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T cell recognition of myelin proteolipid protein and myelin proteolipid protein peptides in the peripheral blood of multiple sclerosis and control subjects
Journal of Neuroimmunology 84 Ž1998. 172–178
T cell recognition of myelin proteolipid protein and myelin proteolipid protein peptides in the peripheral blood of multiple sclerosis and control subjects John L. Trotter a,) , Clara M. Pelfrey b, Amy L. Trotter a , Jacqueline A. Selvidge a , Kelly C. Gushleff a , T. Mohanakumar b, Henry F. McFarland c a
c
Department of Neurology and Neurological Surgery, Washington UniÕersity School of Medicine, St. Louis, MO 63110-1093, USA b Department of Pathology, Washington UniÕersity School of Medicine, St. Louis, MO 63110-1093, USA Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA Received 3 September 1997; received in revised form 19 November 1997; accepted 24 November 1997
Abstract Myelin proteolipid protein ŽPLP. is a prime candidate autoantigen for multiple sclerosis. In order to define potential immunodominant epitopes, T cell lines ŽTCL. from the peripheral blood of HLA-DR 15Ž2. MS patients were established which responded to the intact molecule of PLP. These TCL were then tested in individual proliferation assays with a variety of PLP peptides spanning most of the PLP molecule. Multiple peptides were recognized by TCL from the MS population, with more than one peptide often recognized by lines from the same individual. Three immunodominant peptides were identified which were recognized by the majority of MS patients. Estimated frequency analyses were then performed on the peripheral blood of HLA-DR15Ž2.-positive MS and control subjects using TCL initiated by the three immunodominant peptides, 40–60, 95–117, and 185–206. TCL from HLA-DR15 MS subjects recognized peptide 95–117 significantly more often than TCL from control subjects. q 1998 Elsevier Science B.V. Keywords: Multiple sclerosis; T cells; Myelin proteolipid protein
1. Introduction A variety of clinical and laboratory-derived clues indicate that immunological mechanisms are involved in the etiology and pathogenesis of multiple sclerosis ŽMS. ŽMcFarlin and McFarland, 1982; Martin et al., 1992.. For instance, the most common animal model for MS, relapsing experimental autoimmune Žallergic. encephalomyelitis ŽEAE., resembles MS both clinically and pathologically. EAE can be induced by active immunization with, or transfer of T cells specific for any of at least three proteins specific to myelin, or peptides derived from each of these peptides, including myelin basic protein ŽMBP. ŽPettinelli and McFarlin, 1981., myelin proteolipid protein ŽPLP. )
Corresponding author. Department of Neurology and Neurological Surgery, Washington University School of Medicine, Box 8111, 660 S. Euclid, St. Louis, MO, 63110, USA. Tel.: q1 312 3623293; fax: q1 312 7471345. 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 6 0 - 9
ŽYamamura et al., 1986; Trotter et al., 1987; Tuohy et al., 1988; van der Veen et al., 1989, 1990; Whitham et al., 1991; Greer et al., 1992; van der Veen et al., 1992; Amor et al., 1993; ., and myelin–oligodendrocyte glycoprotein ŽMOG. ŽJohns et al., 1995; Devaux et al., 1997.. The human cellular immune response to MBP has been extensively studied in efforts to determine whether it is an autoantigen in humans ŽLisak et al., 1981; Richert et al., 1989; Allegretta et al., 1990; Ota et al., 1990; Martin et al., 1990, 1992; Chou et al., 1992; Zhang et al., 1994., but studies to date have not definitely confirmed MBP to be an autoantigen in MS. Evidence supports the relevance of another candidate autoantigen, PLP. PLP is encephalitogenic in animals and specific to myelin. T lymphocytes recognizing PLP or PLP peptides have been found in the peripheral blood and cerebrospinal fluid of MS patients ŽOta et al., 1990; Sun et al., 1991; Trotter et al., 1991; Chou et al., 1992;Pelfrey et al., 1993, 1994; Zhang et al., 1994; Markovic-Plese et al.,
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1995; .. HPRT mutant clones recognizing PLP peptides have been identified in the peripheral blood of MS patients ŽTrotter et al., 1997.. We have previously identified two PLP peptide sequences as human T cell epitopes that induce T cell-mediated cytotoxicity when presented by activated B cells ŽPelfrey et al., 1993, 1994.. The present studies extend this work by studying human T cell recognition of peptides spanning most of the PLP molecule. In this study, immunodominant ŽSercarz et al., 1993. PLP peptide sequences, defined as such by their recognition by the majority of T cell lines ŽTCL. raised against the whole PLP molecule, were identified. Estimated frequency analyses performed on TCL initiated with these peptides demonstrated higher frequencies among MS when compared to control subjects.
troscopy. The PLP peptides used in the study had the following amino acid sequences ŽDiehl et al., 1986.: 40–60, TGTEKLIETYFSKNYQDYEYL; 44–76, KLIETYFSKNYQDYEYLINVIHAFQYVIYGTAS; 89–106, GFYTTGAYRQIFGDYLTT; 95–117, AVRQIFGDYKTTICGKGLSATVT; 117–150, TGGQKGRGSRGQHQAHSLERVCHCLGKWLGHPDK; 139–151, HCLGKWLGHPDKF; 185–206, SIAFPSKTSASIGSLCADARMY; 207–231, GVLPWNAFPGKVCGSNLLSICKTAE; and 254–276, LTFMIAATYNFAVLKLMGRGTKF It should be noted that peptides 1–47 and 232–255 were not studied because they could not be extracted from their solid supports after synthesis.
2. Materials and methods
2.3. Establishment of 2 ml cultures
2.1. Human subjects
Mononuclear cells ŽMNC. were isolated from heparinized whole blood on Histopaquexe ŽSigma Chemical, St. Louis, MO, USA. gradients and washed. Two ml cultures were established with 5 = 10 6 MNC in Hepesbuffered RPMI 1640 with antibiotics and 5% human male AB serum ŽCM.. Cultures were maintained weekly with fresh PLP-pulsed, irradiated adherent MNC; 100 m grml of PLP in 1 ml RPMI was added to irradiated Ž4500R. adherent syngeneic MNC, incubated 2 h at 378C, and washed prior to the addition to the TCL. Proliferation assays were performed using each of the selected peptides on TCL in which the cell concentration was at least 1 = 10 6rwell after a culture duration of 6 weeks. Five lines were randomly sampled for phenotypic analysis; all were ) 90% CD4 q .
The MS patients involved in the present studies had clinically definite disease with a relapsing–remitting course, with a disease duration of 2–10 yr, and had suffered at least one exacerbation within the previous 2 yr. All patients were in remission at the time of phlebotomy. The patients ranged in age from 33 to 55 yr; there were 8 females and 4 males. The Kurtzke disability status scale levels of disability ranged from 3.5 to 6.0. Patients had not received corticosteroids for at least 3 months, and none had received other immunomodulatory or immunosuppressive agents, including b-interferons. Normal control subjects were volunteers recruited among employees of Washington University School of Medicine in St. Louis, MO and spouses of MS patient volunteers. All subjects were HLA-typed by the Barnes Hospital Department of Laboratory Medicine or Smith-Kline Laboratories. Patients and control subjects with the HLA-DR15Ž2. type, which is the most common DR type among North American Caucasians with MS, were used exclusively in these experiments. The blood donations for these studies were approved by the Human Studies Committee ŽIRB. of the Washington University School of Medicine. 2.2. Reagents Myelin PLP was prepared by a method modified from that described by Folch et al. Ž1957. as modified by Hampson and Poduslo Ž1986.. PLP was shown to be free of MBP ŽTrotter et al., 1981.. Only preparations of PLP which were active in proliferation assays of TCL known to recognize the intact antigen were used in these studies. Synthetic peptides of PLP were prepared by Monsanto, or the Washington University Department of Biochemistry using FastMOC chemistry. The peptides were purified by HPLC, and the sequences confirmed by mass spec-
2.4. Proliferation assays on PLP stimulated TCL Proliferation assays were performed in quadruplicate in flat-bottom microtiter tissue culture plates. Each well contained 1 = 10 4 cells from each TCL and 1 = 10 5 irradiated MNC as feeders in the presence of 20 m grml PLP peptides in CM. The doses of antigen selected had previously been shown to be optimal by proliferation assays, and represented plateau values when peptide-initiated TCL had been tested in concentrations ranging from 5–25 m grml. Twenty m grml represented a plateau response for all peptides tested. Cells were incubated for 72 h before being pulsed with 2 m Cirwell Žmethyl- 3 H . thymidine ŽAmersham, Arlington Heights, IL, USA. for 18 h. The cells were harvested and the incorporated radioactivity counted using a Betaplatee system. 2.5. Estimated frequency analyses Following the identification of immunodominant peptides of PLP, estimated frequency analysis was performed
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Table 1 Recognition of PLP peptides by PLP-initiated T cell lines derived from MS patients Patients
MS a1
PLP lines 6r17 a Synthetic Peptidesb 40–60 5 44–76 2 89–106 0 95–117 4 117–150 2 139–151 2 185–206 5 207–231 0 254–276 0
MS a2
MS a3
MS a4
MS a5
MS a6
MS a7
MS a8
MS a9
MS a10
MS a11
MS a12
Total
6r17
6r18
2r17
2r17
3r17
5r20
4r17
3r17
8r17
2r17
3r17
50r208
5 2 1 4 3 2 5 0 0
4 1 0 4 4 0 4 0 0
2 0 0 1 2 1 2 0 0
1 0 0 0 1 0 2 0 0
2 0 0 0 1 0 1 0 0
3 0 1 2 2 0 3 0 0
3 0 0 3 3 0 3 0 0
3 3 1 0 1 0 2 0 0
7 0 2 5 3 0 6 0 0
0 0 0 2 1 2 1 0 0
0 0 3 0 0 3 1 0 0
35r50 8r50 8r50 25r50 23r50 10r50 35r50 0r50 0r50
a
Positive linesrtotal lines after 6 weeks. T cell lines initiated with the whole PLP molecule, and which recognized the whole molecule after 6 weeks in culture were tested for recognition of PLP synthetic peptides. b Synthetic peptides 1–47 and 232–255 could not be extracted from their solid supports.
on the peripheral blood of MS and control subjects in round-bottom microtiter tissue culture plates, using a method modified from that described by Ota et al., 1990. A total of 2 = 10 5 MNC were incubated with each of the selected synthetic peptides Ž20 m grml. at 378C in CM. Four to 15 96-well plates were used to test each antigen. Twenty units recombinant human interleukin 2 ŽIL-2. were added to the cultures every 4 days beginning at day 8. The last dose of IL-2 was administered on day 16. Proliferation assays were performed on day 24 using a modification of the ‘split-well’ technique described by Ota et al. This technique involves splitting the culture wells into three new wells, with the addition of fresh irradiated feeders. Antigen reactivity is determined using two of the wells, with the third saved for perpetuation.
3. Results Our initial studies revealed that high doses of PLP Žon the order of 50–100 m grml. often suppressed the proliferation of TCL, especially when the TCL had been cultured for several weeks. This finding, as well as the inherent difficulty dealing with the insoluble antigen, PLP, led us to adopt a method where excess antigen is removed from
pulsed APC, since high concentrations of antigen inhibit proliferation. Two ml TCL which recognized intact PLP were derived from the peripheral blood MNC of 12 DR15Ž2. MS patients ŽTable 1.. Seventeen to 20 TCL per patient were established Žsee Section 2. for a total of 208 lines. Of these, 50 TCL survived at least 6 weeks, at which time they were analyzed for recognition of each of the selected synthetic peptides. A stimulation index of 3.0 was considered positive, if the Dcpm was G 500. These criteria resulted in cpm which were greater than 3 standard deviations ŽS.D.. above the mean of the unstimulated cultures in all cases. The antigenic repertoire of the lines was heterogeneous, i.e., many lines recognized more than one peptide ŽTable 1.. Peptides 207–231 and 254–276 were recognized by none of the lines. Peptides 40–60, 95–117, and 185–206 were recognized by the majority of MS subjects and yielded high stimulation indices. Therefore, these peptides were selected to be studied by estimated frequency analyses to determine if the T cells which recognized them were more frequent in MS than control subjects. When comparing frequencies in the peripheral blood, the greatest difference between MS patients and control subjects in our series appeared to be for PLP peptide 95–117 ŽTable 2; Fig. 1.. The estimated frequencies in the
Table 2 Estimated frequency of T cell recognition of PLP peptides in MS and control subjects Subjects Ž n.
Peptide
Positive wellsrplate Žmean " S.D..
Frequency Žrange.
MS Ž9. NC Ž5. MS Ž12. NC Ž6. MS Ž9. NC Ž9.
40–60 40–60 95–117 95–117 185–206 185–206
1.0 " 1.3 0.3 " 0.2 2.5 " 2.3 0.9 " 0.8 3.2 " 3.5 1.4 " 1.8
0–1r4,512,000 0–1r38,400,000 0–1r2,553,600 1r7,680,000–1r9,600,000 1r8,524,800–1r2,035,200 0–1r3,552,000
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Fig. 1. The estimated frequencies for the three immunodominant peptides tested are shown in Figure 1 for MS patients and control subjects. The mean and standard deviations of the frequencies for the normal control subjects are demonstrated in the columns for the MS patients. The p-values resulting from the Wilcoxin Rank Sums comparisons are shown for each peptide.
peripheral blood for T cells from the MS subjects that recognized this peptide ranged from 0 to 1r2,553,600 whereas the estimated frequencies for the control subjects ranged from 1r76,800,000 to 1r9,600,000 Ž p s 0.033, Mann–Whitney Rank Sums test.. Examining the estimated frequencies in a different manner, five of 12 MS patients had frequencies 2 standard deviations above the mean of the control subjects when PLP peptide 95–117 was studied, whereas three of nine and two of nine MS patients fulfilled these criteria for peptides 40–60 and 185–206, respectively ŽFig. 1..
4. Discussion We have here confirmed and extended our previous work and that of others, that intact PLP protein is recognized by T cells from the peripheral blood of MS and control subjects. Thus, PLP remains a candidate antigen in MS, with as much potential relevance as the more frequently cited candidate, MBP. We elected to determine ‘immunodominant’ peptides for MS subjects ŽTable 1. based on the premise that such peptides might have pathogenic relevance. The present studies demonstrated that there are multiple candidate epitopes of PLP. The estimated frequencies of recognition of peptides 40–60, 95–117, and 185–206, which were im-
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munodominant in the majority of TCL initiated to intact PLP and derived from MS patients, were higher among MS patients when compared to control subjects. The question arises whether the frequencies of T cell recognition we have demonstrated for PLP peptides is sufficient to induce human disease. The following facts taken from the literature regarding EAE may be pertinent. 1. In studies on the initiation of EAE using the adoptive transfer method, Cross et al. demonstrated that a very small number of T cells is needed to initiate a lesion. After development of an inflammatory lesion, only 1%–4% of T cells within the lesion were from the original inoculate of cells ŽCross et al., 1990.. 2. Vandenbark et al. have shown that as few as 5 million cells in a TCL recognizing PLP peptide 139–151 can transfer severe relapsing–remitting EAE to naive recipient rats ŽJones et al., 1990.. 3. Vandenbark et al. Ž1992. found that the frequencies of MBP-specific T cells in the peripheral blood of Lewis rats immunized with MBP and CFA ranged between 2r1,000,000 prior to disease onset and 1r100,000 at the peak of clinical EAE. Thus, the human lymphoid system certainly contains sufficient cells recognizing each of the immunodominant peptides to cause disease. Furthermore, we have demonstrated that there is recognition of multiple peptides of PLP by T cells from MS subjects, especially in the peptide span of 95–150, so that multiple interactions with PLP in the myelin membrane are possible. Of note in our studies is the fact that individual patients recognize multiple peptides when multiple TCL’s are initiated with PLP or individual peptides. These findings agree with the findings of another group of investigators who identified ‘immunodominant’ PLP peptides which overlap with two we have identified. However, in that study ŽMarkovic-Plese et al., 1995., PLP peptide 95–117 was not studied, and frequency analyses comparing the recognition of PLP peptides among MS and control subjects were not performed. A more recent study found that PLP peptide 95–116 was recognized significantly more frequently by Japanese DR15 MS patients compared to DR15 controls subjects ŽOhashi et al., 1995.. Results have varied in the literature when the percentage of MS subjects vs. controls recognizing the whole molecule PLP is considered ŽChou et al., 1992; Sun et al., 1991; Trotter et al., 1991, 1997; Zhang et al., 1994.. It is clear from our work and that of others, that recognition of PLP is not restricted to MS subjects. If a single epitope of PLP were responsible for disease induction in a given individual, one might still observe multiple recognition sites in that patient through the phenomenon of antigen determinant spreading. We and others have previously demonstrated in inbred mice with EAE induced by a single peptide or protein antigen that antigen determinant spreading occurs and increases the antigenic repertoire over time ŽCross et al., 1993; Lehmann et al.,
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1992; McCarron et al., 1990; McRae et al., 1995; Miller et al., 1995; Perry et al., 1991; Perry and Barzaga, 1987; Yu et al., 1996.. This may explain the recognition of multiple peptides by PLP TCL. The situation is further complicated by the possible relevance of cryptic epitopes to the disease pathogenesis since these peptides may also be pathogenic ŽLipham et al., 1991.. Although the patients in the present studies were limited to those with a relapsing–relapsing course, and controlled for HLA type, they were not controlled for disease duration, MRI plaque burden, or remote disease history, which may affect antigen determinant spreading.
Acknowledgements
5. Conclusion
References
In the studies we present here, recognition of the PLP peptide 95–117 was significantly greater among MS compared to control subjects. An epitope recognized at relatively higher frequencies might be more likely to be associated with the initiation of pathology than an epitope with a lower T cell frequency. This notion remains highly speculative, since the criteria for determining relevance to the pathogenesis of disease have not been established. The methods we used in this report involved prolonged culture and repeated stimulation with antigen, suggesting the possibility of in vitro sensitization. We previously used a different approach to identify HPRT mutant clones from MS patients which recognized PLP peptides ŽTrotter et al., 1997.. Other methods, such as testing for antigen recognition after stimulating T cells bearing IL-2 receptors, have been adopted in attempts to better reflect in vivo conditions and relevance, but these too have the disadvantage of requiring cell culture ŽZhang et al., 1994.. Studies using more subjects, inclusion of a series of shorter overlapping peptides and studies of disease controls are needed to confirm our findings. Such studies are underway in our laboratory. PLP recognition by T cells from concordant vs. discordant twins with MS might further elucidate the relevance of individual epitopes, at least within families. However, the ultimate method of proving a pathogenic epitope in humans would derive from results of clinical trials with reagents directed to the individual epitopeŽs.. The present studies included peptides which span most but not all of the PLP molecule. Further studies with additional overlapping peptides may reveal more candidates for antigens relevant to MS. Exhaustive studies with shorter peptides and amino acid substitutions may be necessary to determine therapeutic agents, which could be used to treat MS using methods similar to those described in EAE ŽAcha-Orbea et al., 1988; Gaur et al., 1992; Hashim et al., 1990; Howell et al., 1989; Lamont et al., 1990; Kennedy et al., 1990; Owhashi and Heber-Katz, 1988; Sakai et al., 1989; Sharma et al., 1991; Smilek et al., 1991; Vandenbark et al., 1989; Wraith et al., 1989..
Acha-Orbea, H., Mitchell, D.J., Timmerman, L., Wraith, D.C., Tausch, G.S., Waldor, M.K., Zamvil, S.S., McDevitt, H.O., Steinman, L., 1988. Limited heterogeneity of T cell receptors from T lymphocytes mediating encephalomyelitis allows specific immune intervention. Cell 54, 263–273. Allegretta, M., Nicklas, J.A., Sriram, S., Albertini, R.J., 1990. T cells responsive to myelin basic protein in patients with multiple sclerosis. Science 247, 718–721. Amor, S., Baker, D., Groome, N., Turk, J.L., 1993. Identification of a major encephalitogenic epitope of proteolipid protein Žresidues 56–70. for the induction of experimental allergic encephalomyelitis in Biozzi ABrH and nonobese diabetic mice. J. Immunol. 150, 5666–5672. Chou, Y.K., Bourdette, D.N., Offner, H., Whitham, R., Wang, R.Y., Hashim, G.A., Vandenbark, A.A., 1992. Frequency of T cells specific for myelin basic protein and myelin proteolipid protein in blood and CSF in MS. J. Neuroimmunol. 38, 105–114. Cross, A.H., Cannella, B., Brosnan, C.F., Raine, C.S., 1990. Homing to central nervous system vasculature by antigen-specific lymphocytes: I. Localization of 14 C-labeled cells during acute, chronic, and relapsing experimental allergic encephalomyelitis. Lab. Invest. 63, 162–170. Cross, A.H., Tuohy, V.K., Raine, C.S., 1993. Development of reactivity to new myelin antigens during chronic relapsing autoimmune demyelination. Cell. Immunol. 146, 261–269. Devaux, B., Enderlin, F., Wallner, B., Smilek, D.E., 1997. Induction of EAE in mice with recombinant human MOG, and treatment of EAE with a MOG peptide. J. Neuroimmunol. 75, 169–173. Diehl, H.J., Schaich, M., Budzinski, R.M., Stoffel, W., 1986. Individual exons encode the integral membrane domains of human myelin proteolipid protein. Proc. Natl. Acad. Sci. U.S.A. 83, 9807–9811. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Gaur, A., Wiers, B., Liu, A., Rothbard, J., Fathman, C.G., 1992. Amelioration of autoimmune encephalomyelitis by myelin basic protein synthetic peptide-induced anergy. Science 258, 1491–1494. Greer, J.M., Kuchroo, V.K., Sobel, R.A., Lees, M.B., 1992. Identification and characterization of a second encephalitogenic determinant of myelin proteolipid protein Žresidues 178–191. for SJL mice. J. Immunol. 149, 783–788. Hampson, D.R., Poduslo, S.E., 1986. Purification of proteolipid protein and production of specific antiserum. J. Neuroimmunol. 11, 117–129. Hashim, G.A., Vandenbark, A., Galang, A.B., Diamanduros, T., Carvalho, E., Srinivasan, J., Jones, R., Vainiene, M., Morrison, W.J., Offner, H., 1990. Antibodies specific for Vb 8 receptor peptide suppress experimental autoimmune encephalomyelitis. J. Immunol. 144, 4621–4627. Howell, M.D., Winters, S.T., Olee, T., Powell, H.C., Carlo, D.J., Brostoff,
We thank Larry Sulze, Joan Bryan and Cheryl Damico for excellent technical assistance. Drs. Anne H. Cross, Amy Lovett-Racke, and Michael K. Racke reviewed the manuscript and provided thoughtful discussion. This work was supported in part by grant RG 2681 from the National Multiple Sclerosis Society, a grant from the Washington University-Monsanto Research Program, grant RO1 NS29851 from the National Institutes of Health, the Barnes-Jewish Hospital Foundation, and gifts from the Joanne Kirberg and the Frala Osherow Funds.
J.L. Trotter et al.r Journal of Neuroimmunology 84 (1998) 172–178 S.W., 1989. Vaccination against experimental allergic encephalomyelitis with T cell receptor peptides. Science 246, 668–670. Johns, T.G., Kerlero de Rosbo, N., Menon, K., Abo, S., Gonzales, M.F., Bernard, C.C.A., 1995. Demyelinating encephalomyelitis resembling multiple sclerosis after immunization with a peptide from myelin oligodendrocyte glycoprotein. J. Immunol. 154, 5536–5541. Jones, R.E., Bourdette, D.N., Offner, H., Vandenbark, A.A., 1990. Myelin basic protein-specific T cells induce demyelinating experimental autoimmune encephalomyelitis in Buffalo rats. J. Neuroimmunol. 30, 61–69. Kennedy, M.K., Tan, L.J., DalCanto, M.C., Miller, S.D., 1990. Inhibition of murine relapsing experimental autoimmune encephalomyelitis by immune tolerance to proteolipid protein and its encephalitogenic peptide. J. Immunol. 1441, 909–915. Lamont, A.G., Sette, A., Fujinami, R., Colon, S.M., Miles, C., Grey, H.M., 1990. Inhibition of experimental autoimmune encephalomyelitis induction in SJLrJ mice by using a peptide with high affinity for Ias molecules. J. Immunol. 145, 1687–1693. Lehmann, P.V., Forsthuber, T., Miller, A., Sercarz, E.E., 1992. Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen. Nature 358, 155–157. Lipham, W.J., Redmond, T.M., Takahashi, H., Berzofsky, J.A., Wiggert, B., Chader, G.J., Gery, I., 1991. Recognition of peptides that are immunopathogenic but cryptic. J. Immunol. 146, 3757–3762. Lisak, R.P., Zweiman, B., Whitaker, J.N., 1981. Spinal fluid basic protein immunoreactive material and spinal fluid lymphocyte reactivity to basic protein. Neurology 31, 180–182. Markovic-Plese, S., Fukaura, H., Zhang, J., al-Sabbagh, A., Southwood, S., Sette, A., Kuchroo, V.K., Hafler, D.A., 1995. T cell recognition of immunodominant and cryptic proteolipid protein epitopes in humans. J. Immunol. 155, 982–992. Martin, R., Jaraquemada, D., Flerlage, M., Richert, J., Brostoff, S., Long, E.O., McFarlin, D.E., McFarland, H.F., 1990. Fine specificity and HLA restriction of myelin basic protein specific cytotoxic T cell lines from multiple sclerosis patients and healthy individuals. J. Immunol. 145, 540–548. Martin, R., McFarland, H.F., McFarlin, D.E., 1992. Immunological aspects of demyelinating diseases. Annu. Rev. Immunol. 10, 153–187. McCarron, R.M., Fallis, R.J., McFarlin, D.E., 1990. Alterations in T cell antigen specificity and class II restriction during the course of chronic relapsing experimental allergic encephalomyelitis. J. Neuroimmunol. 29, 73–79. McFarlin, D.E., McFarland, H.F., 1982. Multiple sclerosis ŽReview.. New. Eng. J. Med. 307, 1183–1188, 1246–1251. McRae, B.L., Vanderlugt, C.L., Dal Canto, M.C., Miller, S.D., 1995. Functional evidence for epitope spreading in the relapsing pathology of experimental autoimmune encephalomyelitis. J. Exp. Med. 182, 75–85. Miller, S.D., McRae, B.L., Vanderlugt, C.L., Nikcevich, K.M., Pope, J.G., Pope, L., Karpus, W.J., 1995. Evolution of the T-cell repertoire during the course of experimental immune mediated demyelinating disease. Immunol. Rev. 144, 226–244. Ohashi, T., Yamamura, T., Inobe, J., Kondo, T., Kunishita, T., Tabira, T., 1995. Analysis of proteolipid protein ŽPLP.-specific T cells in multiple sclerosis; identification of PLP 95–116 as an HLA-DR2, w15-associated determinant. Int. Immunol. 7, 1771–1778. Ota, K., Matsui, M., Milford, E.L., Mackin, G.A., Weiner, H.L., Hafler, D.A., 1990. T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature 346, 183–187. Owhashi, M., Heber-Katz, E., 1988. Protection from experimental allergic encephalomyelitis conferred by a monoclonal antibody directed against a shared idiotype on rat T cell receptors. J. Exp. Med. 168, 2153–2163. Pelfrey, C.M., Trotter, J.L., Tranquill, L.R., McFarland, H.F., 1993. Identification of a novel T cell epitope of human proteolipid protein Žresidues 40–60. recognized by proliferative and cytolytic CD4q T cells from MS patients. J. Neuroimmunol. 46, 33–42.
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Pelfrey, C.M., Trotter, J.L., Tranquill, L.R., McFarland, H.F., 1994. Identification of a second T cell epitope of human proteolipid protein ŽResidues 89–106. recognized by proliferative and cytolytic CD4q T cells from multiple sclerosis patients. J. Neuroimmunol. 53, 153–162. Perry, L.L., Barzaga, M.E., 1987. Kinetics and specificity of T and B cell responses in relapsing experimental allergic encephalomyelitis. J. Immunol. 138, 1434–1441. Perry, L.L., Barzaga, E., Trotter, J.L., 1991. T cell sensitization to proteolipid protein in myelin basic protein-induced relapsing experimental allergic encephalomyelitis. J. Neuroimmunol. 33, 7–15. Pettinelli, C.B., McFarlin, D.E., 1981. Adoptive transfer of experimental allergic encephalomyelitis in SJLrJ mice after in vitro activation of lymph node cells by myelin basic protein: requirement for Lyt 1q 2-T lymphocytes. J. Immunol. 127, 1420–1426. Richert, J.R., Robinson, E.D., Deibler, G.E., Martenson, R.E., Dragovic, L.J., Kies, M.W., 1989. Evidence for multiple human T cell recognition sites on myelin basic protein. J. Neuroimmunol. 23, 55–66. Sakai, K., Zamvil, S.S., Mitchell, D.J., Hodgkinson, S., Rothbard, J.B., Steinman, L., 1989. Prevention of experimental encephalomyelitis with peptides that block interaction of T cells with major histocompatibility complex proteins. Proc. Natl. Acad. Sci. 86, 9470–9474. Sercarz, E.E., Lehmann, P.V., Amentani, A., Benichou, G., Miller, A., Moudgil, K., 1993. Dominance and crypticity of T cell antigenic determinants. Annu. Rev. Immunol. 11, 729–766. Sharma, S.C., Nag, B., Su, X.M., Green, D., Spack, E., Clark, B.R., Sriram, S., 1991. Antigen-specific therapy of experimental allergic encephalomyelitis by soluble class II major histocompatibility complex—peptide complexes. Proc. Natl. Acad. Sci. 88, 11465–11469. Smilek, D.E., Wraith, D.C., Hodgkinson, S., Dwivedy, S., Steinman, L., McDevitt, H.O., 1991. A single amino acid change in a myelin basic protein peptide confers the capacity to prevent rather than induce experimental autoimmune encephalomyelitis. Proc. Natl. Acad. Sci. 88, 9633–9637. Sun, J.B., Olsson, T., Wang, W.Z., Xiao, B.G., Kostulas, V., Fredrikson, S., Ekre, H.P., Link, H., 1991. Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls. Eur. J. Immunol. 21, 1461–1468. Trotter, J.L., Lieberman, L., Margolis, F.L., Agrawal, H.C., 1981. Radioimmunoassay for central nervous system myelin specific proteolipid protein. J. Neurochem. 32, 1256–1262. Trotter, J.L., Clark, H.B., Collins, K.G., Wegenscheide, C.L., Scarpellini, J.D., 1987. Myelin proteolipid protein induces demyelinating disease in mice. J. Neurol. Sci. 79, 173–188. Trotter, J.L., Hickey, W.F., van der Veen, R.C., Sulze, L., 1991. Peripheral blood mononuclear cells from multiple sclerosis patients recognize myelin proteolipid protein and selected peptides. J. Neuroimmunol. 33, 55–62. Trotter, J.L., Damico, C.A., Cross, A.H., Pelfrey, C.M., Karr, R.W., Fu, X., McFarland, H.F., 1997. HPRT mutant T-cell lines from multiple sclerosis patients recognize myelin proteolipid protein peptides. J. Neuroimmunol. 75, 95–103. Tuohy, V.K., Lu, Z., Sobel, R.A., Laursen, R.A., Lees, M.B., 1988. A synthetic peptide from myelin proteolipid protein induces experimental allergic encephalomyelitis. J. Immunol. 141, 1226–1230. Vandenbark, A., Hashim, G., Offner, H., 1989. Immunization with a synthetic T-cell receptor V-region against experimental autoimmune encephalomyelitis. Nature 341, 541–544. Vandenbark, A.A., Vainiene, M., Celnik, B., Hashim, G., Offner, H., 1992. TCR peptide therapy decreases the frequency of encephalitogenic T cells in the periphery and the central nervous system. J. Neuroimmunol. 39, 251–260. van der Veen, R.C., Trotter, J.L., Clark, H.B., Kapp, J.A., 1989. The adoptive transfer of chronic relapsing experimental allergic encephalomyelitis with lymph node cells sensitized to myelin proteolipid protein. J. Neuroimmunol. 21, 183–191. van der Veen, R.C., Trotter, J.L., Hickey, W.F., Kapp, J.A., 1990. The development and characterization of encephalitogenic cloned T cells
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specific for myelin proteolipid protein. J. Neuroimmunol. 26, 139– 145. van der Veen, R.C., Trotter, J.L., Kapp, J.A., 1992. Immune processing of proteolipid protein by subsets of antigen-presenting spleen cells. J. Neuroimmunol. 38, 139–146. Whitham, R.H., Jones, R.E., Hashim, G.A., Hoy, C.M., Wang, R.Y., Vandenbark, A.A., Offner, H., 1991. Location of a new encephalitogenic epitope Žresidues 43 to 64. in proteolipid protein that induces relapsing experimental autoimmune encephalomyelitis. J. Immunol. 147, 3803–3808. Wraith, D.C., Smilek, D.E., Mitchell, D.J., Steinman, L., McDevitt, H.O., 1989. Antigen recognition in autoimmune encephalomyelitis and the potential for peptide-mediated immunotherapy. Cell 59, 247–255.
Yamamura, T., Namikawa, T., Endoh, M., Kunishita, T., Tabira, T., 1986. Experimental allergic encephalomyelitis induced by proteolipid apoprotein in Lewis rats. J. Neuroimmunol. 12, 143–146. Yu, M., Johnson, J.M., Tuohy, V.K., 1996. A predictable sequential determinant spreading cascade invariably accompanies progression of experimental autoimmune encephalomyelitis: a basis for peptidespecific therapy after onset of clinical disease. J. Exp. Med. 183, 1777–1788. Zhang, J., Markovic-Plese, S., Lacet, B., Raus, J., Weiner, H.L., Hafler, D.A., 1994. Increased frequency of interleukin 2-responsive T cells specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. J. Exp. Med. 179, 973–984.
T cell recognition of myelin proteolipid protein and myelin proteolipid protein peptides in the peripheral blood of multiple sclerosis and control subjects John L. Trotter a,) , Clara M. Pelfrey b, Amy L. Trotter a , Jacqueline A. Selvidge a , Kelly C. Gushleff a , T. Mohanakumar b, Henry F. McFarland c a
c
Department of Neurology and Neurological Surgery, Washington UniÕersity School of Medicine, St. Louis, MO 63110-1093, USA b Department of Pathology, Washington UniÕersity School of Medicine, St. Louis, MO 63110-1093, USA Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA Received 3 September 1997; received in revised form 19 November 1997; accepted 24 November 1997
Abstract Myelin proteolipid protein ŽPLP. is a prime candidate autoantigen for multiple sclerosis. In order to define potential immunodominant epitopes, T cell lines ŽTCL. from the peripheral blood of HLA-DR 15Ž2. MS patients were established which responded to the intact molecule of PLP. These TCL were then tested in individual proliferation assays with a variety of PLP peptides spanning most of the PLP molecule. Multiple peptides were recognized by TCL from the MS population, with more than one peptide often recognized by lines from the same individual. Three immunodominant peptides were identified which were recognized by the majority of MS patients. Estimated frequency analyses were then performed on the peripheral blood of HLA-DR15Ž2.-positive MS and control subjects using TCL initiated by the three immunodominant peptides, 40–60, 95–117, and 185–206. TCL from HLA-DR15 MS subjects recognized peptide 95–117 significantly more often than TCL from control subjects. q 1998 Elsevier Science B.V. Keywords: Multiple sclerosis; T cells; Myelin proteolipid protein
1. Introduction A variety of clinical and laboratory-derived clues indicate that immunological mechanisms are involved in the etiology and pathogenesis of multiple sclerosis ŽMS. ŽMcFarlin and McFarland, 1982; Martin et al., 1992.. For instance, the most common animal model for MS, relapsing experimental autoimmune Žallergic. encephalomyelitis ŽEAE., resembles MS both clinically and pathologically. EAE can be induced by active immunization with, or transfer of T cells specific for any of at least three proteins specific to myelin, or peptides derived from each of these peptides, including myelin basic protein ŽMBP. ŽPettinelli and McFarlin, 1981., myelin proteolipid protein ŽPLP. )
Corresponding author. Department of Neurology and Neurological Surgery, Washington University School of Medicine, Box 8111, 660 S. Euclid, St. Louis, MO, 63110, USA. Tel.: q1 312 3623293; fax: q1 312 7471345. 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 6 0 - 9
ŽYamamura et al., 1986; Trotter et al., 1987; Tuohy et al., 1988; van der Veen et al., 1989, 1990; Whitham et al., 1991; Greer et al., 1992; van der Veen et al., 1992; Amor et al., 1993; ., and myelin–oligodendrocyte glycoprotein ŽMOG. ŽJohns et al., 1995; Devaux et al., 1997.. The human cellular immune response to MBP has been extensively studied in efforts to determine whether it is an autoantigen in humans ŽLisak et al., 1981; Richert et al., 1989; Allegretta et al., 1990; Ota et al., 1990; Martin et al., 1990, 1992; Chou et al., 1992; Zhang et al., 1994., but studies to date have not definitely confirmed MBP to be an autoantigen in MS. Evidence supports the relevance of another candidate autoantigen, PLP. PLP is encephalitogenic in animals and specific to myelin. T lymphocytes recognizing PLP or PLP peptides have been found in the peripheral blood and cerebrospinal fluid of MS patients ŽOta et al., 1990; Sun et al., 1991; Trotter et al., 1991; Chou et al., 1992;Pelfrey et al., 1993, 1994; Zhang et al., 1994; Markovic-Plese et al.,
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1995; .. HPRT mutant clones recognizing PLP peptides have been identified in the peripheral blood of MS patients ŽTrotter et al., 1997.. We have previously identified two PLP peptide sequences as human T cell epitopes that induce T cell-mediated cytotoxicity when presented by activated B cells ŽPelfrey et al., 1993, 1994.. The present studies extend this work by studying human T cell recognition of peptides spanning most of the PLP molecule. In this study, immunodominant ŽSercarz et al., 1993. PLP peptide sequences, defined as such by their recognition by the majority of T cell lines ŽTCL. raised against the whole PLP molecule, were identified. Estimated frequency analyses performed on TCL initiated with these peptides demonstrated higher frequencies among MS when compared to control subjects.
troscopy. The PLP peptides used in the study had the following amino acid sequences ŽDiehl et al., 1986.: 40–60, TGTEKLIETYFSKNYQDYEYL; 44–76, KLIETYFSKNYQDYEYLINVIHAFQYVIYGTAS; 89–106, GFYTTGAYRQIFGDYLTT; 95–117, AVRQIFGDYKTTICGKGLSATVT; 117–150, TGGQKGRGSRGQHQAHSLERVCHCLGKWLGHPDK; 139–151, HCLGKWLGHPDKF; 185–206, SIAFPSKTSASIGSLCADARMY; 207–231, GVLPWNAFPGKVCGSNLLSICKTAE; and 254–276, LTFMIAATYNFAVLKLMGRGTKF It should be noted that peptides 1–47 and 232–255 were not studied because they could not be extracted from their solid supports after synthesis.
2. Materials and methods
2.3. Establishment of 2 ml cultures
2.1. Human subjects
Mononuclear cells ŽMNC. were isolated from heparinized whole blood on Histopaquexe ŽSigma Chemical, St. Louis, MO, USA. gradients and washed. Two ml cultures were established with 5 = 10 6 MNC in Hepesbuffered RPMI 1640 with antibiotics and 5% human male AB serum ŽCM.. Cultures were maintained weekly with fresh PLP-pulsed, irradiated adherent MNC; 100 m grml of PLP in 1 ml RPMI was added to irradiated Ž4500R. adherent syngeneic MNC, incubated 2 h at 378C, and washed prior to the addition to the TCL. Proliferation assays were performed using each of the selected peptides on TCL in which the cell concentration was at least 1 = 10 6rwell after a culture duration of 6 weeks. Five lines were randomly sampled for phenotypic analysis; all were ) 90% CD4 q .
The MS patients involved in the present studies had clinically definite disease with a relapsing–remitting course, with a disease duration of 2–10 yr, and had suffered at least one exacerbation within the previous 2 yr. All patients were in remission at the time of phlebotomy. The patients ranged in age from 33 to 55 yr; there were 8 females and 4 males. The Kurtzke disability status scale levels of disability ranged from 3.5 to 6.0. Patients had not received corticosteroids for at least 3 months, and none had received other immunomodulatory or immunosuppressive agents, including b-interferons. Normal control subjects were volunteers recruited among employees of Washington University School of Medicine in St. Louis, MO and spouses of MS patient volunteers. All subjects were HLA-typed by the Barnes Hospital Department of Laboratory Medicine or Smith-Kline Laboratories. Patients and control subjects with the HLA-DR15Ž2. type, which is the most common DR type among North American Caucasians with MS, were used exclusively in these experiments. The blood donations for these studies were approved by the Human Studies Committee ŽIRB. of the Washington University School of Medicine. 2.2. Reagents Myelin PLP was prepared by a method modified from that described by Folch et al. Ž1957. as modified by Hampson and Poduslo Ž1986.. PLP was shown to be free of MBP ŽTrotter et al., 1981.. Only preparations of PLP which were active in proliferation assays of TCL known to recognize the intact antigen were used in these studies. Synthetic peptides of PLP were prepared by Monsanto, or the Washington University Department of Biochemistry using FastMOC chemistry. The peptides were purified by HPLC, and the sequences confirmed by mass spec-
2.4. Proliferation assays on PLP stimulated TCL Proliferation assays were performed in quadruplicate in flat-bottom microtiter tissue culture plates. Each well contained 1 = 10 4 cells from each TCL and 1 = 10 5 irradiated MNC as feeders in the presence of 20 m grml PLP peptides in CM. The doses of antigen selected had previously been shown to be optimal by proliferation assays, and represented plateau values when peptide-initiated TCL had been tested in concentrations ranging from 5–25 m grml. Twenty m grml represented a plateau response for all peptides tested. Cells were incubated for 72 h before being pulsed with 2 m Cirwell Žmethyl- 3 H . thymidine ŽAmersham, Arlington Heights, IL, USA. for 18 h. The cells were harvested and the incorporated radioactivity counted using a Betaplatee system. 2.5. Estimated frequency analyses Following the identification of immunodominant peptides of PLP, estimated frequency analysis was performed
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Table 1 Recognition of PLP peptides by PLP-initiated T cell lines derived from MS patients Patients
MS a1
PLP lines 6r17 a Synthetic Peptidesb 40–60 5 44–76 2 89–106 0 95–117 4 117–150 2 139–151 2 185–206 5 207–231 0 254–276 0
MS a2
MS a3
MS a4
MS a5
MS a6
MS a7
MS a8
MS a9
MS a10
MS a11
MS a12
Total
6r17
6r18
2r17
2r17
3r17
5r20
4r17
3r17
8r17
2r17
3r17
50r208
5 2 1 4 3 2 5 0 0
4 1 0 4 4 0 4 0 0
2 0 0 1 2 1 2 0 0
1 0 0 0 1 0 2 0 0
2 0 0 0 1 0 1 0 0
3 0 1 2 2 0 3 0 0
3 0 0 3 3 0 3 0 0
3 3 1 0 1 0 2 0 0
7 0 2 5 3 0 6 0 0
0 0 0 2 1 2 1 0 0
0 0 3 0 0 3 1 0 0
35r50 8r50 8r50 25r50 23r50 10r50 35r50 0r50 0r50
a
Positive linesrtotal lines after 6 weeks. T cell lines initiated with the whole PLP molecule, and which recognized the whole molecule after 6 weeks in culture were tested for recognition of PLP synthetic peptides. b Synthetic peptides 1–47 and 232–255 could not be extracted from their solid supports.
on the peripheral blood of MS and control subjects in round-bottom microtiter tissue culture plates, using a method modified from that described by Ota et al., 1990. A total of 2 = 10 5 MNC were incubated with each of the selected synthetic peptides Ž20 m grml. at 378C in CM. Four to 15 96-well plates were used to test each antigen. Twenty units recombinant human interleukin 2 ŽIL-2. were added to the cultures every 4 days beginning at day 8. The last dose of IL-2 was administered on day 16. Proliferation assays were performed on day 24 using a modification of the ‘split-well’ technique described by Ota et al. This technique involves splitting the culture wells into three new wells, with the addition of fresh irradiated feeders. Antigen reactivity is determined using two of the wells, with the third saved for perpetuation.
3. Results Our initial studies revealed that high doses of PLP Žon the order of 50–100 m grml. often suppressed the proliferation of TCL, especially when the TCL had been cultured for several weeks. This finding, as well as the inherent difficulty dealing with the insoluble antigen, PLP, led us to adopt a method where excess antigen is removed from
pulsed APC, since high concentrations of antigen inhibit proliferation. Two ml TCL which recognized intact PLP were derived from the peripheral blood MNC of 12 DR15Ž2. MS patients ŽTable 1.. Seventeen to 20 TCL per patient were established Žsee Section 2. for a total of 208 lines. Of these, 50 TCL survived at least 6 weeks, at which time they were analyzed for recognition of each of the selected synthetic peptides. A stimulation index of 3.0 was considered positive, if the Dcpm was G 500. These criteria resulted in cpm which were greater than 3 standard deviations ŽS.D.. above the mean of the unstimulated cultures in all cases. The antigenic repertoire of the lines was heterogeneous, i.e., many lines recognized more than one peptide ŽTable 1.. Peptides 207–231 and 254–276 were recognized by none of the lines. Peptides 40–60, 95–117, and 185–206 were recognized by the majority of MS subjects and yielded high stimulation indices. Therefore, these peptides were selected to be studied by estimated frequency analyses to determine if the T cells which recognized them were more frequent in MS than control subjects. When comparing frequencies in the peripheral blood, the greatest difference between MS patients and control subjects in our series appeared to be for PLP peptide 95–117 ŽTable 2; Fig. 1.. The estimated frequencies in the
Table 2 Estimated frequency of T cell recognition of PLP peptides in MS and control subjects Subjects Ž n.
Peptide
Positive wellsrplate Žmean " S.D..
Frequency Žrange.
MS Ž9. NC Ž5. MS Ž12. NC Ž6. MS Ž9. NC Ž9.
40–60 40–60 95–117 95–117 185–206 185–206
1.0 " 1.3 0.3 " 0.2 2.5 " 2.3 0.9 " 0.8 3.2 " 3.5 1.4 " 1.8
0–1r4,512,000 0–1r38,400,000 0–1r2,553,600 1r7,680,000–1r9,600,000 1r8,524,800–1r2,035,200 0–1r3,552,000
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Fig. 1. The estimated frequencies for the three immunodominant peptides tested are shown in Figure 1 for MS patients and control subjects. The mean and standard deviations of the frequencies for the normal control subjects are demonstrated in the columns for the MS patients. The p-values resulting from the Wilcoxin Rank Sums comparisons are shown for each peptide.
peripheral blood for T cells from the MS subjects that recognized this peptide ranged from 0 to 1r2,553,600 whereas the estimated frequencies for the control subjects ranged from 1r76,800,000 to 1r9,600,000 Ž p s 0.033, Mann–Whitney Rank Sums test.. Examining the estimated frequencies in a different manner, five of 12 MS patients had frequencies 2 standard deviations above the mean of the control subjects when PLP peptide 95–117 was studied, whereas three of nine and two of nine MS patients fulfilled these criteria for peptides 40–60 and 185–206, respectively ŽFig. 1..
4. Discussion We have here confirmed and extended our previous work and that of others, that intact PLP protein is recognized by T cells from the peripheral blood of MS and control subjects. Thus, PLP remains a candidate antigen in MS, with as much potential relevance as the more frequently cited candidate, MBP. We elected to determine ‘immunodominant’ peptides for MS subjects ŽTable 1. based on the premise that such peptides might have pathogenic relevance. The present studies demonstrated that there are multiple candidate epitopes of PLP. The estimated frequencies of recognition of peptides 40–60, 95–117, and 185–206, which were im-
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munodominant in the majority of TCL initiated to intact PLP and derived from MS patients, were higher among MS patients when compared to control subjects. The question arises whether the frequencies of T cell recognition we have demonstrated for PLP peptides is sufficient to induce human disease. The following facts taken from the literature regarding EAE may be pertinent. 1. In studies on the initiation of EAE using the adoptive transfer method, Cross et al. demonstrated that a very small number of T cells is needed to initiate a lesion. After development of an inflammatory lesion, only 1%–4% of T cells within the lesion were from the original inoculate of cells ŽCross et al., 1990.. 2. Vandenbark et al. have shown that as few as 5 million cells in a TCL recognizing PLP peptide 139–151 can transfer severe relapsing–remitting EAE to naive recipient rats ŽJones et al., 1990.. 3. Vandenbark et al. Ž1992. found that the frequencies of MBP-specific T cells in the peripheral blood of Lewis rats immunized with MBP and CFA ranged between 2r1,000,000 prior to disease onset and 1r100,000 at the peak of clinical EAE. Thus, the human lymphoid system certainly contains sufficient cells recognizing each of the immunodominant peptides to cause disease. Furthermore, we have demonstrated that there is recognition of multiple peptides of PLP by T cells from MS subjects, especially in the peptide span of 95–150, so that multiple interactions with PLP in the myelin membrane are possible. Of note in our studies is the fact that individual patients recognize multiple peptides when multiple TCL’s are initiated with PLP or individual peptides. These findings agree with the findings of another group of investigators who identified ‘immunodominant’ PLP peptides which overlap with two we have identified. However, in that study ŽMarkovic-Plese et al., 1995., PLP peptide 95–117 was not studied, and frequency analyses comparing the recognition of PLP peptides among MS and control subjects were not performed. A more recent study found that PLP peptide 95–116 was recognized significantly more frequently by Japanese DR15 MS patients compared to DR15 controls subjects ŽOhashi et al., 1995.. Results have varied in the literature when the percentage of MS subjects vs. controls recognizing the whole molecule PLP is considered ŽChou et al., 1992; Sun et al., 1991; Trotter et al., 1991, 1997; Zhang et al., 1994.. It is clear from our work and that of others, that recognition of PLP is not restricted to MS subjects. If a single epitope of PLP were responsible for disease induction in a given individual, one might still observe multiple recognition sites in that patient through the phenomenon of antigen determinant spreading. We and others have previously demonstrated in inbred mice with EAE induced by a single peptide or protein antigen that antigen determinant spreading occurs and increases the antigenic repertoire over time ŽCross et al., 1993; Lehmann et al.,
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1992; McCarron et al., 1990; McRae et al., 1995; Miller et al., 1995; Perry et al., 1991; Perry and Barzaga, 1987; Yu et al., 1996.. This may explain the recognition of multiple peptides by PLP TCL. The situation is further complicated by the possible relevance of cryptic epitopes to the disease pathogenesis since these peptides may also be pathogenic ŽLipham et al., 1991.. Although the patients in the present studies were limited to those with a relapsing–relapsing course, and controlled for HLA type, they were not controlled for disease duration, MRI plaque burden, or remote disease history, which may affect antigen determinant spreading.
Acknowledgements
5. Conclusion
References
In the studies we present here, recognition of the PLP peptide 95–117 was significantly greater among MS compared to control subjects. An epitope recognized at relatively higher frequencies might be more likely to be associated with the initiation of pathology than an epitope with a lower T cell frequency. This notion remains highly speculative, since the criteria for determining relevance to the pathogenesis of disease have not been established. The methods we used in this report involved prolonged culture and repeated stimulation with antigen, suggesting the possibility of in vitro sensitization. We previously used a different approach to identify HPRT mutant clones from MS patients which recognized PLP peptides ŽTrotter et al., 1997.. Other methods, such as testing for antigen recognition after stimulating T cells bearing IL-2 receptors, have been adopted in attempts to better reflect in vivo conditions and relevance, but these too have the disadvantage of requiring cell culture ŽZhang et al., 1994.. Studies using more subjects, inclusion of a series of shorter overlapping peptides and studies of disease controls are needed to confirm our findings. Such studies are underway in our laboratory. PLP recognition by T cells from concordant vs. discordant twins with MS might further elucidate the relevance of individual epitopes, at least within families. However, the ultimate method of proving a pathogenic epitope in humans would derive from results of clinical trials with reagents directed to the individual epitopeŽs.. The present studies included peptides which span most but not all of the PLP molecule. Further studies with additional overlapping peptides may reveal more candidates for antigens relevant to MS. Exhaustive studies with shorter peptides and amino acid substitutions may be necessary to determine therapeutic agents, which could be used to treat MS using methods similar to those described in EAE ŽAcha-Orbea et al., 1988; Gaur et al., 1992; Hashim et al., 1990; Howell et al., 1989; Lamont et al., 1990; Kennedy et al., 1990; Owhashi and Heber-Katz, 1988; Sakai et al., 1989; Sharma et al., 1991; Smilek et al., 1991; Vandenbark et al., 1989; Wraith et al., 1989..
Acha-Orbea, H., Mitchell, D.J., Timmerman, L., Wraith, D.C., Tausch, G.S., Waldor, M.K., Zamvil, S.S., McDevitt, H.O., Steinman, L., 1988. Limited heterogeneity of T cell receptors from T lymphocytes mediating encephalomyelitis allows specific immune intervention. Cell 54, 263–273. Allegretta, M., Nicklas, J.A., Sriram, S., Albertini, R.J., 1990. T cells responsive to myelin basic protein in patients with multiple sclerosis. Science 247, 718–721. Amor, S., Baker, D., Groome, N., Turk, J.L., 1993. Identification of a major encephalitogenic epitope of proteolipid protein Žresidues 56–70. for the induction of experimental allergic encephalomyelitis in Biozzi ABrH and nonobese diabetic mice. J. Immunol. 150, 5666–5672. Chou, Y.K., Bourdette, D.N., Offner, H., Whitham, R., Wang, R.Y., Hashim, G.A., Vandenbark, A.A., 1992. Frequency of T cells specific for myelin basic protein and myelin proteolipid protein in blood and CSF in MS. J. Neuroimmunol. 38, 105–114. Cross, A.H., Cannella, B., Brosnan, C.F., Raine, C.S., 1990. Homing to central nervous system vasculature by antigen-specific lymphocytes: I. Localization of 14 C-labeled cells during acute, chronic, and relapsing experimental allergic encephalomyelitis. Lab. Invest. 63, 162–170. Cross, A.H., Tuohy, V.K., Raine, C.S., 1993. Development of reactivity to new myelin antigens during chronic relapsing autoimmune demyelination. Cell. Immunol. 146, 261–269. Devaux, B., Enderlin, F., Wallner, B., Smilek, D.E., 1997. Induction of EAE in mice with recombinant human MOG, and treatment of EAE with a MOG peptide. J. Neuroimmunol. 75, 169–173. Diehl, H.J., Schaich, M., Budzinski, R.M., Stoffel, W., 1986. Individual exons encode the integral membrane domains of human myelin proteolipid protein. Proc. Natl. Acad. Sci. U.S.A. 83, 9807–9811. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Gaur, A., Wiers, B., Liu, A., Rothbard, J., Fathman, C.G., 1992. Amelioration of autoimmune encephalomyelitis by myelin basic protein synthetic peptide-induced anergy. Science 258, 1491–1494. Greer, J.M., Kuchroo, V.K., Sobel, R.A., Lees, M.B., 1992. Identification and characterization of a second encephalitogenic determinant of myelin proteolipid protein Žresidues 178–191. for SJL mice. J. Immunol. 149, 783–788. Hampson, D.R., Poduslo, S.E., 1986. Purification of proteolipid protein and production of specific antiserum. J. Neuroimmunol. 11, 117–129. Hashim, G.A., Vandenbark, A., Galang, A.B., Diamanduros, T., Carvalho, E., Srinivasan, J., Jones, R., Vainiene, M., Morrison, W.J., Offner, H., 1990. Antibodies specific for Vb 8 receptor peptide suppress experimental autoimmune encephalomyelitis. J. Immunol. 144, 4621–4627. Howell, M.D., Winters, S.T., Olee, T., Powell, H.C., Carlo, D.J., Brostoff,
We thank Larry Sulze, Joan Bryan and Cheryl Damico for excellent technical assistance. Drs. Anne H. Cross, Amy Lovett-Racke, and Michael K. Racke reviewed the manuscript and provided thoughtful discussion. This work was supported in part by grant RG 2681 from the National Multiple Sclerosis Society, a grant from the Washington University-Monsanto Research Program, grant RO1 NS29851 from the National Institutes of Health, the Barnes-Jewish Hospital Foundation, and gifts from the Joanne Kirberg and the Frala Osherow Funds.
J.L. Trotter et al.r Journal of Neuroimmunology 84 (1998) 172–178 S.W., 1989. Vaccination against experimental allergic encephalomyelitis with T cell receptor peptides. Science 246, 668–670. Johns, T.G., Kerlero de Rosbo, N., Menon, K., Abo, S., Gonzales, M.F., Bernard, C.C.A., 1995. Demyelinating encephalomyelitis resembling multiple sclerosis after immunization with a peptide from myelin oligodendrocyte glycoprotein. J. Immunol. 154, 5536–5541. Jones, R.E., Bourdette, D.N., Offner, H., Vandenbark, A.A., 1990. Myelin basic protein-specific T cells induce demyelinating experimental autoimmune encephalomyelitis in Buffalo rats. J. Neuroimmunol. 30, 61–69. Kennedy, M.K., Tan, L.J., DalCanto, M.C., Miller, S.D., 1990. Inhibition of murine relapsing experimental autoimmune encephalomyelitis by immune tolerance to proteolipid protein and its encephalitogenic peptide. J. Immunol. 1441, 909–915. Lamont, A.G., Sette, A., Fujinami, R., Colon, S.M., Miles, C., Grey, H.M., 1990. Inhibition of experimental autoimmune encephalomyelitis induction in SJLrJ mice by using a peptide with high affinity for Ias molecules. J. Immunol. 145, 1687–1693. Lehmann, P.V., Forsthuber, T., Miller, A., Sercarz, E.E., 1992. Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen. Nature 358, 155–157. Lipham, W.J., Redmond, T.M., Takahashi, H., Berzofsky, J.A., Wiggert, B., Chader, G.J., Gery, I., 1991. Recognition of peptides that are immunopathogenic but cryptic. J. Immunol. 146, 3757–3762. Lisak, R.P., Zweiman, B., Whitaker, J.N., 1981. Spinal fluid basic protein immunoreactive material and spinal fluid lymphocyte reactivity to basic protein. Neurology 31, 180–182. Markovic-Plese, S., Fukaura, H., Zhang, J., al-Sabbagh, A., Southwood, S., Sette, A., Kuchroo, V.K., Hafler, D.A., 1995. T cell recognition of immunodominant and cryptic proteolipid protein epitopes in humans. J. Immunol. 155, 982–992. Martin, R., Jaraquemada, D., Flerlage, M., Richert, J., Brostoff, S., Long, E.O., McFarlin, D.E., McFarland, H.F., 1990. Fine specificity and HLA restriction of myelin basic protein specific cytotoxic T cell lines from multiple sclerosis patients and healthy individuals. J. Immunol. 145, 540–548. Martin, R., McFarland, H.F., McFarlin, D.E., 1992. Immunological aspects of demyelinating diseases. Annu. Rev. Immunol. 10, 153–187. McCarron, R.M., Fallis, R.J., McFarlin, D.E., 1990. Alterations in T cell antigen specificity and class II restriction during the course of chronic relapsing experimental allergic encephalomyelitis. J. Neuroimmunol. 29, 73–79. McFarlin, D.E., McFarland, H.F., 1982. Multiple sclerosis ŽReview.. New. Eng. J. Med. 307, 1183–1188, 1246–1251. McRae, B.L., Vanderlugt, C.L., Dal Canto, M.C., Miller, S.D., 1995. Functional evidence for epitope spreading in the relapsing pathology of experimental autoimmune encephalomyelitis. J. Exp. Med. 182, 75–85. Miller, S.D., McRae, B.L., Vanderlugt, C.L., Nikcevich, K.M., Pope, J.G., Pope, L., Karpus, W.J., 1995. Evolution of the T-cell repertoire during the course of experimental immune mediated demyelinating disease. Immunol. Rev. 144, 226–244. Ohashi, T., Yamamura, T., Inobe, J., Kondo, T., Kunishita, T., Tabira, T., 1995. Analysis of proteolipid protein ŽPLP.-specific T cells in multiple sclerosis; identification of PLP 95–116 as an HLA-DR2, w15-associated determinant. Int. Immunol. 7, 1771–1778. Ota, K., Matsui, M., Milford, E.L., Mackin, G.A., Weiner, H.L., Hafler, D.A., 1990. T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature 346, 183–187. Owhashi, M., Heber-Katz, E., 1988. Protection from experimental allergic encephalomyelitis conferred by a monoclonal antibody directed against a shared idiotype on rat T cell receptors. J. Exp. Med. 168, 2153–2163. Pelfrey, C.M., Trotter, J.L., Tranquill, L.R., McFarland, H.F., 1993. Identification of a novel T cell epitope of human proteolipid protein Žresidues 40–60. recognized by proliferative and cytolytic CD4q T cells from MS patients. J. Neuroimmunol. 46, 33–42.
177
Pelfrey, C.M., Trotter, J.L., Tranquill, L.R., McFarland, H.F., 1994. Identification of a second T cell epitope of human proteolipid protein ŽResidues 89–106. recognized by proliferative and cytolytic CD4q T cells from multiple sclerosis patients. J. Neuroimmunol. 53, 153–162. Perry, L.L., Barzaga, M.E., 1987. Kinetics and specificity of T and B cell responses in relapsing experimental allergic encephalomyelitis. J. Immunol. 138, 1434–1441. Perry, L.L., Barzaga, E., Trotter, J.L., 1991. T cell sensitization to proteolipid protein in myelin basic protein-induced relapsing experimental allergic encephalomyelitis. J. Neuroimmunol. 33, 7–15. Pettinelli, C.B., McFarlin, D.E., 1981. Adoptive transfer of experimental allergic encephalomyelitis in SJLrJ mice after in vitro activation of lymph node cells by myelin basic protein: requirement for Lyt 1q 2-T lymphocytes. J. Immunol. 127, 1420–1426. Richert, J.R., Robinson, E.D., Deibler, G.E., Martenson, R.E., Dragovic, L.J., Kies, M.W., 1989. Evidence for multiple human T cell recognition sites on myelin basic protein. J. Neuroimmunol. 23, 55–66. Sakai, K., Zamvil, S.S., Mitchell, D.J., Hodgkinson, S., Rothbard, J.B., Steinman, L., 1989. Prevention of experimental encephalomyelitis with peptides that block interaction of T cells with major histocompatibility complex proteins. Proc. Natl. Acad. Sci. 86, 9470–9474. Sercarz, E.E., Lehmann, P.V., Amentani, A., Benichou, G., Miller, A., Moudgil, K., 1993. Dominance and crypticity of T cell antigenic determinants. Annu. Rev. Immunol. 11, 729–766. Sharma, S.C., Nag, B., Su, X.M., Green, D., Spack, E., Clark, B.R., Sriram, S., 1991. Antigen-specific therapy of experimental allergic encephalomyelitis by soluble class II major histocompatibility complex—peptide complexes. Proc. Natl. Acad. Sci. 88, 11465–11469. Smilek, D.E., Wraith, D.C., Hodgkinson, S., Dwivedy, S., Steinman, L., McDevitt, H.O., 1991. A single amino acid change in a myelin basic protein peptide confers the capacity to prevent rather than induce experimental autoimmune encephalomyelitis. Proc. Natl. Acad. Sci. 88, 9633–9637. Sun, J.B., Olsson, T., Wang, W.Z., Xiao, B.G., Kostulas, V., Fredrikson, S., Ekre, H.P., Link, H., 1991. Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls. Eur. J. Immunol. 21, 1461–1468. Trotter, J.L., Lieberman, L., Margolis, F.L., Agrawal, H.C., 1981. Radioimmunoassay for central nervous system myelin specific proteolipid protein. J. Neurochem. 32, 1256–1262. Trotter, J.L., Clark, H.B., Collins, K.G., Wegenscheide, C.L., Scarpellini, J.D., 1987. Myelin proteolipid protein induces demyelinating disease in mice. J. Neurol. Sci. 79, 173–188. Trotter, J.L., Hickey, W.F., van der Veen, R.C., Sulze, L., 1991. Peripheral blood mononuclear cells from multiple sclerosis patients recognize myelin proteolipid protein and selected peptides. J. Neuroimmunol. 33, 55–62. Trotter, J.L., Damico, C.A., Cross, A.H., Pelfrey, C.M., Karr, R.W., Fu, X., McFarland, H.F., 1997. HPRT mutant T-cell lines from multiple sclerosis patients recognize myelin proteolipid protein peptides. J. Neuroimmunol. 75, 95–103. Tuohy, V.K., Lu, Z., Sobel, R.A., Laursen, R.A., Lees, M.B., 1988. A synthetic peptide from myelin proteolipid protein induces experimental allergic encephalomyelitis. J. Immunol. 141, 1226–1230. Vandenbark, A., Hashim, G., Offner, H., 1989. Immunization with a synthetic T-cell receptor V-region against experimental autoimmune encephalomyelitis. Nature 341, 541–544. Vandenbark, A.A., Vainiene, M., Celnik, B., Hashim, G., Offner, H., 1992. TCR peptide therapy decreases the frequency of encephalitogenic T cells in the periphery and the central nervous system. J. Neuroimmunol. 39, 251–260. van der Veen, R.C., Trotter, J.L., Clark, H.B., Kapp, J.A., 1989. The adoptive transfer of chronic relapsing experimental allergic encephalomyelitis with lymph node cells sensitized to myelin proteolipid protein. J. Neuroimmunol. 21, 183–191. van der Veen, R.C., Trotter, J.L., Hickey, W.F., Kapp, J.A., 1990. The development and characterization of encephalitogenic cloned T cells
178
J.L. Trotter et al.r Journal of Neuroimmunology 84 (1998) 172–178
specific for myelin proteolipid protein. J. Neuroimmunol. 26, 139– 145. van der Veen, R.C., Trotter, J.L., Kapp, J.A., 1992. Immune processing of proteolipid protein by subsets of antigen-presenting spleen cells. J. Neuroimmunol. 38, 139–146. Whitham, R.H., Jones, R.E., Hashim, G.A., Hoy, C.M., Wang, R.Y., Vandenbark, A.A., Offner, H., 1991. Location of a new encephalitogenic epitope Žresidues 43 to 64. in proteolipid protein that induces relapsing experimental autoimmune encephalomyelitis. J. Immunol. 147, 3803–3808. Wraith, D.C., Smilek, D.E., Mitchell, D.J., Steinman, L., McDevitt, H.O., 1989. Antigen recognition in autoimmune encephalomyelitis and the potential for peptide-mediated immunotherapy. Cell 59, 247–255.
Yamamura, T., Namikawa, T., Endoh, M., Kunishita, T., Tabira, T., 1986. Experimental allergic encephalomyelitis induced by proteolipid apoprotein in Lewis rats. J. Neuroimmunol. 12, 143–146. Yu, M., Johnson, J.M., Tuohy, V.K., 1996. A predictable sequential determinant spreading cascade invariably accompanies progression of experimental autoimmune encephalomyelitis: a basis for peptidespecific therapy after onset of clinical disease. J. Exp. Med. 183, 1777–1788. Zhang, J., Markovic-Plese, S., Lacet, B., Raus, J., Weiner, H.L., Hafler, D.A., 1994. Increased frequency of interleukin 2-responsive T cells specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. J. Exp. Med. 179, 973–984.