Journal of Neuroimmunology, 39 (1992) 67-74 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00
67
JNI 02198
Myelin proteolipid protein: minimum sequence requirements for active induction of autoimmune encephalomyelitis in SWR/J and SJL/J mice * Vincent K. Tuohy
a,b,d, R a y m o n d A.
Sobel c,e Zhijian Lu and Marjorie B. Lees a,b,d
f, Richard
A. Laursen f
Department of Biochemistry, E.K. Shriver Center, Waltham, MA, USA, ~ Department of Neurology and c Department of Pathology, Harvard Medical School, Boston, MA, USA, a Neurology Service and e Immunopathology and Neuropathology Units, Massachusetts General Hospital, Boston, MA, USA, and f Department of Chemistry, Boston UniL,ersity, Boston, MA, USA
(Received 22 November 1991) (Revised, received20 February 1992) (Accepted 20 February 1992)
Key words." Myelin proteolipid protein; Experimental autoimmune encephalomyelitis; T cell epitope; Encephalitogenic; Demyelination; Multiple sclerosis
Summary Proteolipid protein (PLP) is the major protein constituent of mammalian central nervous system myelin. We have previously identified two different PLP encephalitogenic T cell epitopes in two mouse strains. Murine PLP peptides 103-116 YKTTICGKGLSATV and 139-151 HCLGKWLGHPDKF are encephalitogenic determinants in S W R / J (H-2 q) and SJL/J (H-2 s) mice, respectively. The purpose of the present study was to determine the minimum sequence requirements for each of these PLP encephalitogens. In S W R / J mice, at least two distinct overlapping peptides can induce experimental autoimmune encephalomyelitis (EAE). The eleven residue sequences PLP 105-115 TTICGKGLSAT and PLP 106-116 TICGKGLSATV are encephalitogenic in S W R / J mice, but PLP 106-115 TICGKGLSAT, the decapeptide indigenous to both sequences, is non-encephalitogenic. In contrast, the shortest PLP sequence capable of inducing EAE in SJL/J mice is the nonapeptide 141-149 LGKWLGHPD. These data indicate that encephalitogenic determinants of PLP are short contiguous peptide sequences similar in length and diversity to those of MBP.
Introduction Correspondence to: V.K. Tuohy. Present address: The Cleveland Clinic Foundation, Research Institute, Immunology, NN1, 9500 Euclid Avenue, Cleveland, OH 44195, USA. * This work was presented in abstract form at the Second Annual Meeting of the Society for Experimental Neuropathology, on September 23, 1989 in New Orleans, LA.
Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease model that shares many clinical and histopathological similarities with multiple sclerosis (re-
68 viewed in Alvord et al., 1984). EAE can be induced by immunization of animals with either whole CNS tissue, myelin, or myelin components such as MBP or PLP. Synthetic peptides have been used to identify encephalitogenic MBP determinants in many species including guinea pig (Eylar and Hashim, 1968; Eylar et al., 1970), rabbit (Shapira et al., 1971), monkey (Karkhanis et aI., 1975), rat (Hashim et al., 1978), and mouse (Zamvil et al., 1986; Kono et al., 1988; Sakai et al., 1988a, b). Encephalitogenic peptides of MBP have consistently been described as short, contiguous major histocompatibility complex (MHC) restricted, immunodominant T cell epitopes. We have identified two PLP encephalitogenic T cell epitopes. S W R / J (H-2 q) mice develop EAE after immunization with murine PLP peptide 103-116 YKTTICGKGLSATV (Tuohy et al., 1988b), whereas S J L / J (H-2 ~) mice develop disease when immunized with PLP peptide 139-151 HCLGKW L G H P D K F (Tuohy et al., 1989a; Sobel et al., 1990). Recent studies indicate that PLP encephalitogens may play an immunodominant role in both the induction (Whitham et al., 1991a) and prevention (Kennedy et al., 1990) of whole spinal cord-induced EAE. Therefore, basic information about immune recognition of PLP may lead to a better understanding of the role PLP plays in the pathogenesis of human multiple sclerosis. In the present study, we immunized S W R / J and S J L / J mice with a series of overlapping and truncated peptides within the appropriate determinant. In this way we were able to identify the minimum PLP sequence required for inducing EAE in each mouse strain. The results show that the minimum encephalitogenic sequence in the S W R / J mouse can be either PLP 105-115 TTICGKGLSAT or 106-116 TICGKGLSATV, whereas the minimum sequence in the S J L / J mouse is PLP 141-149 LGKWLGHPD.
Materials and methods
Mice Female S J L / J (H-2 S) and S W R / J (H-2 q) mice were purchased from The Jackson Laboratory, Bar Harbor, ME. They were obtained at 5-8
weeks of age and were immunized between 10 and 14 weeks.
Peptide synthesis All peptides were prepared according to the sequence of murine PLP (Milner et al., 1985; Macklin et al., 1987; Nave et al., 1987). Peptides were synthesized manually as described earlier (Tuohy et al., 1988b, 1989a) by the simultaneous multiple peptide synthesis ('teabag') method (Houghten, 1985; Houghten et al., 1986). In this procedure peptides were synthesized on a pmethylbenzhydrylamine resin which yielded peptides with C-terminal amides. After cleavage of the peptide from the resin, HPLC analysis of the crude product showed greater than 80-85% of the material eluting as a single peak, the identity of which was confirmed by amino acid analysis. Immunization procedure Each mouse was immunized s.c. on day 0 with 100 nmol of peptide and 200 Izg Mycobacterium tuberculosis H37RA in 200 /~1 of an emulsion of equal volumes of water and IFA. Each mouse was also injected i.v. on day 0 and day 3 with Bordetella pertussis bacilli (pertussis vaccine, lot no. WF262, Massachusetts Public Health Biologics Laboratories, Boston, MA). The amount injected was chosen by determining the maximum non-lethal dose. S J L / J mice received 0.75 x 10 l° bacilli each injection, whereas S W R / J mice were injected with 1.0 x 101° each time. Clinical and histologic evaluation All animals were assessed clinically as previously described (Tuohy et al., 1988a) according to the following criteria: 0, no disease; 1, decreased tail tone or slightly clumsy gait; 2, tail atony a n d / o r moderately clumsy gait a n d / o r poor righting ability; 3, limb weakness; 4, limb paralysis; 5, moribund state. Animals were killed within 1 week after showing clinical signs of disease. Mice that did not develop clinical signs were killed at 7 weeks post-immunization. Brains and spinal cords were fixed in 10% 'phosphatebuffered formalin and paraffin-embedded sections were stained with Luxol fast blue-hematoxylin and eosin for light microscopy. Histologic disease was quantitated by counting the inflam-
69
both PLP encephalitogens. However, one mouse did have very mild histologic disease. S W R / J mice never developed clinical or histologic E A E after immunization with PLP 107-115, 108-115, or 109-115. Peptide 139-151, the PLP encephalitogenic determinant for S J L / J mice, did not induce either clinical or histologic EAE in any S W R / J mice. Thus, the two overlapping 11 residue PLP peptides 105-115 T r I C G K G L S A T and 106-116 T I C G K G L S A T V are the shortest sequences capable of inducing EAE in S W R / J mice.
matory loci in meninges and parenchyma as previously described (Sobel et al., 1984). Results
EAE in SWR / J mice Immunization of S W R / J mice with the synthetic PLP peptide 103-116 Y K T T I C G K G L S A TV induced acute clinical and histologic EAE in six out of six mice (Table 1). In order to identify the N- and C-terminal residues of this encephalitogenic determinant, we immunized S W R / J mice with peptides containing the overlapping PLP residues 103-113, 104-114, 105-115, and 106116. S W R / J mice never developed clinical or histologic E A E after immunization with either PLP 103-113 or PLP 104-114. However, seven out of ten mice immunized with PLP 105-115 T T I C G K G L S A T developed severe clinical and histologic disease (Fig. 1A), and the remaining three mice in this group had histologic EAE. Furthermore, two out of ten SWR mice immunized with PLP 106-116 T I C G K G L S A T V developed severe clinical and histologic disease and one of the eight remaining mice in this group had histologic E A E (Fig. 1B). In two subsequent experiments, none out of seven and none out of 11 animals developed clinical E A E following immunization of SWR mice with PLP 106-115 T I C G K G L S A T , the decapeptide indigenous to
EAE in SJL / J mice Immunization of S J L / J mice with synthetic PLP peptides 139-151 H C L G K W L G H P D K F and 141-150 L G K W L G H P D K induced severe clinical and histologic EAE in ten out of ten and ten out of 13 mice, respectively (Table 2). The shortest PLP sequence capable of inducing E A E in S J L / J mice was the nonapeptide 141-149 LGKWLGHPD. In an initial experiment, only one out of ten mice injected with PLP 141-149 developed clinical EAE, but all ten mice had severe histologic disease. This experiment was repeated, and a dramatic increase occurred in the incidence of clinical disease. In the second experiment eight out of ten mice immunized to PLP 141-149 developed acute clinical disease, and all ten mice
TABLE 1 EAE IN S W R / J (H-2 q) MICE I M M U N I Z E D W I T H PLP PEPTIDES Murine PLP peptide sequence
100 nmol dose (/zg)
No. with both clinical and histologic E A E
Mean day of onset
Clinical grade a
No. of inflammatory foci b
No. with histologic EAE only c
No. of inflammatory foci
103-116 103-113 104-114 105-115 106-116 106-115
144 117 108 105 105 95 85 74 63 152
6/6 0/7 0/7 7/10 2/10 0/18 0/6 0/6 0/6 0/8
14 (12-16) 37 (25-48) 31 (30-32) -
5.0 0 0 3.7 4.0 0
81 (53-116)
0 0/7 0/7 3/3 1/8 1/7
0 0 0 16 (4-32) 24 6
107-115
108-115 109-115 139-151
57 (19-166) 97 (65-128) -
-
0
-
0/6
0
-
0 0 0
-
0/6 0/6 0/8
0 0 0
a Values represent mean clinical grade scored for animals showing clinical disease as described in Materials and methods. b Mean number of total (meningeal and parenchymal) inflammatory loci in paraffin sections of CNS. Values in parentheses represent ranges. c These animals never showed any clinical signs and were killed at 7 weeks.
70 had histologic E A E (Fig. 1C). PLP peptides 141148 and 142-150 were not encephalitogenic, thus indicating that Leu-141 and Asp-149 represent the N- and C-terminal residues, respectively, of this PLP encephalitogenic determinant for S J L / J mice. Peptide 103-116, the PLP encephalitogenic determinant for S W R / J mice, did not induce either clinical or histologic E A E in any S J L / J mice.
Discussion In the present study we have determined the minimum sequence requirements of two PLP determinants for active induction of EAE. In S W R / J mice, the shortest encephalitogenic PLP sequences are two overlapping l l - a m i n o acid peptides, PLP 105-115 TT1CGKGLSAT and PLP 106-116 T I C G K G L S A T V , whereas in S J L / J mice the nonapeptide PLP 141-149 LGKWLG H P D is the shortest encephalitogenic PLP peptide. The inability of PLP 106-115 to induce E A E in S W R / J mice indicates the importance of an intact amino acid at either the N-terminal 105 or the C-terminal 116 position. Furthermore, the difference in disease incidence (Table 1) induced by PLP 105-115 (10/10) compared to PLP 106116 (3/10) suggests that these overlapping encephalitogens may represent distinctly different
S W R / J T cell epitopes. The presence of multiple overlapping T cell epitopes within a PLP determinant for S W R / J mice would correspond with their existence in MBP (Kono et al., 1988; Sakai et al., 1988b) and PLP (Kuchroo et al., 1991) determinants for S J L / J mice. Unlike MBP, PLP contains many cysteine residues whose labile sulfhydryl groups can be problematic in immunologic studies. Such difficulties can be circumvented by substitution of serine for cysteine in the synthesis of PLP determinants for S J L / J mice (Tuohy et al., 1989a; Van der Veen et al., 1990; Endoh et al., 1990). This is also true for the S W R / J determinant of PLP, since PLP 103-116 containing a serine substitution for Cys-108 induces severe E A E (data not shown). Furthermore, PLP 105-115 is encephalitogenic despite the fact that Cys-108 is a posttranslationally modified acylation site of PLP (Bizzozero et al., 1990). An acylated Cys-108 in the native protein suggests that this region of PLP may play an important role in determining tertiary structure. Whatever the structural or functional role of acylated Cys-108 may be, it does not interfere with the capacity of PLP 105115 to serve as an autoimmune target in vivo. Perhaps the relatively rapid fatty acid turnover at the acylation site(s) (Bizzozero and Good, 1991) provides a ready availability in vivo of deacylated determinant for Class II binding and immune presentation and recognition. Alternatively, an
TABLE 2 EAE IN SJL/J (H-2~) MICE IMMUNIZED WITH PLP PEPTIDES Murine PLP peptide sequence
100 nmol dose (p.g)
No. with both clinicaland histologic EAE
Mean day of onset
Clinical grade a
No. of No with inflammatory histologic foci b EAE only c
No. of inflammatory foci
139-151 141-150 142-150 141-149 (1) d 141-149 (2) 141-148 103-116
154 115 104 102 102 91 144
10/10 10/13 0/12 1/10 8/10 0/7 0/7
15 (9-23) 31 (24-49)
3.8 2.7 0 2.0 4.0 0 0
83 (26-'140) 95 (45-150) 31 88 (41-170) -
58 (12-107) 0 60 (34-108) 57 (14--100) 0 0
28 13 (10--18)
0 3/3 0/12 8/9 2/2 0/7 0/7
Values represent mean clinical grade scored for animals showing clinical disease as described in Materials and methods. b Mean number of total (meningeal and parenchymal) inflammatory foci in paraffin sections of CNS. Values in parentheses represent ranges. c These animals never showed any clinical signs and were killed at 7 weeks. a Because of disparity between clinical and histological scores, this experiment was repeated.
71 acylated cysteine may not interfere with target recognition and i m m u n e activation. In contrast to the P L P d e t e r m i n a n t for the S W R / J mouse, the M B P d e t e r m i n a n t for the P L / J mouse actually
requires modification for encephalitogenicity. T h e i m m u n o d o m i n a n t M B P 1 - 9 determinant is encephalitogenic in P L / J mice only when the A l a - I residue of the synthetic peptide is N-acetylated as is native M B P in vivo (Zamvil et al., 1986). Thus, protein modification can actually confer encephalitogenicity to a determinant p e r h a p s by altering its c o n f o r m a t i o n or by protecting it from proteolysis. It appears from the current data that longer peptides are m o r e encephalitogenic than their truncated counterparts (Tables 1 and 2). Increasing the 'tail region' amino acid side chains may serve to better stabilize the 'antigenic core region' of encephalitogens within the trimolecular complex and thus e n h a n c e immunogenicity (Reddehase et al., 1989; Wraith et al., 1989). However, increasing the length of encephalitogenic peptides can also decrease their encephalitogenicity. Both P L P peptides 137-151 and 139-154 are not as encephalitogenic as the shorter P L P 139-151 (Tuohy et al., 1989a). Perhaps the increased hydrophobicity of these longer peptides contributes to their poor encephalitogenicity. Alternatively, the longer peptides may contain flanking epitopes involved in immunologic downregulation. T h e latter view is supported by the e n h a n c e d tolerogenicity of P L P 139-154 compared to its truncated peptide counterparts (Kennedy et al., 1990). It is of interest to note that the P L P determinants defined in the present study for S J L / J and S W R / J mice as well as the P L P determinants for C 3 H / H e (H-2 k) mice ( E n d o h et al., 1990), P L / J (H-2 u) mice ( W h i t h a m et al., 1991b), and Lewis
Fig. 1. A. Spinal cord of an SWR/J mouse sensitized with PLP peptide 105-115. The animal developed clinical EAE on day 42, rear limb paralysis by day 44, and was killed on day 49. Perivascular and diffuse mononuclear infiltrates are present. Luxol fast blue-hematoxylin and eosin (LHE), x 200. B. Brain stem of an SWR/J mouse sensitized with PLP peptide 106116 and killed on day 49. The animal showed no clinical signs of EAE. Perivascular mononuclear infiltrates with pallor indicative of demyelination are present. LHE, x 200. C. Spinal cord of an SJL/J mouse sensitized with PLP peptide 141-149. The animal developed clinical EAE on day 10 and was moribund and killed on day 12. Subpial infiltration by both mononuclear cells and neutrophils is present. LHE, x200. (Publisher's magnification is 0.76.)
72 rats (Jones and Vandenbark, 1991) are all coded at the ends of exons (Tuohy et al., 1989b) (Fig. 2). Furthermore, many encephalitogens of MBP are also coded at exon-intron junctions including determinants for the guinea pig (Eylar and Hashim, 1968; Eylar et al., 1970), rabbit (Eylar et al., 1971), monkey (Karkhanis et al., 1975), P L / J (H-2") mouse (Zamvil et al., 1986), S J L / J mouse (Kono et al., 1988; Sakai et al., 1988a,b), and S W R / J mouse (Sakai et al., 1988b; Cross et al., 1991) as well as suspected human autoimmune targets (Baxevanis et al., 1989; Richert et al.,
G LLE
C C A R C L V G A P F A S
1989). The relationship between encephalitogenicity and exon-intron junctions may be more apparent than real since some encephalitogens are clearly not coded at junctional sites, most notably the Lewis rat MBP determinant (Hashim et al., 1978), an I-E u Class II restricted determinant of MBP in P L / J mice (Zamvil et al., 1988), and a PLP determinant in the rabbit (Linington et al., 1990). On the other hand, splice junction peptides may actually represent priority sites as autoimmune targets perhaps because of some underlying structural a n d / o r functional role that
LVATG
L C F F G V A L F C G C G
I ~:'
~2
H E A L T G T E K L I E T Y F S
r--"
K N Y Q D Y E Y L I N V I H A F Q Y V PL/J
I
(H-2 u) m o u s e
~O
loo
0
I YGTAS
TT
F F F L Y G A L L L A E G F Y T T G A V R Q I
I C, G K G L S A T SWR/J
T G G Q K G R G S
FGDYK
R G Q H Q A H S
LERVCH
(H-2 q) m o u s e
"~°
~
C L G K W L G H P D K
"~° VG
I T Y A L T V V W L L V F A C S A V P V Y
S J L / J (H-2 s) m o u s e Lewis Rat (139-151) I Y F N T W T T C Q S
IAFPS
KTSAS
IGS
L C A D A R M Y G V L
I PWNA
F PG
~oS
KV
CG
C3H/He
GAAAT
LVS
I,
N LLS
I C KT
AE
F~Q M T
FH
a.~o L F I AA
FV
(H-2 k) m o u s e
T F M I A A T Y N F A V L K L M G R G T K F
Fig. 2. Amino acid sequence of murine PLP (Milner et al., 1985;Macklin et al., 1987; Nave et al., 1987).The underlined sequences indicate the currently identified murine encephalitogenicdeterminants. Arrows indicate sites of exon-intronjunctions.
73
exon terminal peptides serve, such as their tendency to be exposed sites or hinge locations within proteins (Craik et al., 1982). The present study reinforces the strain specificities of S W R / J and S J L / J PLP determinants (Tables 1 and 2), and ongoing studies indicate that both encephalitogens are MHC Class II restricted (data not shown). Further studies on the characterization of the immune response to PLP encephalitogens are in progress.
Acknowledgement The authors express their gratitude to Mr. Timothy Odykirk for his expert technical assistance. This work was supported in part by National Institutes of Health Grants NS 16945 and HD 04147 (M.B.L.), NS 26773 (R.A.S.), NS 29095 (FIRST Award to V.K.T.), National Multiple Sclerosis Society Grant JF-2045-A-1 (Harry Weaver Neuroscience Scholarship to V.K.T) and National Science Foundation Grant DMB 8503940 (R.A.L.).
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74 central nervous system myelin. Proc. Natl. Acad. Sci. USA 84, 5665-5669. Reddehase, M., Rothbard, J.B. and Ulrich, H.K. (1989) A pentapeptide as minimal antigenic determinant for MHC class I-restricted T lymphocytes. Nature 337, 651-653. Richert, J.R., Robinson, E.D., Deibler, G.E., Martenson, R.E., Dragovic, L.J. and Kies, M.W. (1989) Human cytotoxic T-cell recognition of a synthetic peptide of myelin basic protein. Ann. Neurol. 26, 342-346. Sakai, K., Zamvil, S.S., Mitchell, D.J., Lim, M., Rothbard, J.B. and Steinman, L. (1988a) Characterization of a major encephalitogenic T cell epitope in SJL/J mice with synthetic oligopeptides of myelin basic protein. J. Neuroimmunol. 19, 21-32. Sakai, K., Sinha, A.A., Mitchell, D.J., Zamvil, S.S., Rothbard, J.B., McDevitt, H.O. and Steinman, L. (1988b) Involvement of distinct murine T-cell receptors in the autoimmune encephalitogenic response to nested epitopes of myelin basic protein. Proc. Natl. Acad. Sci. USA 85, 8608-8612. Shapira, R., Chou, F.C., McKneally, S., Urban, E. and Kibler, R.F. (1971) Biological activity and synthesis of an encephalitogenic determinant. Science 173, 736-738. Sobel, R.A., Blanchette, B.W., Bhan, A.K. and Colvin, R.B. (1984) The immunopathology of experimental allergic encephalomyelitis. I. Quantitative analysis of inflammatory cells in situ. J. lmmunol. 132, 2393-2401. Sobel, R.A., Tuohy, V.K., Lu, Z., Laursen, R.A. and Lees, M.B. (1990) Acute experimental allergic encephalomyelitis in SJL/J mice induced by a synthetic peptide of myelin proteolipid protein. J. Neuropathol. Exp. Neurol. 49, 468479. Tuohy, V.K., Sobel, R.A. and Lees, M.B. (1988a) Myelin proteolipid protein-induced experimental allergic encephalomyelitis. Variations of disease expression in different strains of mice. J. Immunol. 140, 1868-1873. Tuohy, V.K., Lu, Z., Sobel, R.A., Laursen, R.A. and Lees, M.B. (1988b) A synthetic peptide from myelin proteolipid protein induces experimental allergic encephalomyelitis. J. Immunol. 141, 1126-1130.
Tuohy, V.K., Lu, Z., Sobel, R.A., Laursen, R.A. and Lees, M.B. (1989a) Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice. J. Immunol. 142, 1523-1527. Tuohy, V.K., Lu, Z., Sobel, R.A., Laursen, R.A. and Lees, M.B. (1989b) Target T cell epitopes for autoimmune encephalomyelitis in two susceptible mouse strains are coded at exon terminal regions of myelin proteolipid protein (PLP). Ann. Neurol. 26, 297. Van der Veen, R.C., Trotter, J.L., Hickey, W.F. and Kapp, J.A. (1990) The development and characterization of encephalitogenic cloned T cells specific for myelin proteolipid protein. J. Neuroimmunol. 26, 139-145. Whitham, R.H., Bourdette, D.N., Hashim, G.A., Herndon, R.M., llg, R.C., Vandenbark, A.A. and Offner, H. (1991a) Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis. J. Immunol. 146, 101-107. Whitham, R.H., Jones, R.E., Hashim, G.A., Hoy, C.M., Wang, R.-Y., Vandenbark, A.A. and Offner, H. (1991b) Location of a new encephalitogenic epitope (residues 43-64) in proteolipid protein that induces relapsing experimental autoimmune encephalomyelitis in P L / J and (SJL×PL)F I mice. J. Immunol. 147, 3803-3808. Wraith, D.C., Smilek, D.E., Mitchell, D.J., Steinman, L. and McDevitt, H.O. (1989) Antigen recognition in autoimmune encephalomyelitis and the potential for peptide-mediated immunotherapy. Cell 59, 247-255. Zamvil, S.S., Mitchell, D.J., Moore, A.C., Kitamura, K., Steinman, L. and Rothbard, J.B. (1986) T-cell epitope of the autoantigen myelin basic protein that induces encephalomyelitis. Nature 324, 258-260. Zamvil, S.S., Mitchell, D.J., Powell, M.S., Sakai, K., Rothbard, J.B. and Steinman, L. (1988) Multiple discrete encephalitogenic epitopes of the autoantigen myelin basic protein include a determinant for I-E class II-restricted T cells. J. Exp. Med. 168, 1181-1186.
67
JNI 02198
Myelin proteolipid protein: minimum sequence requirements for active induction of autoimmune encephalomyelitis in SWR/J and SJL/J mice * Vincent K. Tuohy
a,b,d, R a y m o n d A.
Sobel c,e Zhijian Lu and Marjorie B. Lees a,b,d
f, Richard
A. Laursen f
Department of Biochemistry, E.K. Shriver Center, Waltham, MA, USA, ~ Department of Neurology and c Department of Pathology, Harvard Medical School, Boston, MA, USA, a Neurology Service and e Immunopathology and Neuropathology Units, Massachusetts General Hospital, Boston, MA, USA, and f Department of Chemistry, Boston UniL,ersity, Boston, MA, USA
(Received 22 November 1991) (Revised, received20 February 1992) (Accepted 20 February 1992)
Key words." Myelin proteolipid protein; Experimental autoimmune encephalomyelitis; T cell epitope; Encephalitogenic; Demyelination; Multiple sclerosis
Summary Proteolipid protein (PLP) is the major protein constituent of mammalian central nervous system myelin. We have previously identified two different PLP encephalitogenic T cell epitopes in two mouse strains. Murine PLP peptides 103-116 YKTTICGKGLSATV and 139-151 HCLGKWLGHPDKF are encephalitogenic determinants in S W R / J (H-2 q) and SJL/J (H-2 s) mice, respectively. The purpose of the present study was to determine the minimum sequence requirements for each of these PLP encephalitogens. In S W R / J mice, at least two distinct overlapping peptides can induce experimental autoimmune encephalomyelitis (EAE). The eleven residue sequences PLP 105-115 TTICGKGLSAT and PLP 106-116 TICGKGLSATV are encephalitogenic in S W R / J mice, but PLP 106-115 TICGKGLSAT, the decapeptide indigenous to both sequences, is non-encephalitogenic. In contrast, the shortest PLP sequence capable of inducing EAE in SJL/J mice is the nonapeptide 141-149 LGKWLGHPD. These data indicate that encephalitogenic determinants of PLP are short contiguous peptide sequences similar in length and diversity to those of MBP.
Introduction Correspondence to: V.K. Tuohy. Present address: The Cleveland Clinic Foundation, Research Institute, Immunology, NN1, 9500 Euclid Avenue, Cleveland, OH 44195, USA. * This work was presented in abstract form at the Second Annual Meeting of the Society for Experimental Neuropathology, on September 23, 1989 in New Orleans, LA.
Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease model that shares many clinical and histopathological similarities with multiple sclerosis (re-
68 viewed in Alvord et al., 1984). EAE can be induced by immunization of animals with either whole CNS tissue, myelin, or myelin components such as MBP or PLP. Synthetic peptides have been used to identify encephalitogenic MBP determinants in many species including guinea pig (Eylar and Hashim, 1968; Eylar et al., 1970), rabbit (Shapira et al., 1971), monkey (Karkhanis et aI., 1975), rat (Hashim et al., 1978), and mouse (Zamvil et al., 1986; Kono et al., 1988; Sakai et al., 1988a, b). Encephalitogenic peptides of MBP have consistently been described as short, contiguous major histocompatibility complex (MHC) restricted, immunodominant T cell epitopes. We have identified two PLP encephalitogenic T cell epitopes. S W R / J (H-2 q) mice develop EAE after immunization with murine PLP peptide 103-116 YKTTICGKGLSATV (Tuohy et al., 1988b), whereas S J L / J (H-2 ~) mice develop disease when immunized with PLP peptide 139-151 HCLGKW L G H P D K F (Tuohy et al., 1989a; Sobel et al., 1990). Recent studies indicate that PLP encephalitogens may play an immunodominant role in both the induction (Whitham et al., 1991a) and prevention (Kennedy et al., 1990) of whole spinal cord-induced EAE. Therefore, basic information about immune recognition of PLP may lead to a better understanding of the role PLP plays in the pathogenesis of human multiple sclerosis. In the present study, we immunized S W R / J and S J L / J mice with a series of overlapping and truncated peptides within the appropriate determinant. In this way we were able to identify the minimum PLP sequence required for inducing EAE in each mouse strain. The results show that the minimum encephalitogenic sequence in the S W R / J mouse can be either PLP 105-115 TTICGKGLSAT or 106-116 TICGKGLSATV, whereas the minimum sequence in the S J L / J mouse is PLP 141-149 LGKWLGHPD.
Materials and methods
Mice Female S J L / J (H-2 S) and S W R / J (H-2 q) mice were purchased from The Jackson Laboratory, Bar Harbor, ME. They were obtained at 5-8
weeks of age and were immunized between 10 and 14 weeks.
Peptide synthesis All peptides were prepared according to the sequence of murine PLP (Milner et al., 1985; Macklin et al., 1987; Nave et al., 1987). Peptides were synthesized manually as described earlier (Tuohy et al., 1988b, 1989a) by the simultaneous multiple peptide synthesis ('teabag') method (Houghten, 1985; Houghten et al., 1986). In this procedure peptides were synthesized on a pmethylbenzhydrylamine resin which yielded peptides with C-terminal amides. After cleavage of the peptide from the resin, HPLC analysis of the crude product showed greater than 80-85% of the material eluting as a single peak, the identity of which was confirmed by amino acid analysis. Immunization procedure Each mouse was immunized s.c. on day 0 with 100 nmol of peptide and 200 Izg Mycobacterium tuberculosis H37RA in 200 /~1 of an emulsion of equal volumes of water and IFA. Each mouse was also injected i.v. on day 0 and day 3 with Bordetella pertussis bacilli (pertussis vaccine, lot no. WF262, Massachusetts Public Health Biologics Laboratories, Boston, MA). The amount injected was chosen by determining the maximum non-lethal dose. S J L / J mice received 0.75 x 10 l° bacilli each injection, whereas S W R / J mice were injected with 1.0 x 101° each time. Clinical and histologic evaluation All animals were assessed clinically as previously described (Tuohy et al., 1988a) according to the following criteria: 0, no disease; 1, decreased tail tone or slightly clumsy gait; 2, tail atony a n d / o r moderately clumsy gait a n d / o r poor righting ability; 3, limb weakness; 4, limb paralysis; 5, moribund state. Animals were killed within 1 week after showing clinical signs of disease. Mice that did not develop clinical signs were killed at 7 weeks post-immunization. Brains and spinal cords were fixed in 10% 'phosphatebuffered formalin and paraffin-embedded sections were stained with Luxol fast blue-hematoxylin and eosin for light microscopy. Histologic disease was quantitated by counting the inflam-
69
both PLP encephalitogens. However, one mouse did have very mild histologic disease. S W R / J mice never developed clinical or histologic E A E after immunization with PLP 107-115, 108-115, or 109-115. Peptide 139-151, the PLP encephalitogenic determinant for S J L / J mice, did not induce either clinical or histologic EAE in any S W R / J mice. Thus, the two overlapping 11 residue PLP peptides 105-115 T r I C G K G L S A T and 106-116 T I C G K G L S A T V are the shortest sequences capable of inducing EAE in S W R / J mice.
matory loci in meninges and parenchyma as previously described (Sobel et al., 1984). Results
EAE in SWR / J mice Immunization of S W R / J mice with the synthetic PLP peptide 103-116 Y K T T I C G K G L S A TV induced acute clinical and histologic EAE in six out of six mice (Table 1). In order to identify the N- and C-terminal residues of this encephalitogenic determinant, we immunized S W R / J mice with peptides containing the overlapping PLP residues 103-113, 104-114, 105-115, and 106116. S W R / J mice never developed clinical or histologic E A E after immunization with either PLP 103-113 or PLP 104-114. However, seven out of ten mice immunized with PLP 105-115 T T I C G K G L S A T developed severe clinical and histologic disease (Fig. 1A), and the remaining three mice in this group had histologic EAE. Furthermore, two out of ten SWR mice immunized with PLP 106-116 T I C G K G L S A T V developed severe clinical and histologic disease and one of the eight remaining mice in this group had histologic E A E (Fig. 1B). In two subsequent experiments, none out of seven and none out of 11 animals developed clinical E A E following immunization of SWR mice with PLP 106-115 T I C G K G L S A T , the decapeptide indigenous to
EAE in SJL / J mice Immunization of S J L / J mice with synthetic PLP peptides 139-151 H C L G K W L G H P D K F and 141-150 L G K W L G H P D K induced severe clinical and histologic EAE in ten out of ten and ten out of 13 mice, respectively (Table 2). The shortest PLP sequence capable of inducing E A E in S J L / J mice was the nonapeptide 141-149 LGKWLGHPD. In an initial experiment, only one out of ten mice injected with PLP 141-149 developed clinical EAE, but all ten mice had severe histologic disease. This experiment was repeated, and a dramatic increase occurred in the incidence of clinical disease. In the second experiment eight out of ten mice immunized to PLP 141-149 developed acute clinical disease, and all ten mice
TABLE 1 EAE IN S W R / J (H-2 q) MICE I M M U N I Z E D W I T H PLP PEPTIDES Murine PLP peptide sequence
100 nmol dose (/zg)
No. with both clinical and histologic E A E
Mean day of onset
Clinical grade a
No. of inflammatory foci b
No. with histologic EAE only c
No. of inflammatory foci
103-116 103-113 104-114 105-115 106-116 106-115
144 117 108 105 105 95 85 74 63 152
6/6 0/7 0/7 7/10 2/10 0/18 0/6 0/6 0/6 0/8
14 (12-16) 37 (25-48) 31 (30-32) -
5.0 0 0 3.7 4.0 0
81 (53-116)
0 0/7 0/7 3/3 1/8 1/7
0 0 0 16 (4-32) 24 6
107-115
108-115 109-115 139-151
57 (19-166) 97 (65-128) -
-
0
-
0/6
0
-
0 0 0
-
0/6 0/6 0/8
0 0 0
a Values represent mean clinical grade scored for animals showing clinical disease as described in Materials and methods. b Mean number of total (meningeal and parenchymal) inflammatory loci in paraffin sections of CNS. Values in parentheses represent ranges. c These animals never showed any clinical signs and were killed at 7 weeks.
70 had histologic E A E (Fig. 1C). PLP peptides 141148 and 142-150 were not encephalitogenic, thus indicating that Leu-141 and Asp-149 represent the N- and C-terminal residues, respectively, of this PLP encephalitogenic determinant for S J L / J mice. Peptide 103-116, the PLP encephalitogenic determinant for S W R / J mice, did not induce either clinical or histologic E A E in any S J L / J mice.
Discussion In the present study we have determined the minimum sequence requirements of two PLP determinants for active induction of EAE. In S W R / J mice, the shortest encephalitogenic PLP sequences are two overlapping l l - a m i n o acid peptides, PLP 105-115 TT1CGKGLSAT and PLP 106-116 T I C G K G L S A T V , whereas in S J L / J mice the nonapeptide PLP 141-149 LGKWLG H P D is the shortest encephalitogenic PLP peptide. The inability of PLP 106-115 to induce E A E in S W R / J mice indicates the importance of an intact amino acid at either the N-terminal 105 or the C-terminal 116 position. Furthermore, the difference in disease incidence (Table 1) induced by PLP 105-115 (10/10) compared to PLP 106116 (3/10) suggests that these overlapping encephalitogens may represent distinctly different
S W R / J T cell epitopes. The presence of multiple overlapping T cell epitopes within a PLP determinant for S W R / J mice would correspond with their existence in MBP (Kono et al., 1988; Sakai et al., 1988b) and PLP (Kuchroo et al., 1991) determinants for S J L / J mice. Unlike MBP, PLP contains many cysteine residues whose labile sulfhydryl groups can be problematic in immunologic studies. Such difficulties can be circumvented by substitution of serine for cysteine in the synthesis of PLP determinants for S J L / J mice (Tuohy et al., 1989a; Van der Veen et al., 1990; Endoh et al., 1990). This is also true for the S W R / J determinant of PLP, since PLP 103-116 containing a serine substitution for Cys-108 induces severe E A E (data not shown). Furthermore, PLP 105-115 is encephalitogenic despite the fact that Cys-108 is a posttranslationally modified acylation site of PLP (Bizzozero et al., 1990). An acylated Cys-108 in the native protein suggests that this region of PLP may play an important role in determining tertiary structure. Whatever the structural or functional role of acylated Cys-108 may be, it does not interfere with the capacity of PLP 105115 to serve as an autoimmune target in vivo. Perhaps the relatively rapid fatty acid turnover at the acylation site(s) (Bizzozero and Good, 1991) provides a ready availability in vivo of deacylated determinant for Class II binding and immune presentation and recognition. Alternatively, an
TABLE 2 EAE IN SJL/J (H-2~) MICE IMMUNIZED WITH PLP PEPTIDES Murine PLP peptide sequence
100 nmol dose (p.g)
No. with both clinicaland histologic EAE
Mean day of onset
Clinical grade a
No. of No with inflammatory histologic foci b EAE only c
No. of inflammatory foci
139-151 141-150 142-150 141-149 (1) d 141-149 (2) 141-148 103-116
154 115 104 102 102 91 144
10/10 10/13 0/12 1/10 8/10 0/7 0/7
15 (9-23) 31 (24-49)
3.8 2.7 0 2.0 4.0 0 0
83 (26-'140) 95 (45-150) 31 88 (41-170) -
58 (12-107) 0 60 (34-108) 57 (14--100) 0 0
28 13 (10--18)
0 3/3 0/12 8/9 2/2 0/7 0/7
Values represent mean clinical grade scored for animals showing clinical disease as described in Materials and methods. b Mean number of total (meningeal and parenchymal) inflammatory foci in paraffin sections of CNS. Values in parentheses represent ranges. c These animals never showed any clinical signs and were killed at 7 weeks. a Because of disparity between clinical and histological scores, this experiment was repeated.
71 acylated cysteine may not interfere with target recognition and i m m u n e activation. In contrast to the P L P d e t e r m i n a n t for the S W R / J mouse, the M B P d e t e r m i n a n t for the P L / J mouse actually
requires modification for encephalitogenicity. T h e i m m u n o d o m i n a n t M B P 1 - 9 determinant is encephalitogenic in P L / J mice only when the A l a - I residue of the synthetic peptide is N-acetylated as is native M B P in vivo (Zamvil et al., 1986). Thus, protein modification can actually confer encephalitogenicity to a determinant p e r h a p s by altering its c o n f o r m a t i o n or by protecting it from proteolysis. It appears from the current data that longer peptides are m o r e encephalitogenic than their truncated counterparts (Tables 1 and 2). Increasing the 'tail region' amino acid side chains may serve to better stabilize the 'antigenic core region' of encephalitogens within the trimolecular complex and thus e n h a n c e immunogenicity (Reddehase et al., 1989; Wraith et al., 1989). However, increasing the length of encephalitogenic peptides can also decrease their encephalitogenicity. Both P L P peptides 137-151 and 139-154 are not as encephalitogenic as the shorter P L P 139-151 (Tuohy et al., 1989a). Perhaps the increased hydrophobicity of these longer peptides contributes to their poor encephalitogenicity. Alternatively, the longer peptides may contain flanking epitopes involved in immunologic downregulation. T h e latter view is supported by the e n h a n c e d tolerogenicity of P L P 139-154 compared to its truncated peptide counterparts (Kennedy et al., 1990). It is of interest to note that the P L P determinants defined in the present study for S J L / J and S W R / J mice as well as the P L P determinants for C 3 H / H e (H-2 k) mice ( E n d o h et al., 1990), P L / J (H-2 u) mice ( W h i t h a m et al., 1991b), and Lewis
Fig. 1. A. Spinal cord of an SWR/J mouse sensitized with PLP peptide 105-115. The animal developed clinical EAE on day 42, rear limb paralysis by day 44, and was killed on day 49. Perivascular and diffuse mononuclear infiltrates are present. Luxol fast blue-hematoxylin and eosin (LHE), x 200. B. Brain stem of an SWR/J mouse sensitized with PLP peptide 106116 and killed on day 49. The animal showed no clinical signs of EAE. Perivascular mononuclear infiltrates with pallor indicative of demyelination are present. LHE, x 200. C. Spinal cord of an SJL/J mouse sensitized with PLP peptide 141-149. The animal developed clinical EAE on day 10 and was moribund and killed on day 12. Subpial infiltration by both mononuclear cells and neutrophils is present. LHE, x200. (Publisher's magnification is 0.76.)
72 rats (Jones and Vandenbark, 1991) are all coded at the ends of exons (Tuohy et al., 1989b) (Fig. 2). Furthermore, many encephalitogens of MBP are also coded at exon-intron junctions including determinants for the guinea pig (Eylar and Hashim, 1968; Eylar et al., 1970), rabbit (Eylar et al., 1971), monkey (Karkhanis et al., 1975), P L / J (H-2") mouse (Zamvil et al., 1986), S J L / J mouse (Kono et al., 1988; Sakai et al., 1988a,b), and S W R / J mouse (Sakai et al., 1988b; Cross et al., 1991) as well as suspected human autoimmune targets (Baxevanis et al., 1989; Richert et al.,
G LLE
C C A R C L V G A P F A S
1989). The relationship between encephalitogenicity and exon-intron junctions may be more apparent than real since some encephalitogens are clearly not coded at junctional sites, most notably the Lewis rat MBP determinant (Hashim et al., 1978), an I-E u Class II restricted determinant of MBP in P L / J mice (Zamvil et al., 1988), and a PLP determinant in the rabbit (Linington et al., 1990). On the other hand, splice junction peptides may actually represent priority sites as autoimmune targets perhaps because of some underlying structural a n d / o r functional role that
LVATG
L C F F G V A L F C G C G
I ~:'
~2
H E A L T G T E K L I E T Y F S
r--"
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(H-2 u) m o u s e
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loo
0
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TT
F F F L Y G A L L L A E G F Y T T G A V R Q I
I C, G K G L S A T SWR/J
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LERVCH
(H-2 q) m o u s e
"~°
~
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"~° VG
I T Y A L T V V W L L V F A C S A V P V Y
S J L / J (H-2 s) m o u s e Lewis Rat (139-151) I Y F N T W T T C Q S
IAFPS
KTSAS
IGS
L C A D A R M Y G V L
I PWNA
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~oS
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CG
C3H/He
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F~Q M T
FH
a.~o L F I AA
FV
(H-2 k) m o u s e
T F M I A A T Y N F A V L K L M G R G T K F
Fig. 2. Amino acid sequence of murine PLP (Milner et al., 1985;Macklin et al., 1987; Nave et al., 1987).The underlined sequences indicate the currently identified murine encephalitogenicdeterminants. Arrows indicate sites of exon-intronjunctions.
73
exon terminal peptides serve, such as their tendency to be exposed sites or hinge locations within proteins (Craik et al., 1982). The present study reinforces the strain specificities of S W R / J and S J L / J PLP determinants (Tables 1 and 2), and ongoing studies indicate that both encephalitogens are MHC Class II restricted (data not shown). Further studies on the characterization of the immune response to PLP encephalitogens are in progress.
Acknowledgement The authors express their gratitude to Mr. Timothy Odykirk for his expert technical assistance. This work was supported in part by National Institutes of Health Grants NS 16945 and HD 04147 (M.B.L.), NS 26773 (R.A.S.), NS 29095 (FIRST Award to V.K.T.), National Multiple Sclerosis Society Grant JF-2045-A-1 (Harry Weaver Neuroscience Scholarship to V.K.T) and National Science Foundation Grant DMB 8503940 (R.A.L.).
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