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The Journal of Immunology, 2002, 168: 2554-2559.
Copyright © 2002 by The American Association of Immunologists

A Single-Chain Class II MHC-IgG3 Fusion Protein Inhibits Autoimmune Arthritis by Induction of Antigen-Specific Hyporesponsiveness1

Li Zuo*, Constance M. Cullen*, Monica L. DeLay*, Sherry Thornton*, Linda K. Myers{ddagger}, Edward F. Rosloniec§, Gregory P. Boivin{dagger} and Raphael Hirsch*,2

* Division of Rheumatology, Children’s Hospital Medical Center, and {dagger} Division of Comparative Pathology, University of Cincinnati, Cincinnati, OH 45229; and {ddagger} Department of Pediatrics, University of Tennessee, and § Veterans Affairs Medical Center, Memphis, TN 38163


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cells play a central role in many autoimmune diseases. A method to specifically target the function of autoreactive T cell clones would avoid the global immunosuppression associated with current therapies. To develop a molecule capable of inhibiting autoreactive T cell responses in vivo, single-chain peptide-I-A-IgG3 fusion proteins were constructed and expressed in both mammalian and insect cells. The fusion proteins were designed with an IgG3 Fc moiety to make them divalent, allowing TCR cross-linking, while lacking FcR binding and costimulation. The fusion proteins stimulated T cell hybridomas in vitro in a peptide-specific, MHC-restricted manner but failed to do so in soluble form. In vivo administration of an I-Aq fusion protein, containing an immunodominant collagen II peptide, significantly delayed the onset and reduced the severity of collagen-induced arthritis in DBA/1 mice by induction of Ag-specific hyporesponsiveness. Such fusion proteins may be useful to study novel therapeutic approaches for T cell-mediated autoimmune diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tcells, specifically Th cells, play an essential role in the initiation and perpetuation of autoimmune diseases such as rheumatoid arthritis (RA)3 (1, 2, 3, 4). The T cell immune response is normally exquisitely regulated to control against autoaggression through negative selection of self-reactive cells in the thymus. However, the process of negative selection is leaky and autoreactive cells do reach the periphery. Mechanisms exist to prevent these cells from inducing autoimmunity. However, the appearance of increased autoantigen, the exposure of protected self proteins, and the disregulation of immunity are potential means by which anergic autoreactive cells can become activated and mount an autoaggressive response.

Agents that can down-regulate autoreactive T cells provide a therapeutic approach to the treatment of these diseases. Abs against the TCR complex can block T cell function, although they also have the potential to induce T cell activation. The in vivo T cell-activating properties of these Abs depend on multivalent TCR cross-linking mediated via binding of the Ab Fc domains to FcR-bearing cells (5). F(ab')2 (6), or Abs with isotypes that do not bind FcR (7, 8, 9), allow only divalent TCR cross-linking, leading to partial signaling, Th cell hyporesponsiveness, and suppression of autoimmunity in mice (10, 11). However, these nonmitogenic Abs affect all Th cells and therefore may have undesirable global immunosuppressive effects. A therapy that can specifically target autoreactive T cell clones would be more attractive.

One approach toward this goal would be to target the complementarity-determining region of the TCR, which recognizes unique peptide-MHC structures. Soluble class I (12, 13, 14, 15, 16) and class II (17, 18, 19, 20, 21) MHC molecules have been produced that can be loaded with specific peptides and are able to signal T cells in vitro. Soluble class II have been reported to inhibit experimental allergic encephalomyelitis (22, 23). However, in general, soluble MHC molecules have not been effective at delivering inhibitory T cell signals in vivo. This likely relates to a number of factors. First, monovalent engagement of TCR is insufficient to induce T cell signaling (6, 13, 24, 25). Second, soluble MHC molecules are likely to have short half-lives in vivo. Divalent MHC (generated by cross-linking soluble H-2Kd molecules with anti-H-2Kd Ab) is required to induce T cell signaling in vitro, and higher order aggregates were even more effective (13). A dimeric class I MHC-IgG fusion protein was recently shown to induce Ag-specific blockade in vivo (26). A few studies demonstrating signaling by soluble class II may have used aggregated MHC molecules (20, 22, 23).

To combine the clonal specificity of soluble MHC molecules with the desirable signaling properties of nonmitogenic anti-TCR Abs, we have designed MHC-IgG3 fusion protein molecules which are divalent, allowing TCR cross-linking, while lacking FcR binding. MHC-IgG fusion proteins have been described and studied in vitro (18, 27, 28, 29), and a divalent I-Ed molecule fused to an IgG2a Fc was recently shown to induce differentiation of T cells toward a Th2 phenotype (30). MHC-IgG fusion proteins have not, to our knowledge, been used in vivo to inhibit T cell autoimmune responses. The present study was designed to test the hypothesis that class II MHC-IgG3 fusion proteins can mimic the in vivo T cell inhibitory effect of nonmitogenic anti-TCR Abs, but at a clonal level.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Design and expression of fusion proteins

Type II collagen (CII)-I-Aq-IgG3 was generated through a series of nested and overlapping PCR, as diagrammed in Fig. 6Go for CII-N. Primers A and B were used to amplify a 150-bp fragment from an I-Aq {beta}-chain cDNA clone. Primer A encodes a 5' NotI restriction site to facilitate subcloning of the final PCR product into the expression vectors. The PCR product from this reaction was reamplified with primer A and primer C to generate the A-C PCR product, which was then reamplified with primer A and D to generate the A-D PCR product. In a separate reaction, primers E and F were used to amplify the extracellular portion ({beta}1 and {beta}2 domains) from the I-Aq cDNA clone, beginning with the fourth amino acid. The A-D and E-F PCR products were reamplified using primers A and F to generate the A-F product. In a separate reaction, primers G and H were used to amplify the extracellular portion ({alpha}1 and {alpha}2 domains) from an I-Aq {alpha}-chain cDNA clone. Linker 2 was constructed using four overlapping oligonucleotides (O1–O4) and ligated to the 5' end of the G-H product, using an existing Bbs1 site in the {alpha}1 domain. The product of this reaction was then ligated to the A-F product using an Asc1 site to generate the A-H product. The A-H product was amplified using primers I and J to generate the I-J product. In a separate reaction, primers K and L were used to amplify the hinge, CH2, and CH3 domains of murine IgG3 using cDNA from a murine plasma cell known to produce an Ig of the IgG3 subclass (BP107.2.2, ATCC). Primer L encodes a 3' ApaI restriction site to facilitate subcloning of the final PCR product into the expression vectors. The product of this reaction was reamplified with the I-J product using primers I and L to generate the I-L product. The A-F and I-L products were amplified with primers A and L to generate the final fusion protein construct. The final 2.12-kb fragment was gel purified, digested with NotI and ApaI, ligated into the pRc/CMV expression vector (Invitrogen, Carlsbad, CA) and sequenced to ensure no mutations. CII-A and CII-I were derived from CII-N by site-directed mutagenesis. HSV-I-Ad-IgG3 was constructed by connecting a cDNA construct encoding a single chain HSV-I-Ad{beta}-I-Ad{alpha} (18), generously provided by P. Rhode and H. Wong (Sunol Molecular, Miami, FL), to the hinge, CH2, and CH3 domains of murine IgG3, using the same strategy described above. The final 2.04-kb fragment was gel purified, digested with NotI and ApaI, ligated into the pRc/CMV expression vector, and sequenced to ensure no mutations.



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  		FIGURE 6. Design of fusion protein constructs.

 
The sequences of the primers and oligonucleotides, from 5' to 3', are: primer A, 5'-CTGCGGCCGCGGATGGCTCTGCAGAT-3'; primer B, 5'-GGGGCCTTGTTCGCCTTTGAAGCCAGCAATACCCAGCTCGGAGTTTCCGCCCTC-3'; primer C, 5'-GCCCCGTGGCACTAGTGAGCCACCACCTCCGGGGCCTTGTTCGCC-3'; primer D, 5'-CGAAATGCCTTTCGGACCCACCTCCTCCAGAGCCCCGTGGCACT-3'; primer E, 5'-GAACAAGGCCCCGGAGGTGGTGGCTCACTAGTGCCACGGGGCCT-3'; primer F, 5'-CTTGCTCCGGGCAGA-3'; primer G, 5'-AAAGATCTAGGATGCCGCGCAGCAGA-3'; primer H, 5'-AACCCGGGTTAAGTCTCTGTCAGCTC-3'; primer I, 5'-CGGTGGTGGATCCGGTGGCGG-3'; primer J, 5'-GGGTATTCTAGGAGTCTCTGTCAGCTCTGA-3'; primer K, 5'-CTGACAGAGACTCCTAGAATACCCAAG-3'; primer L, 5'-AAGCATGGGCCCTCTAGATTATTTACCAGGGGAGCG-3'; O1, 5'-GGCCGCGAATGGCGCGCCCAGTCCGAGTCTGCCCGGAGCAAGACCTCAGGTGGCGGTGGCA-3'; O2, 5'-CGCCGCTGCCACCGCCACCTGAGGTCTTGCTCCGGGCAGACTCGGACTGGGCGCGCCATTCGC-3'; O3, 5'-GCGGCGGTGGTGGATCCGGTGGCGGCGGTTCCGGAGGTGGCGGTTCCTCGAGTGAGGATGA-3'; O4, 5'-AATGTCATCCTCACTCGAGGAACCGCCACCTCCGGAACCGCCGCCACCGGATCCACCAC-3'.

Transfection, biochemical analysis, and purification of fusion proteins

For transient transfections, COS-7 cells were transfected with fusion protein-encoding plasmids using Lipofectamine (Life Technologies, Gaithersburg, MD). After 48 h, cells were cultured overnight with 100 µCi/ml Tran35S-label (ICN Pharmaceuticals, Costa Mesa, CA) in 2 ml medium lacking methionine and cysteine. Supernatants were collected and incubated for 1 h at 4°C with Sepharose G (Pharmacia, Peapack, NJ). Sepharose beads were collected by centrifugation and washed three times with 5% sucrose, 1% Nonidet P-40, 0.5 M NaCl, 50 mM Tris, and 5 mM EDTA (pH 7.2), and once with 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.15% SDS, and 1% sodium deoxycholate. Following the last wash, 40 µl of SDS-PAGE sample buffer, with or without 2-ME, was added, samples were boiled for 5 min, and 20 µl of each sample was electrophoresed on 4–20% SDS Tris-glycine gels (NOVEX, San Diego, CA). Gels were fixed in 10% acetic acid, dried, and scanned on a Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

To generate stable transfectants, plasmid constructs were linearized and SP2/0 cells were transfected by electroporation using a gene pulser with a capacitance extender (Bio-Rad, Hercules, CA) at 25 µF, 360 V. Cells were rested for 24 h after electroporation, followed by selection in 900 µg/ml G418 (Life Technologies). For insect cell expression, constructs were inserted between NotI and XbaI sites of pFastBac (Life Technologies), which was then used to transform DH10Bac cells (Life Technologies). Recombinant bacmid was isolated and transfected into SF9 cells using CellFection (Life Technologies).

To purify fusion proteins, supernatants were adjusted to pH 8 with 1 N NaOH and passed over an ImmunoPure protein A-Sepharose column (Pierce, Rockford, IL). The column was washed with ImmunoPure binding buffer and the bound fusion protein was eluted with 50 mM glycine-HCl, pH 11, immediately neutralized to pH 8 with 3 M Tris-HCl, and dialyzed against PBS. Purified fusion protein was stored at 4°C until used.

Analysis of fusion protein conformation

Ninety-six-well, flat-bottom ELISA plates (Nalge Nunc, Rochester, NY) were coated with 250 ng/well anti-mouse IgG3 (BD PharMingen, San Diego, CA) in PBS and incubated overnight at 4°C. The plates were washed with PBS-Tween, blocked with 1% BSA in PBS, washed again, and incubated in duplicate wells with 250 ng/well purified fusion protein. Plates were washed again, followed by addition of 1 µg/ml biotin-labeled 2G9 (31) or Y3P (32). Plates were washed, followed by addition of avidin-peroxidase (BD PharMingen). Plates were washed, developed with Peroxidase Substrate System ABTS (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and read at 410 nm with a Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA), and duplicates were averaged.

Activation of T cell hybridomas

Plates were coated with 250 ng/well goat anti-mouse IgG3 (BD PharMingen) and incubated overnight at 4°C. The plates were then washed with PBS, blocked with 1% BSA in PBS, washed again, and incubated for 2 h with 250 ng/well fusion protein. Plates were washed with PBS to remove unbound fusion protein, followed by addition of 1 x 105 QCII.92.33 (33) or GD12 (34) T-hybridoma cells/well. In some cases, 250 ng/well KH116 (anti-I-Aq) was added before addition of cells. For assays involving soluble fusion protein, 250 ng of fusion protein was added to wells that had not been preincubated with anti-IgG3, and the fusion protein was not washed out. Supernatants were collected after 24 h and stored at -20°C for subsequent IL-2 quantitation, as previously described (35), using the indicator cell line, CT.4R (36). All assays were performed in triplicate.

Treatment protocols

Arthritis was induced with bovine CII (Elastin Products, Owensville, MO), as previously described (11). Male DBA/1 mice (The Jackson Laboratory, Bar Harbor, ME) 6–10 wk of age were injected intradermally with 100 µg of CII in CFA at the base of the tail on days 0 and 21. The booster immunization was given in CFA to obtain a higher incidence and synchronized onset of arthritis (11, 37, 38, 39). Fusion proteins (20 µg in 1 ml of PBS) or PBS alone was administrated on day 17 by i.p. injection. Mice were evaluated several times a week for arthritis using an established macroscopic scoring system (11) ranging from 0 to 4 (0, no detectable arthritis; 1, swelling and/or redness of paw or one digit; 2, two joints involved; 3, three or four joints involved; and 4, severe arthritis of the entire paw and digits). The arthritic index for each mouse was calculated by adding the four scores of the individual paws. The Mann-Whitney U test (two-tailed, independent) was used to test the significance of intergroup differences in the arthritic indices.

Histology

Paws were fixed in 10% neutral buffered formalin and decalcified in calrite (R. Allen, Richland, MI). Tissues were then dehydrated in a gradient of alcohols, paraffin embedded, sectioned at 5 µm, mounted on glass slides, and stained with H&E. A blinded observer performed histologic analysis.

T cell function assays

A total of 5 x 105 spleen cells/well were plated in triplicate at 37°C in 96-well flat-bottom microtiter plates in DMEM containing 0.5% normal DBA/1 mouse serum in the presence of 100 µg/ml heat-denatured (56°C for 10 min) CII, 25% anti-CD3 (2C11)-containing supernatant, or medium alone. For proliferation assays, cells were incubated for 48 (2C11 stimulation) or 72 h (CII stimulation), followed by addition of 1 µCi of [3H]TdR for an additional 18 h. Cells were harvested and [3H]TdR incorporation per well was measured and averaged for each triplicate. The stimulation index was calculated by dividing the counts from cells incubated with CII or 2C11 by the counts of cells incubated in medium alone. For IL-2 assays, supernatants were harvested after 24 h for subsequent IL-2 quantitation and values were subtracted from counts of spleen cells in medium alone.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Design and characterization of MHC-IgG3 fusion proteins

Class II MHC-IgG3 fusion proteins were constructed by multiple rounds of PCR. CII-I-Aq-IgG3 contains the immunodominant peptide (amino acids 257–269) from chick CII (40) and the extracellular domains of the murine class II I-Aq molecule, which is the restricting element for murine collagen-induced arthritis (CIA). Three CII-I-Aq-IgG3 fusion proteins were constructed with slightly different CII-derived peptides. Fusion protein CII-N contains the native peptide. Fusion protein CII-A has a substitution at position 257 (A for E) which confers a higher affinity for I-Aq and up to a 200-fold increase in stimulation of CII257–261-specific T cells (33). Fusion protein CII-I, which was used as a negative control, has a substitution at position 261 (I for A), which results in binding to I-Aq with equal affinity as the native peptide but poor stimulation of CII257–261-specific T cells (33). An additional control fusion protein, HSV-I-Ad-IgG3, contains an immunodominant peptide (amino acids 246–261) from HSV-1 glycoprotein D and the extracellular domains of the murine class II I-Ad molecule.

The constructs were ligated into the expression vector, pRc/CMV, and transiently transfected into COS-7 cells. Cells were pulsed with [35S]methionine and supernatants were immunoprecipitated with protein G-Sepharose. Electrophoresis on 4–20% gradient gels (Fig. 1GoA) demonstrated that the fusion proteins are homodimers composed of two 80-kDa polypeptides, approximating the predicted size of the fusion protein. Cell lysates also contained the fusion protein (data not shown); however, the majority was found in the supernatants, demonstrating that the fusion protein is secreted as a soluble product.



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  		FIGURE 1. Characterization of fusion proteins. A, SDS-PAGE analysis of the CII-I-Aq-IgG3 fusion protein, CII-N, under nonreducing and reducing conditions. Supernatants from [35S]methionine-pulsed COS-7 cells transfected with the plasmid pRc/CMV (lanes 1 and 3) or transfected with CII-I-Aq-IgG3-pRc/CMV (lanes 2 and 4) were immunoprecipitated with protein G-Sepharose. B, Analysis of fusion protein conformation by ELISA. Fusion proteins were analyzed by ELISA, as described.

 
To generate stable transfectants, the constructs were either transfected into SP2/0 cells by electroporation followed by selection in G418 or expressed in insect Sf9 cells using the baculovirus expression system. Fusion proteins were purified by passage of supernatants over a protein A column. On average, 2 mg of fusion protein were obtained per liter of supernatant from transfected SP2/0 cells. All subsequent experiments were performed with purified fusion protein.

The fusion proteins were further characterized for proper folding by ELISA, using I-A-specific Abs. The CII-I-Aq-IgG3 and HSV-I-Ad-IgG3 fusion proteins were recognized by the anti-I-A Ab, 2G9 (Fig. 1GoB). The CII-I-Aq-IgG3, but not the HSV-I-Ad-IgG3, fusion proteins were recognized by the conformation-dependent anti-I-Aq Ab, Y3P. Thus, the fusion proteins appeared to be conformationally intact.

Immobilized, but not soluble, fusion proteins activate T cell hybridomas in vitro

Based on the observation that immobilized anti-TCR complex Abs can induce T cell activation, we tested the ability of immobilized fusion proteins to activate T cell hybridomas in a peptide-specific, MHC-restricted manner. Fusion proteins were immobilized onto microtiter plates that had been precoated with anti-IgG3, followed by addition of T cell hybridomas. CII-N and CII-A induced IL-2 secretion from QCII92.33 cells (restricted to I-Aq and specific for CII257–269), but not from GD12 cells (restricted to I-Ad and specific for HSV246–261) (Fig. 2Go). The control CII-I fusion protein did not stimulate QCII92.33 cells. HSV-I-Ad-IgG3 induced IL-2 secretion from GD12 cells but not from QCII92.33 cells. Stimulation of QCII92.33 could be blocked by anti-I-Aq Ab. These data demonstrate that the fusion proteins could signal T cells in an MHC-restricted, peptide-specific manner. CII-I-Aq-IgG3 fusion proteins in soluble form failed to induce IL-2 secretion, consistent with previous observations with soluble anti-TCR Abs that multivalent TCR cross-linking is necessary for effective T cell activation.



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  		FIGURE 2. Immobilized fusion protein activates T cell hybridomas in an MHC-restricted, peptide-specific manner. QCII.92.33 (CII-specific, I-Aq-restricted) or GD12 (HSV-specific, I-Ad-restricted) T cell hybridomas were incubated in fusion protein-coated plates and supernatants were collected for IL-2 measurement using the indicator cell line, CT.4R. Fusion protein-induced T cell activation could be blocked by the anti-I-A Ab, KH116. Each bar represents the mean ± SEM of three wells.

 
In vivo administration of CII-I-Aq-IgG3 inhibits CIA

To determine whether the fusion proteins could inhibit T cell autoimmune responses in vivo, CIA-susceptible DBA/1 (H-2q) mice were immunized on days 0 and 21 with CII (10 mice per group). On day 17, mice received a single i.p. injection of 20 µg of purified CII-I-Aq-IgG3 fusion proteins or PBS. The dose was chosen based on earlier studies with anti-CD3 mAbs (41). Day 17 was chosen based on earlier studies demonstrating that blocking T cell function before the booster can inhibit CIA (11). Mice were evaluated for arthritis from days 24 to 41. CII-N and CII-A significantly delayed the onset and reduced the severity of CIA, while CII-I failed to inhibit disease (Fig. 3Go). HSV-I-Ad-IgG3 also had no effect on CIA (data not shown). These findings were also confirmed by histopathology. Mice treated with PBS had severe arthritis, characterized by inflammatory cell infiltrate in the synovium and cartilage erosion (Fig. 4GoA), while mice treated with CII-N and CII-A had minimal inflammation (Fig. 4Go, B and C). Mice treated with the control CII-I fusion protein had similar histopathologic appearance to that of PBS-treated mice (Fig. 4GoD). No effect was observed following fusion protein administration after disease onset (data not shown), consistent with the T cell independence of established CIA (11, 42).



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  		FIGURE 3. Effect of fusion proteins on CIA. Mice were immunized with CII, as described. On day 17, 10 mice per group received i.p. injections of purified fusion proteins or PBS. *, p < 0.05 and **, p < 0.01, compared with PBS.

 


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  		FIGURE 4. Effect of fusion proteins on CIA. Mice were immunized with CII, as described. On day 17, mice received i.p. injections of PBS (A), CII-N (B), CII-A (C), or CII-I (D). Mice were sacrificed on day 40 and hind paw joints were analyzed by histology. T, Tendon; S, synovium; B, bone. Magnification, x50.

 
In vivo administration of CII-I-Aq-IgG3 induces Ag-specific T cell hyporesponsiveness

To determine the mechanism of the observed inhibition of CIA, mice were sacrificed on day 40 and spleens cells were harvested and stimulated in vitro with denatured CII or anti-CD3 mAb (2C11). Spleen cells from mice treated with PBS or the control CII-I fusion protein proliferated (Fig. 5GoA) and secreted IL-2 (Fig. 5GoB) in response to CII, whereas spleen cells from mice treated with CII-N or CII-A were hyporesponsive. This inhibition was Ag specific, as the response to a pan-T cell stimulus (2C11) was normal (Fig. 5Go, C and D). Thus, administration of CII-I-Aq-IgG3 to DBA/1 mice induced Ag-specific hyporesponsiveness. No inhibitory effects on anti-CII Ab titers was observed (data not shown), consistent with earlier observations that blocking T cell function can suppress CIA without affecting anti-CII Ab titers (11, 37, 43).



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  		FIGURE 5. In vivo administration of fusion proteins induces Ag-specific T cell hyporesponsiveness. Mice were immunized with CII, as described. On day 17, mice received i.p. injections of PBS or fusion proteins. Mice were sacrificed on day 40 and spleen cells were incubated with heat-denatured CII (A and B) or 25% anti-CD3 (2C11)-containing supernatant (C and D). The stimulation index represents the proliferation ([3H]thymidine incorporation) of cells incubated with CII or 2C11 divided by the proliferation of cells incubated in medium alone. Each bar represents the mean ± SEM of three mice. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To our knowledge, this is the first demonstration of the use of class II MHC-IgG fusion proteins to induce Ag-specific hyporesponsiveness and suppression of autoimmune responses in vivo. The fusion proteins were designed with an IgG3 Fc moiety to make the molecule divalent, allowing TCR cross-linking, while lacking FcR binding and costimulation. The IgG CH2 and CH3 domains provide interchain disulfide bonds, resulting in a divalent molecule that can deliver a down-regulatory signal. Biochemical analysis confirmed that the majority of the fusion protein molecules exist as homodimers. The degree of valence required for T cell signaling is still controversial. Triggering of T cells with anti-TCR mAb Fab has been reported (44); however, peptide/MHC ligands are nonstimulatory as monomers and optimal T cell activation requires trimeric or tetrameric ligands (45). Divalent TCR cross-linking has been demonstrated to induce T cell hyporesponsiveness in a number of studies (6, 7, 8, 9, 10). The ability of the two stimulatory fusion proteins, CII-N and CII-A, but not the nonstimulatory CII-I, to inhibit CIA suggests that induction of hyporesponsiveness requires signal delivery rather than mere binding to the TCR.

The second property conferred by the IgG3 Fc moiety is the lack of FcR binding. Studies with anti-TCR Abs demonstrate that the activating properties of anti-TCR mAbs administered in vivo result from TCR cross-linking mediated by FcR-bearing cells (5). Anti-CD3 mAbs that bind FcR induce in vivo T cell activation; however, F(ab')2 of such mAbs (35), or mAbs of an isotype that do not bind FcR (8), induce T cell hyporesponsiveness rather than activation. In a recent study in mice comparing the in vivo effects of whole and F(ab')2 of anti-CD3 Ab (46), whole Ab primed T cells to subsequent in vitro stimulation. In contrast, F(ab')2 induced T cell hyporesponsiveness reflected by a reduced secretion of IL-2 and IFN-{gamma} upon subsequent in vitro stimulation. In addition to promoting multivalent TCR cross-linking, bridging of T cells to FcR-bearing cells might also promote costimulation, which is avoided by use of the IgG3 Fc moiety in the fusion protein molecule.

A final consideration in designing the fusion protein with an Fc moiety was the possibility that the Fc moieties might prolong the in vivo half-life. Although we were not able to detect circulating fusion protein in treated mice, this principle has been demonstrated with many mAbs. For example, the anti-murine CD3 Ab, 2C11, has a half-life in C57BL/10 mice of 2 wk, while F(ab')2 of the Ab have a half-life of 2 days (6). The fusion protein itself is unlikely to induce a strong humoral response, because the only potential nonself epitopes include the peptide and the novel epitope created by the fusion between the MHC and the hinge of the IgG.

The lack of efficacy in established disease is not surprising, given that T cell depletion after established CIA does not alter the course of disease (11, 42). The role of T cells in established human RA is probably more important because large numbers of CD4+ T cells are found in RA synovial tissues (1, 2, 3, 4). Potential T cell autoantigens have been described in RA, including CII (47, 48), suggesting that the approach described herein may have clinical utility in human disease. It might also be applied to other T cell-mediated processes, such as transplantation.

In summary, the current study demonstrates the construction of CII-I-Aq-IgG3 fusion proteins with the following unique features: 1) they can be produced in large quantities from both mammalian and insect cells and easily purified using protein A affinity chromatography; 2) their divalent structure allows T cell signaling in an Ag-specific, MHC-restricted manner; and 3) in vivo administration significantly reduces the severity of CIA in mice by induction of Ag-specific hyporesponsiveness. Such fusion proteins might be useful for treatment of T cell-mediated autoimmune diseases.


   Acknowledgments
 
We thank Drs. P. Rhode and H. Wong for supplying the HSV-I-Ad construct used to construct HSV-I-Ad-IgG3, and Drs. D. Glass and A. Grom for helpful discussion and critical review of the manuscript.


   Footnotes
 
1 This work was supported, in part, by National Institutes of Health Grants AI34958 and AR47363, the Schmidlapp Foundation, and the Children’s Hospital Research Foundation of Cincinnati. Back

2 Address correspondence and reprint requests to Dr. Raphael Hirsch, Division of Rheumatology, Children’s Hospital Medical Center, Pav 2-129, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail address: hirschr{at}chmcc.org Back

3 Abbreviations used in this paper: RA, rheumatoid arthritis; CIA, collagen-induced arthritis; CII, type II collagen. Back

Received for publication September 21, 2001. Accepted for publication December 27, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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