Major T-cell responses in multiple sclerosis

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Major T-cell responses in multiple sclerosis

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emerging that the major T- and B-cell multiple sclerosis (MS) is directed to a ifelinbasicprotein (MBP) between residues ~~en~p

t models of MS, immunization f MBP evokes experimental alomyelitis (EAE). T cells found hOW similarities in the VJ and VDJ alpha and beta chains with T cells in ;ions, and with T cells that are specificfor §4-103 isolated from patients with MS. If this region of MBP is critical in the pathogenesis of MS, then therapy aimed at controlling the immune response to this immunodominant region of MBP may be beneficial in treating MS.

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-Lawren e Steinman Department of Neurolo~ ciences, Beckman Center for Molecular: Genetic Medicine, Stanford Universil CA 94305, USA, and Deparn Biology, Weizmann Institute

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Ari Waisman Stanford University, CA, USA and Weizmann Institute of Science, Rehovot, Israel. Daniel Altmann Clinical Sciences Centre, Hammersmith Hospital, UK.

MULTIPLE sclerosis (MS) is a chronic inflammatory disease of the central nervous system. The disease is characterized by demyelination in the white matter of the brain and spinal cord. Lesions in the myelin sheath contain T ceils, B cells, macrophages and immunoglobulin. Pro-inflammatory cytokines, such as interferon ",/(IFN-3,), tumor necrosis factor a (TNF-a) and interleukin 6 (IL-6) are found at the site of inflammation and demyelination.The disease begins most often in early adulthood, and is characterized by episodes of paralysis, blindness, or sensory disturbances, which often remit. These episodes are often preceded by infectious illnesses. Over a period of 10 to 15 years, in about one quarter to one half Of affected patients, the disease progresses, resulting in increasing disability, including loss of ambulation, severe visual impairment and problems with autonomic function, including bowel and bladder control. The disease affects over one million individuals in North America and Europe. Two thirds of MS patients are female and certain HLA genes confer an elevated risk of contracting the disease (see Box 1). The identity of the antigens that provoke the inflammatory response in MS is unknown. Intensive searches for an infrctious agent that induces MS have failed to identify a single convincing candidate. Investigations on the autoantigens that elicit MS have centered on the components of the myelin sheath, including its fipids and myelin proteins. Neuroimmunologists have explored the specificity of the T- and B-ceil m

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response at the site of inflammation in the central nervous system (CNS), in the cerebrospinal fluid (CSF) that drains the brain and spinal cord, and in peripheral blood. Studies on the immune response to the major proteins of myelin have focused on myelin basic protein (MBP), which constitutes ~30% of the protein in myelin, myelin-associated glycoprotein (MAG), myelin oligodendroglial glycoprotein (MOG), transaldolase, 2',3' cyclic nucleotide 3' phosphodiesterases (CNP), and proteolipid protein (PLP), as the major targets in MS. Clear answers are emerging about the specificity of the immune response, including the detailed analysis of the T- and B-cell responses at the site of the disease.

Glossary Altered-peptide ligand - Once a specific antigenic peptide is

isolated that can stimulate a T cell, it is possible to modify the peptide and to use this altered peptide to induce tolerance. Discordant, concordant - I n studies on identical twins, individ-

uals can either be concordant for a trait; for example, both have MS, or they are discordant, with one having MS while the other does not. EAE - Experimental allergic encephalomyelitis. Immunizing an

experimental animal against a component of its own myelin will result in inflammation in the nervous system, often with demyelination and paralysis. Elispot assay - An assay combining enzyme-linked immune

reactions with histology, enabling one to enumerate cytokine production by T cells responding to a specific protein fragment. hu-SCID mice - These are mice containing a mutation in the

machinery for Ig and T-cell receptor gene rearrangements. When human lymphoid cells are engrafted into these mice, the ensuing chimeric animal is referred to as a hu-SClD. Immunodominant - The major response to a complex protein

M B P p 8 4 - 1 0 3 : the i m m u n o d o m i n a n t epitope in MS A group directed by Luciano Adorini, then at Sandoz (Basel, Switzerland), and now at Roche (Milan, Italy) and Alex Sette from Cytel (La Jolla, CA, USA) used a panel of overlapping 20-mers (1-20, I3-32, 23-42, etc.) to show that MBPp84-103 is the strongest binder of all MBP peptides to HLA class H molecules, and that it elicits the strongest response in peripheral blood T cells taken from patients with MS (Ref. 1). All MBP peptides bound some HLA-DR molecule with high or medium affinit3, (defined as Kn = 5-500 riM), but peptides 13-31, 84-103 and 144-163 bound with the highest affinity to several HLA-DR molecules (see Box 1). Widespread binding to several different HLA class H molecules was most evident for p84-103, which bound eight out of ten HLA-DR molecules tested. Moreover, the highest binding affinities were recorded for MBPp84-103. This peptide bound particularly well to DR molecules associated with increased susceptibility to MS, such as HLA-DRBI* 1501 (KD= 5 riM). Lower-affinity binding to DR2 alleles, which are not associated with increased susceptibility to MS, was also observed. These observations on the stronger binding of MBPp84-103 to certain alldes of DR2 may explain, in part, why these subtypes of HLA-DR2 confer a higher risk for development of MS than subtypes associated with weak binding. These investigators established 61 T-cell lines, which responded to 78 human MBP peptides from 20 patients with MS. They noted that 'two MBP epitopes are clearly immunodominant. One is included in the hMBP sequence 84-103, inducing 25/78 (32%) peptide-specific T-cell responses. A second immunodominant epitope, comprising 20/78 (25%) peptide-specific T cell responses, is located in the hMBP sequence 144-163 (Ref. 1). It should be noted that MBPp144-163 (VDAQGTLSKIFKLGGRDSRS) has a critical Ile-Phe-Lys sequence, and that ]]e-Phe is critical in HLA-DR2 binding for this epitope (see below). Hailer and colleagues (Harvard University, Boston, MA, USA) used a set of overlapping 20-residue oligomers from MBP to study the response of T-cell lines to MBP (Ref. 2). They studied 23 patients with MS and made 15 824 short-term T-cell lines. Of a total of 302 lines that could be expanded, 140 (46%) reacted with residues 84-102. In healthy individuals, only 11% (11/100 lines) reacted to this epitope. T-cell lines responding

antigen is directed to a single domain. In T cells, this is often a linear sequence of contiguous peptides, called the immunodominant epitope. Limiting-dilution analysis-This is a methodology for measuring the frequency of a T cell with a given specificity in a complex population. Cells are plated at various dilutions in microtitre plates. An arbitrary definition is given in order to score a well as positive but, commonly, this is a stimulation, measured by incorporation of radiolabelled thymidine, of three times above background. Estimation of the frequency of antigen-specific T cells is then calculated by applying the Poisson distribution, FN= (~/N!) × e-~, where FNis the probability of obtaining N-specific T cells per well, when u is the concentration of responding cells per well. Thus, when the mean number of responding cells per well is one, e-~= 37% of the wells are negative. Extrapolation to this point allows one to calculate the frequency of responding cells to a specific antigen. Membrane-attack complex -This is a supramolecular structure, which carries out the final phase of the complement cascade. It comprises C5b, C6, C7, (28, and a multimeric aggregate of C9. This supramolecular structure generates pores in membranes including the oligodendroglial cell.

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HLA is a cluster of genes located on the short arm of chromosome 6 that encodes several families of proteins. Susceptibility to multiple sclerosis (MS) is associated with certain HLA class II molecules, HLA-DR and HLA-DQ. These molecules are heterodimers comprising an alpha chain and a beta chain. With the exception of the HLA-DR alpha chain, these molecules are the most polymorphic proteins encoded in the human genome. The class I1 molecules have a cleft measuring ~10 x 10 x 20/~. that binds foreign molecules or selfconstituents. The polymorphisms encoded in the HLA complex are reflected in the diversity of amino acids fining this binding cleft. This permits certain HLA molecules to bind some polypeptides better than other HLA molecules. This difference in binding is critical for susceptibility to autoimmunedisease. The association between MS and HLA-DR2 is well established, particularly in Northern European Caucasoidpopulations, where certain DR2 types confer a four- to fivefold relative risk of developing MS. Nevertheless, even in identical twins who share this susceptibleHLA-DR2 subtype, only 50% are concordant for the disease. This implies that environmental factors are also critical for the developmentof MS.

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to MBPp61-82 and p124-142 were also isolated from patients with MS, though less frequently T cell than lines to p84-I02. Both patients and controls responded to p143-168. In this study, a family with one afflicted sibling was studied. Transmission of responsiveness to MBPp84-102 segregated with HLA-DR2 phenotype, indicating that this trait was linked to HLA-DR2. At the time they published their work in 1990, similar results were reported by Roland Martin and John Richert with Henry McFarland's group at the National Institutes of Health (NIH) (Bethesda, MD, USA) (Ref. 3), and by Hartmut Wekerle's group in Germany (Max Planck Institute, Martinsreid, Germany) (Ref. 4). Hailer has gone on to show an increased frequency of the T cells that are reactive to MBP p84-102 in the spinal fluid of patients with MS (Ref. 5). MBP-reactive T cells generated with stimulation with recombinant interleukin 2 (IL-2) accounted for 7% of the IL-2-responsive ceils, Figure 1. MHC and TCR contact sites for myelin basic protein peptide p84-103. The key MHC (blue) and T-cell receptor (red) contacts are shown for two HLA-DR2 subtypes associated with MS. which was more than tenfold higher than paired blood samples. These T cells selectively recognized MBPp84-102 and p143-168. By limitingdilution analysis, 7% of the T cells from the CSF were reactive to MBP. Hauser's group at the University of California, San Francisco (San This is extraordinarily high in an inflammatory infiltrate where most of Francisco, CA, USA) studied the response ofT cells to MBP in familial the T cells are recruited non-specifically. Such MBP-reactive T cells, with MS (Ref. 8). In this study, three families, each with several individuals specificity for p84-102, were not detectable in any CSF samples from affected by MS, were analysed. Although the paper fails to show an aspatients with other neurological disease (n = 7). These results demonstrate sociation of specific HLA types or TCR V regions with particular epitopes, that T ceils that are reactive to MBPp84-102 are dominant in the fluid there is solid confirmation of the immunodominance of MBPp82-101. Given the degeneracy of binding of MBPp84-102 to various HLA types draining the inflamed brain in MS. observed by Adorini and Sette I, this was not unexpected. The results of the study are fully compatible with the immunodominance of MBPp84--102 Cytoxic T cells in MS target MBPp84-103 Similar results have been obtained by the groups of Martin and when MBP clones are isolated from individuals, with and without MS. Richert at NIH and Georgetown (Washington DC, USA), and Wekerle at In Family one, all three members investigated had lines to MBPp82-101. the Max Planck Institute (Martinsreid, Germany) (Refs 3,4). In the Seven out of 12 T-cell lines isolated from the affected individuals were Martin study, 26 cytotoxic T-cell lines were derived from 22 patients with directed to this epitope. One line was made from the CSF of a patient, and MS and 16 T-cell lines were derived from normal individuals. Nine this was directed to MBPp82-101. In Family two, there was pronounced out of 17 cytotoxic T-cell lines tested from patients with MS, and six out diversity in the response to MBP. Even so, in three out of six patients, of seven lines from unaffected individuals recognized MBPp87-106, multiple lines were derived to MBPp82-101 and, in two patients, which was the highest frequency of any peptide tested. One of the cyto- p82-101 predominated. In Family three, two affected half-sisters were toxic T-cell lines recognizing MBPp84-102, utilized the same TCR studied. In one patient, multiple independent responses to different MBP Va and VI3 regions as TCR-gene rearrangements observed by our group epitopes were seen, including to p82-101. In patient two, the predominant in brain lesions of patients with MS and in EAE (Ref. 6). This implies response was to MBPp82-101, which occurred in 22 out of 32 lines. Both the immunodominance and the persistence of MBPp84-102 that T-cell responses to MBPp84-102 are occurring in MS lesions in the CNS, as well as in lesions of EAE. Six out of 15 T-cell lines from have been demonstrated in several other studies. Wekerle's group patients with MS, and four out of ten control samples, recognized MBP explored the diversity of MBP responses in 13 patients with MS (Ref. 9). p154--172, the second most common reactivity. In the work of Pette et al., In this study, the immunodominant MBP epitope was MBPp80-105 from Wekerle's group, T-cell lines reactive to MBPp80-99 were restricted (31% of the T-cell lines). Ollgoclonal and persistent and stable responses via the DRB 1' 1501 (DR2b) product. A systematic analysis of all clones for periods of two years were noted to MBPp80-105. A second group in Belgium 0hrdlems Instituut, Diepenbeek, Belgium), to overlapping synthetic peptides was not undertaken in that study4. directed by Jef Raus and Jingwu Zhang, studied T-cell vaccination in patients with MS (Ref. 10). Immunodominant clones, including clones M B P p 8 4 - 1 0 3 is immunodominant in twin pairs and to the two most dominant peptides p84-102 and p143-168, were selected multiplex families Investigations have been undertaken on pairs of twins with MS, and for vaccination. At a recent workshop on T-cell recep!or usage in human on families with multiple members afflicted with MS. Martin and col- and experimental demyelinating disease, held October 1994 in Leiden, leagues recently extended these fmdings on the immunodominance of The Netherlands, Zhang reported that the TCR VDJ[3 for the p84-102 MBPp84-102 in studies on identical twins discordant or concordant for clones, used for T-cell vaccination, was Ala-Arg-Gly-Ala-Asn, which is MS (Ref. 7). T-cell lines derived from these twin pairs showed the similar to the dominant TCR VDJ[3 motif reported in MS lesions 6. The same tendency to mount an immunodominant response to MBPp84-102. T-cell vaccination protocol led to a depletion of the MBPp84-102 m

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clones. Finally, Pozzilli's group (Rome, Italy), in collaboration with Wekerle's group, described the immunodominance of MBPp86-99 (Ref. 11). Two out of three patients who carried HLA-DR2 had predominant responses to MBPp86-99.

Structural requirements for binding of HLA-DR2 to MBPp84-103 Detailed investigations of the structural requirements for the binding of p84-102 to HLA class II molecules have been performed by a number of independent groups12-14.All of these studies are in strong agreement with the structural binding requirements for MBP to class II molecules, as described by Adorini, Sette and colleagues I. The requirement for Ph%oin the binding to HLA DRBI*1501 and DRB5*0101, the two DR[3 gene products of the HLA-DR2 haplotype associated with 75% of MS cases is of particular note (Fig. 1). Rothbard extended this requirement for Ph%oto two other HLA alleles, DRBI*0101 and DRBI*0401 (Ref. 15). It should be emphasized that those groups who studied the immune response to MBP using proteolytic cleavage products, clipped the molecule at its core binding site to HLA between residues Phes9and Ph%oI This observation suggests that the p84-103 fragment may be immunodominant because, when bound to HLA class II, it resists cleavage by proteases in the CNS.

B-cell epitope in MS

MOLECULAR MEDICINETODAY

One of the issues raised by the evidence in favor of p84-103 as a pathogenic epitope of MBE is the significance of responses to the other dominant region p139-162. Transgenic mice with HLA-DR2 alleles have been constructed. The pathogenicity of MBP epitopes in these DR and CD4 transgenic animals is under investigation. HLA-DR1 transgenic mice mounted a T-cell response to MBPp139-154, yet no pathology was seen in the CNS. Such a negative result does not rule out the potential pathogenic role of MBPp139--154 in MS, of course ~s. A new generation of experiments can be devised using these transgenic animals, including those made transgenic for CD4, TCR and HLA genes of interest. Other experiments involving transfer of MS T-cell clones into hu-SCID mice could illuminate the pathological significance of certain T-cell epitopes in MS.

Evidence against MBPp84-103 as the dominant epitope in MS T-cell clones that reacted to p84-103 were not seen in work by Ben Nun, Liblau and colleagues on French patients with MS (Ref. 19), nor by Kotzin, Vandenbark and colleagues on American patients with MS (Ref. 20). Both groups used proteolytic enzymes that cleaved MBP at Phe89_9oand, hence, the clones were targeted to pl-38, p45-89, p90-170, p149-170 and p149-162. Olsson and colleagues employed an Elispot assay to enumerate T cells that reacted to various epitopes of MBP (Ref. 21). They used a synthetic fragment, VI-IFFKNIVTPRTP,representing the dominant epitope that we had described in inbred SJL mice in 1988, p89-101 (Ref. 22). This residue binds the HLA class II molecules DRBI*1501 and DRB5*0101 with a five- to tenfold lower alfinity than p84-i02 (Ref. 12). Nevertheless, T cells reactive to this epitope were the most frequently represented of the six MBP peptides chosen, although the results were not statistically different from the other five MBP peptides. HLA-DR2 patients had the highest number of p89-101 T cells, although again there were no statistical differences in the frequency of these p89-101 T cells in patients with different HLA types. Repetition of these studies using the sophisticated Elispot technology developed by Olsson and colleagues is worth undertaking with the optimal MBP epitope, p84-103, in humans.

Further confirmation of the importance of p84-103 comes from a set of remarkable studies by Warren and Catz (Edmonton, Canada), on the specificity of antibody in the CSF of patients with MS and in brain material. In MS, a hallmark of the disease is the presence of oligoclonal bands of immunoglobulin (Ig) when CSF Ig from MS patients is electrophoresed. Before the days of magnetic resonance scanning, this was an important diagnostic test for MS. Furthermore, the synthesis of Ig in the CNS may be involved in the pathogenesis of MS. Immunoglobulin can be isolated from myelin lesions, and membrane-attack complexes can be found in the CSF of MS patients. As a consequence, the B-cell response to MBP in MS has been studied extensively. The majority of MS patients have Ig that reacts to MBP in the CNS. Recently, Warren and colleagues reported that 111 out of 116 patients with chronic progressive MS had anti-MBP titres in their CSF, the remaining five patients reactedto PLP. Therapeutic possibilities: tolerizing to the AtTanity-purified IgG from CNS lesions reacted with the same region of immunodominant epitope In 1988, McDevitt and our group at Stanford were able to block EAE MBP, p75-106, which is the imm~odominant T-cellepitope in MS (Ref. 15). In solid-phase radioimmunoassays using anti-MBP purified from by tolerizing with altered peptide ligands to the immunodominant epitope the CSF and brain tissue of patients with MS and in liquid-phase com- of MBP. Subsequent work at Stanford by Gaur and Fathman clearly petition with synthetic peptides of hMBP, the B-cell epitope was located demonstrated that if one tolerizes or suppresses responses to the immunobetween residues 61-106 for free antibody, and residues 75-106 for both dominant peptldes of MBP, then one can suppress disease induced by the free and bound anti-MBP. Similar results have been reported in an animal whole MBP molecule~zt. Further support of this concept comes from model system in which a major epitope for rabbit anti-bovine MBP Wauben et al., who demonstrated that, in the Lewis rat, tolerization to the was also associated with residues corresponding to bMBP 81-90. Rabbit immunodominant epitope p72-85 could prevent EAE induced by whole antisera to the residue MBPp81-90 did not bind native MBP, indicating MBP (Ref. 27). Recent studies from Karin and colleagues demonstrate that a soluble that antigen processing was critical in the induction of the response to this form of an altered peptide ligand for MBPp87-99 can reverse EAE in the part of the molecule. The discovery that both B- and T cells found in the CNS of patients Lewis rat, and downregulate production of TNF-et and IFN-3, (Ref. 28). with MS react to the same area of the MBP molecule emphasizes the These cytokines are critical in the pathogenesis of both MS and EAE. It involvement of this MBP segment in the pathogenesis of the disease. has been possible to reverse EAE induced with MBE and even with Although antibody responses to other myelin antigens, such as MOG, proteolipid protein, by infusion of a soluble seven-mer peptide that PLP and transaldolase are seen in the spinal fluid of some patients with induces tolerance to a single immunodominant peptide of MBE This MS, the widespread antibody activity directed to MBPp75-106 in over implies that, in MS, if one knew the immunodominant epitope, then toler90% of patients is noteworthy tt. Olsson's group has studied the specificity izing to this epitope with an altered peptide ligand might reverse the pathoof the antibody response in the CSF of MS patients. Antibody specific for genic process, as one is able to do in experimental animals. Moreover, this MBPp70-89 was predominant in the CSF of patients with MS. would allow the downregulation of subdominant immune responses to Unfortunately, in that study, MBPp84--103 was not checked, and over- other myelin antigens. This approach will be tested in patients with MS, lapping peptides covering the whole MBP molecule were not assessed 17. by inducing tolerance to MBPp84-102 with an altered peptide ligand. l

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Conclusions Several conclusions can be drawn from the studies reviewed here: (1) The role of other antigens such as MOG, transaldolase or PLP in the pathogenesis of MS is much less well understood than the evidence available for the role of MBP (Refs 28-30). Studies of the immune response to these other antigens of myelin should be pursued vigorously with sets of overlapping peptides. Responses to these antigens may arise from intemaolecular spreading of the immune response to MBP at the site of inflammation~6. (2) Peptide p84-I03 is the immunodominant T- and B-cell epitope of MBP in MS. (3) No major contradictory evidence disputes the demonstration that p84-103 is imrnunodominantfor MBP, when sets of overlapping peptides are used to map T-cell epitopes. (4) This epitope of MBP, p84-103, overlaps with the main antibody response observed in the brain and spina[ fluid of patients with MS. (5) Blockade of the immunodominant epitope will suppress ongoing EAE, in experimental animals, even when disease is induced by the whole MBP. A similar strategy might be developed in MS, enabling the control of MS, by targeting the inrnmnodominantepitope of MBP with altered peptide ligands3~.

The outstanding questions • By inducing tolerance to the dominant autoimmune response in multiple sclerosis (MS) directed to myelin-basic protein (MBP) p84-103, can one influence responses to other epitopes of MBP, and even to other myelin antigens? If so, then one might be able to treat MS by tolerizing to the dominant epitope, MBPp84-103. • Can induction of tolerance with altered peptide ligands to epitopes of MBP change the cytokine profile of responding T cells, transforrning a pathogenic response to a suppressive response? If so, then one might be able to turn off the production of pathogenic cytokines such as interferon 3' (IFN-3') and tumor necrosis factor (TNF) and, instead produce suppressive cytokines such as interleukin 10 (IL-10) and transforming ~owth factor 13 (TGF-13). • What are the major B-cell epitopes in MS, and what is the role of the antibody response to myelfla antigens in the pathogenesis of the disease? • Detailed epitope mapping of the response to other myelin ,antigens must be undertaken. These antigens include myelin oligodendroglial glycoprotein (MOG), myelin-associated glycoprotein (MAG), proteolipid protein (PLP), transaldolase, various manlmalian heat shock proteins, and 2',3' cyclic nucleotide 3' phosphodiesterases (CNP). • T-cell responses to lipid antigens must be studied.

Acknowledgements. Thisworkwas supportedby grantsfromthe NationalInstitutesof Health (NIH)and the UK MedicalResearchCouncil(MRC). References 1 Valli, A. et al. (1993) HLA class II moleculesand their recognitionby T cellsfrom

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