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Preferential association of Vλx light chains with γ2a heavy chains in naturally occurring human myelin basic protein reactive antibodies
Journal of Neuroimmunology 70 Ž1996. 15–20
Preferential association of Vl x light chains with g2a heavy chains in naturally occurring human myelin basic protein reactive antibodies F. Shawn Galin a , Shan-Ren Zhou a
b,c
, John N. Whitaker
b,c,d,e
, J. Edwin Blalock
a,b,c,)
Department of Physiology and Biophysics, UniÕersity of Alabama at Birmingham, Birmingham, AL 35294, USA b Center for Neuroimmunology, UniÕersity of Alabama at Birmingham, Birmingham, AL 35294, USA c ComprehensiÕe Cancer Center, UniÕersity of Alabama at Birmingham, Birmingham, AL 35294, USA d Department of Neurology, UniÕersity of Alabama at Birmingham, Birmingham, AL 35294, USA e Neurology and Research SerÕices of the Birmingham Veterans Medical Center, Birmingham, AL 35233, USA Received 24 January 1996; revised 10 April 1996; accepted 12 April 1996
Abstract Active immunization with myelin basic protein ŽMBP. induces experimental allergic encephalomyelitis ŽEAE. in a variety of animal species, including rats and mice. We have previously described the ability of the newly described mouse lambda Ž l . variable ŽV. region, Vl x, to confer MBP reactivity to an Ab. In this report, we have evaluated the heavy ŽH. chain isotype distribution of Vl x-bearing Abs in normal mouse serum. We demonstrate a biased H chain isotype association with Vl x light ŽL. chains with a skewing towards g2a and 2b isotypes. The IgG2a restriction in normal mouse Igs is even more evident in Vl x-containing Abs that bind MBP. This was confirmed by the ability of purified polyclonal IgG2a Abs to bind MBP and the finding that most or all of the IgG2a Abs that bind MBP seem to harbor a Vl x L chain. The specificity of naturally-occurring Vl x-bearing Abs with MBP can be localized to a particular epitope encompassing residues 25–34 of the MBP molecule. Furthermore, virtually all of the reactivity of Vl x-containing Abs with MBP peptide 25–34 is associated with the g2a isotype. Collectively, these results suggest that the interaction of Vl x with MBP seems to be facilitated by an association with g2a which may reflect preferred VH usage by this isotype. Such unique pairing of particular H chains with Vl x L chains in Abs that bind MBP may be indicative of a new B-cell component involved in the pathogenesis of EAE. Keywords: Experimental allergic encephalomyelitis; B-lymphocytes; Autoimmunity; Antibodies; Multiple sclerosis
1. Introduction Experimental allergic encephalomyelitis ŽEAE. is known to be mediated by CD4q T-cells and susceptibility is linked to genes of the MHC. EAE is considered to be the prime animal model for the study of the human autoimmune demyelinating disease, multiple sclerosis ŽMS.. One of the hallmarks of EAE has been the observation of biased or restricted usage of TCR genes, notably Vb8.2, in the recognition of myelin basic protein ŽMBP. or encephalitogenic MBP peptides ŽBurns et al., 1989; Acha-Orbea et al., 1988.. Administration of mAb against Vb8 or its peptides has been shown to be effective in preventing EAE ŽAcha-Orbea et al., 1988.. Furthermore, anti-idiotypic Žanti-Id. or anticlonotypic T-cells can also prevent EAE ) Corresponding author: Tel.: q1-205-9346439; fax: q1-205-9341446; e-mail: [email protected]
ŽLider et al., 1988.. These results have led to the notion that an Id-anti-Id network may be involved in the pathogenesis or immunoregulation of EAE. Evidence for this includes the expression of crossreactive idiotopes ŽCRI. on Abs as well as TCR against encephalitogenic epitopes of MBP ŽZhou and Whitaker, 1992, 1993; Zhou et al., 1994; Maier et al., 1994.. One particularly relevant CRI was discovered on a mAb, F28C4, raised in the PLrJ mouse against MBP residues acetyl ŽAc. 1–9. The F28C4 Id was found to be shared with PLrJ Vb8q TCR against the same peptide ŽZhou and Whitaker, 1992. and encephalitogenic Lewis rat Vb8q TCR against MBP 68–88 ŽZhou et al., 1994.. The CRI is defined by an anti-Id mAb ŽF30C7. which can block both peptide recognition by F28C4 ŽZhou and Whitaker, 1992. and peptide stimulation of MBP Ac1-9specific PLrJ T-cells ŽZhou and Whitaker, 1993. or encephalitogenic MBP 68–88 specific T-cells ŽZhou et al.,
0165-5728r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 6 . 0 0 0 7 2 - 0
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F.S. Galin et al.r Journal of Neuroimmunology 70 (1996) 15–20
1994.. This effect on T-cell reactivity strongly suggests that the Id resides at or near the combining sites of F28C4 and the TCR. Furthermore, the CRI is not a public Id but rather appears to be EAE-related because F30C7 specifically immunoprecipitates the TCR from PLrJ MBP acetyl 1–9 and Lewis rat MBP 68–88 reactive CD4q Vb8q T-cells but not Vb8q cells of other specificities, and F30C7 does not bind isotype-matched mouse Ig ŽZhou et al., 1994.. Moreover, the CRI is probably very relevant to EAE, since F30C7 lessens clinical disease in the adoptive transfer model of EAE in PLrJ mice ŽZhou and Whitaker, 1993. and apparently causes anergy in Lewis rat encephalitogenic T-cells ŽZhou et al., 1994.. To further characterize this CRI, we have previously cloned and sequenced the variable regions encoding F28C4. Sequence homology Ž75% overall. was found between the regions containing complementary determining region ŽCDR. 3 of F28C4 VL and VH and the V–D–J junction of certain TCR Vb8 from encephalitogenic PLrJ ŽMaier et al., 1994. and Lewis rat T-cells ŽJ.E. Blalock, unpublished observation.. This similarity seems, in part, to explain the fine epitope specificity shared between F28C4 and PLrJ encephalitogenic TCR ŽMaier et al., 1994.. This sequence homology is not shared by other Ig CDR3 regions and arises, in part, because F28C4 uses an unusual V lambda Ž l . L chain, Vl x. Prior to this finding, there was no Ab described with a known specificity that contained Vl x. Vl x is a newly described Vl gene segment that was discovered in 1986 in polyclonally activated B-cells ŽDildrop et al., 1987; Sanchez et al., 1987.. Vl x rearranges with Jl2–Cl2 and displays at least three unique features: its amino acid sequence is only 30–33% homologous to any other known Vl andror V-kappa Žk . gene; the last codon of the Vl x gene is a TAA termination codon that must be disrupted by Jl2 to create a functional codon; and such appropriate joining at the V–J junction results in a third hypervariable region extended by an additional four amino acids. It is interesting to note that homologous sequences have been found in various mammalian species, including man, thus suggesting that Vl x apparently existed before the speciation of mammals ŽSanchez et al., 1990.. Considering the rarity of Vl x usage, less than 0.5% of all Ig in normal mice sera ŽSanchez et al., 1987., it was tempting to speculate that the expression of Vl x in response to an encephalitogenic immunogen may not have been a coincidental event but may rather represent an underlying component involved in the pathogenesis of disease. Consistent with this idea was the observation that the five known Vl x-containing IgM mAbs, each of previously unknown specificity, as well as recombinant Vl x alone, were able to bind human MBP. Since mAbs are somewhat artificial in their selection and might not occur in a more natural setting, we purified the Vl x-containing Ig from normal BALBrc sera and found that these Abs also bind human MBP. Interestingly, the average affinity of these Abs was higher than the five Vl x-containing IgM
mAbs and was closer to that of F28C4 ŽIgG2a, Vl x.. Collectively, such studies demonstrate that Vl x can confer MBP reactivity to an Ig ŽGalin et al., 1996.. In an effort to understand the apparently higher MBP reactivity of naturally occurring Vl x-containing Igs relative to the Vl x-positive IgMs, we have evaluated the heavy chain isotype distribution of Vl x-containing Abs in normal mouse sera. In this report, we show a biased H chain isotype association with Vl x with a skewing toward IgG2a and b. The IgG2a restriction is even more evident in Vl x-bearing Abs that bind human MBP. Thus, the interaction of Vl x with MBP seems to be facilitated by an association with g2a which may reflect preferred VH usage by this isotype. This suggests that, as with MBP reactive TCR in EAE, restricted V region usage may extend to Abs to MBP.
2. Materials and methods 2.1. Antigens Human MBP was prepared by previously described techniques ŽZhou and Whitaker, 1992.. Briefly, human MBP Ž1-170. was isolated from human brain by delipidation, acid extraction at pH 3, and carboxymethylcellulose chromatography at pH 10.6. The human MBP peptide acetyl ŽAc. 1–9 was synthesized in our laboratory on a Biosearch Peptide Synthesizer, Model 9500, and purified by reverse-phase high-performance liquid chromatography. Human MBP peptides 10–19, 14–24, 25–34, and 80–89 were synthesized by Peninsula Laboratories ŽSan Carlos, CA.. 2.2. Polyclonal monospecific Ab to Vl x A New Zealand white rabbit ŽMyrtle Laboratories, Thompson Station, TN. was injected subcutaneously Žfour sites, every two weeks over a six week period. with purified F28C4 Ž50 mgrinjection., first in Freund’s complete adjuvant and twice in incomplete adjuvant. Serum was obtained prior to the first injection and two weeks after each booster to monitor anti-F28C4 titers by ELISA. F28C4 is of the IgG2a isotype and, as with all Vl x-bearing Abs, is a l2 isotype. In order to make the antiserum light chain Ži.e., Vl x. specific, it was passed over an IgG2a Sepharose 4B affinity column to deplete heavy chain specificity. Next, the Jl2–Cl2 reactivity was removed using a MOPC 315 Ž a , l2. Sepharose 4B affinity column. Monospecific Ab to Vl x was collected after binding to and elution from an F28C4 affinity column. The specificity of the resulting anti-Vl x Ab was determined by Western analysis Ždata not shown..
F.S. Galin et al.r Journal of Neuroimmunology 70 (1996) 15–20
2.3. Isolation of Vl x-bearing Ig from normal BALBr c mouse serum Vl x-containing Ig from normal BALBrc mouse sera ŽSigma, St. Louis, MO. was purified over a Sepharose 4B anti-Vl x Ab affinity column prepared using the polyclonal monospecific rabbit anti-Vl x Ab described above. Vl x-containing Abs represented approximately 0.5% of total Ig as previously described ŽSanchez et al., 1987.. 2.4. Polyclonal IgG2a Abs Purified polyclonal IgG2a Ab preparations were purchased from Sigma, St. Louis, MO. 2.5. ELISA Microtiter wells Ž96-well ELISA plates, Corning Glass Works, Corning, NY. were coated with human MBP Ž10.0 mgrml in PBS. or synthetic MBP peptides Ž10 mgrml in PBS. at 48C overnight and blocked with 2% casein in PBS. Washings were performed with PBS containing 0.05% Tween 20. Abs were diluted in PBS containing 1.0% BSA and allowed to bind for two hours at room temperature. Ab binding to MBP or MBP peptides was detected with an appropriate alkaline phosphatase conjugated anti-mouse heavy chain-specific Ab ŽSouthern Biotech, Birmingham, AL., or with anti-mouse H chain isotype specific Abs as described below Žsee Ab Capture ELISA.. Reactions were developed using p-nitrophenyl ŽSigma. and color development was quantitated as absorbance at 405 nm on a UVmax Kinetic Microplate Reader ŽMolecular Devices, Menlo Park, CA.. Background optical density was the mean of wells where only coating Ag was omitted. Controls were also performed with Ag-coated wells and all ELISA components except the primary Ab. These were always negative.
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ing 1% BSA and allowed to bind for 1 h at room temperature. Reactions were developed as described above.
3. Results We first purified the Ig fraction containing Vl x light chains from normal mouse sera and determined whether there was a preferred isotype usage in the Abs that bound human MBP. Fig. 1 shows that human MBP is predominantly bound by IgG2a Abs which have Vl x light chains. To determine whether this may have resulted from a skewed association of Vl x with g2a heavy chains, we assessed the ratio of isotypes in normal mouse sera as compared to the Vl x-containing fraction. Fig. 2 demonstrates that there is a 3.7-fold enrichment of g2a heavy chains in Vl x-bearing Ig. The ratio of other isotypes remained essentially the same except for g2b which was increased by 66% and a which was under- represented by 66%. The increase of 3.7-fold is not sufficient to account for the 14-fold increase in IgG2a binding to MBP as compared to IgG1, IgG3, or IgA Žcompare Figs. 1 and 2.. It does, however, account for the difference with IgG2b. Thus, association of g2a or b with Vl x results in enhanced binding to human MBP although Vl x apparently prefers to associate with g2a. Since five out of five IgM mAb with Vl x light chains bound human MBP, it was quite unexpected to observe such a low level of binding by naturally occurring IgM with Vl x. It seems likely that this was either due to insufficient amounts of IgM in the Vl x fraction or possibly due to an artificial selection for the IgM mAbs. To confirm the ability of IgG2a Abs to bind human MBP, we next tested a purified preparation of polyclonal
2.6. Ab capture ELISA ELISA microtiter wells Ž96-well ELISA plates, Corning Glass Works. were coated with a goat anti-mouse Ig ŽIgM, IgG, IgA. specific Ab ŽSouthern Biotechnology Associates. at a constant concentration Ž5 mgrml. at 48C overnight and blocked with 2% casein in PBS. Washings were performed with PBS containing 0.05% Tween 20. The purified Vl x-bearing Ab fraction or total serum Ig was diluted in PBS containing 1% BSA to a final concentration of 2 mgrml and allowed to bind for 2 h at room temperature. The reactions were set up in replicates of six to allow for detection of Ab binding with a panel of alkaline phosphatase-labelled goat anti-mouse H chainspecific Abs ŽSouthern Biotechnology Associates. that are specific for six murine H chain isotypes Žm, g1, g2a, g2b, g3, a .. Labelled Abs were diluted 1:300 in PBS contain-
Fig. 1. H chain isotype distribution of the Vl x-containing Abs purified from normal BALBrc mouse sera that bind human MBP. ELISA microtiter wells were coated with human MBP Ž10 mgrml.. The purified Vl x-bearing Abs were diluted to a final concentration of 2 mgrml. The reactions were set up in replicates of six to allow for detection of Ab binding with a panel of alkaline phosphatase labelled goat anti-mouse H chain isotype specific Abs that are specific for six murine H isotypes Žm, g1, g2a, g2b, g3, and a .. The results are the mean"SEM of three experiments, each done in duplicate.
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F.S. Galin et al.r Journal of Neuroimmunology 70 (1996) 15–20
Fig. 2. Comparison of the H chain isotype distribution of Vl x-containing Abs purified from normal BALBrc mouse sera versus that of total BALBrc mouse sera Ig. ELISA microtiter wells were coated with a goat anti-mouse Ig ŽIgM, IgG, IgA. specific Ab at 5 mgrml. The purified Vl x-bearing Ab fraction or total serum Ig were diluted to a final concentration of 2 mgrml. The reactions were set up in replicates of six to allow for detection of Ab binding with a panel of alkaline phosphatase-labelled goat anti-mouse H chain-specific Abs that are specific for six murine H chain isotypes Žm, g1, g2a, g2b, g3, a .. The results are presented as the ratio of the percent of isotype representation in the Vl x-bearing Ab fraction versus total serum Ig.
Fig. 4. Reactivity of purified polyclonal IgG2a Abs with various human MBP peptides. ELISA microtiter wells were coated with synthetic MBP peptides representing MBP regions Ac 1–9, 10–19, 14–24, 25–34, and 80–89 at equal concentrations Ž10 mgrml.. The purified IgG2a Abs were diluted to a final concentration of 10 mgrml. Ab reactivity was detected using an alkaline phosphatase labelled goat anti-mouse g2a-specific Ab. The results are the mean"SEM of three experiments, each done in duplicate.
IgG2a. Fig. 3 shows a strong interaction of a subpopulation of IgG2a Abs with human MBP as determined with Ab to mouse IgG. When this same ELISA was developed with polyclonal monospecific Ab to Vl x and corrected for secondary Ab reactivity, the dose responses were superimposable. This strongly suggests that whenever a IgG2a molecule interacts with human MBP, it invariably has a Vl x light chain. When the polyclonal IgG2a preparation was evaluated for MBP epitope specificity, we found that IgG2a molecules with Vl x light chains were predomi-
nantly interactive with human MBP 25–34 ŽFig. 4.. This is particularly interesting since this is the same epitope that is recognized by IgM mAb with Vl x light chains and Vl x alone ŽGalin et al., 1996.. Based on this, it is tempting to speculate that IgM and IgG2a Abs which contain Vl x and bind MBP may use an overlapping group of VH regions. The isotype preference of Vl x-bearing antibodies for MBP 25–34 was confirmed by assessing the total Vl x-bearing Ig fraction for interaction with this peptide. Fig. 5 shows that virtually all reactivity of Vl x-containing Abs with MBP 25–34 resided with the g2a isotype. Once again, the lack of reactivity of Vl x-bearing IgM Abs with the peptide is most likely due to an inadequate amount of this isotype in the Vl x-positive fraction.
Fig. 3. Reactivity of purified mouse polyclonal IgG2a Abs with human MBP. ELISA microtiter wells were coated with human MBP Ž10 mgrml.. The purified polyclonal IgG2a Abs were diluted at various concentrations ranging from 1 mgrml to 100 mgrml. The reactions were set up in duplicate to allow for detection of Ab binding with an alkaline phosphatase conjugated goat anti-mouse IgG specific Ab or monitored with rabbit polyclonal monospecific Ab to Vl x. The latter was detected using a goat anti-rabbit IgG-specific Ab diluted 1:1000 in PBS containing 1% BSA. The results are presented as the optical density at 405 nm.
Fig. 5. H chain isotype distribution of naturally occurring Vl x-containing Abs purified from normal BALBrc mouse sera that are reactive with human MBP peptide 25–34. ELISA microtiter wells were coated with human MBP peptide 25–34 Ž10 mgrml.. The Vl x-containing Ab fraction isolated from total sera was diluted to a final concentration of 2 mgrml. The reactions were set up in replicates of six to allow for detection of Ab binding with a panel of alkaline phosphatase labelled goat anti-mouse H chain isotype specific Abs that are specific for six murine H chain isotypes Žm, g1, g2a, g2b, g3, and a .. The results are the mean"SEM of three experiments, each done in duplicate.
F.S. Galin et al.r Journal of Neuroimmunology 70 (1996) 15–20
4. Discussion In this report, we investigated the association of particular H chain isotypes in the normal mouse Vl x-bearing Ig population as well as those Abs reactive with MBP. Analysis of the isotype distribution in naturally occurring Vl xbearing Abs revealed a preferential usage of g, particularly g2a, isotypes. Thus the fact that the mAb, F28C4 ŽIgG2a, Vl x., is of the g2a isotype may not have been coincidental. This is particularly intriguing considering some of the characteristics associated with g isotypes. For instance, IgG Ab is thought to most often arise in response to Ag stimulation and is in a sense not representative of the normal B-cell repertoire before Ag selection ŽGearhart et al., 1981.. This suggests that there may be a selective pressure by an endogenous Ag during ontogeny that results in the expansion of clones expressing Vl x. In regard to H and L chain pairing, a restriction in the autoreactive Ab repertoire is suggested in autoimmune diseases. This ‘lack of promiscuity’, with regard to H and L chain association, has been documented in both systemic lupus erythematosus ŽRadic et al., 1991. and thyroid disease ŽPortolano et al., 1993.. Such studies have provided insight into the nature of H and L chain contribution to Ag specificity. For example, a recent study analyzing H and L chain contribution to thyroid peroxidase ŽTPO. reactivity found that restricted H and L chain pairing is crucial for high affinity binding to a particular epitope on TPO ŽCostante et al., 1994.. Interestingly, such epitope specificity is lost with different L chain pairings but is maintained in the presence of different D and nonidentical VH regions. These findings suggest that it is the L chain that plays a role in defining TPO epitope recognition. This is similar to our present and previous findings in that the preferential association of Vl x with g isotypes was required for high affinity binding to MBP but the Ag specificity resides with Vl x. These studies also raise the possibility that Vl x predominately associates with g2a isotypes because of a particular VH domain. Interestingly, a recent report described the preferential association of a H chain Id, termed F4 , with an L chain Id in Abs binding DNA ŽDavidson et al., 1990.. Extensive analysis of F4 revealed that it was present almost exclusively on Abs bearing g isotypes. This would suggest that the biased usage of a restricted VH may be indicative of preferential association with a particular C H . Thus, it is possible that the preferential association of g2a and b isotypes with Vl x L chains may underlie a unique pairing of particular VH with g2a and b in these Abs. The existence of isotype-restricted Id has been previously reported in both IgA ŽNishinarita et al., 1985. and IgM ŽRinfret et al., 1985. Abs. In these studies, the first domain of the constant region is thought to contribute in some way to the tertiary conformation of the variable region. This is of particular interest considering nuclear magnetic resonance ŽNMR. studies on F28C4 ŽIgG2a, Vl x. ŽMaier et al., 1994.. In these studies, the area of
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contact residues between the MBP peptide Ac 1–9 complexed with F28C4 was inferred by NMR. The results showed that, like MBP peptide–TCR interactions, F28C4 contacts MBP peptide Ac 1–9 in an extended conformation and that the central residues are more tightly bound than the terminal residues. These NMR studies confirmed and extended epitope mapping and cDNA sequencing which showed homologous sequences and identical fine specificity of F28C4 and encephalitogenic TCR against the same peptide. The contribution of the g2a region to those unique characteristics of F28C4 remains to be determined. Though, as previously mentioned, the constant region in certain instances can contribute to the variable region conformation. Considering that F28C4 and encephalitogenic TCR share a CRI, it is plausible that the shared Id on F28C4 results from a unique, yet biased, VH C H and Vl x combinations as we detected. Although it is often assumed that Ab responses to self Ags result only in pathogenicity, there is also evidence to the contrary. For instance, sera obtained from animals that have recovered from EAE are able to suppress EAE when given to naive recipients at the time of active disease induction ŽKillen and Swanborg, 1982.. This is entirely consistent with the ability of antiserum from animals immunized with MBP or its encephalitogenic peptides to inhibit the disease ŽMacphee et al., 1990.. Considering that mAbs specific for MBP can inhibit the proliferation and cytotoxicity of MBP-specific T-cell lines, in vitro ŽJingwu et al., 1989., an IgG2arVl x humoral response to MBP, in vivo, could potentially modulate the T-cell-mediated immune response through a T-cellrB-cell network. This notion is consistent with the finding that the F28C4 anti-Id mAb, F30C7, can suppress EAE. Furthermore, our preliminary results suggest that the Vl x containing Ab, F28C4, is a potent idiotypic vaccine against both adoptively transferred and actively induced EAE ŽF.S. Galin et al., manuscript in preparation.. If extended to humans, such results could suggest a new therapeutic regimen for MS. Delineation of the regulation of Vl x expression and its preferential association with g2a in the response to an epitope of MBP becomes an important step in linking V region genes and CRI to the inflammatory demyelination in EAE and MS. Acknowledgements This work was supported by a Multiple Sclerosis Society Postdoctoral Fellowship Award to F.S.G.; National Institutes of Health Grant PPG P01 NS29719 to J.E.B., S.R.Z., and J.N.W.; National Institutes of Health Grant DK38024, Tobacco Council Grant 2222, and a Multiple Sclerosis Society Pilot Grant to J.E.B.; and National Institutes of Health Grant NS23240 and the Research Program of the Veterans Administration to J.N.W. The authors wish to thank Diane Weigent for editorial assistance and typing of this manuscript.
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F.S. Galin et al.r Journal of Neuroimmunology 70 (1996) 15–20
References Acha-Orbea, H., Mitchell, D.J., Timmermann, L., Wraith, D.C., Tausch, G.S., Waldor, M.K., Zamvil, S.S., McDevitt, H.O. and Steinman, L. Ž1988. Limited heterogeneity of T cell receptors from lymphocytes mediating autoimmune allows specific immune intervention. Cell 54, 263–273. Burns, F.R., Li, X., Shen, N., Offner, H., Chou, V.K., Vandenbark, A., and Heber-Katz, E. Ž1989. Both rat and mouse T cell receptors specific for the encephalitogenic determinants of myelin basic protein use similar Va and Vb genes. J. Exp. Med. 169, 27–39. Costante, G., Portolano, S., Nishikawa, T., Jaume, J.C., Chazenbalk, G.D., Rapoport, B. and McLachlan, S.M. Ž1994. Recombinant thyroid peroxidase-specific autoantibodies. II. Role of individual heavy and light chains in determining epitope recognition. Endocrinology 135, 25–30. Davidson, A., Smith, A., Katz, J., Preud’homme, J.-L., Solomon, A. and Diamond, B. Ž1990. A cross-reactive idiotype on anti-DNA antibodies defines a H chain determinant present almost exclusively on IgG antibodies. J. Immunol. 143, 174–180. Dildrop, R., Gause, A., Muller, W. and Rajewsky, K. Ž1987. A new V ¨ gene expressed in lambda-2 light chains of the mouse. Eur. J. Immunol. 17, 731–734. Galin, F.S., Maier, C.C., Zhou, S.-R., Whitaker, J.N. and Blalock, J.E. Ž1996. Murine Vl x and Vl x-containing antibodies bind human myelin basic protein. J. Clin. Invest. 97, 486–492. Gearhart, P.J., Johnson, N.D., Douglas, R. and Hood, L. Ž1981. IgG antibodies to phosphorylcholine exhibit more diversity than their IgM counterparts. Nature ŽLondon. 291, 29–34. Jingwu, Z., Vandenbark, A.A., Jacobs, M.P., Offner, H. and Raus, J.C.M. Ž1989. Murine monoclonal anti-myelin basic protein ŽMBP. antibodies inhibit the proliferation and cytotoxicity of MBP-specific human T cell clones. J. Neuroimmunol. 24, 87–94. Killen, J.A. and Swanborg, R.H. Ž1982. Regulation of experimental allergic encephalomyelitis. IV. Further characterization of postrecovery suppressor cells. J. Neuroimmunol. 3, 159–166. Lider, O., Reshef, T., Beraud, E., Ben-Nun, A. and Cohen, I.R. Ž1988. Anti-idiotypic network induced by T cell vaccination against experimental autoimmune encephalomyelitis. Science 239, 181–183.
Macphee, I.A.M., Day, M.J. and Mason, D.W. Ž1990. The role of serum factors in the suppression of experimental allergic encephalomyelitis: Evidence for immunoregulation by antibody to the encephalitogenic peptide. Immunol. 70, 527–534. Maier, C.C., Galin, F.S., Jarpe, M.A., Jackson, P., Krishna, R.N., Gautam, A.M., Zhou, S.-R., Whitaker, J.N. and Blalock, J.E. Ž1994. A Vl x-bearing monoclonal antibody with similar specificity and sequence to encephalitogenic T cells. J. Immunol. 153, 1132–1140. Nishinarita, S., Claflin, J.L. and Lieberman, R. Ž1985. IgA isotype restricted idiotypes associated with T15 Idq Pc antibodies. J. Immunol. 134, 2544–2549. Portolano, S., Chazenbalk, G.D., Hutchinson, J.S., McLachlan, S.M. and Rapoport, B. Ž1993. Lack of promiscuity in autoantigen-specific H and L chain combinations as revealed by human H and L chain "roulette." J. Immunol. 150, 880–887. Radic, M.Z., Mascelli, M.A., Erikson, J., Shan, H. and Weigert, M. Ž1991. Ig H and L chain contributions to autoimmune specificities. J. Immunol. 146, 176–182. Rinfret, A., Horne, C., Dorrington, J. and Klein, M. Ž1985. Noncovalent association of heavy and light chains of human immunoglobulins. IV: the roles of the C and C L domains in idiotype expression. J. Immunol. 135, 2574–2581. Sanchez, P., Marche, P.N., LeGuern, C. and Cazenave, P.A. Ž1987. Structure of a third murine immunoglobulin l light chain variable region that is in laboratory mice. Proc. Natl. Acad. Sci. 8, 9185–9188. Sanchez, P., Marche, P.N., Rueff-Juy, D. and Cazenave, P.A. Ž1990. Mouse Vl x gene sequence generates no junctional diversity and is conserved in mammalian species. J. Immunol. 144, 2816–2820. Zhou, S.-R. and Whitaker, J.N. Ž1992. Interstrain cross-reactive idiotypes on monoclonal antibodies to an encephalitogenic myelin basic protein peptide. Clin. Immunol. Immunopathol. 63, 74–83. Zhou, S.-R. and Whitaker, J.N. Ž1993. Specific modulation of T cells and murine experimental allergic encephalomyelitis by monoclonal antiidiotypic antibodies. J. Immunol. 150, 1629–1642. Zhou, S.-R., Whitaker, J.N., Han, Q., Maier, C.C. and Blalock, J.E. Ž1994. A cross-reactive idiotope on T cells from PLrJ mice and Lewis rats recognizing different myelin basic protein encephalitogenic epitopes but restricted by T cell receptor Vb8.2. J. Immunol. 153, 2340–2351.
Preferential association of Vl x light chains with g2a heavy chains in naturally occurring human myelin basic protein reactive antibodies F. Shawn Galin a , Shan-Ren Zhou a
b,c
, John N. Whitaker
b,c,d,e
, J. Edwin Blalock
a,b,c,)
Department of Physiology and Biophysics, UniÕersity of Alabama at Birmingham, Birmingham, AL 35294, USA b Center for Neuroimmunology, UniÕersity of Alabama at Birmingham, Birmingham, AL 35294, USA c ComprehensiÕe Cancer Center, UniÕersity of Alabama at Birmingham, Birmingham, AL 35294, USA d Department of Neurology, UniÕersity of Alabama at Birmingham, Birmingham, AL 35294, USA e Neurology and Research SerÕices of the Birmingham Veterans Medical Center, Birmingham, AL 35233, USA Received 24 January 1996; revised 10 April 1996; accepted 12 April 1996
Abstract Active immunization with myelin basic protein ŽMBP. induces experimental allergic encephalomyelitis ŽEAE. in a variety of animal species, including rats and mice. We have previously described the ability of the newly described mouse lambda Ž l . variable ŽV. region, Vl x, to confer MBP reactivity to an Ab. In this report, we have evaluated the heavy ŽH. chain isotype distribution of Vl x-bearing Abs in normal mouse serum. We demonstrate a biased H chain isotype association with Vl x light ŽL. chains with a skewing towards g2a and 2b isotypes. The IgG2a restriction in normal mouse Igs is even more evident in Vl x-containing Abs that bind MBP. This was confirmed by the ability of purified polyclonal IgG2a Abs to bind MBP and the finding that most or all of the IgG2a Abs that bind MBP seem to harbor a Vl x L chain. The specificity of naturally-occurring Vl x-bearing Abs with MBP can be localized to a particular epitope encompassing residues 25–34 of the MBP molecule. Furthermore, virtually all of the reactivity of Vl x-containing Abs with MBP peptide 25–34 is associated with the g2a isotype. Collectively, these results suggest that the interaction of Vl x with MBP seems to be facilitated by an association with g2a which may reflect preferred VH usage by this isotype. Such unique pairing of particular H chains with Vl x L chains in Abs that bind MBP may be indicative of a new B-cell component involved in the pathogenesis of EAE. Keywords: Experimental allergic encephalomyelitis; B-lymphocytes; Autoimmunity; Antibodies; Multiple sclerosis
1. Introduction Experimental allergic encephalomyelitis ŽEAE. is known to be mediated by CD4q T-cells and susceptibility is linked to genes of the MHC. EAE is considered to be the prime animal model for the study of the human autoimmune demyelinating disease, multiple sclerosis ŽMS.. One of the hallmarks of EAE has been the observation of biased or restricted usage of TCR genes, notably Vb8.2, in the recognition of myelin basic protein ŽMBP. or encephalitogenic MBP peptides ŽBurns et al., 1989; Acha-Orbea et al., 1988.. Administration of mAb against Vb8 or its peptides has been shown to be effective in preventing EAE ŽAcha-Orbea et al., 1988.. Furthermore, anti-idiotypic Žanti-Id. or anticlonotypic T-cells can also prevent EAE ) Corresponding author: Tel.: q1-205-9346439; fax: q1-205-9341446; e-mail: [email protected]
ŽLider et al., 1988.. These results have led to the notion that an Id-anti-Id network may be involved in the pathogenesis or immunoregulation of EAE. Evidence for this includes the expression of crossreactive idiotopes ŽCRI. on Abs as well as TCR against encephalitogenic epitopes of MBP ŽZhou and Whitaker, 1992, 1993; Zhou et al., 1994; Maier et al., 1994.. One particularly relevant CRI was discovered on a mAb, F28C4, raised in the PLrJ mouse against MBP residues acetyl ŽAc. 1–9. The F28C4 Id was found to be shared with PLrJ Vb8q TCR against the same peptide ŽZhou and Whitaker, 1992. and encephalitogenic Lewis rat Vb8q TCR against MBP 68–88 ŽZhou et al., 1994.. The CRI is defined by an anti-Id mAb ŽF30C7. which can block both peptide recognition by F28C4 ŽZhou and Whitaker, 1992. and peptide stimulation of MBP Ac1-9specific PLrJ T-cells ŽZhou and Whitaker, 1993. or encephalitogenic MBP 68–88 specific T-cells ŽZhou et al.,
0165-5728r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 6 . 0 0 0 7 2 - 0
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1994.. This effect on T-cell reactivity strongly suggests that the Id resides at or near the combining sites of F28C4 and the TCR. Furthermore, the CRI is not a public Id but rather appears to be EAE-related because F30C7 specifically immunoprecipitates the TCR from PLrJ MBP acetyl 1–9 and Lewis rat MBP 68–88 reactive CD4q Vb8q T-cells but not Vb8q cells of other specificities, and F30C7 does not bind isotype-matched mouse Ig ŽZhou et al., 1994.. Moreover, the CRI is probably very relevant to EAE, since F30C7 lessens clinical disease in the adoptive transfer model of EAE in PLrJ mice ŽZhou and Whitaker, 1993. and apparently causes anergy in Lewis rat encephalitogenic T-cells ŽZhou et al., 1994.. To further characterize this CRI, we have previously cloned and sequenced the variable regions encoding F28C4. Sequence homology Ž75% overall. was found between the regions containing complementary determining region ŽCDR. 3 of F28C4 VL and VH and the V–D–J junction of certain TCR Vb8 from encephalitogenic PLrJ ŽMaier et al., 1994. and Lewis rat T-cells ŽJ.E. Blalock, unpublished observation.. This similarity seems, in part, to explain the fine epitope specificity shared between F28C4 and PLrJ encephalitogenic TCR ŽMaier et al., 1994.. This sequence homology is not shared by other Ig CDR3 regions and arises, in part, because F28C4 uses an unusual V lambda Ž l . L chain, Vl x. Prior to this finding, there was no Ab described with a known specificity that contained Vl x. Vl x is a newly described Vl gene segment that was discovered in 1986 in polyclonally activated B-cells ŽDildrop et al., 1987; Sanchez et al., 1987.. Vl x rearranges with Jl2–Cl2 and displays at least three unique features: its amino acid sequence is only 30–33% homologous to any other known Vl andror V-kappa Žk . gene; the last codon of the Vl x gene is a TAA termination codon that must be disrupted by Jl2 to create a functional codon; and such appropriate joining at the V–J junction results in a third hypervariable region extended by an additional four amino acids. It is interesting to note that homologous sequences have been found in various mammalian species, including man, thus suggesting that Vl x apparently existed before the speciation of mammals ŽSanchez et al., 1990.. Considering the rarity of Vl x usage, less than 0.5% of all Ig in normal mice sera ŽSanchez et al., 1987., it was tempting to speculate that the expression of Vl x in response to an encephalitogenic immunogen may not have been a coincidental event but may rather represent an underlying component involved in the pathogenesis of disease. Consistent with this idea was the observation that the five known Vl x-containing IgM mAbs, each of previously unknown specificity, as well as recombinant Vl x alone, were able to bind human MBP. Since mAbs are somewhat artificial in their selection and might not occur in a more natural setting, we purified the Vl x-containing Ig from normal BALBrc sera and found that these Abs also bind human MBP. Interestingly, the average affinity of these Abs was higher than the five Vl x-containing IgM
mAbs and was closer to that of F28C4 ŽIgG2a, Vl x.. Collectively, such studies demonstrate that Vl x can confer MBP reactivity to an Ig ŽGalin et al., 1996.. In an effort to understand the apparently higher MBP reactivity of naturally occurring Vl x-containing Igs relative to the Vl x-positive IgMs, we have evaluated the heavy chain isotype distribution of Vl x-containing Abs in normal mouse sera. In this report, we show a biased H chain isotype association with Vl x with a skewing toward IgG2a and b. The IgG2a restriction is even more evident in Vl x-bearing Abs that bind human MBP. Thus, the interaction of Vl x with MBP seems to be facilitated by an association with g2a which may reflect preferred VH usage by this isotype. This suggests that, as with MBP reactive TCR in EAE, restricted V region usage may extend to Abs to MBP.
2. Materials and methods 2.1. Antigens Human MBP was prepared by previously described techniques ŽZhou and Whitaker, 1992.. Briefly, human MBP Ž1-170. was isolated from human brain by delipidation, acid extraction at pH 3, and carboxymethylcellulose chromatography at pH 10.6. The human MBP peptide acetyl ŽAc. 1–9 was synthesized in our laboratory on a Biosearch Peptide Synthesizer, Model 9500, and purified by reverse-phase high-performance liquid chromatography. Human MBP peptides 10–19, 14–24, 25–34, and 80–89 were synthesized by Peninsula Laboratories ŽSan Carlos, CA.. 2.2. Polyclonal monospecific Ab to Vl x A New Zealand white rabbit ŽMyrtle Laboratories, Thompson Station, TN. was injected subcutaneously Žfour sites, every two weeks over a six week period. with purified F28C4 Ž50 mgrinjection., first in Freund’s complete adjuvant and twice in incomplete adjuvant. Serum was obtained prior to the first injection and two weeks after each booster to monitor anti-F28C4 titers by ELISA. F28C4 is of the IgG2a isotype and, as with all Vl x-bearing Abs, is a l2 isotype. In order to make the antiserum light chain Ži.e., Vl x. specific, it was passed over an IgG2a Sepharose 4B affinity column to deplete heavy chain specificity. Next, the Jl2–Cl2 reactivity was removed using a MOPC 315 Ž a , l2. Sepharose 4B affinity column. Monospecific Ab to Vl x was collected after binding to and elution from an F28C4 affinity column. The specificity of the resulting anti-Vl x Ab was determined by Western analysis Ždata not shown..
F.S. Galin et al.r Journal of Neuroimmunology 70 (1996) 15–20
2.3. Isolation of Vl x-bearing Ig from normal BALBr c mouse serum Vl x-containing Ig from normal BALBrc mouse sera ŽSigma, St. Louis, MO. was purified over a Sepharose 4B anti-Vl x Ab affinity column prepared using the polyclonal monospecific rabbit anti-Vl x Ab described above. Vl x-containing Abs represented approximately 0.5% of total Ig as previously described ŽSanchez et al., 1987.. 2.4. Polyclonal IgG2a Abs Purified polyclonal IgG2a Ab preparations were purchased from Sigma, St. Louis, MO. 2.5. ELISA Microtiter wells Ž96-well ELISA plates, Corning Glass Works, Corning, NY. were coated with human MBP Ž10.0 mgrml in PBS. or synthetic MBP peptides Ž10 mgrml in PBS. at 48C overnight and blocked with 2% casein in PBS. Washings were performed with PBS containing 0.05% Tween 20. Abs were diluted in PBS containing 1.0% BSA and allowed to bind for two hours at room temperature. Ab binding to MBP or MBP peptides was detected with an appropriate alkaline phosphatase conjugated anti-mouse heavy chain-specific Ab ŽSouthern Biotech, Birmingham, AL., or with anti-mouse H chain isotype specific Abs as described below Žsee Ab Capture ELISA.. Reactions were developed using p-nitrophenyl ŽSigma. and color development was quantitated as absorbance at 405 nm on a UVmax Kinetic Microplate Reader ŽMolecular Devices, Menlo Park, CA.. Background optical density was the mean of wells where only coating Ag was omitted. Controls were also performed with Ag-coated wells and all ELISA components except the primary Ab. These were always negative.
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ing 1% BSA and allowed to bind for 1 h at room temperature. Reactions were developed as described above.
3. Results We first purified the Ig fraction containing Vl x light chains from normal mouse sera and determined whether there was a preferred isotype usage in the Abs that bound human MBP. Fig. 1 shows that human MBP is predominantly bound by IgG2a Abs which have Vl x light chains. To determine whether this may have resulted from a skewed association of Vl x with g2a heavy chains, we assessed the ratio of isotypes in normal mouse sera as compared to the Vl x-containing fraction. Fig. 2 demonstrates that there is a 3.7-fold enrichment of g2a heavy chains in Vl x-bearing Ig. The ratio of other isotypes remained essentially the same except for g2b which was increased by 66% and a which was under- represented by 66%. The increase of 3.7-fold is not sufficient to account for the 14-fold increase in IgG2a binding to MBP as compared to IgG1, IgG3, or IgA Žcompare Figs. 1 and 2.. It does, however, account for the difference with IgG2b. Thus, association of g2a or b with Vl x results in enhanced binding to human MBP although Vl x apparently prefers to associate with g2a. Since five out of five IgM mAb with Vl x light chains bound human MBP, it was quite unexpected to observe such a low level of binding by naturally occurring IgM with Vl x. It seems likely that this was either due to insufficient amounts of IgM in the Vl x fraction or possibly due to an artificial selection for the IgM mAbs. To confirm the ability of IgG2a Abs to bind human MBP, we next tested a purified preparation of polyclonal
2.6. Ab capture ELISA ELISA microtiter wells Ž96-well ELISA plates, Corning Glass Works. were coated with a goat anti-mouse Ig ŽIgM, IgG, IgA. specific Ab ŽSouthern Biotechnology Associates. at a constant concentration Ž5 mgrml. at 48C overnight and blocked with 2% casein in PBS. Washings were performed with PBS containing 0.05% Tween 20. The purified Vl x-bearing Ab fraction or total serum Ig was diluted in PBS containing 1% BSA to a final concentration of 2 mgrml and allowed to bind for 2 h at room temperature. The reactions were set up in replicates of six to allow for detection of Ab binding with a panel of alkaline phosphatase-labelled goat anti-mouse H chainspecific Abs ŽSouthern Biotechnology Associates. that are specific for six murine H chain isotypes Žm, g1, g2a, g2b, g3, a .. Labelled Abs were diluted 1:300 in PBS contain-
Fig. 1. H chain isotype distribution of the Vl x-containing Abs purified from normal BALBrc mouse sera that bind human MBP. ELISA microtiter wells were coated with human MBP Ž10 mgrml.. The purified Vl x-bearing Abs were diluted to a final concentration of 2 mgrml. The reactions were set up in replicates of six to allow for detection of Ab binding with a panel of alkaline phosphatase labelled goat anti-mouse H chain isotype specific Abs that are specific for six murine H isotypes Žm, g1, g2a, g2b, g3, and a .. The results are the mean"SEM of three experiments, each done in duplicate.
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F.S. Galin et al.r Journal of Neuroimmunology 70 (1996) 15–20
Fig. 2. Comparison of the H chain isotype distribution of Vl x-containing Abs purified from normal BALBrc mouse sera versus that of total BALBrc mouse sera Ig. ELISA microtiter wells were coated with a goat anti-mouse Ig ŽIgM, IgG, IgA. specific Ab at 5 mgrml. The purified Vl x-bearing Ab fraction or total serum Ig were diluted to a final concentration of 2 mgrml. The reactions were set up in replicates of six to allow for detection of Ab binding with a panel of alkaline phosphatase-labelled goat anti-mouse H chain-specific Abs that are specific for six murine H chain isotypes Žm, g1, g2a, g2b, g3, a .. The results are presented as the ratio of the percent of isotype representation in the Vl x-bearing Ab fraction versus total serum Ig.
Fig. 4. Reactivity of purified polyclonal IgG2a Abs with various human MBP peptides. ELISA microtiter wells were coated with synthetic MBP peptides representing MBP regions Ac 1–9, 10–19, 14–24, 25–34, and 80–89 at equal concentrations Ž10 mgrml.. The purified IgG2a Abs were diluted to a final concentration of 10 mgrml. Ab reactivity was detected using an alkaline phosphatase labelled goat anti-mouse g2a-specific Ab. The results are the mean"SEM of three experiments, each done in duplicate.
IgG2a. Fig. 3 shows a strong interaction of a subpopulation of IgG2a Abs with human MBP as determined with Ab to mouse IgG. When this same ELISA was developed with polyclonal monospecific Ab to Vl x and corrected for secondary Ab reactivity, the dose responses were superimposable. This strongly suggests that whenever a IgG2a molecule interacts with human MBP, it invariably has a Vl x light chain. When the polyclonal IgG2a preparation was evaluated for MBP epitope specificity, we found that IgG2a molecules with Vl x light chains were predomi-
nantly interactive with human MBP 25–34 ŽFig. 4.. This is particularly interesting since this is the same epitope that is recognized by IgM mAb with Vl x light chains and Vl x alone ŽGalin et al., 1996.. Based on this, it is tempting to speculate that IgM and IgG2a Abs which contain Vl x and bind MBP may use an overlapping group of VH regions. The isotype preference of Vl x-bearing antibodies for MBP 25–34 was confirmed by assessing the total Vl x-bearing Ig fraction for interaction with this peptide. Fig. 5 shows that virtually all reactivity of Vl x-containing Abs with MBP 25–34 resided with the g2a isotype. Once again, the lack of reactivity of Vl x-bearing IgM Abs with the peptide is most likely due to an inadequate amount of this isotype in the Vl x-positive fraction.
Fig. 3. Reactivity of purified mouse polyclonal IgG2a Abs with human MBP. ELISA microtiter wells were coated with human MBP Ž10 mgrml.. The purified polyclonal IgG2a Abs were diluted at various concentrations ranging from 1 mgrml to 100 mgrml. The reactions were set up in duplicate to allow for detection of Ab binding with an alkaline phosphatase conjugated goat anti-mouse IgG specific Ab or monitored with rabbit polyclonal monospecific Ab to Vl x. The latter was detected using a goat anti-rabbit IgG-specific Ab diluted 1:1000 in PBS containing 1% BSA. The results are presented as the optical density at 405 nm.
Fig. 5. H chain isotype distribution of naturally occurring Vl x-containing Abs purified from normal BALBrc mouse sera that are reactive with human MBP peptide 25–34. ELISA microtiter wells were coated with human MBP peptide 25–34 Ž10 mgrml.. The Vl x-containing Ab fraction isolated from total sera was diluted to a final concentration of 2 mgrml. The reactions were set up in replicates of six to allow for detection of Ab binding with a panel of alkaline phosphatase labelled goat anti-mouse H chain isotype specific Abs that are specific for six murine H chain isotypes Žm, g1, g2a, g2b, g3, and a .. The results are the mean"SEM of three experiments, each done in duplicate.
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4. Discussion In this report, we investigated the association of particular H chain isotypes in the normal mouse Vl x-bearing Ig population as well as those Abs reactive with MBP. Analysis of the isotype distribution in naturally occurring Vl xbearing Abs revealed a preferential usage of g, particularly g2a, isotypes. Thus the fact that the mAb, F28C4 ŽIgG2a, Vl x., is of the g2a isotype may not have been coincidental. This is particularly intriguing considering some of the characteristics associated with g isotypes. For instance, IgG Ab is thought to most often arise in response to Ag stimulation and is in a sense not representative of the normal B-cell repertoire before Ag selection ŽGearhart et al., 1981.. This suggests that there may be a selective pressure by an endogenous Ag during ontogeny that results in the expansion of clones expressing Vl x. In regard to H and L chain pairing, a restriction in the autoreactive Ab repertoire is suggested in autoimmune diseases. This ‘lack of promiscuity’, with regard to H and L chain association, has been documented in both systemic lupus erythematosus ŽRadic et al., 1991. and thyroid disease ŽPortolano et al., 1993.. Such studies have provided insight into the nature of H and L chain contribution to Ag specificity. For example, a recent study analyzing H and L chain contribution to thyroid peroxidase ŽTPO. reactivity found that restricted H and L chain pairing is crucial for high affinity binding to a particular epitope on TPO ŽCostante et al., 1994.. Interestingly, such epitope specificity is lost with different L chain pairings but is maintained in the presence of different D and nonidentical VH regions. These findings suggest that it is the L chain that plays a role in defining TPO epitope recognition. This is similar to our present and previous findings in that the preferential association of Vl x with g isotypes was required for high affinity binding to MBP but the Ag specificity resides with Vl x. These studies also raise the possibility that Vl x predominately associates with g2a isotypes because of a particular VH domain. Interestingly, a recent report described the preferential association of a H chain Id, termed F4 , with an L chain Id in Abs binding DNA ŽDavidson et al., 1990.. Extensive analysis of F4 revealed that it was present almost exclusively on Abs bearing g isotypes. This would suggest that the biased usage of a restricted VH may be indicative of preferential association with a particular C H . Thus, it is possible that the preferential association of g2a and b isotypes with Vl x L chains may underlie a unique pairing of particular VH with g2a and b in these Abs. The existence of isotype-restricted Id has been previously reported in both IgA ŽNishinarita et al., 1985. and IgM ŽRinfret et al., 1985. Abs. In these studies, the first domain of the constant region is thought to contribute in some way to the tertiary conformation of the variable region. This is of particular interest considering nuclear magnetic resonance ŽNMR. studies on F28C4 ŽIgG2a, Vl x. ŽMaier et al., 1994.. In these studies, the area of
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contact residues between the MBP peptide Ac 1–9 complexed with F28C4 was inferred by NMR. The results showed that, like MBP peptide–TCR interactions, F28C4 contacts MBP peptide Ac 1–9 in an extended conformation and that the central residues are more tightly bound than the terminal residues. These NMR studies confirmed and extended epitope mapping and cDNA sequencing which showed homologous sequences and identical fine specificity of F28C4 and encephalitogenic TCR against the same peptide. The contribution of the g2a region to those unique characteristics of F28C4 remains to be determined. Though, as previously mentioned, the constant region in certain instances can contribute to the variable region conformation. Considering that F28C4 and encephalitogenic TCR share a CRI, it is plausible that the shared Id on F28C4 results from a unique, yet biased, VH C H and Vl x combinations as we detected. Although it is often assumed that Ab responses to self Ags result only in pathogenicity, there is also evidence to the contrary. For instance, sera obtained from animals that have recovered from EAE are able to suppress EAE when given to naive recipients at the time of active disease induction ŽKillen and Swanborg, 1982.. This is entirely consistent with the ability of antiserum from animals immunized with MBP or its encephalitogenic peptides to inhibit the disease ŽMacphee et al., 1990.. Considering that mAbs specific for MBP can inhibit the proliferation and cytotoxicity of MBP-specific T-cell lines, in vitro ŽJingwu et al., 1989., an IgG2arVl x humoral response to MBP, in vivo, could potentially modulate the T-cell-mediated immune response through a T-cellrB-cell network. This notion is consistent with the finding that the F28C4 anti-Id mAb, F30C7, can suppress EAE. Furthermore, our preliminary results suggest that the Vl x containing Ab, F28C4, is a potent idiotypic vaccine against both adoptively transferred and actively induced EAE ŽF.S. Galin et al., manuscript in preparation.. If extended to humans, such results could suggest a new therapeutic regimen for MS. Delineation of the regulation of Vl x expression and its preferential association with g2a in the response to an epitope of MBP becomes an important step in linking V region genes and CRI to the inflammatory demyelination in EAE and MS. Acknowledgements This work was supported by a Multiple Sclerosis Society Postdoctoral Fellowship Award to F.S.G.; National Institutes of Health Grant PPG P01 NS29719 to J.E.B., S.R.Z., and J.N.W.; National Institutes of Health Grant DK38024, Tobacco Council Grant 2222, and a Multiple Sclerosis Society Pilot Grant to J.E.B.; and National Institutes of Health Grant NS23240 and the Research Program of the Veterans Administration to J.N.W. The authors wish to thank Diane Weigent for editorial assistance and typing of this manuscript.
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