Jun 27, 1988 - Kappler, J. W., Wade, T., White, J., Kushnir, E., Blackman,. M., Bill, J. ... Hafler, D. A., Duby, A. D., Lee, S. J., Benjamin, D., Seid- man, J. G. ...
Proc. Nad. Acad. Sci. USA Vol. 85, pp. 8608-8612, November 1988 Immunology
Involvement of distinct murine T-cell receptors in the autoimmune encephalitogenic response to nested epitopes of myelin basic protein (autoimmunity/immunotherapy/experimental allergic encephalomyelitis)
KoICHIRO SAKAI*t, ANIMESH A. SINHAt, DENNIS J. MITCHELL*t, SCOTT S. ZAMVIL*t JONATHAN B. ROTHBARD§, HUGH 0. MCDEVITT§, AND LAWRENCE STEINMANt¶ Departments of *Neurology, tGenetics, and tMicrobiology and Immunology, Stanford University, Stanford, CA 94305; and hthe Imperial Cancer Research Fund, London WC2A3PX, United Kingdom
Contributed by Hugh 0. McDevitt, June 27, 1988
ABSTRACT The peptide p89-101 (Val-His-Phe-Phe-LysAsn-lle-Val-Thr-Pro-Arg-Thr-Pro) of myelin basic protein is encephalitogenic in mice expressing H-2q and H-2S antigens. Six of 13 encephalitogen-specific T-cell clones were shown to express the variable a-chain (Va) 17a gene product (KJ23a'), whereas seven clones were KJ23a-. Both KJ23a' and KJ23asubpopulations were encephalitogenic in'SJL/J mice when adoptively transferred. Depletion of KJ23a+ cells in vivo with the administration of the antibody KJ23a suppresses expenmental allergic encephalomyelitis induced with KJ23a+ T-cell lines. However, experimental allergic encephalomyelitis induced with either (i) encephalitogenic peptide p89-101, (u) intact myelin basic protein, or (iii) KJ23a- T cells reactive to p89-101 cannot be prevented with monoclonal antibody KJ23a. These data indicate that in spite of the V. 17a gene expression in a relatively large proportion of p89-101-specific T cells, such Vp, gene use is not essential for the induction of experimental allergic encephalomyelitis in SJL/J mice. These results contrast with the predominance of V,9 gene use (V, 8.2) in T cells reactive to the encephalitogenic fragment (pR1-11) in PL/J mice. One reason for this lack of dominant use of a particular T-cell receptor V.3 gene family in the autoimmune response to myelin basic protein in SJL/J mice stems from the observation that two encephalitogenic epitopes exist in p89-101. KJ23a- T cells are stimulated by the deleted peptide p89-100, whereas KJ23a+ T cells are not. Thus, in the response to an encephalitogenic fragment of myelin basic protein containing two nested epitopes, at least two distinct T-cell receptor Vl genes are expressed. These distinct T-cell subpopulations can each trigger experimental allergic encepbalomyelitis. These rmdings have implications for therapy of autoimmune disease with antibodies to the T-cell receptor gene products.
our laboratory (7) showed a preferential expression of a single TCR variable B-chain (V,3) gene family in encephalitogenic T cells from PL/J mice, a strain highly susceptible to EAE. This indicated that such a preferential use of a TCR V,3 gene in autoimmune effector T cells might correlate with susceptibility to EAE and raised the question whether such a predominant use of a single TCR VB gene could be generalized to EAE in other genetic strains. In this communication we examined another highly susceptible homozygous mouse strain, SJL/J, which lacks the 8 gene family (8) that is predominantly expressed in VP3 encephalitogenic T-cell clones from PL/J mice. In SJL/J mice, an encephalitogenic epitope (p89-101) of MBP was characterized (9). Encephalitogenic T-cell clones specific for this determinant are available (10). Furthermore, a monoclonal antibody, KJ23a, against the TCR V.3 17a gene product has been produced by Kappler et al. (11). The initial studies revealed that the SJL/J-derived encephalitogenic T-cell clone 4b.14a (14a) was positively stained with KJ23a antibody. To determine whether preferential use of a single V3 gene exists in SJL/J mice, we examined the contribution of this V.3 gene to the pathogenesis of EAE in this strain. It was found that about half of the independently derived p89-101specific T-cell clones are KJ23a+. Moreover, the KJ23a- T cells are also encephalitogenic. In addition, we show that there is more than one encephalitogenic epitope within p89101; KJ23a- T cells recognize a nested epitope within p89101-namely, p89-100.
MATERIALS AND METHODS Mice. All female mice were purchased from The Jackson Laboratory. Antigens. Synthetic MBP peptides p89-101 and p89-100 were synthesized by solid-phase techniques. The purity was determined by high-pressure liquid chromatography and by amino acid analysis. Monoclonal Antibodies. KJ23a (11) was a gift from P. Marrack (National Jewish'Hospital, Denver), F23.1 (12) was a gift from M. Bevan (Scripps Clinic and Research Foundation, La Jolla, CA), and GK1.5 (13) was a gift from F. Fitch (University of Chicago). Hybridomas were grown as ascites in BALB/c mice. Ascites were purified over DEAE-Sephacel. T-Cell Clones.' MBP peptide-specific T-cell clones were isolated as described (10, 14), using intact MBP or synthetic MBP peptides. Briefly, SJL/J mice were injected on the flank with an emulsion containing 400 ,tg of intact rat MBP or 200
Experimental allergic encephalomyelitis (EAE) is a murine model of autoimmune inflammatory diseases in the central nervous system; it has been clearly demonstrated that this disorder is mediated by CD4+ T cells (1). EAE can be induced by a monoclonal population of myelin basic protein (MBP)-specific T cells (2) and is prevented or ameliorated by in vivo injection with anti-Ia (3) or anti-L3T4 antibody (4, 5). Although T cells clearly play an essential role in the pathogenesis of this disease, the availability of encephalitogenic T-cell clones has permitted studies of the mofecular genetic basis of EAE. Recently, various rearrangement patterns of the T-cell receptor (TCR) genes have been clarified, and differences in antigen or major histocompatibility complex specificity could be attributed to sequence changes in particular regions ofthe TCR genome (6). A previous report from
Abbreviations: EAE, experimental allergic encephalomyelitis; MBP, myelin basic protein; TCR, T-cell receptor; FACS, fluorescence-activated cell sorter; V,,, variable , chain. $To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
8608
Proc. Natl. Acad. Sci. USA 85 (1988)
Immunology: Sakai et al. nmol of synthetic peptide p89-101 and complete Freund's adjuvant. After 10-14 days a single-cell suspension was made from the draining inguinal and axillary lymph nodes and cultured for 5 days with the immunizing antigen (MBP at 100 Iug/ml or 6.7 ttM of p89-101). The cells were then cultured in RPMI 1640 medium containing 5 x 10-5 M 2-mercaptoethanol, 2 mM L-glutamine, penicillin at 100 units per ml, streptomycin at 100 mg/ml, and 8-15% supernatant of rat spleen cells cultured with Con A every 3 or 4 days. Antigenic stimulation was done every 14 days in the presence of y-irradiated syngeneic splenic antigen-presenting cells. T-cell clones were established from these T-cell lines by limiting dilution at 0.3 cell per well in 96-well microtiter plates in the presence of antigen-presenting cells, antigen, and Con A supernatant. The cloning efficiencies were
