Autoantigen-induced self lysis of human myelin basic protein-specific T lymphocytes

Journal of Neuroimmunology, 35 (1991) 227-236 227 © 1991 Elsevier Science Publishers B.V. All rights reserved 0165-5728/91/$03.50

JNI 02095

Autoantigen-induced self lysis of human myelin basic protein-specific T lymphocytes

James Burns 1, Kim Lit t lef ield ~, Janice Gill ~ and John Tro t t e r 2

I HA. Medical Center, Neurovirology Research, University of Utah School of Medicine, Salt Lake City, UT, U.S.A., and 2 Department of Neurology, Washington Uni~'ersity School of Medicine, St. Louis, MO, U.S.A.

(Received 16 April 1991) (Revised, received 30 July 1991)

(Accepted 31 July 1991)

Key words: Myelin basic protein; Multiple sclerosis; Autoimmunity; Cytotoxicity; Proteolipid protein

Summary

Cytotoxic T cells reactive with myelin basic protein (MBP) may be isolated from most human subjects. Since activated T cells express major histocompatibility complex (MHC) class II antigens, we assessed whether MBP-specific, CD4 + T cells could present MBP or synthetic MBP peptides to themselves and whether this provoked self lysis. We examined two MBP-specific cell lines and eight T cell clones recognizing four different MBP epitopes. All T cell populations presented MBP as well as synthetic peptides to themselves eliciting self lysis of the T cell clones. CD4 ÷ T cell populations recognizing another central nervous system (CNS) protein, proteolipid protein (PLP), or the recall antigen, Candida, did not exhibit this antigen-induced, autocytolytic activity. However, activated, PLP-reactive T cells were susceptible to lysis by cytotoxic MBP-specific T cells in the presence of MBP. These results suggest that antigen-induced self lysis of activated human T cells might limit an autoimmune response within a target organ independent of other immunoregulatory mechanisms.

Introduction

Human, CD4 + T cells generally are not cytotoxic although the immune response against mi- croorganisms such as Mycobacterium leprae or certain viruses is mediated in part by cytotoxic, major histocompatibility complex (MHC) class II-restricted T cells (Ottenhoff et al., 1988; Jacob-

Address for correspondence: Dr. James Burns, Neurovi- rology Research - 151B, V.A. Medical Center, 500 Foothill Drive, Salt Lake City, UT 84148, U.S.A.

son et al., 1989). The human T cell response directed against a major protein component of central nervous system (CNS) myelin, myelin basic protein (MBP), is also mediated predominantly by CD4 + cytotoxic T cells (Weber et al., 1988; Richert et al., 1989; Martin et al., 1990). While a cytotoxic T cell response against infec- tious agents may have some utility, the immunologic role of a cytotoxic response to MBP is less obvious since immunization with MBP induces an inflammatory autoimmune disease, experimental allergic encephalomyelitis (EAE) (Waksman and

228

Adams, 1962; Raine, 1984). In Lewis rats and S J L / J mice, EAE is mediated by MBP-specific CD4 + T cells that display cytotoxic activity and will lyse ceils capable of presenting MBP (Sun and Wekerle, 1986; Fallis and McFarlin, 1989). Although it is uncertain whether MBP is a target antigen for multiple sclerosis (MS), MBP is probably involved in the pathogenesis of the human disorder, postvaccinal encephalomyelitis, induced by vaccination with CNS antigen-containing rabies vaccine (Hemachudha et al., 1987).

The immunoregulatory mechanisms in humans that limit any potential autoimmune activity of MBP-specific T cells are unknown. In experimental animals, clonal deletion or anergy has been demonstrated for the response to certain antigens (Gammon et al., 1986; Kappler et al., 1987; Schwartz, 1990). However, MBP-specific T cells clearly are not deleted from the immunologic repertoire in most human subjects (Martin et al., 1990; Burns et al., 1991). Further, if MBP-specific T cells are anergic in vivo in humans, this anergy is very easily abrogated by relatively short term in vitro culture with MBP and interleukin-2 (IL-2) (Martin et al., 1990; Burns et al., 1991). Active suppressor mechanisms may play a role in regu- lating the response to MBP in experimental animals (Pesoa et al., 1984; Lider et al., 1988, 1989; Sun et al., 1988). In humans such mechanisms are controversial even though antigen nonspecific suppressor cells or suppressor-inducer T cells have been reported to be abnormal in MS patients (Antel et al., 1979; Morimoto et al., 1987; Sobel et al., 1988).

Human, activated T cells express MHC class I1 antigens and thus have the potential to act as antigen-presenting cells (APC). However, T cells generally are not capable of efficient antigen processing and only denatured antigen or small peptides can be presented to other T cells (Ger- rard et al., 1986; Hewitt and Feldman, 1989; Ottenhoff and Mutis, 1990). In the current study we noted that human, MBP-specific T cells can present MBP as well as specific MBP peptides to other MBP-specific T cells. Since these T cells are cytotoxic, presentation of MBP also provoked killing of the MBP-specific T cells by themselves. If autoantigen-induced self lysis occurs in vivo, a dampening of an autoimmune response might

occur within a target organ independent of other immunoregulatory, influences.

Materials and methods

Antigens Human MBP was prepared by the method o1

Deibler (1972). Since some batch preparations of MBP contain low molecular weight MBP breakdown products (Deibler et al., 1984), additional MBP was prepared by a modification of the above method adding protease inhibitors during certain steps of the purification procedure. Specifically, phenylmethanesulfonyl fluoride (1 raM) and eth- ylenediaminetetraacetic acid (1 mM) were added to aqueous wash and incubation solutions in the purification steps immediately following delipida- tion of the whole myelin. Synthetic, overlapping peptides duplicating the entire mouse MBP molecule were generously provided by Drs. Ku- mar and Gomez, Division of Biology, California Institute of Technology. The 16 peptides were synthesized using a solid phase technique on a Model 430A Applied Biosystems peptide synthe- sizer with sequences for all peptides confirmed after synthesis either by amino acid analysis or sequencing (Clark-Lewis et al., 1986). Previous studies have shown recognition of a wide range of these peptides by human T cell clones reactive with human MBP (Burns et al., 1991). Proteolipid protein (PLP) was isolated as previously described (Hampson and Poduslo, 1986; Van der Veen et al., 1990). Candida antigen was purchased as an extract of Candida albicans (Der- matophytin O, Miles, Elkhart, IN, U.S.A.), dia- lyzed, and used at a 5% dilution in proliferation assays.

T cell lines and clones T cell lines specific for human MBP and Can-

dida antigens were isolated from the peripheral blood mononuclear cells (PBMC) of two neurologically normal subjects by the in vitro sensitiza- tion method previously described (Burns et al., 1991). T cells specific for PLP were isolated by the same technique with PEP used at a concentration of 25 p,g/ml in the initial culture of freshly isolated PBMC. T cell lines and clones

were maintained in culture by use of IL-2 and periodic restimulation at 1- to 2-week intervals by irradiated PBMC (3000 Rad) and the appropriate antigen. T cells were cloned as previously described by limiting dilution in round-bottomed microwells at 0.3 T cells per well with 2.5 × 104 irradiated PBMC and phytohemagglutinin (PHA) 0.5/xg/ml (Burns et al., 1991). Cloning efficiency was usually 30-50%. MBP- or PLP-reactive T cells were used in antigen-induced proliferation or cytotoxicity studies between 7 and 14 days after the last restimulation by irradiated APC and antigen. T cell phenotype was determined by epifluorescent microscopy using a panel of monoclonal antibodies purchased from Ortho Pharma- ceutical Co., Raritan, N J, U.S.A. These included antibodies recognizing CD3, CD4, and CD8. Monoclonal antibodies reactive with the MHC class II antigens, HLA-DR, HLA-DQ, and HLA- DP were purchased from Becton-Dickinson, Mountain View, CA, U.S.A. The uncloned T cell lines reactive with MBP and PLP were greater than 95% OKT-3 positive and OKT-4 positive with less than 5% expressing OKT-8. Cloned T cells were less than 1% OKT-8 positive. T cell lines and clones examined for MHC class II expression 5 days following antigen restimulation were at least 60% positive for HLA-DR, -DQ, and -DP. MBP-specific clones 5 and 6 were noted to be restricted by HLA-DQwl through use of HLA-typed APC (not shown).

Antigen-induced proliferation Antigen-induced proliferation of the T cell

lines and clones was determined by culture of T cells with irradiated PBMC plus antigen without added IL-2. MBP-reactive T cells (1.5 x 104) were incubated with 1 × 105 irradiated autologous PBMC in 0.2 ml medium. MBP, synthetic peptides, or PLP was added to cultures at a concentration of 1 -2 /zM and proliferation measured by [3H]thymidine incorporation for the last 18 h of a 72 h culture.

Cytotoxicity assay Two types of cell populations were used as

targets in these experiments: (1) Epstein-Barr virus (EBV)-transformed B cell lines; and (2) T cells reactive with MBP, PLP, or Candida anti-

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gen. B cell lines were labeled with 5~Cr by incubation of 1 × 106 ceils in 0.2 ml medium with 300 /zCi 5~Cr for 1.5 h at 37°C. Two aliquots were used with one cell population incubated with 5~Cr plus antigen, and the other cell population in 51Cr medium alone. When T cells were used as targets, these were labeled with 5~Cr alone, without added antigen, for the initial step of the assay. Each cell population was then washed ex- tensively before counting and use in the cytotoxicity assay. 1 × 104 target cells were used in assays with B cell line and the T cell line targets. Tripli- cate cultures were established in round-bottomed microwells and the supernatants were collected after 4-6 h for B cell line targets and after 10-15 h for T cell targets. Spontaneous and maximal release were measure for all assays and used to determine the percentage specific release. For the 12-15 h 51Cr release assays, the lowest spontaneous release was obtained with culture media containing IL-2 and 10% human serum. With healthy T cell populations under these condi- tions, spontaneous release was usually not greater the 25% of maximal release.

Results

T cell lines recognizing MBP were isolated from two neurologically intact subjects. In addition, the following T cell populations were isolated from one of the two subjects: (1) eight T cell clones reactive against MBP; (2) four T cell clones reactive with Candida antigen; and (3) one T cell line specific for human proteolipid protein (PLP), another CNS antigen capable of inducing EAE. We and others have noted that MBP-specific human T cells will lyse EBV-transformed B cell lines that present MBP (Richert et al., 1989; Burns et al., 1991). Since activated T cells also can present antigens, we determined whether MBP-specific T cells could present MBP to themselves and whether this elicited autocytolytic activity.

Preliminary experiments suggested that T cell- T cell autocytotoxic activity could be measured after 6 h but that optimal killing occurred after 10-16 h (not shown). We also compared the extent of lysis induced by different effector : target

230

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Fig. 1. The degree of MBP-induced, T ce t l -T cell lysis is no t

proportional to the effector:target ratio. The effect of in- creasing effector:target ratios on the extent of MBP-induced lysis of a T cell clone and a B cell line was determined. MBP-pulsed B cells or MBP-reactive T cells were labeled with SICr and were incubated (1 × 1 0 4 / w e l l ) w i th C D 4 +, cytotoxic ME{P-specific T cells at the effector:target ratios indicated. Percent specific Iysis was measured in triplicate cultures as described in Materials and methods. Spontaneous release was

18% for the B cell line and 23% for the T cell clone.

ratios using either B cell lines or MBP-specific T ceils as targets. Specifically we compared the lysis of ] X 10 4 MBP-specific T cells incubated with MBP to the lysis of 1 x 10 4 autologous, MBP- pulsed EBV-transformed B ceils (Fig. 1). In experiments using the same MBP-specific T cell line as the 'effector' ceils, different effector : target ratios from 0 : 1 to 10 : 1 were studied. The results show that MBP-reactive T cells are subject to lysis by themselves in the presence of MBP and that this effect is not proportional to the effector : target ratio. Since these T cells are both targets and effectors, killing occurs as efficiently when 1 × 10 4 labeled 'targets' are incubated alone as when unlabeled MBP-reactive T cells are added as additional effector cells to the cultures. This pattern is in sharp contrast to the usual enhanced killing of B cell targets proportional to the number of cytolytic T cells added to cytotoxicity assays (Fig. 1). Additional experiments established that the intensity of cytotoxic activity is

proportional to antigen concentration for both MBP and synthetic peptides (Fig. 2). As shown in Fig. 2, the peptides often provoked a level of cytotoxicity at least as great as that seen with equimolar concentrations of whole MBP.

MBP-specific T cell clones isolated from this subject recognized four different epitopes within the MBP molecule as determined by proliferation assays using synthetic MBP peptides (Table 1). Autocytolytic activity and proliferation showed the same antigenic fine specificity as demonstrated by Fig. 3. In these experiments, 16 synthetic overlapping peptides spanning the entire MBP rnolecule were used to determine antigen specificity for proliferation and cytotoxic activity. Fig. 3 presents the proliferative and cytotoxic response to whole MBP by clone 3 and clone 7 with the response to each of the 16 synthetic peptides shown below the response to whole MBP. Clone 7 responded to whole MBP and to peptides (87-114) and (101-120) with proliferation (Fig. 3, left panel) and cytotoxic activity (Fig. 3, right panel). By comparison, clone 3 recognized MBP and peptide (11-34)wi th both prolif-

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ANTIGEN CONCENTRATION ( /~M )

Fig. 2. T ce l l -T cell lysis is proportional to the concentration of MBP or specific MBP peptide. 1 × 104 5~Cr-labeled T cells recognizing whole MBP and the MBP peptide (121 -140)were incubated in triplicate, round-bottomed mierowells with the indicated concentration of antigen. The values shown repre- sent the percent specific lysis. Spontaneous release was 2 0 %

of the total release.

TABLE 1

AUTOCYTOLYTIC ACTIVITY OF T CELL LINES AND CLONES

T cell-T cell autocytolytic activity was assessed for each of the T cell populations listed below as described in the legend to Fig. 1. Candida antigen was used at a 5% dilution and PLP at a 1 /~M concentration.

T cell Antigen Percent specific population specificity autocytolytic killing

MBP-specific Clone 1 MBP (151-168) 71 Clone 2 MBP (11-34) 45 Clone 3 MBP (11-34) 41 Clone 5 MBP (101-114) 56 Clone 6 MBP 55 Clone 7 MBP (101-114) 51 Clone 8 MBP (121-140) 47 Clone 9 MBP (121-140) 34

Candida-specific Clone 1 Candida - 2 Clone 2 Candida - 1 Clone 3 Candida 4

PLP-specific Cell line Human PLP 1

231

eration and cytotoxic activity. No cytolytic activity or proliferation was elicited for clone 3 by peptides (87-114) or (101-120) as noted with clone 7. All MBP-specific T cell clones showed some degree of autocytolytic activity as noted in Table 1. Three Candida specific T cell clones displayed no autocytotoxicity.

LaSalle and colleagues (1991) recently reported that MBP-reactive T ceils were capable of presenting batch-purified MBP but not high per- formance liquid chromatography (HPLC)-puri- fled MBP to other T cells. This observation suggests that intact MBP could not be processed and presented by T cells. Thus the antigens in the batch-purified MBP presented by T cells may be the lower molecular weight MBP breakdown products often found in these preparations. To determine the purity of the MBP used in the current study, a sodium dodecyl sulfate-poly- acrylamide gel electrophoresis (SDS-PAGE) analysis was performed as shown in Fig. 4, column B, showing some breakdown products as we have previously noted (Burns et al., 1989). Also

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PROLIFERATION ( CPM X 10 -3 ) PERCENT SPECIFIC LYSIS

[ ~ Clone 7

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Fig. 3. Antigenic fine specificity of MBP-reactive T cell clones for induction of proliferation or cytotoxic activity. The antigenic fine specificities of T cell clone 3 and clone 7 were determined by use of overlapping synthetic peptides duplicating specific portions of MBP. For induction of proliferation, 1 × 104 T cells were cultured with 1 × 105 irradiated PBMC in triplicate microwells with 1 /xM concentration of MBP or the synthetic peptide indicated. Proliferation was determined by [3H]thymidine incorporation following a 72 h culture; background was below 900 cpm. T ce l l -T cell cytotoxic activity was assessed by culture of 1 x 104 51Cr-labeled T cells in round-bottomed microwells with 1 / z M of MBP or synthetic peptide. Isotope release was measure after 14 h

and the percent specific lysis determined; spontaneous release was 26% of total release.

232

A B C

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Fig. 4. SDS-PAGE of MBP preparations used in T cell autocytolytic assays. Lane B shows the results of electrophoresis of MBP prepared by the batch method described by Deibler (1972). The MBP shown in lane C was prepared by a modification of this method using protease inhibitors as described in Materials and methods. Equal amounts of MBP were used in the lanes shown. Molecular weight markers are

shown in lane A.

shown in Fig. 4, column C, is a similar analysis of MBP prepared by a method incorporating protease inhibitors in the purification protocol as described above. This preparation shows virtually no breakdown products by SDS-PAGE analysis. In three separate cytotoxicity assays, similar levels of T cell autocytolytic activity were observed using these two MBP preparations at identical concentrations (not shown).

PLP is the major protein component of CNS myelin and will also induce EAE (Sobel et al., 1986; Tuohy et at., 1988; Van der Veen et al., 1989). Therefore, we isolated a PLP-specific T cell line from subject 2 to determine whether autocytolytic activity was a characteristic of T cells reactive with another CNS autoantigen. As shown in Table 2, when PBMC were used as APC, the PLP-specific cell line proliferated in response to PLP at a 1 / ,M concentration but did not respond to MBP. Similarly, an MBP-reactive T cell line responded solely to MBP and did not recognize PLP. In Fig. 5, PLP-reactive T cells were used as target cells to determine whether

"FABLE 2

SPECIFICITY OF CNS AUTOANTIGEN-REACTIVE T CELLS

Uncloned T cell lines (1 × l() 4) recognizing MBP or PEP were incubated with 1 × 10:' irradiated PBMC as described in the legend to Fig. 3. MBP or PEP was added at the indicated concentration and proliferation determined after 72 h.

Specificity of cell line

Antigen present in culture

None MBP (30 # g / m l ) PLP (25 #g / ro l l

Proliferation (cpm [~H]thymidine)

(1) MBP 778 34,104 589 (2) PLP 3,778 3,995 26,187

these would be lysed by PLP-specific effector T cells in the presence of PLP. We also determined whether PLP-specific target T ceils would be lysed by MBP-reactive effector T cells in the presence of MBP. As shown in Fig. 5, when PLP-specific target T cells were incubated with PLP and PLP-reactive effector T cells, no autocytolytic activity was apparent. However, when

EFFECTOR T CELLS ANTIGEN ~ 0 20 30 40 50 60 70

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PLP PLP ~

MBP NONE ~

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PERCENT SPECIFIC LYSIS OF PLP REACTIVE T CELLS

Fig. 5. Absence of autocytolytic activity of PLP-specific "l- cells. PEP-reactive T cells were used as target cells to determine whether these were lysed by PLP-specific effector 1- cells when incubated with PLP. In addition, lysis of PLP-reactive target T cells by MBP-reactive effector T cells when incubated with MBP was assessed. 5~Cr-labeled, PLP-specific T cells (1 × 104) were incubated for 14 h in round-bonomed microwells with either PLP or MBP and 5 × 104 unlabeled "I cells reactive with either MBP (clone 7) or PLP. Isotope release was measured and the percent specific lysis was deter-

mined for triplicate cultures.

PLP-specific target T cells were incubated with both MBP-reactive effector T cells and MBP, the PLP-reactive cells were susceptible to lysis. No lysis occurred when MBP alone was added without effector T cells present. In experiments that are not shown, B cell lines were not able to present PLP to PLP-specific T cells in either proliferation or cytotoxicity assays. Since the same B cell line could present MBP and Candida in proliferation assays, this suggests that this B cell line was not fully competent for antigen-presentation of PLP. Thus we were not able to determine in these experiments whether PLP-reactive T cells possess cytolytic activity.

Discussion

This study demonstrates a T ce l l -T cell interaction that potentially may down-regulate an on- going autoimmune response within tissue containing the target antigen. CD4 +, cytolytic MBP- specific T cells can present MBP and specific MBP peptides to themselves. Since these are cytotoxic T cells, this antigen presentation pro- vokes autoantigen-induced killing of these MBP- reactive T cells. Antigen-induced proliferation and autocytolytic activity occur at the same antigen concentrations and with the same antigenic fine specificity. Among the eight MBP-specific T cell clones and two T cell lines tested, all displayed autocytolytic activity while other cell lines reactive with a common recall antigen, Candida, or another CNS protein, PLP, did not display this antigen-driven, autocytotoxic activity.

Other investigators have speculated that active suppression may be a function of class II-restricted, cytotoxic T cells (Lanzavecchia, 1989). In some instances dampening of immune responsiveness by cytotoxic T cells represents specific targeting through receptor-mediated uptake by target cells of antigen recognized by cytotoxic T cells (Shinohara et al., 1988; Siliciano et al., 1988). An example of this is the very efficient killing of keyhole limpet hemocyanin (KLH)-specific B cells by KLH-reactive, cytotoxic T cells at concentrations of KLH 1000-fold lower than needed for other MHC class II positive targets (Shinohara et al., 1988). Alternatively an antigen-nonspecific ly-

233

sis of antigen-presenting cells in the target organ could also down-modulate responses by impairing antigen-presentation that may have a role in per- petuating an immune response. Since an autologous T cell population not reactive with MBP was also subject to lysis, the T ce l l -T cell interaction reviewed in this study falls in the latter category of nonspecific killing of MHC class II positive antigen-presenting cells. However, the novel as- pect of this T cell killing is that the cytotoxic, autoantigen-reactive T cell population is itself subject to lysis.

Autopresentation of MBP by T cells to T cells also may interfere with immune responsiveness through induction of T cell anergy (Lamb et al., 1983; Schwartz, 1990). This possibility is based on the observation that T cells may be 'defective' antigen presenters providing incomplete signaling for full activation of T cells (Schwartz et al., 1990). 'Defective' antigen presentation by human T cells has been noted to induce tolerance or anergy thus limiting restimulation of T cells by antigen and fully competent antigen-presenting cells (Lamb et al., 1983). In a previous study we also noted that autopresentation of MBP to MBP-specific T cells induces a short-term tolerance to antigen restimulation while the response to IL-2 remains intact (Burns et al., 1986). Thus in vitro autopresentation of MBP by T cells can lead to self lysis of a portion of MBP-specific T cells and possibly generates tolerance to restimulation in some of the remainder. For these mechanisms to be active in vivo, an established inflammatory lesion with myelin breakdown is probably required. Therefore, these consequences of autopresentation of MBP by activated, cytotoxic T cells might help to restrict the extent of autoimmune-mediated damage within the CNS but other mechanisms must maintain self tolerance to pre- vent the initial occurrence of such lesions.

Other investigators have noted that activated T cells can present antigen (Gerrard et al., 1986; Hewitt et al., 1989; Ottenhoff et al., 1990). Anti- gen presentation appears to depend on the expression of MHC class II antigens by T cells although class II expression alone is not always sufficient for antigen presentation. For example, Hewitt et al. (1989) noted that human T cells could present synthetic peptides of influenza but

234

not whole influenza hemagglutinin. Ottenhoff et al. (1990) recently reported that synthetic peptide autopresentat ion by human, cytotoxic CD4 + T cells can result in lysis of a T cell population by itself. The T cells studied by these investigators recognized the mycobacterial antigen, heat shock protein, and could present synthetic peptides but not the whole antigen (Ottenhoff et al., 1990). This inability of T cells to present intact antigen is presumably due to inadequate conventional antigen processing by T cells. Preliminary data reported by LaSalle and colleagues (1991) suggests that human T cells are not able to present intact MBP although small MBP peptides can be presented to other human T cells. By comparison, the MBP-specific T cells examined in this report were able to present whole MBP as well as synthetic MBP peptides. The reason for this dispar- ity in results may be the presence of small amounts of MBP breakdown products in our MBP even though these were not obvious by SDS- P A G E analysis of the MBP batch prepared using protease inhibitors. Alternatively there may be different processing requirements for different epitopes of MBP or variations in the ability of T cell populations to process MBP. This distinction might not be critical in vivo since numerous pro- teases may be released within inflammatory CNS lesions producing extracellular proteolysis not only of MBP but possibly for other CNS autoanti- gens as well.

Although MBP is the CNS protein most often used to induce EAE, PLP also induces this disorder in a variety of experimental animals (Sobel et al., 1986; Tuohy et al., 1988; Van der Veen et al., 1989). PLP-induced EAE also appears to be mediated by CD4 + T cells and the isolation of PLP-reactive T cells from humans has been described (Martin et al., 1989; Ota et al., 1990). In the current study we did not observe autocytolytic activity with PLP-specific T cells. This difference between PLP- and MBP-reactive T cells could result from either a lack of cytotoxic activity in the PLP-specific population or inadequate processing or presentation of PLP by T cells. Prelim- inary data suggest that inadequate processing or presentation may be responsible since this subject 's B cell line was able to present MBP and Candida but not PLP in proliferation assays. Thus

antigen processing requirements may be more stringent for PLP than for MBP. Since this subject 's B cell line did not present PLP, the cytotoxic activity of the PLP-reactive T cell line was not assessed.

The major implication of this study is that activated, autoantigen-specific, cytolytic T cells may participate in the down-regulation of autoimmune responses independent of systemic influences. For this to occur, the autoantigen-reactive cells must possess cytolytic activity and the autoantigen must be present in a form easily presented with minimal processing by activated T cells. In recent MS clinical studies, magnetic resonance imaging of CNS myelin lesions demonstrated regression of some lesions occurring si- multaneously with the appearance and progres- sion of others (Kesselring et al., 1989). This suggests that local and not systemic events may gov- ern the extent of myelin lesions in MS. Overall the current study proposes a mechanism by which class II-restricted cytotoxic T cells might influ- ence the modulation of immune responsiveness.

Acknowledgements

This work was supported by grants from the National Multiple Sclerosis Society (RG 1894-A to J.B. and PP 0137 to J.T.), the NIH (NS 27556), and by Veteran Administration Research Funds. We would like to thank Dr. V. Kumar for critical review of the manuscript and Mr. Ken Hill for assistance with MBP preparat ion and electrophoresis studies.

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Autoantigen-induced self lysis of human myelin basic protein-specific T lymphocytes