iiiiiii!!iiiiii!iii!iiiiiiiiiiii!ii iiiIiiiiii!iiii!ii!iiiiiiiiii!i
T-cell recognition of myelin basic protein Kai W. Wucherpfennig, Howard L. Weiner and David A. Hailer Multiple sclerosis is a chronic inflammatory disease of the central nervous system which has been hypothesized to be autoimmune in nature. To test whether this is the case, Kai Wucherpfennig and colleagues have developed a set of criteria that must be met to satisfy the hypothesis. Here, they present these criteria and assess the extent to which studies to date satisfy them. Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system characterized by focal T-cell and macrophage infiltration into white matter. The inflammatory lesions are distributed around small venules in the white matter, preferentially in the spinal cord, cerebellum and around ventricles. This pathology is frequently associated with neurologic dysfunction referable to these areas of inflammation, in particular paralysis, sensory deficits and visual problems 1. Studies on twins suggest that there is a strong genetic component to MS susceptibility2,3: monozygotic twins have a more than 10-fold higher concordance rate for MS than dizygotic twins while the concordance rate among dizygotic twins is similar to that of siblings 3. Two major theories of pathogenesis of MS have been proposed: one, that MS is a viral disease of the central nervous system (CNS), the inflammatory response in the brain being an anti-viral immune response, and two, that MS is an autoimmune disease in which infiltrating T cells recognize self antigens and attack normal tissue. These two possibilities are not mutually exclusive: the autoimmune response may be triggered by environmental factors such as viral infections. However, the inability to transfer the disease to primates or to isolate virus from CNS tissue of MS patients, despite tremendous efforts, lends indirect support to the theory that the inflammatory process may be autoimmune in nature 4. The critical experiment that would prove this, namely the transfer of antigen-specific T cells to a major histocompatibility complex (MHC)-identical recipient, is impossible to perform in humans. A second approach to define the potential role of myelin-reactive T cells in MS was thought to be the isolation of autoreactive T cells from patients and the determination of their antigen specificity. However, this cannot define the role of myelin-reactive T cells in the disease since cells of the same specificity can also be cultured from the blood of normal individuals s,6. To address conclusively the role of autoreactive T cells in MS, we have defined a set of criteria that can be tested (Box 1)7. If these cells are pathogenic it should be possible to demonstrate that disease susceptibility is associated with immune response (Ir) genes involved in the recognition of the autoantigen(s). Specifically, MHC and T-cell receptor (TCR) gene products that define susceptibility to MS should be involved in the immune recognition of the autoantigen. Autoreactive T cells should be activated in patients or should have undergone clonal expansion in vivo and the specific elimination of auto-
reactive T cells or tolerance induction to the autoantigen will improve the clinical course of the disease (Box l). In this review these postulates are used as a basis to examine the pathophysiology of MS. Postulate 1: association of susceptibility to MS with MHC and TCR genes Susceptibility to MS is associated with MHC molecules - primarily class II (DR and DQ) 8-13. The classical association is with DR2: approximately 50-70% of MS patients carry the DR2 allele found in 20-30 % of normal individuals. Among certain ethnic groups, MS susceptibility is more strongly associated with DR4 (Italian and Jordanian Arabs) or DRw6 (Japanese patients) 8-13. Based on DNA sequence data, four DR2 alleles have been defined in normal individuals, namely DR2Dw2, DR2Dw12, DR2Dw21 and DR2Dw22 (Ref. 14). DR2Dw2 is found in most DR2 + individuals and in most DR2 + MS patients 11. The association of MHC genes with relapsingremitting MS and primary progressive MS has also been compared. Both disease types were found to be associated with the DR2/DQw6 haplotype (DQ6 is a split of DQ1). Interestingly, relapsing-remitting and primary progressive MS could be discriminated by additional
© 1991, Elsevier Science Publishers Ltd, UK. 0167-4919/91/$02.00
Immunology Today
277
Vol 12 No. 8 1991
polymorphisms; the DR4/DQw8 haplotype is associated with primary progressive disease and the DRw17/DQw2 haplotype with relapsing-remitting disease 11. Thus, HLA-DR2, which is shared by both relapsing-remitting and primary progressive patients, may be involved in the initiation of the disease, whereas additional MHC class tI molecules may determine disease progression. Inheritance patterns for MS suggest that several genes are involved in determining susceptibility. In a population-based study of MS patients from Australia and the US17, TCR ~ gene polymorphisms were found to be associated with disease susceptibility. A Ca polymorphism was associated with MS susceptibility in both Australian and US MS patients, while a V~12.1 polymorphism was found to be disease associated only in the US population 15. In a second study 16, the linkage of TCR gene polymorphisms to disease susceptibility was examined in families of relapsing-remitting MS patients. Segregation analysis demonstrated that affected siblings share parental haplotypes with a higher frequency than expected by random distribution 16. Thus, MS is associated with both TCR e~and TCR ~ genes. In the context of known MHC associations, these observations are intriguing as they support the concept of disease determination by several Ir genes that act in concert during the initiation and progression of the disease. Nevertheless, these data by themselves do not prove that the inflammatory process is autoimmune in nature because the outcome of a viral infection could also be determined by the same set of Ir genes. Postulate 2: disease-associated MHC and TCR molecules are recognition elements for T-cell epitopes of the autoantigen(s) MBP in inflammatory white matter disease Several lines of evidence suggest that T-cell reactivity to myelin basic protein (MBP) may be important in human inflammatory white matter disease. First, an acute, disseminated encephalomyelitis can be observed after rabies vaccination with inactivated virus prepared from infected rabbit brains, which strongly suggests that myelin components may be encephalitogenic in humans. Autoreactivity to MBP has been observed in these cases, both at the humoral and the cellular level. Autoantibodies to MBP were found in patients with postvaccine encephalomyelitis but not in normal vaccine recipients. Moreover, antibodies to MBP were synthesized intrathecally, indicating an ongoing inflammatory response in the CNS 17. Second, an autoimmune response against MBP has also been demonstrated in post-viral encephalomyelitis (another form of acute disseminated encephalomyelitis) which follows measles, rubella and other viral infections 18. These post-viral inflammatory diseases are localized to the white matter, with pathology that can vary from inflammation without demyelination to major losses of myelin. There is a striking absence of detectable virus in the brain, indicating that T cells are attacking normal, not virally-altered, myelin tissue 2°. T-cell responsiveness to MBP can be detected in peripheral blood lymphocytes of these patients ~9,2°. These observations suggest that there may be a group of clinical disorders that represent a spectrum of auto-
Immunology Today
immune demyelinating CNS diseases: acute versions may include post-viral and post-vaccine encephalomyelitis 21 and classic MS (relapsing-remitting and primary progressive forms) may represent more chronic versions of this clinical entity. There may also be a relatively large pool of subclinical disease, as suggested by magnetic resonance imaging studies of twins with an affected and an unaffected twin 3. In some cases, the triggering event is relatively obvious (recent viral infection or vaccination), whereas in other circumstances the nature of, and temporal relationship to, the triggering event is not clear. One factor affecting the development of long-term disease after an acute (clinical or subclinical) attack is the inherited set of Ir genes which determine reactivity to myelin antigens. Thus, individuals carrying the DR2 antigen and a certain TCR repertoire may mount a stronger response to autoantigens, leading to clonal expansion of autoreactive T cells and chronic disease. In other individuals autoreactive T cells may be efficiently downregulated after a single attack; interestingly, antigen-nonspecific suppression is impaired in MS patients with chronic progressive disease, as reviewed recently22. T-cell reactivity to MBP
Investigations of MBP reactivity using standard sevenday proliferation assays in subjects with MS suggest that there is a small but consistent increase in proliferation to human MBP in subjects with MS compared with normal subjects or patients with other neurologic diseases 6,23. A number of investigators have compared the selective recognition of particular MBP epitopes in MS patients and control subjects using T-cell lines or clones. One study demonstrated multiple T-cell epitopes on MBP24, while another showed that MBP-reactive lines derived from the blood of patients with MS preferentially recognized human, as opposed to bovine, rat or monkey MBP2s. A third investigation demonstrated significant T-cell responses in MS patients to the human MBP peptides (15-31, 75-96 and 83-96) but not to (131141). The most prominent response, to MBP(83-96), was blocked by an anti-DR antibody 26. In a fourth study, DR2 was found to possess the unusual capacity to restrict all of the T-cell epitopes identified on MBP27. Specificity of T-cell responses to MBP
In the experimental allergic encephalitis (EAE) model in rodents, disease can be transferred by T cells reactive with immunodominant regions of MBP. A number of studies have been performed to delineate the immunodominant regions of human MBP. The immunodominant sites of human MBP were determined in our laboratory with a series of MS patients and control subjects7. Among 302 MBP-reactive T-cell lines generated from peripheral blood of MS patients, 140 (46.4%) reacted with an MBP peptide located in the center of the MBP molecule (residues 84-102). In control subjects, 11 out of 100 T-cell lines were reactive with that region. A second immunodominant region was found between MBP residues 143-168. T-cell lines with reactivity to MBP(61-82), MBP(124-142) and MBP(31-50) could also be generated. MHC restriction experiments using transfected cell lines and Epstein-Barr virus (EBV)-
278
vol 12 No. 8 1991
transformed B-cell lines as antigen-presenting cells demonstrated that the immunodominant MBP(84-102) peptide is presented to T cells by HLA-DR2a and HLADQ1 (K. Wucherpfennig and D. Hailer, unpublished). Similar immunodominant regions were found in another study among long-term CD4 ÷ cytotoxic T-cell lines grown from the peripheral blood of MS patients and control subjects28: MBP(87-106) and MBP(154-172) were recognized by most cell lines. MHC restriction experiments indicated that DR2a and DR4w4 could present MBP(87-106) to T cells and both of these alleles are associated with disease susceptibility. It is clear that the immunodominant MBP(84-102) peptide can be presented by different MHC class II molecules and that HLA-DR2a and HLA-DR2b, the dominant restriction elements, can present a number of different MBP epitopes to T cells29: of 20 lines examined, ten were found to be restricted by DR2a or DR2b. DR2a can present MBP(76-91), MBP(139-145) and MBP(131-145), whereas DR2b was found to present MBP(80-99) and MBP(148-162). These data indicate that disease-associated DR2 antigens can present a variety of MBP peptides to T cells and that there may be competition for peptide binding between different MBP regions which may, in part, determine the immunodominant sites of an autoantigen. The frequency of MBP-reactive T cells in peripheral blood of MS patients was found to be relatively low (less than one MBP-reactive T-cell per million mononuclear cells), but these numbers may have been underestimated owing to culture conditions 7. The frequency of MBPreactive T cells has also been found to be low in the peripheral blood of animals with EAE during ongoing disease 30.
MBP(84-102)-reactive T cells in the blood of this MS patient. The presence of antigen-specific, clonally-expanded T-cell populations suggests that MBP(84-102)specific T cells may have been activated by MBP in vivo 31. Postulate 3: in-vivo-activated and clonally-expanded T-cell populations are reactive with MBP Autoreactive T cells specific for the same immunodominant regions of MBP can be detected in normal individuals and in patients with MS, so what leads to autoimmune disease? Although it is clear that large populations of T cells are clonally deleted in the thymus as a consequence of exposure to certain self antigens, such as minor lymphocyte stimulating antigen (Mls) 33, organ-specific, autoreactive T cells are usually present in the circulation and lymph nodes of normal animals 34. Multiple signals may be required to activate a resting T cell, allowing the presence of circulating autoreactive T cells without associated autoimmune disease3s. Furthermore, the investigation of animal models of autoimmunity has shown that the minimal requirement for inducing an autoimmune disease is the presence of activated, autoreactive T cells36. This begins to shift the focus of autoimmune disease from the issue of clonal deletion to issues of T-cell signaling and activation of autoreactive T cells. Activated MBP-reactive T cells in MS
The major challenge in demonstrating that MS is indeed an autoimmune disease specific for myelin antigen(s) is to demonstrate that these autoreactive T cells actually play a role in the pathogenesis of the disease. As discussed above, the demonstration of autoreactive T cells in the peripheral blood of MS patients is not sufficient by itself since these cells have also been cultured T-cell receptor V gene use from normal individuals. It is therefore important to The TCR repertoire of human MBP-specific T cells has demonstrate that MBP- or proteolipid protein (PLP)been analysed 31. T-cell lines and clones specific for the reactive T cells have been activated in vivo and that they immunodominant region of MBP(84-102) and for have undergone clonal expansion due to antigen exMBP(143-168) were examined by polymerase chain re- posure in vivo. An hprt- mutant-cell assay for culturing in vivo actiaction amplification of cDNAs using a panel of TCR V~3specific primers. Certain TCR segments were used prefer- vated T cells from MS patients was used to address this entially to recognize immunodominant regions of MBP. question 37. The assay is based on the observation that TCR V~17.1 was found on a large number of indepen- dividing cells acquire random mutations during DNA dent T-cell lines generated from one DR2/DQ1 + MS synthesis. Some of these mutations occur in the hprt gene patient and was represented by one to two T-cell lines and result in inactivation of the hprt enzyme. Thus, generated from every patient. Other TCR V-gene seg- mutant cells do not metabolize thioguanine to a cytotox!c ments frequently used to recognize MBP(84-102) were metabolite, which allows a very effective selection of V~312, V~7 and V~8. In another recent investigation 34, these mutants in culture. Eleven of the 25 8 mutant T-cell TCR V~3gene usage of four MBP( 87-106)-reactive T-cell clones cultured with mitogen from the peripheral blood lines that were restricted by different MHC molecules of five of six MS patients showed strong reactivity to was analysed. Interestingly, two cell lines were found to MBP, while none of the 114 clones grown from blood of use the same V~ segment, V~8 (Ref. 32). normal subjects did37. These data strongly suggest that It was of interest to determine the sequence of the MBP-reactive T cells are activated in MS patients. junction of variable-diversity (VD) and joining (J) segWhat would lead to triggering of an autoreactive T cell ments which is thought to be involved in peptide recog- from resting to activated state? The mechanism is of nition. VDJ junctional sequences were different among particular interest in an organ-specific autoimmune discell lines generated from three MS patients and two ease, such as MS, where the target antigen may not be control subjects. In contrast, shared VDJ junctional se- readily accessible to resting T cells. Since resting T cells quences were observed among MBP(84-102)-reactive appear unable to cross the intact blood-brain barrier, an T-cell lines from one patient. These data and sequence autoimmune attack against brain antigens may be predata of TCR ~ chains (K. Wucherpfennig and D. Hailer, vented in a normal state by excluding resting T cells from unpublished) indicate that there is clonal expansion of being activated locally in the brain. However, when
Immunology Today
279
Vol 12 No. 8 1991
presenting cells (APCs). In the brain of MS patients, microglial cells and astrocytes may present peptides derived from myelin antigens to T cells4s,46. Presentation of peptides derived from autoantigens is therefore an important initial event in the development of an autoimmune response. Recent investigations indicate that approximately 0.1% of a specific MHC class II protein has to be occupied with a specific peptide for T-cell activation to occur 47. Since there is competition by a large variety of self and foreign peptides for binding to MHC class II proteins 48,49, this requirement is likely to be met only by peptides that are present in relatively large quantities in APCs, processed efficiently and bound with high affinity to class II molecules. In this context, it is important to note that both MBP and PLP are the most abundant proteins of myelin 53. MBP, as a cationic protein s°, may be relatively efficiently processed by APCs since cationization of antigens has been shown to increase their uptake and processing by macrophages5L This raises the issue that the physical characteristics and processing of the antigen in the tissue may limit the number of potential autoantigens that the immune system can recognize. It is likely that experimental autoimmune diseases are predominantly caused by immunodominant antigens and peptides because a certain threshold of local immune activation has to be reached for clinical dysfunction and disease to be apparent. In summary, resting autoreactive T cells may be activated in the periphery by molecular mimicry or by superantigens, cross the blood-brain barrier in an activated state and initiate an inflammatory response after exposure to self antigen in the CNS white matter. Postulate 4: treatment of the autoimmune disease
The final criterion requires that the autoimmune disease is effectively treated by tolerance induction to the autoantigen or by specific elimination of autoreactive T cells. Although this goal has not yet been accomplished in MS, therapeutic studies in EAE have demonstrated the potential feasibility of a variety of approaches. Tolerance induction to MBP by oral administration of myelin or MBP can prevent the induction of EAE in Lewis rats and treat ongoing clinical disease in guinea-pigs s2,s3. Specific elimination of autoreactive T cells has been achieved using monoclonal antibodies against V~8.2 which is the predominant TCR V~ used by MBP-specific T-cell clones in BIO.PL and PL/J mice s4,ss. The ongoing molecular definition of autoimmune processes in MS may make it possible to design a specific approach for the treatment of the disease. In this regard, we have recently completed a phase one investigation in patients with early relapsing-remitting MS where we have attempted to induce tolerance to myelin antigens by oral administration of bovine myelin. Such investigations may allow us to definitively demonstrate the importance of autoreactivity to myelin antigens in the pathogenesis of MS. Kai Wucherpfennig, Howard Weiner and David Hailer are at the Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Immunology Today
References
1 McFarlin, D.E. and McFarland, H.F. (1982) New Engl. J. Med. 307, 1183-1188
2 Williams, A., Eldridge, R., McFarland, H. et al. (1980) Neurology 30, 1139-1147 3 Ebers, G.C., Bulman, D.E., Sadvnick, A.D. et al. (1986) New Engl. J. Med. 315, 1638-1642 4 Booss, J. and Kim, J.H. (1990) in Handbook of Multiple Sclerosis (Cook, S.D., ed.), pp. 41-61, Marcel Dekker 5 Burns, J., Rosenzweig, A., Zweiman, B. et al. (1983) Cell. Immunol. 81,435-440 6 Johnson, D., Hailer, D.A., Fallis, R. et al. (1986) J. Neuroimmunol. 13, 99-108 7 0 t a , K., Matsui, M., Milford, E. et al. (1990) Nature 346, 183-187 8 Ho, H.Z., Tiwari, J.L., Halle, R.W. et al. (1981) Immunogenetics 15,509-517 9 Spielman, R.S. and Nathenson, N. (1982) Epidemiol. Rev. 4, 45-65 10 Francis, D.A., Batchelor, J.R., McDonald, W.I. et al. (1986) Lancet i, 211 11 Olerup, O., Hillert, J., Fredrickson, S. et al. (1989) Proc. Natl Acad. Sci. USA 86, 7113-7117 12 Vartdal, F., Sollid, L.M., Vandvik, B. et al. (1988) Hum. Immunol. 25, 103-110 13 Clerici, N., Hernandez, M., Fernandez, M. et al. (1989) Tissue Antigens 34, 309-311 14 Gregersen, P.K., Todd, J.A., Ehrlich, H.A. et al. (1987) in Immunobiology ofHLA (Dupont, B., ed.), pp. 1027-1031, Springer-Verlag 15 Oksenberg, J.R., Sherritt, M., Begovich, A.B. et al. (1989) Proc. Natl Acad. Sci. USA 86, 988-992 16 Seboun, E., Robinson, M.A., Doolittle, T.H. et al. (1989) Cell 57, 1095-1100 17 Hemachudha, T., Griffin, D.E., Giffels, J.J. et al. (1987) New Engl. J. Med. 316, 369-374 18 Johnson, R.T., Griffin, D.E., Hirsch, R.L. et al. (1984) New Engl. J. Med. 310, 137-141 19 Hailer, D.A., Benjamin, D.S., Burks, J. et al. (1987) J. Immunol. 139, 68-72 20 Martin, R., Marquadt, P., O'Shea, S. et al. (1989) J. Neuroimmunol. 23, 1-10 21 Hailer, D.A. and Sobel, R. (1990) New Engl. J. Med. 323, 1123-1135 22 Hailer, D.A. and Weiner, H.L. (1989) Immunol. Today 10, 104-107 23 Lisak, R.P. and Zweiman, B. (1977) New Engl. J. Med. 297, 850-853 24 Richert, J.R., Robinson, E.D., Deibler, G.E. et al. (1989) J. Neuroimmunol. 23, 55-66 25 Tournier-Lasserve, E., Hashim, G.A. and Bach, M.A. (1988) J. Neurosci. Reg. 19, 149-156 26 Baxevanis, C.N., Reclos, G.J., Servis, C. et al. (1989) J. Neuroimmunol. 22, 23-30 27 Chou, Y.K., Vainiene, M., Whitman, R. et al. (1989) J. Neurosci. Res. 23,207-216 28 Martin, R., Jaraquemada, D., Flerlage, M. et al. (1990) J. Immunol. 145,540-548 29 Pette, M., Fujita, K., Wilkinson, D. et al. (1990) Proc. Natl Acad. Sci. USA 87, 7968-7972 30 Fallis, R.J., Powers, M.L., Sy, M. et al. (1987) J. Neuroimmunol. 14, 205-219 31 Wucherpfennig, K.W., Ota, K., Endo, N. et al. (1990) Science 248, 1016-1019 32 Martin, R., Howell, M.D., Jaraquemada, D. et al. (1991) J. Exp. Med. 173, 19-24 33 Blackman, M.A., Burgert, H.G. and Woodland, D.L. (1990) Nature 345, 540-542
281
Vol 12 No. 8 1991
34 Schluesener, H.J. and Wekerle, H. (1985) J. lmmunol. 135, 3128-3133 35 Jenkins, M.K., Pardoll, D.M. and Mizuguchi, J. (1987) Immunol. Rev. 95, 114-135 36 Panitch, H.S. and McFarlin, D.E. (1977)J. Immunol. 119, 1134-1137 37 Allegretta, M., Nicklas, J.A., Sriram, S. et al. (1990) Science 247, 718-721 38 Fujinami, R.S. and Oldstone, M. (1985) Science 230, 1043-1045 39 Mollick, J., Cook, R.G. and Rich, R.R. (1989) Science 244, 817-819 40 Choi, Y., Herman, A., DiGusto, D. et al. (1990) Nature 346, 471-473 41 Weiss, A., Imboden, J., Hardy, K. et al. (1986) Annu. Rev. Immunol. 4, 593-619 42 Meuer, S.C., Hussey, R.E., Fabbi, M. et al. (1984) Cell 36, 897-906 43 Hfinig T., Tiefenthaler, G., Meyer zum B6schenfelde, K-H. et al. (1987) Nature 326, 298-301 44 Brod, S.A., Purvee, M., Benjamin, D. et al. (1990) Eur. J. Immunol. 20, 2259-2268
45 Fontana, A., Fierz, W. and Wekerle, H. (1984) Nature 304, 273-276 46 Hickey, W.F. and Kimura, H. (1988) Science 239, 290-292 47 Harding, C. and Unanue, E.R. (1990) Nature 346, 574-576 48 Adorini, L., Muller, S., Cardinaux, F. et al. (1988) Nature 334, 623-625 49 Buus, S., Sette, A., Colon, S.M. et al. (1988) Science 242, 1045-1047 50 Lees, M.B. and Brostoff, S.W. (1984) in Myelin (Morell, P., ed.), pp. 197-217, Plenum Press 51 Muckerheide, A., Apple, R.J., Pesce, A.J. et al. (1987) J. lmmunol. 138,833-837 52 Higgins, P.J. and Weiner, H.L. (1988) J. Immunol. 140, 440-445 53 Brod, S.A., A1-Sabbagh, A., Sobel, R.A. et al. Ann. Neurol. (in press) 54 Urban, J.L., Kumar, V., Kono, D.H. et al. (1988) Cell 54, 577-592 55 Acha-Orbea, H., Mitchell, D.J., Timmermann, L. et al. (1988) Cell 54, 263-273
The International Union of Immunological Societies (IUIS) will administer a program of limited financial assistance* to those who would otherwise find it difficult to attend the VIIIth Congress. Those selected for an award will receive a complimentary registration and, in some cases, a cash award in addition ($200-750 (US) depending upon the awardee's estimated travel costs). Applications must be receivedby November 30, 1991. Decision letters will be mailed by December 15, 1991, but funds will be distributed only at the Congress site. The assistance program is supported by the Hungarian Organizing Committee [100 registrations), the IUIS ($15.000 and the Europ. Fed. Immunol. Soc. ($I0,000) To apply:
I. Complete the form (below) legibly and in English 2. Enclose two (2) self-addressed adhesive mailing labels. 3. Send all material to: IUIS Bursaries, c/o AAI 9650 Rockville Pike Bethesda, MD 20814 USA .
• PPLICATION FORM Doctoral Degree (Yr)(Department, Institution) ~¢ademic Rank:
Teaching responsibilities? No Yes (Student? Faculty?) ,, , (Circle one) ublished research: No Yes (If 'yes', list five (5) most recently published papers on a separate sheet~ • ' (Circle one)
3vited Participant? Svmpos. Spk? Wkshop Chr? If neither, write "NOName of Department Head Signature of D~artment Head
Signature of app icant (Photocopies of this form may be usedt
LATE OR INCOMPLETE APPLICATIONS CANNOT BE CONSIDERED
Immunology Today
282
Vol 12 No. 8 1991
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
T-cell recognition of myelin basic protein Kai W. Wucherpfennig, Howard L. Weiner and David A. Hailer Multiple sclerosis is a chronic inflammatory disease of the central nervous system which has been hypothesized to be autoimmune in nature. To test whether this is the case, Kai Wucherpfennig and colleagues have developed a set of criteria that must be met to satisfy the hypothesis. Here, they present these criteria and assess the extent to which studies to date satisfy them. Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system characterized by focal T-cell and macrophage infiltration into white matter. The inflammatory lesions are distributed around small venules in the white matter, preferentially in the spinal cord, cerebellum and around ventricles. This pathology is frequently associated with neurologic dysfunction referable to these areas of inflammation, in particular paralysis, sensory deficits and visual problems 1. Studies on twins suggest that there is a strong genetic component to MS susceptibility2,3: monozygotic twins have a more than 10-fold higher concordance rate for MS than dizygotic twins while the concordance rate among dizygotic twins is similar to that of siblings 3. Two major theories of pathogenesis of MS have been proposed: one, that MS is a viral disease of the central nervous system (CNS), the inflammatory response in the brain being an anti-viral immune response, and two, that MS is an autoimmune disease in which infiltrating T cells recognize self antigens and attack normal tissue. These two possibilities are not mutually exclusive: the autoimmune response may be triggered by environmental factors such as viral infections. However, the inability to transfer the disease to primates or to isolate virus from CNS tissue of MS patients, despite tremendous efforts, lends indirect support to the theory that the inflammatory process may be autoimmune in nature 4. The critical experiment that would prove this, namely the transfer of antigen-specific T cells to a major histocompatibility complex (MHC)-identical recipient, is impossible to perform in humans. A second approach to define the potential role of myelin-reactive T cells in MS was thought to be the isolation of autoreactive T cells from patients and the determination of their antigen specificity. However, this cannot define the role of myelin-reactive T cells in the disease since cells of the same specificity can also be cultured from the blood of normal individuals s,6. To address conclusively the role of autoreactive T cells in MS, we have defined a set of criteria that can be tested (Box 1)7. If these cells are pathogenic it should be possible to demonstrate that disease susceptibility is associated with immune response (Ir) genes involved in the recognition of the autoantigen(s). Specifically, MHC and T-cell receptor (TCR) gene products that define susceptibility to MS should be involved in the immune recognition of the autoantigen. Autoreactive T cells should be activated in patients or should have undergone clonal expansion in vivo and the specific elimination of auto-
reactive T cells or tolerance induction to the autoantigen will improve the clinical course of the disease (Box l). In this review these postulates are used as a basis to examine the pathophysiology of MS. Postulate 1: association of susceptibility to MS with MHC and TCR genes Susceptibility to MS is associated with MHC molecules - primarily class II (DR and DQ) 8-13. The classical association is with DR2: approximately 50-70% of MS patients carry the DR2 allele found in 20-30 % of normal individuals. Among certain ethnic groups, MS susceptibility is more strongly associated with DR4 (Italian and Jordanian Arabs) or DRw6 (Japanese patients) 8-13. Based on DNA sequence data, four DR2 alleles have been defined in normal individuals, namely DR2Dw2, DR2Dw12, DR2Dw21 and DR2Dw22 (Ref. 14). DR2Dw2 is found in most DR2 + individuals and in most DR2 + MS patients 11. The association of MHC genes with relapsingremitting MS and primary progressive MS has also been compared. Both disease types were found to be associated with the DR2/DQw6 haplotype (DQ6 is a split of DQ1). Interestingly, relapsing-remitting and primary progressive MS could be discriminated by additional
© 1991, Elsevier Science Publishers Ltd, UK. 0167-4919/91/$02.00
Immunology Today
277
Vol 12 No. 8 1991
polymorphisms; the DR4/DQw8 haplotype is associated with primary progressive disease and the DRw17/DQw2 haplotype with relapsing-remitting disease 11. Thus, HLA-DR2, which is shared by both relapsing-remitting and primary progressive patients, may be involved in the initiation of the disease, whereas additional MHC class tI molecules may determine disease progression. Inheritance patterns for MS suggest that several genes are involved in determining susceptibility. In a population-based study of MS patients from Australia and the US17, TCR ~ gene polymorphisms were found to be associated with disease susceptibility. A Ca polymorphism was associated with MS susceptibility in both Australian and US MS patients, while a V~12.1 polymorphism was found to be disease associated only in the US population 15. In a second study 16, the linkage of TCR gene polymorphisms to disease susceptibility was examined in families of relapsing-remitting MS patients. Segregation analysis demonstrated that affected siblings share parental haplotypes with a higher frequency than expected by random distribution 16. Thus, MS is associated with both TCR e~and TCR ~ genes. In the context of known MHC associations, these observations are intriguing as they support the concept of disease determination by several Ir genes that act in concert during the initiation and progression of the disease. Nevertheless, these data by themselves do not prove that the inflammatory process is autoimmune in nature because the outcome of a viral infection could also be determined by the same set of Ir genes. Postulate 2: disease-associated MHC and TCR molecules are recognition elements for T-cell epitopes of the autoantigen(s) MBP in inflammatory white matter disease Several lines of evidence suggest that T-cell reactivity to myelin basic protein (MBP) may be important in human inflammatory white matter disease. First, an acute, disseminated encephalomyelitis can be observed after rabies vaccination with inactivated virus prepared from infected rabbit brains, which strongly suggests that myelin components may be encephalitogenic in humans. Autoreactivity to MBP has been observed in these cases, both at the humoral and the cellular level. Autoantibodies to MBP were found in patients with postvaccine encephalomyelitis but not in normal vaccine recipients. Moreover, antibodies to MBP were synthesized intrathecally, indicating an ongoing inflammatory response in the CNS 17. Second, an autoimmune response against MBP has also been demonstrated in post-viral encephalomyelitis (another form of acute disseminated encephalomyelitis) which follows measles, rubella and other viral infections 18. These post-viral inflammatory diseases are localized to the white matter, with pathology that can vary from inflammation without demyelination to major losses of myelin. There is a striking absence of detectable virus in the brain, indicating that T cells are attacking normal, not virally-altered, myelin tissue 2°. T-cell responsiveness to MBP can be detected in peripheral blood lymphocytes of these patients ~9,2°. These observations suggest that there may be a group of clinical disorders that represent a spectrum of auto-
Immunology Today
immune demyelinating CNS diseases: acute versions may include post-viral and post-vaccine encephalomyelitis 21 and classic MS (relapsing-remitting and primary progressive forms) may represent more chronic versions of this clinical entity. There may also be a relatively large pool of subclinical disease, as suggested by magnetic resonance imaging studies of twins with an affected and an unaffected twin 3. In some cases, the triggering event is relatively obvious (recent viral infection or vaccination), whereas in other circumstances the nature of, and temporal relationship to, the triggering event is not clear. One factor affecting the development of long-term disease after an acute (clinical or subclinical) attack is the inherited set of Ir genes which determine reactivity to myelin antigens. Thus, individuals carrying the DR2 antigen and a certain TCR repertoire may mount a stronger response to autoantigens, leading to clonal expansion of autoreactive T cells and chronic disease. In other individuals autoreactive T cells may be efficiently downregulated after a single attack; interestingly, antigen-nonspecific suppression is impaired in MS patients with chronic progressive disease, as reviewed recently22. T-cell reactivity to MBP
Investigations of MBP reactivity using standard sevenday proliferation assays in subjects with MS suggest that there is a small but consistent increase in proliferation to human MBP in subjects with MS compared with normal subjects or patients with other neurologic diseases 6,23. A number of investigators have compared the selective recognition of particular MBP epitopes in MS patients and control subjects using T-cell lines or clones. One study demonstrated multiple T-cell epitopes on MBP24, while another showed that MBP-reactive lines derived from the blood of patients with MS preferentially recognized human, as opposed to bovine, rat or monkey MBP2s. A third investigation demonstrated significant T-cell responses in MS patients to the human MBP peptides (15-31, 75-96 and 83-96) but not to (131141). The most prominent response, to MBP(83-96), was blocked by an anti-DR antibody 26. In a fourth study, DR2 was found to possess the unusual capacity to restrict all of the T-cell epitopes identified on MBP27. Specificity of T-cell responses to MBP
In the experimental allergic encephalitis (EAE) model in rodents, disease can be transferred by T cells reactive with immunodominant regions of MBP. A number of studies have been performed to delineate the immunodominant regions of human MBP. The immunodominant sites of human MBP were determined in our laboratory with a series of MS patients and control subjects7. Among 302 MBP-reactive T-cell lines generated from peripheral blood of MS patients, 140 (46.4%) reacted with an MBP peptide located in the center of the MBP molecule (residues 84-102). In control subjects, 11 out of 100 T-cell lines were reactive with that region. A second immunodominant region was found between MBP residues 143-168. T-cell lines with reactivity to MBP(61-82), MBP(124-142) and MBP(31-50) could also be generated. MHC restriction experiments using transfected cell lines and Epstein-Barr virus (EBV)-
278
vol 12 No. 8 1991
transformed B-cell lines as antigen-presenting cells demonstrated that the immunodominant MBP(84-102) peptide is presented to T cells by HLA-DR2a and HLADQ1 (K. Wucherpfennig and D. Hailer, unpublished). Similar immunodominant regions were found in another study among long-term CD4 ÷ cytotoxic T-cell lines grown from the peripheral blood of MS patients and control subjects28: MBP(87-106) and MBP(154-172) were recognized by most cell lines. MHC restriction experiments indicated that DR2a and DR4w4 could present MBP(87-106) to T cells and both of these alleles are associated with disease susceptibility. It is clear that the immunodominant MBP(84-102) peptide can be presented by different MHC class II molecules and that HLA-DR2a and HLA-DR2b, the dominant restriction elements, can present a number of different MBP epitopes to T cells29: of 20 lines examined, ten were found to be restricted by DR2a or DR2b. DR2a can present MBP(76-91), MBP(139-145) and MBP(131-145), whereas DR2b was found to present MBP(80-99) and MBP(148-162). These data indicate that disease-associated DR2 antigens can present a variety of MBP peptides to T cells and that there may be competition for peptide binding between different MBP regions which may, in part, determine the immunodominant sites of an autoantigen. The frequency of MBP-reactive T cells in peripheral blood of MS patients was found to be relatively low (less than one MBP-reactive T-cell per million mononuclear cells), but these numbers may have been underestimated owing to culture conditions 7. The frequency of MBPreactive T cells has also been found to be low in the peripheral blood of animals with EAE during ongoing disease 30.
MBP(84-102)-reactive T cells in the blood of this MS patient. The presence of antigen-specific, clonally-expanded T-cell populations suggests that MBP(84-102)specific T cells may have been activated by MBP in vivo 31. Postulate 3: in-vivo-activated and clonally-expanded T-cell populations are reactive with MBP Autoreactive T cells specific for the same immunodominant regions of MBP can be detected in normal individuals and in patients with MS, so what leads to autoimmune disease? Although it is clear that large populations of T cells are clonally deleted in the thymus as a consequence of exposure to certain self antigens, such as minor lymphocyte stimulating antigen (Mls) 33, organ-specific, autoreactive T cells are usually present in the circulation and lymph nodes of normal animals 34. Multiple signals may be required to activate a resting T cell, allowing the presence of circulating autoreactive T cells without associated autoimmune disease3s. Furthermore, the investigation of animal models of autoimmunity has shown that the minimal requirement for inducing an autoimmune disease is the presence of activated, autoreactive T cells36. This begins to shift the focus of autoimmune disease from the issue of clonal deletion to issues of T-cell signaling and activation of autoreactive T cells. Activated MBP-reactive T cells in MS
The major challenge in demonstrating that MS is indeed an autoimmune disease specific for myelin antigen(s) is to demonstrate that these autoreactive T cells actually play a role in the pathogenesis of the disease. As discussed above, the demonstration of autoreactive T cells in the peripheral blood of MS patients is not sufficient by itself since these cells have also been cultured T-cell receptor V gene use from normal individuals. It is therefore important to The TCR repertoire of human MBP-specific T cells has demonstrate that MBP- or proteolipid protein (PLP)been analysed 31. T-cell lines and clones specific for the reactive T cells have been activated in vivo and that they immunodominant region of MBP(84-102) and for have undergone clonal expansion due to antigen exMBP(143-168) were examined by polymerase chain re- posure in vivo. An hprt- mutant-cell assay for culturing in vivo actiaction amplification of cDNAs using a panel of TCR V~3specific primers. Certain TCR segments were used prefer- vated T cells from MS patients was used to address this entially to recognize immunodominant regions of MBP. question 37. The assay is based on the observation that TCR V~17.1 was found on a large number of indepen- dividing cells acquire random mutations during DNA dent T-cell lines generated from one DR2/DQ1 + MS synthesis. Some of these mutations occur in the hprt gene patient and was represented by one to two T-cell lines and result in inactivation of the hprt enzyme. Thus, generated from every patient. Other TCR V-gene seg- mutant cells do not metabolize thioguanine to a cytotox!c ments frequently used to recognize MBP(84-102) were metabolite, which allows a very effective selection of V~312, V~7 and V~8. In another recent investigation 34, these mutants in culture. Eleven of the 25 8 mutant T-cell TCR V~3gene usage of four MBP( 87-106)-reactive T-cell clones cultured with mitogen from the peripheral blood lines that were restricted by different MHC molecules of five of six MS patients showed strong reactivity to was analysed. Interestingly, two cell lines were found to MBP, while none of the 114 clones grown from blood of use the same V~ segment, V~8 (Ref. 32). normal subjects did37. These data strongly suggest that It was of interest to determine the sequence of the MBP-reactive T cells are activated in MS patients. junction of variable-diversity (VD) and joining (J) segWhat would lead to triggering of an autoreactive T cell ments which is thought to be involved in peptide recog- from resting to activated state? The mechanism is of nition. VDJ junctional sequences were different among particular interest in an organ-specific autoimmune discell lines generated from three MS patients and two ease, such as MS, where the target antigen may not be control subjects. In contrast, shared VDJ junctional se- readily accessible to resting T cells. Since resting T cells quences were observed among MBP(84-102)-reactive appear unable to cross the intact blood-brain barrier, an T-cell lines from one patient. These data and sequence autoimmune attack against brain antigens may be predata of TCR ~ chains (K. Wucherpfennig and D. Hailer, vented in a normal state by excluding resting T cells from unpublished) indicate that there is clonal expansion of being activated locally in the brain. However, when
Immunology Today
279
Vol 12 No. 8 1991
presenting cells (APCs). In the brain of MS patients, microglial cells and astrocytes may present peptides derived from myelin antigens to T cells4s,46. Presentation of peptides derived from autoantigens is therefore an important initial event in the development of an autoimmune response. Recent investigations indicate that approximately 0.1% of a specific MHC class II protein has to be occupied with a specific peptide for T-cell activation to occur 47. Since there is competition by a large variety of self and foreign peptides for binding to MHC class II proteins 48,49, this requirement is likely to be met only by peptides that are present in relatively large quantities in APCs, processed efficiently and bound with high affinity to class II molecules. In this context, it is important to note that both MBP and PLP are the most abundant proteins of myelin 53. MBP, as a cationic protein s°, may be relatively efficiently processed by APCs since cationization of antigens has been shown to increase their uptake and processing by macrophages5L This raises the issue that the physical characteristics and processing of the antigen in the tissue may limit the number of potential autoantigens that the immune system can recognize. It is likely that experimental autoimmune diseases are predominantly caused by immunodominant antigens and peptides because a certain threshold of local immune activation has to be reached for clinical dysfunction and disease to be apparent. In summary, resting autoreactive T cells may be activated in the periphery by molecular mimicry or by superantigens, cross the blood-brain barrier in an activated state and initiate an inflammatory response after exposure to self antigen in the CNS white matter. Postulate 4: treatment of the autoimmune disease
The final criterion requires that the autoimmune disease is effectively treated by tolerance induction to the autoantigen or by specific elimination of autoreactive T cells. Although this goal has not yet been accomplished in MS, therapeutic studies in EAE have demonstrated the potential feasibility of a variety of approaches. Tolerance induction to MBP by oral administration of myelin or MBP can prevent the induction of EAE in Lewis rats and treat ongoing clinical disease in guinea-pigs s2,s3. Specific elimination of autoreactive T cells has been achieved using monoclonal antibodies against V~8.2 which is the predominant TCR V~ used by MBP-specific T-cell clones in BIO.PL and PL/J mice s4,ss. The ongoing molecular definition of autoimmune processes in MS may make it possible to design a specific approach for the treatment of the disease. In this regard, we have recently completed a phase one investigation in patients with early relapsing-remitting MS where we have attempted to induce tolerance to myelin antigens by oral administration of bovine myelin. Such investigations may allow us to definitively demonstrate the importance of autoreactivity to myelin antigens in the pathogenesis of MS. Kai Wucherpfennig, Howard Weiner and David Hailer are at the Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
Immunology Today
References
1 McFarlin, D.E. and McFarland, H.F. (1982) New Engl. J. Med. 307, 1183-1188
2 Williams, A., Eldridge, R., McFarland, H. et al. (1980) Neurology 30, 1139-1147 3 Ebers, G.C., Bulman, D.E., Sadvnick, A.D. et al. (1986) New Engl. J. Med. 315, 1638-1642 4 Booss, J. and Kim, J.H. (1990) in Handbook of Multiple Sclerosis (Cook, S.D., ed.), pp. 41-61, Marcel Dekker 5 Burns, J., Rosenzweig, A., Zweiman, B. et al. (1983) Cell. Immunol. 81,435-440 6 Johnson, D., Hailer, D.A., Fallis, R. et al. (1986) J. Neuroimmunol. 13, 99-108 7 0 t a , K., Matsui, M., Milford, E. et al. (1990) Nature 346, 183-187 8 Ho, H.Z., Tiwari, J.L., Halle, R.W. et al. (1981) Immunogenetics 15,509-517 9 Spielman, R.S. and Nathenson, N. (1982) Epidemiol. Rev. 4, 45-65 10 Francis, D.A., Batchelor, J.R., McDonald, W.I. et al. (1986) Lancet i, 211 11 Olerup, O., Hillert, J., Fredrickson, S. et al. (1989) Proc. Natl Acad. Sci. USA 86, 7113-7117 12 Vartdal, F., Sollid, L.M., Vandvik, B. et al. (1988) Hum. Immunol. 25, 103-110 13 Clerici, N., Hernandez, M., Fernandez, M. et al. (1989) Tissue Antigens 34, 309-311 14 Gregersen, P.K., Todd, J.A., Ehrlich, H.A. et al. (1987) in Immunobiology ofHLA (Dupont, B., ed.), pp. 1027-1031, Springer-Verlag 15 Oksenberg, J.R., Sherritt, M., Begovich, A.B. et al. (1989) Proc. Natl Acad. Sci. USA 86, 988-992 16 Seboun, E., Robinson, M.A., Doolittle, T.H. et al. (1989) Cell 57, 1095-1100 17 Hemachudha, T., Griffin, D.E., Giffels, J.J. et al. (1987) New Engl. J. Med. 316, 369-374 18 Johnson, R.T., Griffin, D.E., Hirsch, R.L. et al. (1984) New Engl. J. Med. 310, 137-141 19 Hailer, D.A., Benjamin, D.S., Burks, J. et al. (1987) J. Immunol. 139, 68-72 20 Martin, R., Marquadt, P., O'Shea, S. et al. (1989) J. Neuroimmunol. 23, 1-10 21 Hailer, D.A. and Sobel, R. (1990) New Engl. J. Med. 323, 1123-1135 22 Hailer, D.A. and Weiner, H.L. (1989) Immunol. Today 10, 104-107 23 Lisak, R.P. and Zweiman, B. (1977) New Engl. J. Med. 297, 850-853 24 Richert, J.R., Robinson, E.D., Deibler, G.E. et al. (1989) J. Neuroimmunol. 23, 55-66 25 Tournier-Lasserve, E., Hashim, G.A. and Bach, M.A. (1988) J. Neurosci. Reg. 19, 149-156 26 Baxevanis, C.N., Reclos, G.J., Servis, C. et al. (1989) J. Neuroimmunol. 22, 23-30 27 Chou, Y.K., Vainiene, M., Whitman, R. et al. (1989) J. Neurosci. Res. 23,207-216 28 Martin, R., Jaraquemada, D., Flerlage, M. et al. (1990) J. Immunol. 145,540-548 29 Pette, M., Fujita, K., Wilkinson, D. et al. (1990) Proc. Natl Acad. Sci. USA 87, 7968-7972 30 Fallis, R.J., Powers, M.L., Sy, M. et al. (1987) J. Neuroimmunol. 14, 205-219 31 Wucherpfennig, K.W., Ota, K., Endo, N. et al. (1990) Science 248, 1016-1019 32 Martin, R., Howell, M.D., Jaraquemada, D. et al. (1991) J. Exp. Med. 173, 19-24 33 Blackman, M.A., Burgert, H.G. and Woodland, D.L. (1990) Nature 345, 540-542
281
Vol 12 No. 8 1991
34 Schluesener, H.J. and Wekerle, H. (1985) J. lmmunol. 135, 3128-3133 35 Jenkins, M.K., Pardoll, D.M. and Mizuguchi, J. (1987) Immunol. Rev. 95, 114-135 36 Panitch, H.S. and McFarlin, D.E. (1977)J. Immunol. 119, 1134-1137 37 Allegretta, M., Nicklas, J.A., Sriram, S. et al. (1990) Science 247, 718-721 38 Fujinami, R.S. and Oldstone, M. (1985) Science 230, 1043-1045 39 Mollick, J., Cook, R.G. and Rich, R.R. (1989) Science 244, 817-819 40 Choi, Y., Herman, A., DiGusto, D. et al. (1990) Nature 346, 471-473 41 Weiss, A., Imboden, J., Hardy, K. et al. (1986) Annu. Rev. Immunol. 4, 593-619 42 Meuer, S.C., Hussey, R.E., Fabbi, M. et al. (1984) Cell 36, 897-906 43 Hfinig T., Tiefenthaler, G., Meyer zum B6schenfelde, K-H. et al. (1987) Nature 326, 298-301 44 Brod, S.A., Purvee, M., Benjamin, D. et al. (1990) Eur. J. Immunol. 20, 2259-2268
45 Fontana, A., Fierz, W. and Wekerle, H. (1984) Nature 304, 273-276 46 Hickey, W.F. and Kimura, H. (1988) Science 239, 290-292 47 Harding, C. and Unanue, E.R. (1990) Nature 346, 574-576 48 Adorini, L., Muller, S., Cardinaux, F. et al. (1988) Nature 334, 623-625 49 Buus, S., Sette, A., Colon, S.M. et al. (1988) Science 242, 1045-1047 50 Lees, M.B. and Brostoff, S.W. (1984) in Myelin (Morell, P., ed.), pp. 197-217, Plenum Press 51 Muckerheide, A., Apple, R.J., Pesce, A.J. et al. (1987) J. lmmunol. 138,833-837 52 Higgins, P.J. and Weiner, H.L. (1988) J. Immunol. 140, 440-445 53 Brod, S.A., A1-Sabbagh, A., Sobel, R.A. et al. Ann. Neurol. (in press) 54 Urban, J.L., Kumar, V., Kono, D.H. et al. (1988) Cell 54, 577-592 55 Acha-Orbea, H., Mitchell, D.J., Timmermann, L. et al. (1988) Cell 54, 263-273
The International Union of Immunological Societies (IUIS) will administer a program of limited financial assistance* to those who would otherwise find it difficult to attend the VIIIth Congress. Those selected for an award will receive a complimentary registration and, in some cases, a cash award in addition ($200-750 (US) depending upon the awardee's estimated travel costs). Applications must be receivedby November 30, 1991. Decision letters will be mailed by December 15, 1991, but funds will be distributed only at the Congress site. The assistance program is supported by the Hungarian Organizing Committee [100 registrations), the IUIS ($15.000 and the Europ. Fed. Immunol. Soc. ($I0,000) To apply:
I. Complete the form (below) legibly and in English 2. Enclose two (2) self-addressed adhesive mailing labels. 3. Send all material to: IUIS Bursaries, c/o AAI 9650 Rockville Pike Bethesda, MD 20814 USA .
• PPLICATION FORM Doctoral Degree (Yr)(Department, Institution) ~¢ademic Rank:
Teaching responsibilities? No Yes (Student? Faculty?) ,, , (Circle one) ublished research: No Yes (If 'yes', list five (5) most recently published papers on a separate sheet~ • ' (Circle one)
3vited Participant? Svmpos. Spk? Wkshop Chr? If neither, write "NOName of Department Head Signature of D~artment Head
Signature of app icant (Photocopies of this form may be usedt
LATE OR INCOMPLETE APPLICATIONS CANNOT BE CONSIDERED
Immunology Today
282
Vol 12 No. 8 1991
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
