Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids

Jan Grünewald; Grady S. Hunt; Liqun Dong; Frank Niessen; Ben G. Wen; Meng-Lin Tsao; Roshan Perera; Mingchao Kang; Bryan A. Laffitte; Sassan Azarian; Wolfram Ruf; Marc Nasoff; Richard A. Lerner; Peter G. Schultz; Vaughn V. Smider

doi:10.1073/pnas.0900507106

Contributed by Richard A. Lerner, January 15, 2009 (received for review December 12, 2008)

Abstract

For more than 2 centuries active immunotherapy has been at the forefront of efforts to prevent infectious disease [Waldmann TA (2003) Nat Med 9:269–277]. However, the decreased ability of the immune system to mount a robust immune response to self-antigens has made it more difficult to generate therapeutic vaccines against cancer or chronic degenerative diseases. Recently, we showed that the site-specific incorporation of an immunogenic unnatural amino acid into an autologous protein offers a simple and effective approach to overcome self-tolerance. Here, we characterize the nature and durability of the polyclonal IgG antibody response and begin to establish the generality of p-nitrophenylalanine (pNO₂Phe)-induced loss of self-tolerance. Mutation of several surface residues of murine tumor necrosis factor-α (mTNF-α) independently to pNO₂Phe leads to a T cell-dependent polyclonal and sustainable anti-mTNF-α IgG autoantibody response that lasts for at least 40 weeks. The antibodies bind multiple epitopes on mTNF-α and protect mice from severe endotoxemia induced by lipopolysaccharide (LPS) challenge. Immunization of mice with a pNO₂Phe⁴³ mutant of murine retinol-binding protein (RBP4) also elicited a high titer IgG antibody response, which was cross-reactive with wild-type mRBP4. These findings suggest that this may be a relatively general approach to generate effective immunotherapeutics against cancer-associated or other weakly immunogenic antigens.

Keywords:

Critical to the process of immunological self–nonself discrimination is self-tolerance (1), in which a mammal's immune system is “tolerized” to self-proteins to avoid autoimmune disease, primarily due to the absence or inactivation of self-reactive B or T cells. It has been known for years, however, that the immune system can be induced to attack self-proteins. For example, cross-reactive immune responses to self-proteins can be induced by introducing foreign T helper cell epitopes into chimeric antigens (2, 3), by extensive chemical derivatization of self-antigens (4), and by DNA vaccines (5). Furthermore, a number of specific genes and cellular mechanisms involved in self-tolerance have been identified which when disrupted result in breakdown of tolerance and autoimmune disease (1, 6). Despite these advances, the design of effective immunotherapeutics has been a slow process, as exemplified by the fact that only a few vaccines for cancer treatment have reached late stage clinical development (7, 8).

Nitroaryl groups are highly immunogenic, likely due to their ability to form strong stacking and van der Waals interactions. Indeed, the nonspecific derivatization of autologous cancer cells with dinitrophenyl groups has been exploited as a vaccine in melanoma patients (9), and physiological 3-nitrotyrosine formation has been implicated in the pathology of a number of autoimmune diseases (10–12). To test whether this immunogenic group could be used to break tolerance to specific self-proteins, we previously introduced a p-nitrophenylalanine (pNO₂Phe) residue at a single site in murine TNF-α. Genetic substitution of pNO₂Phe for Tyr-86 of mTNF-α created a T cell epitope, which enhanced T cell help to elicit a strong cross-reactive antibody response against this disease-related self-protein (13). Here, we show that immunochemical breakdown of self-tolerance leads to sustained high-titer antibody responses that efficiently protect mice against a lipopolysaccharide (LPS) challenge. Moreover, we demonstrate that this methodology is generalizable to a self-protein unrelated to immune function, namely retinol binding protein 4 (RBP4).

Results and Discussion

Mechanistic Studies of pNO₂Phe-Induced Breakdown of Self-Tolerance.

Previously, we showed that substitution of pNO₂Phe for Tyr-86 in mTNF-α led to a high titer cross reactive antibody response to wildtype (WT) protein. The mutant protein was shown to induce T cell proliferation in immunized animals, whereas WT protein did not (13). To provide further evidence for a T cell-dependent immune response against pNO₂Phe TNF-α we have carried out ELISA analysis of the mTNF-α autoantibodies with either anti-mouse IgM or anti-mouse IgG secondary antibody. The majority of the anti-mTNF-α autoantibodies in sera from Bcl-2 transgenic mice [C57BL/6-TgN(BCL2)22Wehi] immunized with pNO₂Phe⁸⁶ mTNF-α are of the IgG subtype indicating T cell-mediated Ig class switching (Fig. 1A). To determine whether the presence of pNO₂Phe is critical throughout the immunization process, we initially injected 4 mice with pNO₂Phe⁸⁶ mTNF-α in complete Freund's adjuvant (CFA) followed by 7 injections of either WT mTNF-α or pNO₂Phe⁸⁶ mTNF-α in incomplete Freund's adjuvant (IFA). The results shown in Fig. S1 clearly demonstrate that, in contrast to pNO₂Phe⁸⁶ mTNF-α, WT mTNF-α cannot sustain significant titers of cross-reactive anti-mTNF-α antibodies. This result supports the notion that pNO₂Phe-induced breakdown of self-tolerance requires a T cell response mediated by the nitrophenyl group, and is consistent with previous studies that showed that a Tyr86Phe mTNF-α mutant is not able to elicit a strong immune response.

Fig. 1.

pNO₂Phe⁸⁶ mTNF-α immunization promotes class-switching to an IgG response, which displays significant cross-reactivity with WT mTNF-α and lasts for at least 40 weeks in mice. (A) Determination of serum titers for Bcl-2 transgenic mice immunized with pNO₂Phe⁸⁶ mTNF-α or WT mTNF-α over a period of 17 days in the presence of complete Freund's adjuvant (CFA) for the initial injection and incomplete Freund's adjuvant (IFA) for the remainder. ELISAs were measured against WT mTNF-α using either anti-mouse IgM (light blue and dark blue bars) or anti-mouse IgG (orange and red bars) as a secondary antibody. Before measurement, serum samples were diluted 1:100 (light blue and orange bars) or 1:1,000 (dark blue and red bars) with 1% BSA in PBS buffer. (B) ELISA titration to quantify the affinity of polyclonal anti-WT mTNF-α IgG and polyclonal anti-pNO₂Phe⁸⁶ mTNF-α IgG for either pNO₂Phe⁸⁶ mTNF-α or WT mTNF-α. (C) Serum titer durability study of 3 Bcl-2 transgenic mice immunized with pNO₂Phe⁸⁶ mTNF-α. After a sequence of 8 immunizations, bleeds were taken for ELISA analysis against pNO₂Phe⁸⁶mTNF-α at defined time points (Δt corresponds to the time period between the last immunization and the bleed). Before each measurement, serum samples were diluted 1:100 with 1% BSA in PBS buffer.

One question regarding the mechanism of pNO₂Phe-induced breakdown of self-tolerance is whether the antibody response is directed at the epitope that contains pNO₂Phe, or whether epitope spreading occurs, resulting in a polyclonal IgG response against multiple epitopes in the target protein. To address this issue, Bcl-2 transgenic mice were immunized with pNO₂Phe⁸⁶ mTNF-α to generate 50 B cell hybridomas, which were screened by ELISA to identify those clones that produced antibodies against WT mTNF-α. We then assessed the binding of these monoclonal antibodies (mAbs) to a set of 3 mTNF-α fragments that were expressed in E. coli and whose molecular masses were verified by MALDI-TOF (Fig. S2): an N-terminal fragment (amino acid 1–60), an internal fragment (amino acid 61–100), and a C-terminal fragment (amino acid 101–156). Although this assay largely detects specificities against linear (presumably continuous) B cell epitopes, we identified 5 mAbs (3L24, 5K19, 6J22, 7O1, and 7F23) that bound the N-terminal fragment and 1 mAb (1P19) that bound the C-terminal fragment (Fig. S3). Significantly, none of the mAbs bound the internal fragment encoding pNO₂Phe⁸⁶ in the original immunogen. Thus, antibodies that bind more than 1 epitope are produced through pNO₂Phe⁸⁶ mTNF-α immunization, and these epitopes do not necessarily include the pNO₂Phe residue of the immunogen. The polyclonal IgGs from pNO₂Phe⁸⁶ mTNF-α immunized mice cross react with native mTNF-α with K_d values in the nanomolar range (Fig. 1B). Together, these results further support the hypothesis that a cross-reactive neutralizing antibody response can be generated against a self-protein by simply substituting a pNO₂Phe residue into its sequence.

Sustainability of pNO₂Phe-Induced Antibody Response.

To determine the durability of anti-mTNF-α IgG antibody titers, we immunized 3 Bcl-2 transgenic mice with the pNO₂Phe⁸⁶ mTNF-α. After the last boost injection, bleeds were analyzed by ELISA against pNO₂Phe⁸⁶mTNF-α at defined time points. Remarkably, antibody levels were maintained at >80% their initial levels for at least 40 weeks (Fig. 1C), after which time the mice were killed. In contrast, in a previous anti-mTNF-α vaccination study based on immunization with mTNF-α mutant containing a hen egg-white lysozyme T cell epitope, titers declined 4 weeks after the last boost, and after 26 weeks the anti-mTNF-α antibody titers had dropped by 80–87% (2). Thus, our pNO₂Phe-based vaccine strategy is effective in inducing persistent immunity and long-term protection against TNF-α as a disease-associated self-antigen.

Extension to Mutations at Other Surface Sites Within mTNF-α.

To examine the generality of the pNO₂Phe-induced breakdown of self-tolerance, 4 additional surface-exposed residues of mTNF-α were mutated to pNO₂Phe: Lys¹¹, Gln²¹, Asp⁴², and Val⁴⁹ (Fig. S4A). These residues are also structurally distinct from pNO₂Phe. After confirming the composition and homogeneity of pNO₂Phe¹¹ mTNF-α, pNO₂Phe²¹ mTNF-α, pNO₂Phe⁴² mTNF-α, and pNO₂Phe⁴⁹ mTNF-α by SDS/PAGE and mass spectrometry (Fig. S4B and Table 1), the quaternary structure of these mutant proteins was shown to be trimeric by size exclusion chromatography (Table S1). Furthermore, an NFκB-luciferase reporter gene assay showed that pNO₂Phe¹¹ mTNF-α has 9%, pNO₂Phe²¹ mTNF-α has 22%, pNO₂Phe⁴² mTNF-α has 22%, and pNO₂Phe⁴⁹ mTNF-α has 10% of the activity of WT mTNF-α (Table S1 and Fig. S4C). All mutants are therefore significantly more active than the previously characterized pNO₂Phe⁸⁶ mTNF-α, which has only 2% of the activity of the wild-type protein in this assay. To determine the immunogenicity of these pNO₂Phe mTNF-α mutants, 14 C57BL/6 mice were randomized into 5 groups and injected with these mutants, or WT mTNF-α by the RIMMS (repetitive immunization at multiple sites) protocol (14). An ELISA analysis revealed no correlation between mTNF-α activity in the NFκB-luciferase reporter gene assay and the ability to induce an antibody response, ruling out a direct effect on the immune system. As shown in Fig. 2, pNO₂Phe at position 11 induced a high titer IgG response to WT mTNF-α, equivalent to that against the pNO₂Phe⁸⁶ mTNF-α immunogen. In contrast, although mutations of positions 21, 42, and 49 also yielded high titer IgG responses against the pNO₂Phe-containing immunogen, the IgG antibodies had only moderate cross-reactivity to WT mTNF-α. Antibodies generated against all 4 mutant TNF-α's were then used for passive immunization of forty C57BL/6 mice, which were randomized into 5 groups and injected with the anti-pNO₂Phe or anti-WT mTNF-α IgG. Twenty-four hours after passive immunization, the animals were challenged with LPS as described in ref. 15. All mice receiving anti-pNO₂Phe¹¹ mTNF-α IgG survived the lethal LPS challenge (Fig. 3). Even the other groups receiving moderately cross-reactive anti-pNO₂Phe²¹ mTNF-α IgG, anti-pNO₂Phe⁴² mTNF-α IgG, and anti-pNO₂Phe⁴⁹ mTNF-α IgG had survival rates of at least 75%; whereas mice injected with anti-WT mTNF-α IgG showed a survival rate of only 13%. Thus, the ability to break self-tolerance using pNO₂Phe is not dependent on a single amino acid position, since we have shown that at least 5 positions (including position 86) can induce a neutralizing cross-reactive anti-mTNF-α IgG response in vivo. Moreover, the site of substitution does not need to be structurally similar to pNO₂Phe.

View this table:

Table 1.

ESI mass spectrometry analysis of mRBP4 variants

Fig. 2.

Significant immunogenicity of 4 surface-exposed sites on mTNF-α. (A) Serum titers against WT mTNF-α (red bars), pNO₂Phe¹¹ mTNF-α (blue bars), and PBS (yellow bars) for C57BL/6 mice immunized with pNO₂Phe¹¹ mTNF-α or WT mTNF-α. (B) Serum titers against WT mTNF-α (red bars), pNO₂Phe²¹ mTNF-α (blue bars), and PBS (yellow bars) for C57BL/6 mice immunized with pNO₂Phe²¹ mTNF-α or WT mTNF-α. (C) Serum titers against WT mTNF-α (red bars), pNO₂Phe⁴² mTNF-α (blue bars), and PBS (yellow bars) for C57BL/6 mice immunized with pNO₂Phe⁴² mTNF-α or WT mTNF-α. (D) Serum titers against WT mTNF-α (red bars), pNO₂Phe⁴⁹ mTNF-α (blue bars), and PBS (yellow bars) for C57BL/6 mice immunized with pNO₂Phe⁴⁹ mTNF-α or WT mTNF-α. Before each measurement, serum samples were diluted 1/800 (A), 1/200 (B), 1/200 (C), or 1/200 (D) with 1% BSA in PBS buffer.

Fig. 3.

Significant survival benefit for mice immunized with various pNO₂Phe mTNF-α mutants after lipopolysaccharide (LPS) challenge. Male C57BL/6 mice were i.p. injected with 4 mg/kg purified IgG from mice immunized with pNO₂Phe¹¹ mTNF-α and pNO₂Phe⁴⁹ mTNF-α (A), or pNO₂Phe²¹ mTNF-α and pNO₂Phe⁴² mTNF-α (B) 1 day before LPS challenge. Kaplan–Meier survival plots of these mice were compared with mice injected with control IgG (n = 8 per group). Survival advantage of mice immunized with each modified TNF P < 0.01 versus control, log rank test with Bonferroni correction.

Expression and Characterization of Mutant mRBP4 Proteins.

Given that multiple positions within mTNF-α lead to breakdown of self-tolerance when mutated to pNO₂Phe, we then asked whether this methodology could be generalized to other self-proteins. Specifically, we examined the ability of pNO₂Phe to break self-tolerance against another model self-protein found in serum, RBP4 (16, 17). In contrast to TNF-α this is a highly soluble, relatively low molecular mass (20 kDa), monomeric protein. RBP4 knockout mice show no apparent phenotypic abnormalities other than visual deficiency (18), suggesting that mice will survive a neutralizing immune response against self-RBP4. Based on the X-ray crystal structure of monomeric human RBP4 (19), we selected the following surface-exposed residues for mutation to pNO₂Phe: Tyr⁴³ and Tyr¹⁰⁸ (Fig. S5A). These residues are highly conserved among different mammalian RBP4s, including murine RBP4 (mRBP4). These mRBP4 mutants and WT mRBP4 were expressed in E. coli as N-terminal His₆-tagged proteins, purified by Ni²⁺ affinity chromatography under denaturing conditions, and refolded according to a previously described protocol (20). The site-specific incorporation of pNO₂Phe into mRBP4 at positions 43 and 108 was confirmed by SDS/PAGE analysis, and by MS/MS fragmentation of the tryptic fragments containing the unnatural amino acid (Figs. S5 and S6 and Table S2). Analytical size-exclusion chromatography indicated a monomeric structure for all mRBP4 proteins, which is in agreement with the published quaternary structure of human RBP4 (Table S3) (19). Moreover, according to a retinol displacement assay, all pNO₂Phe mRBP4 mutants bind retinol with K_d values in the nanomolar range, which is in good agreement with WT mRBP4 (Table S3).

Generality of pNO₂Phe-Induced Breakdown of Self-Tolerance.

To determine the immunogenicity of the pNO₂Phe mRBP4 mutants, 9 Bcl-2 transgenic mice were randomized into 3 groups and injected with pNO₂Phe⁴³ mRBP4, pNO₂Phe¹⁰⁸ mRBP4 and WT mRBP4 by the RIMMS protocol (14). According to ELISA analysis, mice immunized with either WT mRBP4 or pNO₂Phe¹⁰⁸ mRBP4 had insignificant serum IgG titers against WT mRBP4 (Fig. 4A). In contrast, mice immunized with pNO₂Phe⁴³ mRBP4 were found to display markedly high serum IgG titers (up to 1:100,000), binding both the pNO₂Phe⁴³ mRBP4 immunogen and the wild-type protein. Similar results were obtained with C57BL/6 mice (Fig. S7). Furthermore, in accordance with previous observations with pNO₂Phe⁸⁶ mTNF-α, CD4⁺ T cells specific for pNO₂Phe⁴³ mRBP4 were induced upon immunization with pNO₂Phe⁴³ mRBP4 protein, indicating a mature T cell-dependent immune response (Fig. 4B). Together, these results further support the hypothesis that the introduction of pNO₂Phe into a protein sequence can create a strong T cell epitope, which initiates a sustained cross-reactive IgG antibody response. Not all sites lead to a strong cross-reactive immune response, which is not surprising since it is unlikely that all sites correspond to potential T cell epitopes.

Fig. 4.

Loss of tolerance to a second self-antigen, mRBP4. (A) Serum titers for Bcl-2 transgenic mice immunized with WT mRBP4 (a), pNO₂Phe⁴³ mRBP4 (b), and pNO₂Phe¹⁰⁸ mRBP4 (c), are shown. ELISAs were measured against WT mRBP4 (red bars) and pNO₂Phe⁴³ mRBP4 (blue bars). Before measurement, serum samples were diluted 1:1,000 with 1% BSA in PBS buffer. (B) Proliferation of CD4⁺ T cells from C57BL/6 mice immunized with pNO₂Phe⁴³ mRBP4 and stimulated in vitro with serial dilutions of pNO₂Phe⁴³ mRBP4.

Conclusion

We have shown that the genetic introduction of pNO₂Phe leads to sustained IgG antibody responses against the self-proteins mTNF-α and mRBP4. In terms of mechanism, incorporation of the p-nitrophenyl group at a single position results in T cells that can only be stimulated by the pNO₂Phe mutant but not the WT protein. This pNO₂Phe-induced T cell-dependent response ultimately leads to activation of autoreactive B cells and the production of polyclonal antibodies that are highly cross-reactive to the native self-protein. These results are comparable to recent studies showing that posttranslationally modified proteins can enhance T cell responsiveness (21–23). For example, citrullination and glycosylation are posttranslational modifications reported to be involved in T cell-dependent autoimmune diseases (21–25). Similarly, dinitrofluorobenzene modification of skin antigens has been used for decades as a model of the T cell response in contact hypersensitivity (26). Site-specific incorporation of pNO₂Phe into self-proteins therefore establishes a simple model system to biochemically mimic posttranslationally or chemically mediated loss of self-tolerance. This methodology should therefore also help to understand how the immune system responds to chemically modified antigens during autoimmunity. Furthermore, pNO₂Phe-induced breakdown of self-tolerance should not only afford a robust method for raising neutralizing antibodies against pathogenic self-proteins associated with cancer or degenerative diseases, it may also be applicable to weakly immunogenic foreign antigens of infectious agents.

Materials and Methods

Bacterial Strains and Reagents.

E. coli XL1-Blue and XL10-Gold were used as hosts for cloning, and E. coli BL21(DE3) was used as an expression strain. Restriction enzymes, T4 DNA ligase, dNTPs, and factor Xa protease were obtained from NEB (Beverly, MA). Primers were purchased from Integrated DNA Technologies (Coralville, IA). Plasmid DNA preparation was carried out with PureLink Quick Plasmid Miniprep Kit (Invitrogen), and DNA purification after restriction digestion was performed using PureLink PCR Micro Kit (Invitrogen).

Production of pNO₂Phe-Containing mTNF-α and WT mTNF-α.

WT mTNF-α and pNO₂Phe mTNF-α mutants were produced as described in ref. 13. Briefly, site-specific incorporation of pNO₂Phe into the murine TNF-α gene was carried out by introducing TAG amber codons using standard PCR mutagenesis procedures. To express pNO₂Phe mTNF-α mutants, E. coli BL21(DE3) cells were cotransformed with mutNO₂PheRS, mutRNA_CUA and the mutated mTNF-α gene. The transformed cells were then grown in the presence of 1 mM pNO₂Phe (Alfa Aesar) in minimal medium containing 1% glycerol and 0.3 mM leucine (GMML medium) at 37 °C and protein expression was initiated by the addition of 1 mM IPTG. WT mTNF-α was expressed in 2× YT medium in the absence of pNO₂Phe. Protein purification was carried out by immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC) under either native or denaturing conditions.

All proteins were characterized by MALDI-TOF or ESI mass spectrometry. Successful incorporation of pNO₂Phe into mutant proteins was also verified by tryptic in-gel digestion and subsequent MS/MS fragmentation of the respective tryptic fragment containing this unnatural amino acid. Protein quaternary structures were analyzed by analytical SEC on a Superdex 75 10/300 GL gel filtration column, which was calibrated by a molecular weight gel-filtration standard from Bio-Rad. The activity of pNO₂Phe mTNF-α mutants was determined by an NFκB-luciferase reporter gene assay using HEK293 cells stably expressing NFκB-luciferase as described in ref. 13.

Construction of mRBP4 Expression Vector, pSpeedET-mRBP4.

The cDNA encoding murine RBP4 (amino acid 19–201) (Genomics Institute of the Novartis Research Foundation) was amplified with PCR using 2 primers designed specifically for the Polymerase Incomplete Primer Extension (PIPE) cloning method (27): 5′-CTGTACTTCCAGGGCGAGCGCGACTGCAGGG (5′ insert forward primer) and 5′-AATTAAGTCGCGTTACAAACTGTTTCTGGAGGGCC (3′ insert reverse primer). The pSpeedET vector was amplified using a 5′ vector reverse primer 5′-GCCCTGGAAGTACAGGTTTTCGTGATGATGATGATGATG and a 3′ vector forward primer 5′-TAACGCGACTTAATTAACTCGTTTAAACGGTCTCCAGC. The underlined and italicized bases highlight the 2 distinct complementary regions between primers where annealing occurs. The pSpeedET vector appends an N-terminal His₆-tag sequence (MGSDKIHHHHHH), followed by a TEV protease site (ENLYFQG) immediately before the 19^th codon for mRBP4. The unpurified mRBP4 (amino acid 19–201) insert PCR product was mixed 1:1 (vol/vol) with the unpurified pSpeedET vector PCR product. After mixing, E. coli XL10-Gold cells were transformed with 2 μL of the reaction mixture. Site-specific incorporation of pNO₂Phe into mRBP4 (amino acid 19–201) was performed by mutating the codons for Tyr⁴³ or Tyr¹⁰⁸ to a TAG amber codon. The sequences of all pSpeedET-mRBP4 constructs were confirmed by DNA sequence analysis.

Protein Expression and Purification of pNO₂Phe mRBP4 and WT mRBP4.

To express the pNO₂Phe mRBP4 mutants, E. coli BL21(DE3) cells were cotransformed with mutNO₂PheRS, mutRNA_CUA, and the respective mutant mRBP4 gene. The transformed strains were grown at 37 °C in the presence of 1 mM pNO₂Phe in GMML medium, induced with 0.2% (wt/vol) arabinose when the OD₆₀₀ reached 0.5, and harvested after 12–16 h. In contrast to the pNO₂Phe mRBP4 mutants, WT mRBP4 was expressed in 2x YT medium in the absence of pNO₂Phe for 3 h. The cell pellets were suspended in 8 M urea containing 100 mM NaH₂PO₄, 10 mM Tris (pH 8.0) and lysed by sonication on ice for 3 min. Cell debris was removed by centrifugation at 40,000 × g for 25 min. Five milliliters of 50% Ni-NTA slurry (Novagen) was added to the supernatant and mixed gently by shaking for 60 min. The Ni-NTA beads were washed with 8 M urea, 100 mM NaH₂PO₄, and 10 mM Tris (pH 6.3). Elution was carried out with 8 M urea containing 100 mM NaH₂PO₄, and 10 mM Tris (pH 4.5). The protein was concentrated with a 10K molecular mass cut-off Amicon Ultra-15 centrifugal filter device (Millipore). The mRBP4 protein was precipitated by dialysis against PBS (pH 7.4), and redissolved in 8 M urea containing 20 mM Tris and 20 mM DTT (pH 8.0). In vitro folding of mRBP4 protein was performed according to Greene et al. (20). Briefly, native protein was generated by adding the denatured material in 8 M urea dropwise to folding buffer containing 20 mM Tris, 10 mM β-mercaptoethanol, 1 mM 2-hydroxyethyldisulfide, and 1% glycerol (pH 8.5) at a rate of ≈30 drops/min. Folding was allowed to proceed for 16 h at 4 °C, and the protein solution was then concentrated using a 10K molecular mass cut-off Amicon Ultra-15 centrifugal filter device (Millipore). The protein was further purified by SEC on a Superdex 75 10/300 GL column (GE Healthcare) equilibrated with PBS (pH 7.4) at a flow rate of 0.3 mL/min.

Mouse Model of Severe Systemic Inflammation.

All experiments were carried out in accordance with the National Institutes of Health Animal Protection Guidelines and were approved by The Scripps Research Institute Animal Care and Use Committee. Animal experiments were performed in a room with alternating 12 h light–dark cycles under stable conditions of temperature (20–22 °C) and relative humidity (40–60%) (15). Twenty-four hours before LPS challenge, 9-week old male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were passively immunized by injection into the left half of the peritoneal cavity with 4 mg/kg of IgG purified from serum of mice immunized with pNO₂Phe¹¹ mTNF-α, pNO₂Phe²¹ mTNF-α, pNO₂Phe⁴² mTNF-α, or pNO₂Phe⁴⁹ mTNF-α. IgG derived from nonimmunized wild-type mice was used as a negative control. Mice were then injected into the right half of the peritoneal cavity under 2% isoflurane with 7.5 mg/kg lipopolysaccharide (LPS) (E. coli O111:B4 Calbiochem/EMD Biosciences). For statistical analysis, Kaplan–Meier curves were plotted and survival differences were analyzed using a log rank test with Bonferroni correction.

Acknowledgments

We thank Dr. Diane M. Kubitz for carrying out immunizations of C57BL/6 mice and ELISA experiments; Jason Mah, Lilin Li, and Steven Hinton for performing ELISA experiments; Robin Nevarez for carrying out immunizations of C57BL/6 and Bcl-2 transgenic mice; Monique Stinson for performing T cell proliferation assays; Romerson Dimla for technical assistance; Jon V. Apon and Dr. Sunia A. Trauger for performing MS/MS analyses; Eric J. Koesema (Genomics Institute of the Novartis Research Foundation) for providing pSpeedET; and Drs. Benjamin M. Hutchins and Shoutian Zhu for discussions. This work was supported by the National Institutes of Health Grant R01 GM062159, the Skaggs Institute for Chemical Biology, and postdoctoral fellowships from the Alexander von Humboldt Foundation (to J.G.) and the Deutsche Forschungsgemeinschaft (to F.N.). This is manuscript no. 19908 of The Scripps Research Institute.

Footnotes

⁴To whom correspondence should be addressed. E-mail: vvsmider{at}scripps.edu, schultz{at}scripps.edu, or rlerner{at}scripps.edu
Author contributions: J.G., L.D., M.-L.T., R.P., W.R., M.N., R.A.L., P.G.S., and V.S. designed research; J.G., G.S.H., L.D., F.N., B.G.W., M.-L.T., R.P., and B.A.L. performed research; J.G., G.S.H., L.D., F.N., B.G.W., M.-L.T., R.P., M.K., B.A.L., S.A., W.R., M.N., R.A.L., P.G.S., and V.S. analyzed data; and J.G., P.G.S., and V.S. wrote the paper.
↵¹Present address: Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121.
↵²Present address: School of Natural Sciences, University of California, Merced, CA 95344.
↵³Present address: Department of Chemistry and Biochemistry, University of Texas, Arlington, TX 76019.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0900507106/DCSupplemental.

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(2008) Conversion of tyrosine to the inflammation-associated analog 3′-nitrotyrosine at either TCR- or MHC-contact positions can profoundly affect recognition of the MHC class I-restricted epitope of lymphocytic choriomeningitis virus glycoprotein 33 by CD8 T cells. J Immunol 180:5956–5962.
OpenUrl Abstract/FREE Full Text
↵
1. Grünewald J,
2. et al.
(2008) Immunochemical termination of self-tolerance. Proc Natl Acad Sci USA 105:11276–11280.
OpenUrl Abstract/FREE Full Text
↵
1. Kilpatrick KE,
2. et al.
(1997) Rapid development of affinity matured monoclonal antibodies using RIMMS. Hybridoma 16:381–389.
OpenUrl CrossRef PubMed
↵
1. Niessen F,
2. et al.
(2008) Dendritic cell PAR1–S1P3 signalling couples coagulation and inflammation. Nature 452:654–658.
OpenUrl CrossRef PubMed
↵
1. Zanotti G,
2. Berni R
(2004) Plasma retinol-binding protein: Structure and interactions with retinol, retinoids, and transthyretin. Vitam Horm 69:271–295.
OpenUrl CrossRef PubMed
↵
1. Raghu P,
2. Sivakumar B
(2004) Interactions amongst plasma retinol-binding protein, transthyretin and their ligands: Implications in vitamin A homeostasis and transthyretin amyloidosis. Biochim Biophys Acta 1703:1–9.
OpenUrl CrossRef PubMed
↵
1. Vogel S,
2. et al.
(2002) Retinol-binding protein-deficient mice: Biochemical basis for impaired vision. Biochemistry 41:15360–15368.
OpenUrl CrossRef PubMed
↵
1. Cowan SW,
2. Newcomer ME,
3. Jones TA
(1990) Crystallographic refinement of human serum retinol binding protein at 2A resolution. Proteins 8:44–61.
OpenUrl CrossRef PubMed
↵
1. Greene LH,
2. et al.
(2001) Role of conserved residues in structure and stability: Tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily. Protein Sci 10:2301–2316.
OpenUrl CrossRef PubMed
↵
1. Cantaert T,
2. et al.
(2006) Citrullinated proteins in rheumatoid arthritis: Crucial but not sufficient! Arthritis Rheum 54:3381–3389.
OpenUrl CrossRef PubMed
↵
1. Backlund J,
2. et al.
(2002) Predominant selection of T cells specific for the glycosylated collagen type II epitope (263–270) in humanized transgenic mice and in rheumatoid arthritis. Proc Natl Acad Sci USA 99:9960–9965.
OpenUrl Abstract/FREE Full Text
↵
1. Dzhambazov B,
2. et al.
(2005) The major T cell epitope on type II collagen is glycosylated in normal cartilage but modified by arthritis in both rats and humans. Eur J Immunol 35:357–366.
OpenUrl CrossRef PubMed
↵
1. Klareskog L,
2. Ronnelid J,
3. Lundberg K,
4. Padyukov L,
5. Alfredsson L
(2008) Immunity to citrullinated proteins in rheumatoid arthritis. Annu Rev Immunol 26:651–675.
OpenUrl CrossRef PubMed
↵
1. Sollid LM
(2000) Molecular basis of celiac disease. Annu Rev Immunol 18:53–81.
OpenUrl CrossRef PubMed
↵
1. Toews GB,
2. Bergstresser PR,
3. Streilein JW
(1980) Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J Immunol 124:445–453.
OpenUrl PubMed
↵
1. Klock HE,
2. Koesema EJ,
3. Knuth MW,
4. Lesley SA
(2008) Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 71:982–994.
OpenUrl CrossRef PubMed

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Proceedings of the National Academy of Sciences: 106 (11)

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Arlen PM
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[24] Arlen PM

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[28] Aulak KS,

[29] et al.

[30] ↵
Pacher P,
Beckman JS,
Liaudet L
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[31] Pacher P,

[32] Beckman JS,

[33] Liaudet L

[34] ↵
Hardy LL,
Wick DA,
Webb JR
(2008) Conversion of tyrosine to the inflammation-associated analog 3′-nitrotyrosine at either TCR- or MHC-contact positions can profoundly affect recognition of the MHC class I-restricted epitope of lymphocytic choriomeningitis virus glycoprotein 33 by CD8 T cells. J Immunol 180:5956–5962.
OpenUrl Abstract/FREE Full Text

[35] Hardy LL,

[36] Wick DA,

[37] Webb JR

[38] ↵
Grünewald J,
et al.
(2008) Immunochemical termination of self-tolerance. Proc Natl Acad Sci USA 105:11276–11280.
OpenUrl Abstract/FREE Full Text

[39] Grünewald J,

[40] et al.

[41] ↵
Kilpatrick KE,
et al.
(1997) Rapid development of affinity matured monoclonal antibodies using RIMMS. Hybridoma 16:381–389.
OpenUrl CrossRef PubMed

[42] Kilpatrick KE,

[43] et al.

[44] ↵
Niessen F,
et al.
(2008) Dendritic cell PAR1–S1P3 signalling couples coagulation and inflammation. Nature 452:654–658.
OpenUrl CrossRef PubMed

[45] Niessen F,

[46] et al.

[47] ↵
Zanotti G,
Berni R
(2004) Plasma retinol-binding protein: Structure and interactions with retinol, retinoids, and transthyretin. Vitam Horm 69:271–295.
OpenUrl CrossRef PubMed

[48] Zanotti G,

[49] Berni R

[50] ↵
Raghu P,
Sivakumar B
(2004) Interactions amongst plasma retinol-binding protein, transthyretin and their ligands: Implications in vitamin A homeostasis and transthyretin amyloidosis. Biochim Biophys Acta 1703:1–9.
OpenUrl CrossRef PubMed

[51] Raghu P,

[52] Sivakumar B

[53] ↵
Vogel S,
et al.
(2002) Retinol-binding protein-deficient mice: Biochemical basis for impaired vision. Biochemistry 41:15360–15368.
OpenUrl CrossRef PubMed

[54] Vogel S,

[55] et al.

[56] ↵
Cowan SW,
Newcomer ME,
Jones TA
(1990) Crystallographic refinement of human serum retinol binding protein at 2A resolution. Proteins 8:44–61.
OpenUrl CrossRef PubMed

[57] Cowan SW,

[58] Newcomer ME,

[59] Jones TA

[60] ↵
Greene LH,
et al.
(2001) Role of conserved residues in structure and stability: Tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily. Protein Sci 10:2301–2316.
OpenUrl CrossRef PubMed

[61] Greene LH,

[62] et al.

[63] ↵
Cantaert T,
et al.
(2006) Citrullinated proteins in rheumatoid arthritis: Crucial but not sufficient! Arthritis Rheum 54:3381–3389.
OpenUrl CrossRef PubMed

[64] Cantaert T,

[65] et al.

[66] ↵
Backlund J,
et al.
(2002) Predominant selection of T cells specific for the glycosylated collagen type II epitope (263–270) in humanized transgenic mice and in rheumatoid arthritis. Proc Natl Acad Sci USA 99:9960–9965.
OpenUrl Abstract/FREE Full Text

[67] Backlund J,

[68] et al.

[69] ↵
Dzhambazov B,
et al.
(2005) The major T cell epitope on type II collagen is glycosylated in normal cartilage but modified by arthritis in both rats and humans. Eur J Immunol 35:357–366.
OpenUrl CrossRef PubMed

[70] Dzhambazov B,

[71] et al.

[72] ↵
Klareskog L,
Ronnelid J,
Lundberg K,
Padyukov L,
Alfredsson L
(2008) Immunity to citrullinated proteins in rheumatoid arthritis. Annu Rev Immunol 26:651–675.
OpenUrl CrossRef PubMed

[73] Klareskog L,

[74] Ronnelid J,

[75] Lundberg K,

[76] Padyukov L,

[77] Alfredsson L

[78] ↵
Sollid LM
(2000) Molecular basis of celiac disease. Annu Rev Immunol 18:53–81.
OpenUrl CrossRef PubMed

[79] Sollid LM

[80] ↵
Toews GB,
Bergstresser PR,
Streilein JW
(1980) Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J Immunol 124:445–453.
OpenUrl PubMed

[81] Toews GB,

[82] Bergstresser PR,

[83] Streilein JW

[84] ↵
Klock HE,
Koesema EJ,
Knuth MW,
Lesley SA
(2008) Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 71:982–994.
OpenUrl CrossRef PubMed

[85] Klock HE,

[86] Koesema EJ,

[87] Knuth MW,

[88] Lesley SA

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Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids

Abstract

Results and Discussion

Mechanistic Studies of pNO₂Phe-Induced Breakdown of Self-Tolerance.

Sustainability of pNO₂Phe-Induced Antibody Response.

Extension to Mutations at Other Surface Sites Within mTNF-α.

Expression and Characterization of Mutant mRBP4 Proteins.

Generality of pNO₂Phe-Induced Breakdown of Self-Tolerance.

Conclusion

Materials and Methods

Bacterial Strains and Reagents.

Production of pNO₂Phe-Containing mTNF-α and WT mTNF-α.

Construction of mRBP4 Expression Vector, pSpeedET-mRBP4.

Protein Expression and Purification of pNO₂Phe mRBP4 and WT mRBP4.

Mouse Model of Severe Systemic Inflammation.

Acknowledgments

Footnotes

References

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Abstract

Results and Discussion

Mechanistic Studies of pNO2Phe-Induced Breakdown of Self-Tolerance.

Sustainability of pNO2Phe-Induced Antibody Response.

Extension to Mutations at Other Surface Sites Within mTNF-α.

Expression and Characterization of Mutant mRBP4 Proteins.

Generality of pNO2Phe-Induced Breakdown of Self-Tolerance.

Conclusion

Materials and Methods

Bacterial Strains and Reagents.

Production of pNO2Phe-Containing mTNF-α and WT mTNF-α.

Construction of mRBP4 Expression Vector, pSpeedET-mRBP4.

Protein Expression and Purification of pNO2Phe mRBP4 and WT mRBP4.

Mouse Model of Severe Systemic Inflammation.

Acknowledgments

Footnotes

References

Citation Manager Formats

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You May Also be Interested in

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Articles

PNAS Portals

Information

Mechanistic Studies of pNO₂Phe-Induced Breakdown of Self-Tolerance.

Sustainability of pNO₂Phe-Induced Antibody Response.

Generality of pNO₂Phe-Induced Breakdown of Self-Tolerance.

Production of pNO₂Phe-Containing mTNF-α and WT mTNF-α.

Protein Expression and Purification of pNO₂Phe mRBP4 and WT mRBP4.