was a harbinger of neurological signs, preceding the latter by 1-2 days. Neurological signs in SWR mice during the acute disease consisted of floppy tail and hindlimb weakness with occasional turning behavior, similar to signs observed in the other two strains. The acute illness was followed by recovery (usually partial), generally leaving the animal with a static neurologic deficit. Relapses were not seen in ten mice followed for 30 or more days. A second dose of sublethal irradiation given to three SWR recipients of MBP-immune cells at 100 days post-transfer failed to induce a second attack. To examine whether the effects of sublethal irradiation on EAE induction and relapse rate in the SWR strain might be responsible for the lack of relapses and disease progression, four SJL recipients of MBP-immune LNC, derived from MBP-immunized SJL donors, were irradiated prior to cell transfer. Two of the four died during the acute disease. Of the remaining two, one had a

TABLE 2 SPLEEN CELL P R O L I F E R A T I O N TO MBP A N D PEPTIDES S W R mice immunized with: MBP Proliferation to: Media alone MBP (50 # g / m l ) b 21-166 (50 ,ug/ml) 87-99 (10 ~ g / m l ) c 88-99 (10 ~tg/ml) c 90-99 (10/~g/ml) d 90-101 (10 p.g/ml) ~

7,624 _-2- 116 28,697 ± 1,266 27,371± 815 nd nd nd 8,062 ± 834

87-99

88-99

3,357 __+215 (1.0) (1.0) ~ 7,025 + 587 (1.0) (3.7) * 11,785 + 770 (1,7) * 1,958 +_ 357 nd (3.6) * nd ** 9,962 + 618 (1.4) * 2,264 ± 211 2,254_+ 670 nd nd nd nd (1.0) nd

" Stimulation indices given in parentheses. b Approximately 3 ~M. Approximately 7.5 p~M. d Approximately 10 ~M. * Significant proliferation, p < 0.05. ** Not done.

90-99 12,200 16,933 nd 12,728 13,695 13,469 nd

90-101 _+ 1,241 (1.0) 14,139 ± 1,054 _+ 1,734 (1.4) * 16,841 ± 994 18,084 +_ 3,787 +_ 1,386 nd ± 333 (1.1) nd ± 677 (1.1) nd 14,138 ± 2,258

(1.0) (1.2) (1.3)

(1.0)

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Fig. ]. The thoracic spinal cord is shown from an SWR mouse which teceived 8.5 x 107 whole myelin basic protein-responsive spleen cells 4 months earlier. Two large lesions are shown in the lateral columns. Note the predilection for subpial white matter and the presence of nerve fiber destruction as evidenced by the large osmiophilic droplets. Clinical grade 1.5. × 40.

clear remission followed b y a relapse. This animal, which h a d been g r a d e 4 at d a y 10 post-transfer, r e m i t t e d to g r a d e 1 b y d a y 31 a n d then r e l a p s e d to g r a d e 4 b y d a y 39. The other m o u s e r e m a i n e d c h r o n i c a l l y ill at g r a d e 4.

Fig. 2. Detail from the lesion on the right of Fig. 1. The subpial white matter is intensely gliotic and contains some infiltrating cells around the blood vessels in the center (asterisks). Elsewhere in the field, demyelinated (small arrows) and remyelinated (large arrows) fibers are evident. The meningeal surface of the cord lies above, x 1000.

the C N S also d e m o n s t r a t e d a d e e p l y infiltrating, c i r c u m f e r e n t i a l p a t t e r n , suggesting that this pattern was s e c o n d a r y to i r r a d i a t i o n effect. E x a m i n a t i o n of C N S tissue o b t a i n e d f r o m recipients o f cells d i r e c t e d against M B P p e p t i d e s 8 8 - 9 9 , 9 0 - 9 9 , a n d 9 9 - 1 0 1 revealed no p a t h o l o g i c changes. N o changes a t t r i b u t a b l e to i r r a d i a t i o n were f o u n d in these recipients.

Pathology T h e C N S of i r r a d i a t e d recipients of M B P - i m m u n e L N C or SC showed extensive i n f l a m m a t o r y cell infiltration penetrating, m o r e d e e p l y i n t o the C N S p a r e n c h y m a t h a n in u n i r r a d i a t e d SJL or P L mice with EAE. T h e lesions were often confluent a n d d i s t r i b u t e d s u b p i a l l y in a c i r c u m f e r e n t i a l p a t tern. D e m y e l i n a t i o n a n d r e m y e l i n a t i o n were r e a d ily a p p a r e n t in c h r o n i c a l l y afflicted a n i m a l s (Figs. 1 a n d 2). W a l l e r i a n d e g e n e r a t i o n was noted, also in a circumferential pattern. W h e n p e p t i d e 8 7 - 9 9 was used as b o t h the i m m u n o g e n a n d the stimulus in vitro, the p a t h o l o g i c changes o b s e r v e d in four recipients were identical to those seen when whole M B P was used (Fig. 3). N o p a t h o l o g i c changes were n o t e d in either i r r a d i a t e d c o n t r o l S W R mice or n o n - i r r a d i a t e d S W R recipients of M B P - i m m u n e cells. W h e n SJL recipients were i r r a d i a t e d with 500 r a d p r i o r to the transfer o f M B P - i m m u n e ceils, the p a t h o l o g y in

Fig. 3. Detail from the cervical spinal cord of a mouse with EAE adoptively transferred by 5 × 107cells sensitized to peptide 87-99. A perivascular cuff is seen consisting of large mononuclear cells, polymorphonuclear cells and some fibrin deposition. In the adjacent white matter, note the demyelinated axons (arrows) and several nerve fibers with dilated myelin sheaths. Eight days post-transfer; clinical grade 3. x 1000.

64 Discussion

The present study has demonstrated that EAE characterized by CNS inflammation and demyelination can be adoptively transferred in mice of the SWR (H-2 q) strain. In the SWR strain, unlike the more frequently studied SJL and PL strains, sublethal irradiation of recipients was necessary for EAE induction. The mechanism of action of sublethal irradiation of recipients in the present study was not investigated but was perhaps via its effect on the blood-brain barrier (BBB) (Paterson et al., 1975; Dimitrievich et al., 1977; Paterson and Harvey, 1978) and its necessity may reflect the presence of a BBB in the SWR strain which was relatively more resistant to invasion by immune cells. Absence of chronic disease progression or relapses in the SWR strain noted in the present study might support this idea. To this end, it should be noted that in earlier works in which EAE was actively induced in the SWR strain, Bordetella pertussis, an agent known to alter CNS vascular permeability (Bergman et al., 1978), was always included in the induction protocol (Levine and Sowinski, 1973; Raine et al., 1980; Linthicum and Frelinger, 1982; Fritz et al., 1985). Irradiation is not likely to be responsible for the absence of disease progression in the present case because pilot data on irradiated SJL recipients suggest they continue to exhibit disease progression. Alternatively, sublethal irradiation may downregulate suppressor T cell control of EAE effector cells leading to EAE development in an otherwise resistant strain. A recent study by Rodriguez et al. (1990) found that sublethal irradiation (300-600 rad) of normally resistant B10.S (9R) and C 5 7 B L / 1 0 mice rendered them susceptible to Theiler's murine encephalitis virus-induced demyelination. The authors attributed their findings to immunosuppression induced by irradiation because reduced IgM hemagglutination titers to sheep erythrocytes were demonstrated by irradiated mice. In addition, the findings do not fully support the contention of Bourdette et al. (1988) that relapses and cell-mediated demyelination were associated phenomena in that demyelination occurred in the present study in the absence of relapses. However, the extent of demyelination was less than that in the SJL and PL strains.

That the SWR strain recognizes an epitope of MBP located within the 87-99 sequence, also recognized by the SJL mouse, the Lewis rat (Offner et al., 1989), and humans (Wucherpfennig et al., 1990), was not unexpected. In this regard, immunodominant regions have been described for MBP (Offner et al., 1989) and for other large protein antigens (Berkower et al., 1982; Berzofsky and Berkower, 1989). Furthermore, studies of MHC-II-restricted helper responses have shown that as a general rule, only one to three 12-amino acid residue segments per 100 residues of a protein antigen will elicit an immune response (Nagy et al., 1989). It has been speculated that non-immunogenic portions are processed into peptides that fail to fit the binding site of the MHC-II molecule. In the present work, deletion of valine at position 87 led to loss of encephalitogenicity in the SWR strain. Valine is a highly hydrophobic amino acid (Fauchere and Pliska, 1983) and this property might be expected to stabilize the peptide in its interaction with the M H C molecule, perhaps by promoting the folding of the peptide. The 87-99 peptide might also be a dominant product of myelin breakdown and processing and in this manner it may achieve immunodominant status. In earlier work from this laboratory, it was found that the 90-101 epitope required processing prior to its presentation to SJL T cells (Cross et al., 1990). Since the final three amino acids of 90-101 are three prolines which may confer a rigid structure less likely to fit the MHC 'pocket', the question arose whether the lack of response to peptide 90-101 by the SWR strain reflected its inability to process the peptide. However, the 9099 peptide, from which the C-terminal two prolines had been removed, did not prove immunogenic for the SWR strain. Thus, the inability to process peptide 90-101 was not the underlying reason for the lack of encephalitogenicity. The lack of I-E expression and absence of one-half of the T C R B-chain variable region, found in both SJL and SWR mice, might serve to limit their ability to respond to MBP. This combination of deletions is important because current dogma states that CD4 + T cells recognize antigen via the T C R when presented in the cleft of an MHC-II molecule. Susceptibility to EAE in both strains might reflect a limited response to MBP by CD4 +, MHC-II-restricted suppressor-inducer T cells, the

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latter perhaps recognizing different epitopes of MBP (Goodman and Sercarz, 1983). Restricted usage of TCR genes appears to be a common occurrence in EAE (Acha-Orbea et al., 1988; Happ et al., 1988; Sakai et al., 1988; Urban et al., 1988; Burns et al., 1989). The Vfl 17a region has been shown to be utilized by a sizeable proportion of SJL encephalitogenic T cells that recognize peptide 87-99 (Sakai et al., 1988). The SWR strain (I-E negative) expresses the Vfl 17a peptide; I-E positive strains do not (Marrack and Kappler, 1988). Of considerable interest would be whether the SWR strain utilizes the same TCR as the SJL strain in recognizing the same MBP peptide, particularly with regard to therapeutic measures (Wraith et al., 1989). Thus, experiments to define the predominant V-/3 gene usage in encephalitogenic SWR T cells are presently in progress in this laboratory.

Acknowledgements We thank Drs. Dale McFarfin and Robert Fallis of NIH for the gift of peptide 90-101; the Macromolecular Analysis Laboratory at Albert Einstein College of Medicine for synthesis of 8799, 88-99 and 90-99 and for analysis of the purity of all peptides utilized in these studies. We thank Drs. Barbara Cannella, Dale E. McFarlin and Philip Y. Paterson for helpful discussion. Howard Finch, Miriam Pakingan, Rosalie Staine and Earl Swanson provided expert technical assistance. We thank Michele Briggs for careful preparation of the manuscript. A.H.C. was supported by a fellowship from the National Multiple Sclerosis Society, NMSS FG 749-A-1. Supported in part by NS 08952 and NS 11920; and NMSS RG 1001-G-7.

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