Journal of Neuroimmunology, 33 (1991) 55-62 © 1991 Elsevier Science Publishers B.V. 0165-5728/91/$03.50
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JNI 02014
Peripheral blood mononuclear cells from multiple sclerosis patients recognize myelin proteolipid protein and selected peptides John L. Trotter 1, William F. Hickey 2, Roel C. van der Veen 1 and Larry Sulze 1 Departments of I Neurology and 2 Neuropathology, Washington University School of Medicine, St. Louis, MO 63110, U.S.A. (Received 18 December 1990) (Revised, received 23 January 1991) (Accepted 11 February 1991)
Key words: Multiple sclerosis; Cellular immunity; T lymphocyte; Myelin proteolipid protein
Summary Myelin proteolipid protein (PLP) can induce a T cell-mediated chronic relapsing autoimmune encephalomyelitis in animals and therefore is a candidate for an antigen involved in the pathogenesis of multiple sclerosis. In this report, evidence is presented that peripheral blood mononuclear cells from certain multiple sclerosis (MS) patients recognize the intact PLP molecule as well as certain synthetic PLP peptides in proliferation assays. PLP-specific T cell lines could be obtained from six of ten MS patients with early relapsing-remitting disease. These lines recognized more than one PLP peptide and the relevant peptides differed among patients. The relevance of these observations to the pathogenesis of MS remains to be determined.
Introduction Two myelin-specific proteins, myelin basic protein (MBP) and proteolipid protein (PLP) are capable of inducing a chronic, often relapsing autoimmune disease in multiple species of animals (Waksman et al., 1954; Raine, 1984; Trotter et al., 1987). The pathology of the animal model, experimental allergic encephalomyelitis, often resembles multiple sclerosis (MS) and provides evidence that the human disease may have an autoimmune component (reviewed in McFarlin et al., 1982; Raine,
Address for correspondence: John L. Trotter, M.D., Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110, U.S.A. Tel. (314) 362-3293.
1984). Of the two proteins, MBP was the first discovered to have encephalitogenic activity and several models of disease using this protein for induction have been widely studied (Raine, 1984). Evidence is accumulating that MS patients and, interestingly, many controls have peripheral blood T cells which recognize MBP in proliferation assays (Hughes et al., 1968; Colby et al., 1977; Johnson et al., 1986; Vandenbark et al., 1989). Recent studies have utilized knowledge of immunodominant epitopes of MBP and the amino acid sequence of T cell receptors involved in its recognition to provide specific immunotherapy for the MPB-induced inflammatory and demyelinating diseases in animals (Acha-Orbea et al., 1988; Howell et al., 1989; Sakai et al., 1989; Urban et al., 1989; Vandenbark et al., 1989; Wraith et al.,
56
1989). Since the only immunosuppressive therapies that have been tried in multiple sclerosis are nonspecific and have considerable side effects, the possibility of specific immunotherapies for this human disease is quite promising. With these considerations, the possibility that the relevant antigen in some patients with MS may be PLP led us to perform in vitro studies using peripheral blood mononuclear cells from MS patients and to establish PLP-specific T cell lines.
Materials and methods
Preparation of antigens Both myelin antigens were prepared from central nervous system human white matter obtained at autopsy within 18 h of demise. PLP was isolated using chloroform-methanol extraction, followed by washing with water and eventual column chromatography on Sephadex LH-60 (Hampson et al., 1986). The apoprotein was converted to the water-soluble form by solubilization with 2-chloroethanol and dialysis against water. MBP was prepared by the method of Diebler et al. (1972), followed by gel filtration on Sephadex G-150. The purity of the preparations was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblot, radioimmunoassay (Cohen et al., 1975) and lack of cross-reactivity when tested by SJL mouse T cell lines and clones (van der Veen et al., 1989, 1990). Synthetic peptides were prepared as previously reported (van der Veen et al., 1990). In brief, peptides were synthesized employing the FMOC technology for amino acid coupling using the RAMPS system and equipment (New England Nuclear/DuPont Corp., Boston, MA, U.S.A.). Three peptides were created corresponding to amino acid residues 88-108 (EGFYTTGAVRQIFGDYKTTIC), 103-116 ( Y K T T I C G K G L SATV), and 139-154 ( H S L G K W L G H P D K F V G I ) of the human protein sequence (Diehl et al., 1986). Patients and controls Heparinized whole peripheral blood was obtained from four groups of individuals. These
groups were: (1) Sixteen patients with definite multiple sclerosis (Poser et al., 1983) and a rapid chronic progressive course (CPMS). This group had a mean expanded disability status scale (EDSS) rating (Kurtzke, 1983) of 6.0_+ 1.0 and had received no cyclophosphamide at any time and no corticosteroid or other immunosuppressant for 6 months. (2) A group of 14 patients was selected for a diagnosis of definite MS with the onset of symptoms within 2 years and a low disability rating (mean 3.0 +_ 1.0). These patients had a relapsing-remitting course (mean of 1.0 _+ 0.3 exacerbations per year), but had been clinically stable for 6 months (RRMS-S). (3) A group of ten patients with other neurological diseases (OND) (stroke, Parkinson's disease, polymyositis, myasthenia gravis, anoxic encephalopathy at birth) were selected as disease controls. (4) A group of 12 laboratory and other hospital personnel served as normal controls (NC).
In vitro cellular studies on mononuclear cells direct from peripheral blood Peripheral blood mononuclear cells (PBM) were isolated on a discontinuous Ficoll-Hypaque gradient and washed. For the PBM studies the cells were frozen using standard techniques in heparinized RPMI 1640 medium with 10% human AB serum, 30% glucose and 10% dimethyl sulfoxide (DMSO). The thawed cells were 70-100% viable using trypan blue exclusion and responded to concanavalin A in the proliferation assay with a stimulation index of 20 or greater. Prior studies in our laboratory had determined that this degree of proliferation is within the range for freshly isolated PBMs. The antigen sensitivity assays were carried out in triplicate in a final volume of 0.2 ml in flat-bottomed microtiter plates. The cell concentration was 2 × 105/well in Hepes-buffered RPMI with 2% AB (male donor) serum with antibiotics. PBM were cultured for 6 days with PLP, MBP, synthetic peptides, or control media, and allowed to incorporate [3H]thymidine for the final 18 h. The cells were harvested on filter paper and the radioactivity determined in a liquid scintillation spectrometer. A stimulation index was determined by dividing the mean cpm incorporated in anti-
57 gen-stimulated cells by the mean cpm in cells cultured without antigen.
Establishment of lymphocyte lines recognizing PLP T cell lines (TCLs) were initiated using 5 x 106 mononuclear cells from fresh peripheral blood incubated in a 2 ml volume of R P M I containing 5% male donor AB human serum, with fresh glutamine, Hepes, antibiotics, and 5 0 / ~ g / m l PLP. The cells were incubated at 37 ° C for 7 days, washed twice and recultured with 5 x 106 irradiated (2500 R) autologous mononuclear cells with 50 /~g/ml fresh PLP. After 3 more days, recombinant interleukin-2 (IL-2) (50 U / m l ; Sigma Chemical Company, St. Louis, MO, U.S.A.) was added to the culture. Four days later, fresh feeders were again added, with 25 /~g/ml PLP, and IL-2. In the subsequent weeks, fresh feeders, PLP (12.5/~g/ml) and IL-2 were added weekly.
Proliferation assays on cell fines Proliferation assays were performed on cells which had been split from the lines (TCLs) after at least 5 weeks of culture and 'starved', i.e., they did not receive fresh PLP, feeders or IL-2 for 7 - 1 0 days; 2 x 104 cells/0.2 ml well of a flat-bottom plate were incubated with 1 x 105 irradiated autologous mononuclear cells in quadruplicate. 0, 6.25, 12.5 or 25/~g/ml PLP or 0, 0.5, 1 or 3 f f g / m l peptide were added to each well. After 72 h the wells were pulsed with [3H]thymidine for 18 h. The cells were harvested on filter paper and counted in a liquid scintillation spectrometer.
Results Preliminary studies using graded doses of 25, 50, and 100 f f g / m l PLP in the wells during culture
RECOGNITION OF PLPAND MBP : PROLIFERATION ASSAYS
5.0
3.C
4.!6
~b. ~7;4.36
/
V /
C3 Z
z 2.0 0 I--
0.10). The stimulation indices of PBM from fewer mild disability-recent onset patients reached significance as cells from 3/15 (different patients for each protein) recognized each antigen. Note that three CPMS patients had PBM which recognized both antigens and all six patients in this group whose lymphocytes recognized PLP also had relatively high stimulation indices for MBP. This phenomenon was also generally true for the RRMS-S group. Only one individual of the O N D group showed sensitization to a myelin antigen and this 28-yearold patient, who had suffered anoxia at birth, demonstrated recognition of both MBP and PLP. Fig. 1 also demonstrates that the slopes of the lines connecting stimulation indices for MBP and PLP for each patient were not consistently directed positively or negatively, indicating that neither antigen consistently gave a higher stimulation index. However, note that the mean stimulation indices were slightly higher for MBP in the two control groups, although the difference was not statistically significant. Having demonstrated that some patients with MS recognize PLP, the studies were extended to examine in part the fine specificity of the T
TABLE 1 PBM P R O L I F E R A T I V E RESPONSES TO I N T A C T PLP A N D TO PLP PEPTIDES Stimulation indices ± SD. Subjects
PLP
CPMS-1 CPMS-2 CPMS-3 RRMS-1 RRMS-2 RRMS-3 NC-1 NC-2 NC-3 NC-4 NC-5 NC-6 OND
3.2±0.9 5.5±0.6 4.8±0.7 2.7±1.0 2.8±1.3 2.3±0.2 1.7±0.1 1.7±0.7 0.7±0.3 0.9±0.3 0.4±0.3 1.0±0.3 4.6±1.9
a
Amino acids b 88-108
103-116
139-154
3.4±0.4 4.4±0.4 1.7±0.6 0.9±0.0 3.2±0.6 1.0±0.4 0.8±0.3 0.9±0.2 1.0±0.4 0.8±0.3 0.5±0.3 1.3±0.2 10.6±2.6
3.5±0.8 3.9±0.6 2.3±0.8 2.5±1.1 4.0±1.5 6.9±2.0 0.9±0.1 0.7±0.2 1.1±0.3 0.5±0.4 0.9±0.1 1.1±0.3 7.4±2.5
2.1±0.2 6.1±1.4 1.3±0.2 1.8±1.0 6.0±1.6 9.0±1.8 0.6±0.1 0.5±0.4 0.4±0.3 0.5±0.5 0.6±0.2 1.0±0.2 3.0±1.3
The stimulation indices do not match those in Fig. 1, since these were separate experiments. Cultures without antigen incorporated 75 320 cpm. The concentration of PLP used to stimulate lymphocytes was 5 0 / ~ g / m l . b A m i n o acid sequence of peptides of PLP. The stimulation indices of PBM incubated with 3 t.tg/ml peptides are shown•
lymphocyte response. Three peptides of PLP that might have relevance to MS were selected for the in vitro assays on PBM. The peptides which have been demonstrated to be encephalitogenic in SJL (amino acids 139-154) (Tuohy et al., 1989) or SWR (103-116) (Tuohy et al., 1988) mice and another hydrophilic peptide (amino acids 88-108) overlapping the rabbit encephalitogen (Linington et al., 1990) were tested. Preliminary antigen titrations revealed the optimal concentration of peptides to be 3/~g/ml for PBM (data not shown). Further studies were carried out using frozen PBM from three CPMS and three RRMS-S patients who had demonstrated stimulation indices above 2.0 in response to PLP, as well as six normal and one O N D controls. The results (Table 1) demonstrate that each of the three peptides was recognized by T cells of certain MS patients, but none stimulated T cells from the normal controls (Table 1). Two MS patients (CPMS-1 and RPMS-2) recognized all three of the peptides, as did the neurological disease control (OND) who had suffered injury at birth.
59 TABLE 2 PROLIFERATION ASSAYS ON T CELL LINES SPECIFIC FOR PLP Line b 1A 1B 2A 2B 3 4 5 6 NC
NO antigen 177 + 212 + 96+ 55 + 208 + 426 + 203 + 330 + 212 +
28 50 35 6 21 31 80 160 75
PLP a 660 + 206 (3.7) 913 + 300 (4.3) 458 + 85 (4.8) 411 + 77 (7.5) 770 + 242 (3.7) 1 235 + 847 (2.9) 2400 + 5 (11.8) 1 748 + 109 (5.3) 770 + 305 (3.6)
Amino acids a 88-108
103-116
139-154
807 + 220 (4.6) 1 213 + 406 (5.7) 146 + 38(1.5) 106 + 13 (1.9) 734 + 208 (3.5) 291 + 10 (0.7) 353 + 106 (1.2) 245 + 75 (0.7) 160 + 33 (0.8)
826 + 260 (4.7) 2048 ± 502 (9.6) 164+ 83 (1.7) 592 + 132 (11.0) 274 + 28 (1.3) 324 + 57 (0.8) 166 + 79 (0.6) 524 + 291 (1.6) 350+ 30 (1.7)
365 + 55 (2.1) 408 + 52 (1.9) 428 + 19 (4.5) 1 005 + 291 (18.3) 629 + 22 (3.0) 241 + 436 (0.7) 343 + 90 (1.2) 198 + 108 (0.6) 164 + 95 (0.8)
a cpm + SD (stimulation index). All antigens were tested at three concentrations in quadruplicate (0.5, 1, or 3 #g/ml for peptides and 6.25, 12.5, or 25 gg/ml for the whole molecule) in addition to control cultures. The concentration with the highest stimulation index is shown. b The numbers represent six MS patients whose cell lines became PLP-specific. 'A' and 'B' are listed when both lines initiated fulfilled this criterion. 'NC' represents the cell line derived from a laboratory worker which became PLP-specific. Except for the laboratory worker (NC), the patients used for the generation of Table 2 were all 'new' patients, i.e., they are not represented in Fig. I or Table 1 and had not previously been tested for recognition of PLP.
Since we believed that direct testing of m o n o nuclear cells from peripheral b l o o d would u n d e r estimate the n u m b e r of subjects who h a d circulating cells that recognized PLP, we established T cell lines in the presence of PLP, in order to enrich a n y low-frequency cells recognizing this antigen. Preliminary experiments showed n o a d v a n t a g e in starting the lines in varying c o n c e n t r a t i o n s of antigen; however, we soon n o t e d that as the lines matured, the o p t i m a l c o n c e n t r a t i o n of P L P for proliferation of T C L s decreased relative to that of P B M from 50 g g / m l to 12.5 # g / m l , i.e., higher c o n c e n t r a t i o n s suppressed the response (data n o t shown). T cell lines (TCLs) from each of ten low disability R R M S patients a n d six l a b o r a t o r y workers in good general health were initiated i n duplicate. Twelve lines from the MS patients survived more t h a n 6 weeks. T e n of these were P L P specific (Table 2). Six lines from the control patients survived more t h a n 6 weeks. O n l y one of these was PLP specific. M a n y of the PLP-specific T cell lines recognized more than one peptide of P L P (Table 2). Different lines recognized different peptides, i.e., there was n o consistent p a t t e r n of peptide re-
sponse. Some of the lines did n o t r e s p o n d to a n y of the peptides tested.
Discussion W e have d e m o n s t r a t e d that some MS patients a p p e a r to have peripheral b l o o d m o n o n u c l e a r cells which recognize P L P as a n antigen. W e have ext e n d e d the studies to d e m o n s t r a t e recognition of more t h a n o n e p e p t i d e of P L P using either PBLs or TCLs. These results differ somewhat from a previous study e x a m i n i n g the same issue ( J o h n s o n et al., 1986), which required removal of C D 8 + cells before P B M recognizing P L P from MS patients could be detected. This m a y be due to technical factors, such as the physicochemical state of PLP, p a t i e n t s studied, or assay conditions. O u r s t u d y of T cell lines (TCLs) c o n f i r m e d our o b s e r v a t i o n of P L P recognition b y PBMs a n d generally d e m o n s t r a t e d more c o n v i n c i n g stimulation indices. W e have also f o u n d m u l t i p l e peptides recognized b y some MS patients in our simple proliferation assay a n d this was c o n f i r m e d using T cell lines. R e c o g n i t i o n of m u l t i p l e peptides has b e e n n o t e d previously i n the case of M B P i n MS
60 patients (Richert et al., 1989; Pette et al., 1990). The fact that there may be more than one peptide recognized may relate to 'secondary sensitization' (see below). Several studies have demonstrated that T cell lines and clones recognizing MBP using proliferation assays can be derived from the peripheral blood of most MS patients and controls (Burns et al., 1983; Chou et al., 1989; Richert et al., 1989a; Allegretta et al., 1990; Wucherpfennig et al., 1990). Recently, frequency analysis compared MBP T cell lines with lines derived by stimulating with PLP. There was a trend in this preliminary study (Ota et al., 1990) for there to be more reactivity to PLP in MS patients (compared to MBP lines) compared with O N D patients. In some systems, cytotoxicity assays appear to have much more restriction for peptide specificity (Richert et al., 1989b; Weber et al., 1989; Martin et al., 1990). We are currently performing experiments to determine if our lines are cytotoxic. This determination coupled with identification of HLA restriction elements of immunodominant epitopes will better define relevant epitopes. Our studies demonstrate that most patients recognizing PLP also have relatively high stimulation indices for MBP, suggesting that, if those epitopes are important components of an autoimmune response in MS, the disease may have a polyclonal pathophysiology. Recognition of multiple antigens is also suggested in the PBM and TCL peptide studies for some, but not all patients. One explanation for the recognition of multiple epitopes is that one or more antigens represents 'secondary' sensitization, i.e., are recognized as a consequence of the demyelinative process rather than being involved primarily in the cause of the demyelination. We attempted to diminish this possibility by studying the mild disability-recent onset population of RR patients during remission, since they might have fewer 'boosts' of 'secondary' sensitization. The one normal control recognizing MBP in the PBM studies (Fig. 1) had worked with this antigen in the laboratory for over a year and his TCL also recognized PLP (Table 2). Apparent sensitization to MBP by laboratory workers has been noted previously (Uyeda et al., 1976). One O N D patient (anoxic encephalopathy at birth)
had PBMs recognizing both antigens. This patient had suffered a one-time neurologic insult many years previously. This insult presumably did not represent a primary immunologic process, indicating that secondary sensitization to MBP and PLP is indeed possible. However, the presence of 'secondary sensitization', if it is present may not be trivial in MS patients with an immune system which could perpetuate the disease once sensitized. An alternative explanation for 'high' stimulation indices to both protein antigens tested may be that the individual patients react strongly to any antigen (e.g., due to genetic influence on lymphokine production). However, unpublished studies in our laboratory comparing the response to candida or tetanus antigens among the 'high' or 'low' responders to MBP or PLP suggest that this is not the case. Thus we believe that this trivial explanation does not account for our observation. One major factor that may have decreased the number of patients who showed sensitization to one or both antigens in our studies, is that a limited number of relevant cells may be found in the peripheral blood circulation of an MS patient at a given time. This is especially true of proliferation assays performed directly on PBM. Establishment of TCLs allows illumination of antigenspecific cells of lower frequency. Studies of longterm TCLs derived from cerebrospinal fluid or peripheral cells taken at multiple intervals over time with examination of overlapping peptides encompassing the entire PLP sequence may be necessary to establish the 'true' antigenic participation in the MS process. Overall, our findings strongly suggest that myelin proteolipid protein is recognized as antigenic by certain MS patients. Further investigations involving peptides encompassing the entire PLP molecule, with frequency analysis and a more extensive population study, with genetic factor analysis are required, however, to reveal the exact relationship of anti-PEP T lymphocytes to the pathogenesis of MS.
Acknowledgements The authors wish to express appreciation for the continuing patience of Mrs. Patti Nacci who
61 t y p e d the m a n u s c r i p t , a n d to t h e v a l u a b l e assistance of our project coordinator, Mrs. Jane Mclnnis. T h i s w o r k was s u p p o r t e d b y g r a n t s P P 0 1 3 7 a n d R G - 2 2 4 4 f r o m the N a t i o n a l M u l t i p l e Sclerosis Society.
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55
JNI 02014
Peripheral blood mononuclear cells from multiple sclerosis patients recognize myelin proteolipid protein and selected peptides John L. Trotter 1, William F. Hickey 2, Roel C. van der Veen 1 and Larry Sulze 1 Departments of I Neurology and 2 Neuropathology, Washington University School of Medicine, St. Louis, MO 63110, U.S.A. (Received 18 December 1990) (Revised, received 23 January 1991) (Accepted 11 February 1991)
Key words: Multiple sclerosis; Cellular immunity; T lymphocyte; Myelin proteolipid protein
Summary Myelin proteolipid protein (PLP) can induce a T cell-mediated chronic relapsing autoimmune encephalomyelitis in animals and therefore is a candidate for an antigen involved in the pathogenesis of multiple sclerosis. In this report, evidence is presented that peripheral blood mononuclear cells from certain multiple sclerosis (MS) patients recognize the intact PLP molecule as well as certain synthetic PLP peptides in proliferation assays. PLP-specific T cell lines could be obtained from six of ten MS patients with early relapsing-remitting disease. These lines recognized more than one PLP peptide and the relevant peptides differed among patients. The relevance of these observations to the pathogenesis of MS remains to be determined.
Introduction Two myelin-specific proteins, myelin basic protein (MBP) and proteolipid protein (PLP) are capable of inducing a chronic, often relapsing autoimmune disease in multiple species of animals (Waksman et al., 1954; Raine, 1984; Trotter et al., 1987). The pathology of the animal model, experimental allergic encephalomyelitis, often resembles multiple sclerosis (MS) and provides evidence that the human disease may have an autoimmune component (reviewed in McFarlin et al., 1982; Raine,
Address for correspondence: John L. Trotter, M.D., Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO 63110, U.S.A. Tel. (314) 362-3293.
1984). Of the two proteins, MBP was the first discovered to have encephalitogenic activity and several models of disease using this protein for induction have been widely studied (Raine, 1984). Evidence is accumulating that MS patients and, interestingly, many controls have peripheral blood T cells which recognize MBP in proliferation assays (Hughes et al., 1968; Colby et al., 1977; Johnson et al., 1986; Vandenbark et al., 1989). Recent studies have utilized knowledge of immunodominant epitopes of MBP and the amino acid sequence of T cell receptors involved in its recognition to provide specific immunotherapy for the MPB-induced inflammatory and demyelinating diseases in animals (Acha-Orbea et al., 1988; Howell et al., 1989; Sakai et al., 1989; Urban et al., 1989; Vandenbark et al., 1989; Wraith et al.,
56
1989). Since the only immunosuppressive therapies that have been tried in multiple sclerosis are nonspecific and have considerable side effects, the possibility of specific immunotherapies for this human disease is quite promising. With these considerations, the possibility that the relevant antigen in some patients with MS may be PLP led us to perform in vitro studies using peripheral blood mononuclear cells from MS patients and to establish PLP-specific T cell lines.
Materials and methods
Preparation of antigens Both myelin antigens were prepared from central nervous system human white matter obtained at autopsy within 18 h of demise. PLP was isolated using chloroform-methanol extraction, followed by washing with water and eventual column chromatography on Sephadex LH-60 (Hampson et al., 1986). The apoprotein was converted to the water-soluble form by solubilization with 2-chloroethanol and dialysis against water. MBP was prepared by the method of Diebler et al. (1972), followed by gel filtration on Sephadex G-150. The purity of the preparations was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblot, radioimmunoassay (Cohen et al., 1975) and lack of cross-reactivity when tested by SJL mouse T cell lines and clones (van der Veen et al., 1989, 1990). Synthetic peptides were prepared as previously reported (van der Veen et al., 1990). In brief, peptides were synthesized employing the FMOC technology for amino acid coupling using the RAMPS system and equipment (New England Nuclear/DuPont Corp., Boston, MA, U.S.A.). Three peptides were created corresponding to amino acid residues 88-108 (EGFYTTGAVRQIFGDYKTTIC), 103-116 ( Y K T T I C G K G L SATV), and 139-154 ( H S L G K W L G H P D K F V G I ) of the human protein sequence (Diehl et al., 1986). Patients and controls Heparinized whole peripheral blood was obtained from four groups of individuals. These
groups were: (1) Sixteen patients with definite multiple sclerosis (Poser et al., 1983) and a rapid chronic progressive course (CPMS). This group had a mean expanded disability status scale (EDSS) rating (Kurtzke, 1983) of 6.0_+ 1.0 and had received no cyclophosphamide at any time and no corticosteroid or other immunosuppressant for 6 months. (2) A group of 14 patients was selected for a diagnosis of definite MS with the onset of symptoms within 2 years and a low disability rating (mean 3.0 +_ 1.0). These patients had a relapsing-remitting course (mean of 1.0 _+ 0.3 exacerbations per year), but had been clinically stable for 6 months (RRMS-S). (3) A group of ten patients with other neurological diseases (OND) (stroke, Parkinson's disease, polymyositis, myasthenia gravis, anoxic encephalopathy at birth) were selected as disease controls. (4) A group of 12 laboratory and other hospital personnel served as normal controls (NC).
In vitro cellular studies on mononuclear cells direct from peripheral blood Peripheral blood mononuclear cells (PBM) were isolated on a discontinuous Ficoll-Hypaque gradient and washed. For the PBM studies the cells were frozen using standard techniques in heparinized RPMI 1640 medium with 10% human AB serum, 30% glucose and 10% dimethyl sulfoxide (DMSO). The thawed cells were 70-100% viable using trypan blue exclusion and responded to concanavalin A in the proliferation assay with a stimulation index of 20 or greater. Prior studies in our laboratory had determined that this degree of proliferation is within the range for freshly isolated PBMs. The antigen sensitivity assays were carried out in triplicate in a final volume of 0.2 ml in flat-bottomed microtiter plates. The cell concentration was 2 × 105/well in Hepes-buffered RPMI with 2% AB (male donor) serum with antibiotics. PBM were cultured for 6 days with PLP, MBP, synthetic peptides, or control media, and allowed to incorporate [3H]thymidine for the final 18 h. The cells were harvested on filter paper and the radioactivity determined in a liquid scintillation spectrometer. A stimulation index was determined by dividing the mean cpm incorporated in anti-
57 gen-stimulated cells by the mean cpm in cells cultured without antigen.
Establishment of lymphocyte lines recognizing PLP T cell lines (TCLs) were initiated using 5 x 106 mononuclear cells from fresh peripheral blood incubated in a 2 ml volume of R P M I containing 5% male donor AB human serum, with fresh glutamine, Hepes, antibiotics, and 5 0 / ~ g / m l PLP. The cells were incubated at 37 ° C for 7 days, washed twice and recultured with 5 x 106 irradiated (2500 R) autologous mononuclear cells with 50 /~g/ml fresh PLP. After 3 more days, recombinant interleukin-2 (IL-2) (50 U / m l ; Sigma Chemical Company, St. Louis, MO, U.S.A.) was added to the culture. Four days later, fresh feeders were again added, with 25 /~g/ml PLP, and IL-2. In the subsequent weeks, fresh feeders, PLP (12.5/~g/ml) and IL-2 were added weekly.
Proliferation assays on cell fines Proliferation assays were performed on cells which had been split from the lines (TCLs) after at least 5 weeks of culture and 'starved', i.e., they did not receive fresh PLP, feeders or IL-2 for 7 - 1 0 days; 2 x 104 cells/0.2 ml well of a flat-bottom plate were incubated with 1 x 105 irradiated autologous mononuclear cells in quadruplicate. 0, 6.25, 12.5 or 25/~g/ml PLP or 0, 0.5, 1 or 3 f f g / m l peptide were added to each well. After 72 h the wells were pulsed with [3H]thymidine for 18 h. The cells were harvested on filter paper and counted in a liquid scintillation spectrometer.
Results Preliminary studies using graded doses of 25, 50, and 100 f f g / m l PLP in the wells during culture
RECOGNITION OF PLPAND MBP : PROLIFERATION ASSAYS
5.0
3.C
4.!6
~b. ~7;4.36
/
V /
C3 Z
z 2.0 0 I--
0.10). The stimulation indices of PBM from fewer mild disability-recent onset patients reached significance as cells from 3/15 (different patients for each protein) recognized each antigen. Note that three CPMS patients had PBM which recognized both antigens and all six patients in this group whose lymphocytes recognized PLP also had relatively high stimulation indices for MBP. This phenomenon was also generally true for the RRMS-S group. Only one individual of the O N D group showed sensitization to a myelin antigen and this 28-yearold patient, who had suffered anoxia at birth, demonstrated recognition of both MBP and PLP. Fig. 1 also demonstrates that the slopes of the lines connecting stimulation indices for MBP and PLP for each patient were not consistently directed positively or negatively, indicating that neither antigen consistently gave a higher stimulation index. However, note that the mean stimulation indices were slightly higher for MBP in the two control groups, although the difference was not statistically significant. Having demonstrated that some patients with MS recognize PLP, the studies were extended to examine in part the fine specificity of the T
TABLE 1 PBM P R O L I F E R A T I V E RESPONSES TO I N T A C T PLP A N D TO PLP PEPTIDES Stimulation indices ± SD. Subjects
PLP
CPMS-1 CPMS-2 CPMS-3 RRMS-1 RRMS-2 RRMS-3 NC-1 NC-2 NC-3 NC-4 NC-5 NC-6 OND
3.2±0.9 5.5±0.6 4.8±0.7 2.7±1.0 2.8±1.3 2.3±0.2 1.7±0.1 1.7±0.7 0.7±0.3 0.9±0.3 0.4±0.3 1.0±0.3 4.6±1.9
a
Amino acids b 88-108
103-116
139-154
3.4±0.4 4.4±0.4 1.7±0.6 0.9±0.0 3.2±0.6 1.0±0.4 0.8±0.3 0.9±0.2 1.0±0.4 0.8±0.3 0.5±0.3 1.3±0.2 10.6±2.6
3.5±0.8 3.9±0.6 2.3±0.8 2.5±1.1 4.0±1.5 6.9±2.0 0.9±0.1 0.7±0.2 1.1±0.3 0.5±0.4 0.9±0.1 1.1±0.3 7.4±2.5
2.1±0.2 6.1±1.4 1.3±0.2 1.8±1.0 6.0±1.6 9.0±1.8 0.6±0.1 0.5±0.4 0.4±0.3 0.5±0.5 0.6±0.2 1.0±0.2 3.0±1.3
The stimulation indices do not match those in Fig. 1, since these were separate experiments. Cultures without antigen incorporated 75 320 cpm. The concentration of PLP used to stimulate lymphocytes was 5 0 / ~ g / m l . b A m i n o acid sequence of peptides of PLP. The stimulation indices of PBM incubated with 3 t.tg/ml peptides are shown•
lymphocyte response. Three peptides of PLP that might have relevance to MS were selected for the in vitro assays on PBM. The peptides which have been demonstrated to be encephalitogenic in SJL (amino acids 139-154) (Tuohy et al., 1989) or SWR (103-116) (Tuohy et al., 1988) mice and another hydrophilic peptide (amino acids 88-108) overlapping the rabbit encephalitogen (Linington et al., 1990) were tested. Preliminary antigen titrations revealed the optimal concentration of peptides to be 3/~g/ml for PBM (data not shown). Further studies were carried out using frozen PBM from three CPMS and three RRMS-S patients who had demonstrated stimulation indices above 2.0 in response to PLP, as well as six normal and one O N D controls. The results (Table 1) demonstrate that each of the three peptides was recognized by T cells of certain MS patients, but none stimulated T cells from the normal controls (Table 1). Two MS patients (CPMS-1 and RPMS-2) recognized all three of the peptides, as did the neurological disease control (OND) who had suffered injury at birth.
59 TABLE 2 PROLIFERATION ASSAYS ON T CELL LINES SPECIFIC FOR PLP Line b 1A 1B 2A 2B 3 4 5 6 NC
NO antigen 177 + 212 + 96+ 55 + 208 + 426 + 203 + 330 + 212 +
28 50 35 6 21 31 80 160 75
PLP a 660 + 206 (3.7) 913 + 300 (4.3) 458 + 85 (4.8) 411 + 77 (7.5) 770 + 242 (3.7) 1 235 + 847 (2.9) 2400 + 5 (11.8) 1 748 + 109 (5.3) 770 + 305 (3.6)
Amino acids a 88-108
103-116
139-154
807 + 220 (4.6) 1 213 + 406 (5.7) 146 + 38(1.5) 106 + 13 (1.9) 734 + 208 (3.5) 291 + 10 (0.7) 353 + 106 (1.2) 245 + 75 (0.7) 160 + 33 (0.8)
826 + 260 (4.7) 2048 ± 502 (9.6) 164+ 83 (1.7) 592 + 132 (11.0) 274 + 28 (1.3) 324 + 57 (0.8) 166 + 79 (0.6) 524 + 291 (1.6) 350+ 30 (1.7)
365 + 55 (2.1) 408 + 52 (1.9) 428 + 19 (4.5) 1 005 + 291 (18.3) 629 + 22 (3.0) 241 + 436 (0.7) 343 + 90 (1.2) 198 + 108 (0.6) 164 + 95 (0.8)
a cpm + SD (stimulation index). All antigens were tested at three concentrations in quadruplicate (0.5, 1, or 3 #g/ml for peptides and 6.25, 12.5, or 25 gg/ml for the whole molecule) in addition to control cultures. The concentration with the highest stimulation index is shown. b The numbers represent six MS patients whose cell lines became PLP-specific. 'A' and 'B' are listed when both lines initiated fulfilled this criterion. 'NC' represents the cell line derived from a laboratory worker which became PLP-specific. Except for the laboratory worker (NC), the patients used for the generation of Table 2 were all 'new' patients, i.e., they are not represented in Fig. I or Table 1 and had not previously been tested for recognition of PLP.
Since we believed that direct testing of m o n o nuclear cells from peripheral b l o o d would u n d e r estimate the n u m b e r of subjects who h a d circulating cells that recognized PLP, we established T cell lines in the presence of PLP, in order to enrich a n y low-frequency cells recognizing this antigen. Preliminary experiments showed n o a d v a n t a g e in starting the lines in varying c o n c e n t r a t i o n s of antigen; however, we soon n o t e d that as the lines matured, the o p t i m a l c o n c e n t r a t i o n of P L P for proliferation of T C L s decreased relative to that of P B M from 50 g g / m l to 12.5 # g / m l , i.e., higher c o n c e n t r a t i o n s suppressed the response (data n o t shown). T cell lines (TCLs) from each of ten low disability R R M S patients a n d six l a b o r a t o r y workers in good general health were initiated i n duplicate. Twelve lines from the MS patients survived more t h a n 6 weeks. T e n of these were P L P specific (Table 2). Six lines from the control patients survived more t h a n 6 weeks. O n l y one of these was PLP specific. M a n y of the PLP-specific T cell lines recognized more than one peptide of P L P (Table 2). Different lines recognized different peptides, i.e., there was n o consistent p a t t e r n of peptide re-
sponse. Some of the lines did n o t r e s p o n d to a n y of the peptides tested.
Discussion W e have d e m o n s t r a t e d that some MS patients a p p e a r to have peripheral b l o o d m o n o n u c l e a r cells which recognize P L P as a n antigen. W e have ext e n d e d the studies to d e m o n s t r a t e recognition of more t h a n o n e p e p t i d e of P L P using either PBLs or TCLs. These results differ somewhat from a previous study e x a m i n i n g the same issue ( J o h n s o n et al., 1986), which required removal of C D 8 + cells before P B M recognizing P L P from MS patients could be detected. This m a y be due to technical factors, such as the physicochemical state of PLP, p a t i e n t s studied, or assay conditions. O u r s t u d y of T cell lines (TCLs) c o n f i r m e d our o b s e r v a t i o n of P L P recognition b y PBMs a n d generally d e m o n s t r a t e d more c o n v i n c i n g stimulation indices. W e have also f o u n d m u l t i p l e peptides recognized b y some MS patients in our simple proliferation assay a n d this was c o n f i r m e d using T cell lines. R e c o g n i t i o n of m u l t i p l e peptides has b e e n n o t e d previously i n the case of M B P i n MS
60 patients (Richert et al., 1989; Pette et al., 1990). The fact that there may be more than one peptide recognized may relate to 'secondary sensitization' (see below). Several studies have demonstrated that T cell lines and clones recognizing MBP using proliferation assays can be derived from the peripheral blood of most MS patients and controls (Burns et al., 1983; Chou et al., 1989; Richert et al., 1989a; Allegretta et al., 1990; Wucherpfennig et al., 1990). Recently, frequency analysis compared MBP T cell lines with lines derived by stimulating with PLP. There was a trend in this preliminary study (Ota et al., 1990) for there to be more reactivity to PLP in MS patients (compared to MBP lines) compared with O N D patients. In some systems, cytotoxicity assays appear to have much more restriction for peptide specificity (Richert et al., 1989b; Weber et al., 1989; Martin et al., 1990). We are currently performing experiments to determine if our lines are cytotoxic. This determination coupled with identification of HLA restriction elements of immunodominant epitopes will better define relevant epitopes. Our studies demonstrate that most patients recognizing PLP also have relatively high stimulation indices for MBP, suggesting that, if those epitopes are important components of an autoimmune response in MS, the disease may have a polyclonal pathophysiology. Recognition of multiple antigens is also suggested in the PBM and TCL peptide studies for some, but not all patients. One explanation for the recognition of multiple epitopes is that one or more antigens represents 'secondary' sensitization, i.e., are recognized as a consequence of the demyelinative process rather than being involved primarily in the cause of the demyelination. We attempted to diminish this possibility by studying the mild disability-recent onset population of RR patients during remission, since they might have fewer 'boosts' of 'secondary' sensitization. The one normal control recognizing MBP in the PBM studies (Fig. 1) had worked with this antigen in the laboratory for over a year and his TCL also recognized PLP (Table 2). Apparent sensitization to MBP by laboratory workers has been noted previously (Uyeda et al., 1976). One O N D patient (anoxic encephalopathy at birth)
had PBMs recognizing both antigens. This patient had suffered a one-time neurologic insult many years previously. This insult presumably did not represent a primary immunologic process, indicating that secondary sensitization to MBP and PLP is indeed possible. However, the presence of 'secondary sensitization', if it is present may not be trivial in MS patients with an immune system which could perpetuate the disease once sensitized. An alternative explanation for 'high' stimulation indices to both protein antigens tested may be that the individual patients react strongly to any antigen (e.g., due to genetic influence on lymphokine production). However, unpublished studies in our laboratory comparing the response to candida or tetanus antigens among the 'high' or 'low' responders to MBP or PLP suggest that this is not the case. Thus we believe that this trivial explanation does not account for our observation. One major factor that may have decreased the number of patients who showed sensitization to one or both antigens in our studies, is that a limited number of relevant cells may be found in the peripheral blood circulation of an MS patient at a given time. This is especially true of proliferation assays performed directly on PBM. Establishment of TCLs allows illumination of antigenspecific cells of lower frequency. Studies of longterm TCLs derived from cerebrospinal fluid or peripheral cells taken at multiple intervals over time with examination of overlapping peptides encompassing the entire PLP sequence may be necessary to establish the 'true' antigenic participation in the MS process. Overall, our findings strongly suggest that myelin proteolipid protein is recognized as antigenic by certain MS patients. Further investigations involving peptides encompassing the entire PLP molecule, with frequency analysis and a more extensive population study, with genetic factor analysis are required, however, to reveal the exact relationship of anti-PEP T lymphocytes to the pathogenesis of MS.
Acknowledgements The authors wish to express appreciation for the continuing patience of Mrs. Patti Nacci who
61 t y p e d the m a n u s c r i p t , a n d to t h e v a l u a b l e assistance of our project coordinator, Mrs. Jane Mclnnis. T h i s w o r k was s u p p o r t e d b y g r a n t s P P 0 1 3 7 a n d R G - 2 2 4 4 f r o m the N a t i o n a l M u l t i p l e Sclerosis Society.
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