Antigenized antibodies expressing Vβ8.2 TCR peptides immunize against rat experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is an experimentally induced autoimmune disease mediated by T cells. It can be induced in susceptible animals either by immunization with myelin basic protein (MBP) or proteolipid protein PLP, or by immunization with synthetic peptides from the MBP sequence [1]. EAE can also be initiated by the passive transfer of encephalitogenic, MBP-specific T cell lines or clones [2, 3]. In the Lewis rat, EAE is characterized by a self limiting, ascending, hind limb paralysis. Histologically, EAE is hallmarked by perivascular and submeningeal infiltration of inflammatory cells within the brain and spinal cord [4]. After recovery, animals become refractory to further induction of paralysis by immunization with MBP. Owing to similarities in clinical expression and histopathology, EAE has long been recognized as an animal model for multiple sclerosis, a demyelinating chronic inflammatory disease in humans of unknown origin. For this reason, studies on EAE are thought to elucidate aspects of the pathogenesis and indicate possible ways of immune intervention.

EAE is mediated by MHC class II -restricted, MBP-specific CD4⁺ T lymphocytes bearing an antigen receptor (TCR) variable (V) regions belonging to a limited set of TCR V region gene families [5, 6] and restricted Vα-Vβ gene combinations [7]. Several rational approaches have been used to prevent EAE, including passive transfer of monoclonal antibodies that interfere with the recognition of the MHC, TCR and MBP peptide complex [8, 9], antibodies against CD4 [10] and T regulatory cells [11–14]. Active immunity against attenuated encephalitogenic T cells was shown to prevent the induction of disease [15, 16] and vaccination with synthetic peptides of the complementarity-determining regions (CDR) of the TCR of ecephalitogenic T cells, confer resistance to EAE in the rat [17–20]. Together these facts indicated that T cells are crucial to the pathogenesis of EAE and, in converse, immunity to idiotypic determinants of the TCR of encephalitogenic T cells may be protective.

Approaches to directly target the TCR of pathogenetic T cells are an attractive direction for therapy and immunointervention as well as an opportunity to further understand the immunological events involved in protection in vivo. However, limitations exist to methods available for TCR vaccination. Vaccination using attenuated encephalitogenic T cells requires that these are specifically expanded in vitro and can only be used in an autologous system. Synthetic peptides, albeit successful in several instances [17–20], offer no tri-dimensional conformation and may even yield to opposite effect, e.g., worsening of disease [21, 22]. Similarly, vaccination with single chain TCR was shown to either prevent or exacerbate EAE in mice [23].

In previous work from this laboratory we demonstrated the induction of anti-receptor immunity using immunoglobulins (Ig) expressing discrete peptide portions of human CD4 [24]. We refer to such Ig as antigenized antibodies, i.e., Ig molecules in which foreign peptide sequences are conformationally-constrained and expressed in the complementority-determining region (CDR) loops [25]. Immunization with antigenized antibodies is an efficient method to focus the immune response against defined epitopes of foreign antigens. If CDR sequences of TCRs are functionally comparable to Ig idiotypes, antigenized antibodies provide a tool to induce anti-idiotypic responses against TCR. Here, we used antibodies antigenized with TCR sequences as vaccines to control disease. We engineered two antibodies encompassing in the CDR3 of the heavy (H) chain two synthetic peptides from the sequence of rat Vβ8.2 gene product, ³⁹DMGHGLRLIHYSYDVNSTEKG⁵⁹ (CDR2) and ⁹³ASSDSSNTE¹⁰¹ (CDR3, VDJ junction), both reported to confer protection against EAE in the Lewis rat [17–20] when used as vaccines. The results show that vaccination with antigenized antibodies expressing sequences of encephalitogenic T cells induces anti-idiotypic immunity against the TCR and high level resistance against EAE.

In this report we demonstrate that the severity of EAE in the Lewis rat can be greatly attenuated, and in some instances completely prevented, by active immunization with antigenized antibodies expressing amino acid sequences of the rat Vβ8.2 gene product. Immunity against synthetic peptides of the TCR has been shown to be effective in preventing or reducing the severity of EAE in the rat [17–20], suggesting that autoimmunity against the TCR reacting with encephalitogenic sequences of MBP is key to immunoregulatory events. The control of pathogenetic T cells may involve both T cells and antibodies. Autoregulation via T cells in EAE is well established. Thus, spontaneous recovery from EAE is impaired by splenectomy or thymectomy [34] and EAE can be prevented by vaccination with "attenuated" pathogenic T cells [15]. Autoregulation in EAE may involve both CD4⁺ and CD8⁺ T cells [35–38] as well as suppression by cytolytic T-T interactions [39]. A prevailing idea has been that in the rat [40] and in the mouse [41] idiotypic determinants of the TCR may be autoimmunogenic and contribute to mechanisms of immune regulation leading to protection. On the other hand, at least in a few instances, monoclonal antibodies against these TcR Vβ region [9, 42, 43] or against TCR idiotype [44] have been shown to block or attenuate disease.

Here we show that immunity against idiotopes of antibodies engineered to express TCR peptides is effective in generating anti-idiotypic immunity directed against rat Vβ8.2 TCR gene product. Importantly, this type of immunity protected from EAE. The new approach used herein to induce anti-TCR immunity is based on conventional idiotypic immunization in which antigenized antibodies mimic the immunogenic properties of soluble TCR functioning as a surrogate internal image [45] in much the same way as previously demonstrated for a non-self antigen [31]. The present approach is reminiscent of experiments in which induction of anti-idiotypic immunity against TCR with specificity for MHC was obtained by immunization with soluble alloantibodies of relevant specificity [46, 47], or by immunization with autologous idiotype positive molecules that are shed from the cell surface in the serum [48]. Thus, antibodies purposely modified to express selected loops of the TCR obviate the necessity to purify the receptor, isolate idiotypic TCR molecules from the serum, or use antigen-specific T cell blasts.

Antibodies reacting with TCR peptides were detected in every vaccinated rat indicating that immunization with antigenized antibodies is an efficient method to induce an anti-idiotypic response specific for a somatic receptor. The fact that only two out of sixteen vaccinated rats developed antibodies against the native receptor detectable by flowcytometry on Vβ8.2⁺ T cells suggests that cross-reactive anti-idiotypic antibodies may be very low titer. Alternatively, they may be adsorbed on T cells in vivo precluding their detection in the serum. The first possibility is consistent with the self nature of TCR peptides and a predicted paucity of self reactive clonotypes within the natural B cell repertoire. Interestingly, we noted that the anti-idiotypic response against a non-self peptide expressed in an antigenized antibody [31] is much greater than the one observed here against a self peptide. That only rats vaccinated with the antigenized antibody expressing the ⁹³ASSDSSNTE¹⁰¹ sequence developed flowcytometry-reactive autoantibodies could reflect difference in conformation once the two peptides are embedded in the CDR3 loop of an antigenized antibody. For instance, ⁹³ASSDSSNTE¹⁰¹ could be better surface exposed and more stably expressed ³⁹DMGHGLRLIHYSYDVNSTEKG⁵⁹. A computer-assisted comparison of hydrophilicity profiles [49] of the ⁹³ASSDSSNTE¹⁰¹ peptide in the parental. TCR Vβ8.2 gene product and in the antibody V region shows that in both instances the peptide is highly hydrophilic (Figure 5). On the other hand, the Vβ8.2 CDR2 region shows a highly hydrophilic profile alternating with large hydrophobic regions of poorly exposed amino acid residues, both in the parental TCR and in the antibody CDR (data not shown).

Our data show that although the process of antibody antigenization allows one to conformationally-constrain and express discrete peptide sequences of somatic receptors, the induction of anti-receptor antibodies is not directly predictable. Previously, we demonstrated flowcytometry-reactive antibodies to human CD4 in a high proportion (75 %) of cases [24]. We conclude that the physical characteristics of a given receptor peptide (e.g., length, hydrophilicity, etc.) likely determine its ability to induce antibodies cross-reactive with the native receptor.

Interestingly, rats immunized with the antigenized antibody expressing ⁹³ASSDSSNTE¹⁰¹ but not ³⁹DMGHGLRLIHYSYDVNSTEKG⁵⁹ reacted immunologically with a synthetic peptide of human CD4 previously described to bind Ig [32]. Because the two antigenized antibodies reacted equally with the CD4 peptide and only differ by the composition of their CDR3, we suggest that binding to CD4 by anti- ⁹³ASSDSSNTE¹⁰¹ serum antibodies underscores qualitative differences of the immune response between rats immunized with γ1TCR-I and 1TCR-II, respectively. Thus, it appears as if ⁹³ASSDSSNTE¹⁰¹ induced a different immune response than ³⁹DMGHGLRLIHYSYDVNSTEKG⁵⁹. Furthermore, since vaccination with γ1TCR-I also promoted greater protection from EAE, it is tempting to speculate that a component of the anti-idiotypic response against γ1TCR-I is associated with protection.

In conclusion, three points have emerged from this study. First, antigenized antibodies expressing conformationally-constrained loops of the Vβ8.2 TCR can be used as vaccines in the prevention of EAE in the Lewis rat. Our new approach to generate anti-TCR immunity, confirms the relevance of anti-idiotypic regulation in controlling rat EAE [17, 18, 20]. Second, since a weak antibody anti-idiotypic response in the apparent lack of a cell proliferative response was associated with protection, it appears as if a humoral anti-TCR response is relevant to protection from disease. Although this contrasts the relevance of T cell immunity in the regulation of EAE in the rat, reports exist to support the idea that humoral immunity is also important [20, 50, 51]. EAE was shown to be prevented or attenuated by passive transfer of serum from rats recovering from EAE [52], or by passive transfer of monoclonal antibodies against these TCR Vβ region and its idiotypes [9, 42–44]. However, whether anti-idiotypic antibodies against the TCR predispose to anergy, apoptosis or killing of pathogenetic T cells remains to be determined. Finally, our study indicates that antigenized antibodies can be used as vaccines in conditions where immunopathology and disease involve receptors on somatic cells, and anti-receptor immunity alone could prevent or mitigate a pathological condition.