International Immunology, Vol 2, No 6

© 1990 Oxford University Press 0953 8178/90 $3 00

Lymphotoxin and tumor necrosis factor-alpha production by myelin basic protein-specific T cell clones correlates with encephalitogenicity

department of Neurology, Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA 2 Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA department of Epidemiology and Public Health, Yale University Medical School, New Haven, CT 06510, USA Key words: T cell clones, lymphotoxin, tumor necrosis factor

Abstract Lymphoklne activity in seven myelin basic protein (MBP)-speclfic T cell clones was examined. All of the clones recognize MBP peptide 1 - 9 in the context of I-A". A strong positive correlation was found between levels of lymphotoxin (LT) and tumor necrosis factor alpha (TNF-a) mRNA and biological activity on L929 cells and their capacity to Induce paralysis, the clinical hallmark of experimental allergic encephalomyelitls (EAE). No correlation was found between interleukin-2 or gamma Interferon production and encephalitogenicity. LT and/or TNF-a may play a central role in the pathogenesis of EAE. Introduction Immunization with the autoantigen myelin basic protein (MBP) induces experimental allergic encephalomyehtis (EAE) in susceptible mice. EAE is an autoimmune disease mediated by class IIrestricted T lymphocytes (1,2). T cell clones (L3T4 + , Lyt2-) isolated from PL/J and (PLSJ) F, mice immunized with MBP share the same fine specificity to the N-terminal nine residues of MBP in association with the same class II (I-Au) molecules of the MHC (3). Some of the MBP-specific clones mediate relapsing paralysis and demyelination, features associated with the human disease multiple sclerosis. However, not all of the T cell clones that are restricted to I-Au and that recognize the N-terminus of MBP induce EAE with paralysis. To further delineate the pathogenesis of EAE, we are searching for differences that must exist between the encephalitogenic and non-encephalitogenic cells. Recently, we examined a large number of these clones for reactivity with KJ16, a TCR-specific monoclonal antibody. KJ16 phenotype is associated with expression of V08.1 and V^8.2 genes (4,5). There was a predominant use of the TCR V^8 subfamily by these clones, even though only 16% of the peripheral L3T4+ T lymphocytes in PL/J mice

express the KJ16 phenotype (3). Southern analysis of gene rearrangement with TCR-specific probes for V^8 and joining (J^) regions demonstrated that both encephalitogenic and nonencephalitogenic clones use the same TCR V^ gene; but among the clones that mediate disease there are different DNA rearrangements using separate V - D - J combinations. cDNA sequencing of these clones showed that all of the cells utilize the same member of the Va4 gene family and the same J a element (JaTA31) except PJR7.5 (6). Similar findings have recently been reported by Urban and his colleagues with T cell hybridomas that have the same fine specificity and MHC restriction as those we have reported (7). With the information from the molecular studies, we utilized a V^S-specific monoclonal antibody as a treatment for experimental allergic encephalomyelitis induced with T cell clones, myelin basic protein, or its peptide fragments. The antibody therapy not only prevented the disease when given prior to induction but also reversed paralysis when the antibody was given after the mice were paralyzed (3,6). In this report we examine whether differences in lymphokine production by the activated MBP-specific T cell clones may be

Correspondence to: M. B Powell, The Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA Transmrtong editor: P Jones

Received 5 September 1989, accepted 19 March 1990

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Marianne Broome Powell 12 , Dennis Mitchell1, Jennifer Lederman1, Julie Buckmeier2, Scott S. Zamvil1, Mark Graham2, Nancy H. Ruddle3, and Lawrence Steinman1

540

LT and TNF-a in MBP-specific T ceils

correlated with induction or pathogenesis of autoimmune encephalomyelitis Seven MBP-specific T cell clones, that represent cells that cause different degrees of severity of clinical disease or do not induce clinical disease, were analyzed for lymphokine production upon activation with an MBP peptide or with the T cell mitogen Concanavalin A (Con A). Methods Antigens

T cell clones All of the T cell clones were isolated from PL/J or PLSJ F, mice immunized with intact MBP or synthetic peptides in complete Freund's adjuvant. The clones were maintained in RPMI media supplemented with 10% FCS, 5 x 10~5 M 2-mercaptoethanol, 2 mM glutamine, and 1 - 2 U/ml recombinant IL-2 (Cetus Corp.) as described (1). PJR-25, PJB-20, PJB-18, F1-21, and PJpR7.5 were isolated from cell lines derived from different mice. PJpR6.2 and PJpR6.4 were isolated from the same mouse; however, they are not 'sister clones' since these clones differ in their reactivity to V^8-specific antibodies (KJ16/F23.1) and in their patterns of DNA rearrangement (3). Proliferation assay Cloned T cells (1 x 10") were cultured with 5 x 105 irradiated (3000 rad) PL/J splenic APC in 0.2 ml culture media in 96-well, flat-bottomed microtiter plates. At 48 h incubation each well was pulsed with 1 /tCi [3H]thymidine ([3H]TdR) and harvested 16 h later. The mean c.p.m. thymidine incorporation was calculated for triplicate cultures. Induction of EAE Ficoll-separated cells (5 x 10 6 -10 7 ) were injected intravenously into recipient PLSJ F, or PL/J mice that had been given low-dose whole-body irradiation (350 rad) Animals were checked daily and graded for degree of disease Severity of EAE is graded as follows: 0, no sign of EAE; 1, decreased tail tone only; 2, mild paraparesis; 3, moderately severe paraparesis; 4, complete paraplegia; 5, moribund.

Assay for IFN activity The presence of IFN in the culture supernatants was assayed by the protection of L929 cells from the cytopathic effect of vesicular stomatitis virus as described by Dahl and Degre (10). IFN activity in the supernatants was compared with known concentrations of recombinant gamma interferon (Genentech). RNA analysis RNA was isolated by standard methods from T cell clones that had been induced with 5 /ig/ml Con A and 2 U/ml IL-2 for 8 h (11). RNA was analyzed both by slot blot and by gel electrophoresis. For slot blots, serial dilutions (10 /*g to 1 ^9) of total RNA were denatured in 6% formaldehyde/50% formamide at 50°C for 1 h and fixed onto Nytran. Electrophoresis was carried using 25 /tg RNA in a 1 % agarose - formaldehyde gel at 50 V for 5 h. After separation the RNA was transferred to a Nytran filter and baked at 80°C for 1.5 h. Hybridization and washing conditions were as described (12). Results In a previous study, Southern blot analysis and cDNA sequencing revealed that five clones, PJR25, F1 -21, PJpR2.2, PJpR6 2, and PJB20, have the same V^ and Va gene usage and exhibit the same V - D - J rearrangements (6). PJR25, however, is a highly encephalitogenic clone that causes moderate-to-severe disease in a period of 15-40 days when injected intravenously into mice; in contrast, F1-21 and PJB18 have never caused

Table 1. Induction of EAE by encephalitogenic T cell clones Induction of lymphokines T cell clones (2 x 106) were cultured with 1 x 107 APC and 13.3/xM pR1-11 peptide in 2.0 ml media for 48 h. Con A (5 /ig/ml) was added to a second set of cultured clones for 24 h prior to harvesting the culture supernatants. All supernatants were sterile filtered and frozen at - 70°C until assayed.

PJR-25 PJpR6 2 PJpR7 5 PJB-20 PJpR6.4 PJB-18 Measurement of interieukin 2 (IL-2) and inteheukin 4 (IL-4) activity F1-21

IL-2 and IL-4 were measured using the IL-2-dependent T cell line HT-2. HT-2 cells (104) were cultured with the experimental supernatants for 24 h before pulsing for 18 h with 1 /*Ci [3H]TdR. Thymidine incorporation by HT-2 cells cultured with known quantities of recombinant IL-2 were compared with the [3H]TdR incorporation stimulated by the T cell supernatants.

Incidence 40/40

2/3 3/3 2/3 0/3 0/19 0/13

Severity 3.5-5.0 1 0-2.5 1.0-1 5 1.0 — -

Day of onset 15-40 65 90 25 -

Induction of EAE by the encephalitogenic T cell done PJR-25 has been previously described (1). Recipient (PLSJ) F, or PLJ mice are given lowdose whole-body irradiation (350 rad) prior to an intravenous injection with 5 x 106 - 107 Ficoll-separated clones Each animal is checked daily and graded for degree of disease. Severity of EAE is graded as follows: 0, no sign of EAE; 1, decreased tail tone only; 2, mild paraparesis, 3, moderately severe paraparesis; 4, complete paraplegia; 5, moribund.

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N-terminal MBP peptides were synthesized according to the sequences for rat MBP (8) by solid-phase techniques. All peptides used in these studies were kindly provided by Jonathan Rothbard, Imperial Cancer Research Fund, London, UK. Peptide pR1-11 is Ac-ASQKRPSQRHG.

Assay for biological activity of LT/TNF-a The cytotoxic/cytostatic activity of LT/TNF in the supernatants was assessed on L929 murine fibroblasts L929 cells were used for this assay because they are not sensitive to the cytotoxic/cytostatic activity of IFN. T cell supernatants were added to 96-well plates seeded with 1 x 104 L929 cells per well. Fortyeight to 72 h after addition of the LT/TNF, the viability of the target cells was analyzed microscopically, and the surviving cells were enumerated with a Coulter counter (9). LT/TNF titers represent the reciprocal of the endpoint dilution that results in 50% inhibition of target cell growth with the Coulter Counter method. The assay was standardized with recombinant murine TNF (Genentech) and it measures equivalent units.

LT and TNF-a in MBP-spedfic T cells 541

PJR-2S io

natants with those cultured with known amounts of recombinant IL-2. IL-2/IL-4 activity was present in the supernatants of all five of the clones that were assayed (Table 2). The bioassay does not distinguish between IL-2 and IL-4; however, RNA isolated from activated clones was shown to hybridize with a murine IL-2 cDNA probe (pcD mlL-2, kindly provided by Dr Frank Lee, DNAX, PaJo Alto, CA, data not presented). It should be noted that LT in the supernatants can be inhibitory to the HT2 cells. We have demonstrated an inhibitory effect of LT on HT2 cells in earlier studies (13). Thus, the supernatants from clones that are high producers of LT/TNF may actually have higher amounts of IL-2 than we are measuring in our assay. The presence of interferon in the culture supernatants of the clones was detected by protection of L929 cells against the cytopathic effects of vesicular stomatitis virus. The encephalitogenic clone PJR25 and the non-encephalitogenic clone PJB18 both produced high levels of IFN (>200 U/ml; Table 2). Clone PJpR6.4 produced moderate levels of IFN. This lymphokine was only marginally detectable in the supernatants of clones PJpR7.5and PJpR6.2. These findings suggest that IFN production alone is not sufficient to cause clone-induced disease. Further, IFN production does not correlate with the degree of encephalitogencity. RNA isolated from all of the clones hybridized with an IFN-7 cDNA probe (pcD-IFN-7, kindly provided by Dr Frank Lee, DNAX; data not shown). The most striking result of these studies is that PJR25 produces much higher levels of LT/TNF-a than any of the other clones (224 U/ml). There is a strong correlation between the encephalitogenicity of a clone and its level of LT/TNF-a production. This observation was evident in both the assay where biological activity was measured (Table 2) and in compansons of the amount of LT/TNF-a RNA transcripts on slot blots (Fig. 2A and B). The cytotoxic and cytostatic activity of TNF-a and LT in the supernatants were evaluated on L929 cells. Similar levels of LT/TNF-a activity were detected in the supernatants when the cells were stimulated with either antigen or Con A. PJpR7.5, PJpR6.4, and PJpR6.2 produced comparable amounts of

4

Table 2. Lymphokine production by MBP-specificT cell clones

PJR-25 PJpR6.2 PJpR7.5 PJpR6 4 PJB-18

PJB-18 10*

.0067 .067 .67 6.7

«7

.0067 .0(7 .(7

6.7

67

Antigen concentration (pM)

Fig. 1. Act\^lionofMBP-specificTceflconeswithMBPpeptidepR1-11 The encephalitogenic T cell clones PJR-25 and PJpR6.2, and the nonencephalitogenic clones PJB-18 and PJpP.6.4 recognize the N-terminus of MBP in association with class II (I-Au) antigens. For the experiments represented in this figure, activation levels measured by [3H]TdR incorporation were compared when 1 x 104 cloned cells were cultured in 96-well flat-bottomed microtiter plates with various amounts of MBP peptide pR1 -11. The protocol for the proliferation assay is outlined in Methods All of the clones used in this report share the same specificity and have similar activation requirements.

LTn"NF-a(U/ml) 224 ± 64(128->256) 18 ± 9 (4-32) 8 ± 6 (0-16) 65 ± 4 (0-16) 2 ± 4 (0-8)

IFN (U/ml)

>200 4 4

40

>200

IL-2 (U/ml) 254 63 124 240 90

± ± ± ± ±

7 5 2 17 2

Supernatants from 2 x 106 cloned T cells cultured with 1 x 107 irradiated APC and 13.3/iM MBPpR1-11 were harvested 48 h after initiation. Con A (5 uglmf) induced culture supernatants were collected after 24 h. The supernatants were assayed for LT/TNF-a, IFN, and IL-2/IL-4 biological activities as described in Methods. Analogous results were obtained from either antigen- or Con A-activated cells for each rymphokine assayed. No LT/TNF-a, IFN, or IL-2/IL-4 activity was detected in supernatants from unstimulated T cell clones or in supernatants from cultured APC. LT/TNF-a U/ml is presented as the mean ±SD and in parentheses are given the range of units observed in four different tests The amount of IFN did not differ in two separate assays so SD are not presented. The IL-2 U/ml are given as the mean ± SD. The units for each assay were standardized against either recombinant murine TNF, IFN-7, or IL-2. Statistical analysis by ANOVA and by Wilcoxon rank sums showed that the LT/TNF-a produced by PJR25 was significantly greater than the amount of LT/TNF produced by the other clones (F = 0.0001).

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disease when injected into PL/J or PLSJ F, mice (Table 1). We examined three other clones that are mildly encephalitogenic, PJB20, PJpR6.2, and PJpR7.5. They have the same V^8 and Va4 gene usage as the PJR25 and F1-21, but PJR25 and PJpR7.5 use different J^ elements than PJB20 and PJpR6.2. PJpR7.5 also uses a different J a than any of the other clones studied (6). Another clone that we analyzed, PJpR6.4, did not stain with the KJ16 antibody and is non-encephalrtogenic. It does have germline V^8 but these genes are not rearranged or expressed (3). Finally, F1-21, a non-encephalitogenic clone, was analyzed. This clone has V a and V^ and J a and J^ sequences identical to PJB20 and PJpR6.2 (6). The production of lymphokines by these clones activated with antigen or Con A was assessed for lymphotoxin (LT or TNF-/3), tumor necrosis factor (TNF-a), interferon (IFN), and IL-2/IL-4 Quantitative comparisons of lymphokine production and RNA expression were made. Differences in levels of production were not due to different degrees of activation of the cells. Similar levels of stimulation assessed by [3H]TdR incorporation were observed when 1 x 104 cells of each clone were cultured with varying amounts of the synthetic rat MBP peptide pR1-11 (Fig. 1). Optimal stimulation was observed at the peptide concentration of ~ 6.7 - 1 3 /tM for all the clones except PJR 25. Maximal activation of this clone was demonstrated with 5- to 10-fold less antigen. All clones were also stimulated by the MBP nonapeptides, rat pR1-9, and bovine pB1-9 (3). Culture supernatants from both antigen-stimulated and Con A-activated clones were tested for biological activity in LT/TNFa, IFN, and IL-2/IL-4 assays. Supernatants generated on three separate occasions were examined and did not differ significantly from the representative results presented in Table 2. The amount of IL-2/IL-4 in the culture supernatants was determined by comparing [3H]TdR uptake by HT2 cells cultured in the super-

542

LT and TNF-a in MBP-specific T cells

PJR25

PJpR7.5 PJpR6.4

PJpR6.2

PJB18

L929

PJR25

PJB-20 -

-PJpR-2.2 18S-

L929 -

-F.-21

PJpR-6.2 -

-F.-21

PJB-18 -

- PJR-25

PJB-18 -

- PJR-25

Fig. 2. Expression of LT in Con A-stimulated MBP-specific T cell clones. RNA was isolated from T cell clones that had been induced with Con A and 2 U/ml IL-2 for 8 h and analyzed both by slot blot (A and B) and gel electrophoresis (C). For the slot blot (A), serial dilutions (10 ^g to 1 ^g) of total RNA were denatured 1 h and fixed onto Nytran. For slot blot (B), 5 ^g of total RNA were fixed onto the filters. Electrophoresis was carried out using 25 ^g of RNA in a 1 % agarose - formaldehyde gel at 50 V for 5 h. The slot blots and Northern blot were hybridized with a 32Plabeled 700 bp Kpn/Hinc\\ fragment of the mouse LT cDNA, pBSM7A. All of the blots were probed with a 7S DNA to confirm that equivalent amounts of RNA from each clone were loaded onto the blots. The position of the 18S ribosomal RNA indicated in (C) was determined by ethidium bromide staining. RNA from other clones was analyzed; however, only LT mRNA from PJR25 was expressed at a level that was detectable by the described conditions.

PJR25

PJPR64

PJpR6.2

PJB18

L929

DMZ1934

W ^^_

^•r^i

Fig. 3. Expression of TNF-a in Con A-stimulated MBP-specific T cell clones. The RNA slot blot described in Fig. 2(A) was hybridized with a P-labeled 1.2 kb EcoRI/Psd fragment of mouse cachectin (TNF-a) cDNA (kindly provided by Dr Bruce Beutler, University of Texas, Southwestern Medical School). DMZ1934 is a T cell hybridoma between PJR25 and BW5147.

32

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B

LT and TNF-a in MBP-speafic T cells 543

Discussion The results of these studies imply that LT and TNF-a may be contributing to the autoimmune disease experimental allergic encephalomyelitis (EAE). The high level of LT/TNF-a produced by PJR25 is correlated with both early onset and severity of the disease. LT/TNF-a can act synergistically with IFN-7 to enhance its cytotoxic effects (13,17). It may be that this combination of lymphokines produced by PJR25 is necessary for the cloneinduced disease. These studies demonstrate that the combination of IFN and IL-2 produced by the non-encephalitogenic clone PJB18 is not sufficient for the induction of EAE.

Our hypothesis that the high level of LT/TNF-a produced by the T cell clone, PJR25, is responsible for the early onset and severity of the clone-induced disease is also supported by research demonstrating that TNF activates endothelial cells, plays a critical role in inflammatory reactions, and causes destruction of target cells (13,18- 20). The interaction of TNF with endothelial cells could result in leakiness of the blood - brain barrier, thus allowing passage of lymphocytes into the CNS. Once in the CNS the release of lymphokines from the lymphocytes triggers a complex series of reactions. The cytotoxic activity of LT can cause the destruction of cells in the immediate area of release and thereby add to the development of a lesion. Brosnan, Raines, and their colleagues (21,22) have proposed that T cells that mediate EAE may also initiate demyelination. They have demonstrated that in organotypic cultures of mouse spinal cord, recombinant TFN-a causes damage to the myelin sheath and death of the oligodendroglial cell (23). Their findings with TNF-a should hold true for LT as well because the two molecules are functionally indistinguishable and share the same receptor. In EAE it appears that LT/TNF-a can act at two different stages, as an effector molecule involved in the inflammatory lesion or as an afferent molecule, facilitating passage of the T cells into the CNS. N. H. Ruddle and her colleagues (submitted for publication) currently are investigating whether clone-induced disease can be prevented by treatment with antibodies to LT and/or TNFa prior to clinical onset of the disease. In their studies the antibody TN3.19.12, which recognizes both LT and TNF (24) was able to reduce the incidence and severity of EAE in mice injected with MBP-specific T cells (N. H. Ruddle era/., submitted for publication). This therapy also may be effective in delaying progression of the disease. The efficacy of other antj-lymphokine antibodies treatment in another neurological disorder has been reported. Grau and his colleagues demonstrated that treatment of mice with anti-TNF-a antibodies blocked the development of cerebral malaria (25). In addition, it has been noted that strains of mice (C3H/HeJ) which do not produce TNF do not develop the malaria-induced neurological disorder. Understanding the role of lymphokines in autoimmune disorders of the CNS may provide new approaches for treatment of these diseases. Acknowledgements This work was supported by NIH Grant NS18235 (LS), the Swim Foundation (LS), the Rosenthal Foundation (LS), and NIH Grant CA 16885 (NHR).

Abbreviations EAE MBP Con A IL-2 (4) IFN LT TNF [3H]TdR

TCR

experimental allergic encephalomyelitis myelin basic protein Conconavalin A interleukin 2 (4) interferon lymphotoxin tumor necrosis factor [3H]thymidine T cell receptor

References 1 Zamvil, S. S.. Nelson, P , Trotter, J , Mitchell, D., Knobler, R., Fritz, R., and Stetnman, L. 1985 T cell clones for myelin basic protein induced

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cytotoxic activity against L929 cells (Table 2). LT/TNF-a activity in PJB18 supernatants was always the lowest in each assay, ranging from undetectable to 8 U/ml. Unstimulated T cells clones and antigen-presenting cells when cultured alone did not produce any detectable amount of LT/TNF-a. The bioassay alone does not allow one to distinguish between LT and TNF-o because these lymphokines have analogous cytotoxic and cytostatic activity on L929 cells. This was not previously considered a problem for the reason that LT had been reported to be a T cell product and TNF-a was though to be produced only by macrophages (9,14,15). More recently, however, Paul and Ruddle have shown that T cell clones can also produce TNF-a (16). In order to clearly identify the presence of these lymphokines, total RNA was isolated from the clones that had been activated for 8 h with Con A (5 /iCj/ml) and IL-2 (2 U/ml) RNA slot blots were prepared and hybridized with either a 32p.|abeled 700 bp Kpn/Hinc\\ fragment of the mouse LT cDNA, pBSM 7A (Fig. 2A and B) or with a 1.2 kb EcoRI/Psfl fragment of mouse cachectin (TNF-a) cDNA (Fig. 3). As predicted from the bioassay, there were significantly higher levels of LT mRNA expressed by PJR25. Clones PJpR7.5, PJpR6 4, and PJB20 had nearly the same amount of LT RNA. PJpR6.2 RNA weakly hybridized with the LT probe in Fig. 2(A) but when repeated in Fig. 2(B) it hybridized at a level similar to the three previously mentioned clones. LT RNA transcripts were not detectable in the RNA isolated from PJB18, F1-21, PJpR2.2, or the L929 mouse fibroblast cell line. L929 cells have been previously shown to be negative for LT expression (12). Hybridization of the 32P-labeled LT cDNA probe with PJR25 RNA on a Northern Wot detected a hybridizing band at ~ 15S (Fig. 2C). This is the reported transcription size for munne LT mRNA (12). The cDNA probe for TNF-a hybridized most strongly with PJR25 and PJpR7.5 RNA (Fig. 3). The level of TNF mRNA expressed by clone PJpR7.5, however, is not reflected by L929 killing. TNF produced by PJpR7.5 may not be secreted into the culture supernatant but may remain on the cell surface. The membrane-bound form of TNF is not detectable by the bioassay used for these studies. The TNF produced by PJpR6.4 may also be membrane-bound since very little biological activity was observed in the supernatants from this clone even though RNA from PJpR6 4 bound the TNF probe. Clone PJpR6.2 also expressed TNF mRNA. TNF transcripts were not expressed by the non-encephalitogemc clone PJB18 (Fig. 3). RNA isolated from DMZ 1934, a T cell hybrid between PJR25 and BW5147 (a munne thymoma), contained TNF transcript at a level similar to that observed for PJpR6.4 and PJpR6.2. RNA from the T cell hybrid did not hybridize with the LT probe.

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chronic relapsing paralysis and demyelination. Nature 317.355. Ruddle, N. H. 1987. Cloning and expression of murine lymphotoxin 2 Zamvil, S. S., Mitchell, D. J., Moore. A. C , Schwarz, A. J., Steifel, W., cDNA J. Immunol. 138:4496. Nelson, P. A., Rothbard, J. B., and Steinman, L 1987. T cell specificity 13 Powell, M B , Conta, B S., Horowitz, M and Ruddle, N H 1985 for class II (I - A ) and the encephalitogenic N-terminaJ epitope of the The differential inhibitory effect of lymphotoxin and immune interferon autoantigen myelin basic protein J. Immunol. 139'1075. on normal and malignant lymphoid cells. Lymphokine Res, 413 3 Zamvil, S., Mitchell, D. J., Lee, N E., Moore, A C , Waldor, M. K , 14 Beutler, B. and Cerami, A 1986 Cachectin and tumor necrosis factor Sakai, K., Rothbard, J., McDevitt, H O., Steinman, L, and Achaas two sides of the same biological coin. Nature 320584. Orbea, H. 1988. Predominant expression of a T cell receptor V0 gene 15 Old, L. J. 1985 Tumor necrosis factor. Science 230630 subfamily in autoimmune encephalomyelitis. J Exp. Med. 167 1586 16 Paul, N and Ruddle, N H. 1988 Lymphotoxin Annu Rev. Immunol. 4 Haskins, K C , Hannum, C , White, J , Roehm, N , Kubo, J. R , 6:407 Kappler, J., and Marrack, P 1984. The antigen specific, major 17 Stone-Wolff, D S., Yip, Y. P., Chroboczek Kelker, H., Le, J , Hennskenhistocompatibility complex-restricted receptors on T cells VI. An Destefano, D., Rubin, B. Y., Rinderknecht, E , Aggarwal, B. B , and antibody to a receptor allotype. J. Exp. Med. 160:452 Vilcek, J 1984. Interrelationships of human interferon-gamma with 5 Behlke, M. A., Henkel, T. J., Anderson, S , Lan, N , Hood, L , lymphotoxin and monocyte cytotoxm. J Exp Med. 159:828 Braciale, V., Braciale, T., and Loh, D. 1987. Expression of a munne 18 Cavender, D , Saegusa, V., and Ziff, M 1987. Stimulation of endothelia) polyclonal T cell receptor marker correlates with the use of specific cell binding of lymphocytes by tumor necrosis factor. J Immunol. members of the V/38 gene segment subfamily. J. Exp. Med 165257. 139:1855 6 Acha-Orbea, H., Mitchell, D., Timmermann, L, Wraith, D., Tausch, G., 19 Ruddle, N. H. 1987. Tumor necrosis factor and related cytotoxins. Waldor, M., Zamvil, S , McDevitt, H , and Steinman, L 1988 Limited Immunol. Today 8:129. heterogeneity of T cell receptors from lymphocytes mediating auto20 Seelentag, W. K., Mermod, J -J , Montesano, R and Vassalli, P. 1987 immune encephalomyelitis allows specific immune intervention. Cell Additive effects of interleukin and tumor necrosis factor-alpha on the 54:273 accumulation of the three granulocyte and macrophage colony7 Urban, J., Kumer, V , Kono, D., Gomez, C , Horvath, S , Clayton, J , stimulating factor mRNAs in human endothelial cells EMBO J. 6 2261 Ando, O , Sercarz, E., and Hood, L. 1988 Restricted use of TcR V 21 Brosnan, C. F., Selmaj, K., and Raine, C. S. 1988. Hypothesis- a role genes in murine autoimmune encephalomyelitis raises possibilities for tumor necrosis factor in immune-mediated demyelination and its for antibody therapy. Cell 54.577. relevance to multiple sclerosis. J Neuroimmunol. 1887. 8 Martenson, R E 1984 A useful model for multiple scterosts. In Alvord, 22 Lyman, W. D , Roth, G A., Chui, F.-C, Brosnan, C. F., Bornstem, E.C., Jr, ed , Experimental Allergic Encephalomyelitis, Vol 146, p. 511. M B , and Raines, C S 1986. Antigen-specific T cells can mediate Alan R Liss, New York. demyelination in organotypic central nervous system cultures Cell. 9 Conta, B. S., Powell, M. B., and Ruddle, N H 1983 Production of Immunol 102 217 lymphotoxin, IFN-gamma, and IFN-alpha, beta by murine T cell lines 23 Selmaj, K and Raine, C. S 1988 Tumor necrosis factor mediates and clones J Immunol. 130 2231. myelin and digodendrocyte damage in vitro Ann. Neurol. 23339. 10 Dahl, H and Degre, M. 1972 A micro assay for mouse and human 24 Sheehan, K C , Ruddle, N. H., and Schreiber, R. 1989. Generation interferon. Ada Pathol Microbtol. Scand. (B) 80 863. and characterization of hamster monoclonal antbodies that neutralize 11 Chirgwin, J. M , Przybyla, A. E., MacDonald, R J , and Rutter, W J murine tumor necrosis factors J. Immunol. 142:3884. 1979. Isolation of biologically active nbonucletc acid from sources 25 Grau, G. E , Fajardo, L F., Piguet, P.-F., Allet, B , Lambert, P.-H., enriched in nbonuclease Biochemistry 18.5294. and Vassalh, P. 1987 Tumor necrosis factor (cachectin) as an essential 12 Li, C.-B., Gray, P. W., Lin, P.-F , McGrath, K M., Ruddle, F. H., and mediator in murine cerebral malaria. Science 237.1210.

Lymphotoxin and tumor necrosis factor-alpha production by myelin basic protein-specific T cell clones correlates with encephalitogenicity.

Lymphokine activity in seven myelin basic protein (MBP)-specific T cell clones was examined. All of the clones recognize MBP peptide 1-9 in the contex...
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