IgG1 Fusion Protein Induces Antigen-Specific T Cell Activationin Vitroandin Vivo

Cellular Immunology 192, 54 – 62 (1999) Article ID cimm.1998.1434, available online at http://www.idealibrary.com on

A Divalent Major Histocompatibility Complex/IgG1 Fusion Protein Induces Antigen-Specific T Cell Activation in Vitro and in Vivo 1 Constance M. Cullen,* Stephen C. Jameson,† Monica DeLay,* Charles Cottrell,* Eric T. Becken,† Edmund Choi,‡ and Raphael Hirsch* ,2 *Division of Rheumatology, Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229; †Department of Laboratory Medicine and Pathology and Center for Immunology, University of Minnesota Medical School, Minneapolis, Minnesota 55455; and ‡Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 Received August 11, 1998; accepted December 3, 1998

directed against either the ab heterodimer or the CD3 complex can dramatically influence these T cell immune responses in both mice (1–7) and humans (8 –10). Signaling through the TCR complex can result in a full activation signal and the boosting of an immune response if a second costimulatory signal, such as CD28, is provided by an accessory cell (11–14). The activating properties of anti-TCR mAbs administered in vivo result from TCR cross-linking mediated by Fc receptor (FcR) 1 cells capable of delivering a costimulatory signal (15–20). We have previously demonstrated that in vivo administration of anti-CD3 mAbs capable of binding FcR 1 cells leads to initial suppression of T cell responses associated with TCR modulation (1), but ultimately results in T cell priming (20). This is reflected by a shift of the majority of T cells to a memory phenotype and an increased secretion of IL-2 and IFN-g upon subsequent in vitro stimulation. The observation that T cells are fully functional once antiCD3 mAbs are cleared from the circulation suggests that these mAbs do not induce T cell dysfunction, but supress immune function by other means, such as T cell depletion and transient TCR modulation. These observations also suggest that the FcR 1 cell is able to deliver the costimulatory signals required for T cell priming, as treatment with anti-TCR F(ab9) 2 fragments results in T cell anergy (4, 5, 21–23). It is possible to exploit the activating properties of anti-TCR mAbs to boost T cell immune responses to viral or tumor antigens. Indeed, low doses of anti-CD3 mAb can inhibit tumor growth in mice (24). However, mAbs against the TCR complex have no clonal specificity and thus induce potentially deleterious global effects. A more desirable outcome would be to activate antigen-specific T cell clones while leaving the majority of T cells unaffected and able to react normally. While it is possible in animal models to develop peptidespecific tumor vaccines using adjuvants and chemically

Activation of antigen-specific T cell clones in vivo might be possible by generating soluble MHC molecules; however, such molecules do not induce effective T cell responses unless cross-linked. As a first step in generating a soluble MHC molecule that could function as an antigen-specific immunostimulant, the extracellular domains of the murine H-2K b MHC class I molecule were fused to the constant domains of a murine IgG1 heavy chain, resulting in a divalent molecule with both a TCR-reactive and an Fc receptor (FcR)reactive moiety. The fusion protein can be loaded with peptide and can induce T cell activation in a peptidespecific, MHC-restricted manner following immobilization on plastic wells or following cross-linking by FcR 1 spleen cells. The fusion protein induces partial T cell activation in vivo in a mouse transgenic for a TCR restricted to H-2K b. This fusion protein molecule may be useful to study peptide–MHC interactions and may provide a strategy for boosting in vivo antigen-specific T cell responses, such as to viral or tumor antigens. © 1999 Academic Press

INTRODUCTION T cells are major effectors in a variety of immune responses, including those to viruses, tumors, autoantigens, and organ allografts. T cell recognition of antigen occurs via the TCR, composed of polymorphic a and b chains which form a heterodimer associated with invariant CD3 chains. Monoclonal antibodies (mAbs) 1 Supported, in part, by NIH Grants AI34958, AR44059, the Schmidlapp Foundation, the Children’s Hospital Research Foundation of Cincinnati (R. Hirsch), an Arthritis Foundation postdoctoral fellowship (C. Cullen), and the American Cancer Society (S. Jameson). 2 To whom correspondence should be addressed at Division of Rheumatology, Children’s Hospital Medical Center, PAV 2-129, 3333 Burnet Avenue, Cincinnati, OH 45229.

0008-8749/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

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synthesized peptides (25–27), this approach often fails to lead to effective immunity. Soluble MHC molecules have been envisioned as a means to activate T cells in a peptide-specific manner in vivo. The MHC class I antigens are highly polymorphic cell surface proteins which bind peptide antigens for presentation to CD8 1 T cells. The MHC/peptide complex is recognized by T cells through the ab TCR and involves recognition of sequences from both the MHC molecule and the peptide. This highly specific interaction is critical for T cell activation and therefore represents a potential target for immune intervention. Soluble MHC molecules might be loaded with appropriate immunodominant peptides known to bind to the desired MHC allele, with subsequent presentation only to those T cell clones which recognize both the particular MHC and the peptide. Soluble class I molecules have recently been produced. Such molecules can be loaded with class I-specific peptides and are able to stimulate T cells in vitro (28 –30). However, they have generally not been effective at inducing T cell responses in vivo for at least three reasons. First, multivalent TCR cross-linking appears to be necessary for effective T cell activation, as monovalent engagement of TCR, either with anti-TCR mAbs (31) or with soluble class I MHC (30, 32, 33), fails to induce signaling. Second, T cell activation requires a costimulatory signal, in addition to that mediated via the TCR (11–14). This costimulatory signal is generally delivered by the antigen-presenting cell; however, in the case of soluble class I this second signal is absent. Third, soluble class I molecules are likely to have short half-lives in vivo. The present report describes the characterization of a soluble class I molecule generated by fusing the extracellular domains of murine H-2K b to the hinge, CH2, and CH3 domains of IgG1. The molecule is designed to be divalent and to bind FcR 1 cells, allowing for multivalent TCR cross-linking and T cell activation. Similar fusion protein molecules have been reported to inhibit T cell responses (28, 34), but this is the first report of an MHC–Ig fusion protein with activating properties and with the capacity to signal T cells in vivo. MATERIALS AND METHODS Mice. C57BL/6 female mice were purchased from The Jackson Laboratory (Bar Harbor, ME). OT-1 mice transgenic for an ovalbumin 257–264 (OVA)-specific, H-2K b-restricted TCR (35) were a gift of F. Carbone and W. Heath. The OT-1 mice were generated on a C57BL/6 background and carry TCR Vb5 and Va2 transgenes. Heterozygous OT-1 male mice were mated to C57BL/6 females. The resultant offspring were screened for the presence of the TCR transgenes by analysis of peripheral blood using flow cytometry (see below). Heterozygous mice are readily identified, as

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virtually all T cells express the transgenic TCR and are CD8 1. All mice were housed in microisolator cages under specific pathogen-free conditions. Transfection and biochemical analysis. The expression plasmid, pCMV4 (generously provided by D. Russell), allows transgene expression under control of the CMV promoter. pHuActb2 (generously provided by R. Ribaudo) encodes murine b2-microglobulin under the control of the human b-actin promoter and encodes the neomycin resistance gene, allowing for selection in G418. For transient transfections, COS-7 cells were transfected with both plasmids using lipofectamine (Gibco BRL, Gaithersburg, MD). After 48 h, cells were cultured overnight with 100 mCi/ml of Tran 35S-label (ICN, Costa Mesa, CA) in 2 ml of medium lacking methionine and cysteine. Supernatants were collected and incubated for 1 h at 4°C with Y3–Sepharose generated by conjugating the conformationally dependent anti-H-2K b monoclonal antibody Y3 (36 –39) to cyanogen bromide-activated Sepharose 4B (Pharmacia). Sepharose beads were collected by centrifugation and washed three times with 5% sucrose, 1% NP-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, 1% sodium deoxycholate. Following the last wash, 40 ml of SDS–PAGE sample buffer, with or without 2-mercaptoethanol, was added; samples were boiled for 5 min; and 20 ml 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 J558L cells were transfected by electroporation using a gene pulser with a capacitance extender (Bio-Rad) at 960 mF, 260 V. Cells were rested for 24 h after electroporation, followed by selection in 900 mg/ml G418 (Gibco BRL). Preparation and peptide loading of soluble MHC/ IgG1 fusion protein. To purify fusion protein, supernatants were passed over a Protein A–Sepharose column, eluted with 0.1 M sodium acetate, pH 5.0, and dialyzed against PBS. HPLC-purified OVA and vesicular stomatitis virus nuclear protein 52–59 (VSV) peptides were purchased from the University of North Carolina Program in Molecular Biology and Biotechnology Micro Protein Chemistry Facility (Chapel Hill, NC). Purity and expected molecular mass were confirmed by mass spectroscopy. Purified fusion protein, or ammonium sulfate-precipitated J558L supernatants containing fusion protein, were loaded with OVA or VSV peptides at varying concentrations by gentle rotation at 4°C overnight. Assay for detection of peptide-loaded fusion protein. Ninety-six-well, flat-bottom ELISA plates (Corning

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Costar, Cambridge, MA) were coated with antimouse IgG1 (PharMingen, San Diego, CA) at a concentration of 5 mg/ml in PBS and incubated overnight at 4°C. The plates were washed with PBS– Tween, blocked with 1% bovine serum albumin (BSA) in PBS, washed again, and incubated with purified fusion protein, or supernatants containing fusion protein, in triplicate wells. In some cases, fusion protein was previously loaded with peptide, as described above. Plates were washed again, followed by addition of biotin-labeled Y3 (recognizing peptideloaded H-2K b ) at a concentration of 1 mg/ml. Plates were washed, followed by addition of avidin–peroxidase (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), and triplicates were averaged. In vitro T cell assays. In vitro assays were performed in media containing RPMI and 10% fetal calf serum in 96-well polystyrene tissue culture plates (Corning Costar). All assays were performed in triplicate wells in volumes of 200 ml and were repeated a minimum of three times to ensure reproducibility. IL-2 secretion was measured as previously described (21) using the indicator cell line, CT.4R (40). Activation of T hybridomas. Plates were coated with 5 mg/ml goat anti-mouse IgG1 (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 40 min with supernatants containing peptide-loaded fusion protein (OVA or VSV) or the anti-TCR ab mAb H57 (41). Plates were washed with PBS to remove unbound fusion protein or mAb, followed by addition of 5 3 10 4 B3.6.4.5. (H-2K brestricted, OVA-specific T hybridoma; made and kindly contributed by N. Hosken and M. Bevan) or 2B4 (I-E krestricted, pigeon cytochrome C-specific) (42) T hybridoma cells/well. Supernatants were collected after 24 h and stored at -20°C for subsequent IL-2 quantitation. Activation of B3 cytotoxic T lymphocytes (CTL). Purified fusion protein (1 mg/well) was coated directly to wells overnight at 4°C. OVA peptide was added to these wells, or to control wells with no fusion protein, for 2 h. After three washes, 10 5 B3 CTL (OVA-specific CTL line derived from C57BL/6 (H-2K b) mice) (43) was added to each well. As a positive control, B3 CTL were incubated in the continuous presence of 1mM OVA which allows for direct presentation between the CTL in the culture. After 24 h, the supernatants were collected and IL-3/GM-CSF production determined using the reporter cell FDC.P1, as previously described (44). Data are expressed as a percentage of the positive control (B3 1 1 mM OVA).

FcR 1 spleen cell assays. A total (5 3 10 4) B3.6.4.5. cells/well were incubated with 2 3 10 5 irradiated (2500 rad) DBA/1 spleen cells in the presence or absence of 25% 2.4G2-containing (anti-FcR mAb (45)) culture supernatant. Cells were stimulated with medium alone, 250 ng OVA, or 0.5 mg of purified fusion protein previously loaded with 250 ng OVA. Supernatants were collected after 24 h and stored at 220°C for subsequent IL-2 quantitation. Flow cytometry (FCM) analysis. Multicolor FCM was performed using a FACScan (Becton Dickinson, Mountain View, CA) as previously described (1). All procedures were performed in PBS containing 0.2% BSA and 0.01% sodium azide. Fluorescence data were collected using logarithmic amplification on 10,000 viable cells as determined by forward light scatter intensity and/or propidium iodide exclusion. For identification of OT-1 heterozygotes, mice were bled and peripheral blood was stained with FITC-conjugated anti-Vb5 (PharMingen) and PE-conjugated anti-CD8 (PharMingen). For determination of effects of fusion protein on T cell activation, spleen cells from treated mice were stained with PE-conjugated anti-CD8 and FITC-conjugated antibodies to CD69, CD25, CD44, CD45, and CD95 (PharMingen). RESULTS Design and expression of the H-2K b/IgG1 fusion protein. Two rounds of PCR were used to generate a 1591-bp product encoding the fusion protein (Fig. 1). The entire fusion gene was sequenced to ensure that no spurious mutations were introduced during the PCR. The product was gel purified, ligated into the expression vector pCMV4 to generate the plasmid pCMVKbIg, and transiently transfected into COS-7 cells along with pHuActb2 (encoding mouse b2-microglobulin) to ensure sufficient murine b2-microglobulin. Cells were pulsed with [ 35S]methionine and supernatants were immunoprecipitated with Sepharose 4B conjugated to the conformationally dependent anti-H-2K b monoclonal antibody Y3. Electrophoresis on 4 –20% gradient gels showed a novel 145-kDa protein in supernatants from transfected cells (Fig. 2), which reduced to 76-kDa protein, demonstrating that the fusion protein is a homodimer composed of two 76-kDa polypeptides, approximating the predicted size the fusion protein. An 11-kDa protein was also observed, likely representing b2-microglobulin which was predicted to self-associate. Ability to precipitate the fusion protein with Y3 demonstrates that at least some of the fusion protein is conformationally intact and is likely to be loaded with endogenous peptide. 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. To gen-

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FIG. 1. Schematic representation of the strategy used to generate the H-2K b/IgG1 fusion gene construct. During round 1, two separate PCR reactions were used to amplify the appropriate domains of H-2K b and IgG1. The plasmid pBH-2 (generously provided by S. Freeman), encoding the H-2K b cDNA, was used to amplify the extracellular domains of H-2K b using PCR primers 1 and 2, designed to add a ClaI site at the 59 end and a 10-bp sequence on the 39 end homologous to the 59 end of the IgG1 hinge region. The plasmid, pHg1360X (generously provided by J. Capra), encoding the murine IgG 1 heavy chain cDNA, was used to amplify the hinge, CH2, and CH3 regions of IgG1 using primers 3 and 4, designed to add an XbaI site at the 39 end and a 10-bp sequence on the 59 end homologous to the 39 end of the a3 domain of H-2K b. During round 2, the 2 products of the first round of PCR were annealed in a second PCR reaction, using primers 1 and 4, to generate a 1591-bp product encoding the H-2K b/IgG1 fusion protein. The sequences of the PCR primers were: Primer 1, G C G C A T C G A T A T G G T A C C G T G C A C G C T G C T; Primer 2, C C C T G G G C A C C C A T C T C A G G G T G A G G G G C; Primer 3, C C T G A G A T G G G T G C C C A G G G A T T G T G G T; and Primer 4, A A G C A T T C T A G A T C A T T T A C C A G G A G A G T G.

erate stable transfectants, pCMVKbIg and pHuActb2, were cotransfected into J558L cells by electroporation and cells were selected in G418. All subsequent experiments were performed with supernatants or purified fusion protein from these cells. The H-2K b/IgG1 fusion protein can be loaded with exogenous peptide. Immunoprecipitation of the fusion

FIG. 2. SDS–PAGE analysis of the fusion protein molecule under nonreducing and reducing conditions. Supernatants from [ 35S]methionine-pulsed COS-7 cells cotransfected with pCMVKbIg and pHuActb2 (lanes 1 and 3) or transfected with pCMV4 (lanes 2 and 4) were immunoprecipitated with Sepharose conjugated to the conformationally dependent, H-2K b-specific, mAb Y3. Samples were run on a 4 –20% gradient gel which was fixed in 10% acetic acid, dried, and scanned by PhosphorImager.

protein with Y3 suggested that at least a portion of the fusion protein was loaded with peptides, either prior to or following secretion. To determine whether the fusion protein was maximally loaded, culture supernatants containing the fusion protein were incubated with varying concentrations of OVA, which binds with high affinity to H-2K b (46). Addition of OVA, led to a dosedependent increase in Y3 binding (Fig. 3), suggesting that some of the fusion protein existed in an unloaded state and could be loaded by addition of exogenous peptide. Some Y3 binding was observed without exogenous OVA. This binding could be reduced to background levels by incubating the fusion protein at 37°C for 5 h to remove bound peptide and could be recovered by loading with OVA (data not shown). The H-2K b/IgG1 fusion protein activates H-2K b-restricted T cells in a peptide-specific manner. Based on the observation that immobilized mAbs directed against the TCR complex can induce T cell activation, we tested the ability of immobilized fusion protein to activate a T cell hybridoma in a peptide-specific manner. Peptide-loaded fusion protein was immobilized onto microtiter plates which had been precoated with anti-IgG1. Following extensive washing to remove unbound peptide, the T cell hybridoma B3.6.4.5., restricted to H-2K b and specific for OVA, was added. B3.6.4.5. cells secreted IL-2 in response to fusion protein loaded with OVA (Fig. 4A). In contrast, the cells did not secrete IL-2 in response to OVA alone, or fusion protein loaded with VSV, which has similar binding

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FIG. 3. Fusion protein can be loaded with exogenous peptide. Supernatants from J558L cells expressing the fusion protein (F) or from control cell lines transfected with the expression vector pCMV4 (h) were incubated overnight at 4°C with varying amounts of OVA and analyzed by ELISA, as described.

affinity for H-2K b to that of OVA (46). Similarly, IL-3/ GM-CSF secretion was induced by OVA-loaded fusion protein in the OVA-specific CTL line B3, at OVA concentrations as low as 200 pmol (Fig. 4B). The T hybridoma, 2B4, restricted to I-E k and specific for pigeon cytochrome C, failed to secrete IL-2 in the presence of immobilized fusion protein, but did secrete IL-2 in the presence of the anti-TCR ab mAb, H57 (Fig. 4A). While these data suggested that the OVA-loaded fusion protein was able to directly bind to the TCR and mediate signaling, OVA dissociates from the fusion protein following incubation at 37°C for 5 h (data not shown). Thus, it was also possible that the fusion pro-

tein was acting as a “sink” to bind OVA during the washing of the plate, with subsequent release into the medium where it could bind to H-2K b expressed on the surface of the B3.6.4.5. cells and allow for self-presentation between the cells. This possibility was unlikely since the amount of OVA remaining bound following washing was calculated to be less than 0.01 nM, based on the maximal amount of fusion protein that could be bound by the anti-IgG1. However, to rule out this possibility, a separate experiment was performed in which excess OVA was not removed by washing, and B3.6.4.5. cells were incubated at a low density in flat bottom plates to minimize cell– cell contact and peptide pre-

FIG. 4. Immobilized fusion protein activates OVA-specific T cell hybridomas. (A) Round-bottom microtiter plates were coated with anti-IgG1, followed by addition of supernatants containing 2 mg/ml anti-TCR mAb, H57 (h), 5 mM OVA (z), 50 ml of fusion protein-containing supernatant loaded with 5mM OVA (■), or 50 ml of fusion protein-containing supernatant loaded with 5mM VSV (p). Following washing, 5 3 10 4 B3.6.4.5. or 2B4 T cell hybridomas were added for 24 h, at which time supernatants were collected for IL-2 measurement using the indicator cell line CT.4R. (B) Purified fusion protein (1 mg/well) was coated directly to wells overnight at 4°C. OVA was added to these wells (F), or to control wells with no fusion protein (h), for 2 h. After washing, 10 5 B3 CTL were added to each well. As a positive control, B3 CTL were incubated in the continuous presence of 1mM OVA which allows for direct presentation between the CTL in the culture. After 24 h, the supernatants were collected and IL-3/GM-CSF production was determined using the indicator cell line, FDC.P1. Data are expressed as a percentage of the positive control (B3 1 1 mM OVA). (C) 5 3 10 4 B3.6.4.5. cells were added to 96-well flat-bottom plates in the presence of OVA (h) or 0.5 mg purified fusion protein loaded with OVA (F). Following 24 h, supernatants were collected for IL-2 measurement. IL-2 was measured using the indicator cell line CT.4R.

MHC–IgG FUSION PROTEIN INDUCES T CELL RESPONSES

FIG. 5. Soluble fusion protein is cross-linked by FcR 1 spleen cells. 5 3 10 4 B3.6.4.5. cells were incubated for 24 h with 2 3 10 5 irradiated DBA/1 spleen cells and medium (p), 250 ng OVA (z), or0.5 mg fusion protein loaded with 250 ng OVA (■) in the presence or absence of 25% 2.4G2-containing culture supernatant. Supernatants were collected after 24 h and IL-2 was measured using the indicator cell line, CT.4R.

sentation from one cell to the other. Under these conditions, OVA alone minimally activated the B3.6.4.5. cells, while OVA loaded into the fusion protein stimulated well (Fig. 4C). Thus, the fusion protein was able to directly stimulate B3.6.4.5. cells in a peptide-specific, MHC-restricted manner. H-2K b/IgG1 fusion protein can activate T cells following cross-linking by FcR 1 cells. Anti-TCR mAbs can activate T cells in vivo. This activation requires multivalent cross-linking of the mAb by FcR 1 accessory cells, as F(ab9) 2 fragments, or mAbs which do not bind FcR, fail to induce activation. With this in mind, the fusion protein was designed to include an IgG1 Fc region. To determine whether these Fc domains conferred the ability to cross-link FcR, B3.6.4.5. cells were incubated with allogeneic DBA/1 spleen cells incapable of directly presenting OVA. OVA-loaded fusion protein, in the absence of FcR 1 cells, failed to stimulate B3.6.4.5. cells above the level observed with OVA alone (data not shown). However, addition of FcR 1 DBA/1 spleen cells resulted in IL-2 secretion (Fig. 5). This activation could be inhibited by the anti-FcR mAb 2.4G2, suggesting that the fusion protein could be cross-linked by FcR 1 cells, resulting in delivery of an activation signal to the T cell. We were unable to prove that the effect of 2.4G2 was specific because an irrelevant isotype-matched mAb would bind FcR via the Fc portion and thereby also block binding of the fusion protein to FcR. Soluble H-2K b/IgG1 fusion protein activates T cells in vivo. To determine the ability of the fusion protein

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to induce in vivo T cell activation, OVA-loaded fusion protein was administered to OT-1 mice transgenic for an OVA-specific, H-2K b-restricted TCR. Mice were injected iv with 4 mg of fusion protein loaded with 300, 30, or 3 ng of OVA. Controls were injected with OVA alone or with fusion protein not loaded with exogenous peptide. The next day, mice were sacrificed and splenic T cells were analyzed by FCM for expression of the T cell very early activation marker CD69 (47). At a dose of 300 ng of OVA, most T cells were CD69 positive, both in mice injected with OVA alone and in mice injected with OVA-loaded fusion protein (Figs. 6A and 6D). However, at lower doses of OVA, only mice injected with OVA-loaded fusion protein demonstrated significant numbers of activated T cells. At a dose of 30 ng of OVA, mice receiving OVA alone had minimal numbers of activated T cells (2%; Fig. 6E). In contrast, half of the T cells of mice injected with OVA-loaded fusion protein expressed CD69 (Fig. 6B). Even at a 10-fold lower dose of OVA (3 ng), 30% of the T cells of mice injected with OVA-loaded fusion protein expressed CD69 (Fig. 6C). These findings were repeated in three groups of mice with similar findings (data not shown). Fusion protein not loaded with exogenous peptide induced a variable amount of CD69 expression (Figs. 6G and 6H) ranging from none in some mice (data not shown) up to 17%, but in no instances approached the percentages observed when the fusion protein was loaded with OVA. There was no change in the expression of CD25, CD44, CD45, or CD95, nor were anti-OVA CTL detected (data not shown). DISCUSSION The present study demonstrates, for the first time, that a soluble class I–Ig fusion protein can deliver activation signals to T cells. This is also the first demonstration of in vivo T cell activation with a soluble class I molecule in vivo. Similar fusion protein molecules have only been reported to inhibit T cell responses in vitro (28, 34), although it is likely that they would also be found to induce T cell activation under conditions similar to those tested in the present study. The fusion protein was designed with an IgG1 Fc moiety in order to confer three properties to the resultant molecule. First, the IgG CH2 and CH3 domains provide interchain disulfide bonds, resulting in a divalent molecule which can more effectively promote T cell activation. Biochemical analysis confirmed that the majority of the fusion protein molecules exist as a homodimer. The degree of valence required for T cell activation is still controversial. Triggering of T cells with anti-TCR mAb Fab fragments has been reported (48); however, multivalent cross-linking has been found to be essential in most studies and can promote more effective activation than divalent cross-linking. In a recent study by Abastado et al., soluble H-2K d

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FIG. 6. Fusion protein activates T cells in vivo. OT-1 transgenic mice were injected iv with 4 mg of fusion protein loaded with OVA (A–C), OVA alone (D–F), or fusion protein alone (G, H). Twenty-four hours later, splenocytes were removed and analyzed by flow cytometry for the expression of CD69 on CD8 1 T cells. Percentage of CD8 1 T cells expressing CD69 are indicated in the upper right quadrant.

molecules made divalent with anti-H-2K d antibody were sufficient to induce T cell activation in vitro; however, higher order aggregates were more effective (30). In the present study, soluble, divalent fusion protein did not induce T cell activation. T cell activation required multivalent cross-linking, either by anti-IgG1 antibodies or by FcR 1 cells. The difference in the two studies may relate to differing properties of the two class I molecules, differences in the T cell hybridomas used, or to the presence of the IgG moiety in the fusion protein. The second property conferred by the IgG1 moieties is the ability to bind FcR. FcR binding promotes multivalent cross-linking of TCR. In vitro studies demonstrated that soluble fusion protein bound FcR 1 DBA/1 spleen cells and that this was necessary for T cell activation. Studies with anti-TCR mAbs demonstrate that FcR 1 cells can provide multivalent cross-linking in vivo. For example, anti-CD3 mAbs that bind FcR induce in vivo T cell activation; however, F(ab9) 2 fragments of such mAbs (21), or mAbs of an isotype that does not bind FcR (18), fail to activate T cells. Thus, the T cell activation observed in vivo following iv injection of OVA-loaded fusion protein was likely a result of this FcR-mediated cross-linking. The low levels of activa-

tion observed with fusion protein alone might be due to a low-affinity interaction of T cells with the fusion protein in these transgenic mice, since they are H-2K brestricted. Cross-linking by FcR-bearing accessory cells can also allow delivery of costimulatory signals necessary for full T cell activation. While the present study did not directly address the ability of the fusion protein to promote costimulatory signals by FcR 1 cells, other recent studies using anti-TCR mAbs suggest that this can occur. In a recent study in mice comparing the in vivo effects of whole and F(ab9) 2 fragments of anti-CD3 mAb (20), whole mAb primed T cells to subsequent in vitro stimulation. In contrast, F(ab9) 2 fragments induced T cell hyporesponsiveness reflected in a reduced secretion of IL-2 and IFN-g upon subsequent in vitro stimulation. These observations suggest that bridging of T cells to FcR 1 cells can promote costimulation. Soluble class I molecules lacking a cross-linking moiety may be responsible for the observed beneficial effects of blood transfusions on organ allograft survival in humans (49). While a fusion protein similar to that described here was found to inhibit alloreactive CTL function in vitro (28), this might have been due to steric blocking of TCR engagement, rather than anergy in-

MHC–IgG FUSION PROTEIN INDUCES T CELL RESPONSES

duction. The fusion protein described in the present study did not block alloantigen-reactive CTL in vitro (data not shown). A final consideration in designing the fusion protein with an Fc moiety was the possibility that the Fc might prolong the in vivo half-life, as has been demonstrated with mAbs. For example, the anti-murine CD3 mAb 2C11 has a half-life in C57BL/10 mice of 2 weeks, while F(ab9)2 fragments of the mAb have a half-life of 2 days (31). The fusion protein itself is unlikely to induce a strong humoral response since the only potential nonself-epitopes include the loaded peptide and the novel epitope created by the fusion between the MHC and the hinge of the IgG. Indeed, following a single ip injection of 200 mg, the fusion protein could be detected in the serum of mice for at least 2 weeks, although the peptide dissociates within 5 h at physiologic temperatures (data not shown). Minimizing this rapid dissociation of the peptide is likely to be critical for inducing effective in vivo T cell responses. In a recent study, L cells transfected to express cell surface H-2K d with a covalently attached tumor peptide prevented tumor growth; however, no effect was seen when surface H-2K d was exogenously loaded with tumor peptide without covalent attachment (50). While the in vivo activation observed was incomplete, it may be possible to further optimize such proteins to enhance signal delivery by stabilizing the peptide in the MHC groove or by further increasing the valency of the molecule. Stabilization of the peptide in the groove of the class I moiety could be accomplished by covalently attaching the peptide via a short linker (50) or by chemical coupling, for instance, by photoaffinity labeling (51). The valency and resultant crosslinking could be increased by using an IgM Fc moiety. Studies to address these possibilities are presently under way. In summary, the studies described here demonstrate that a soluble, divalent MHC class I/IgG1 fusion protein molecule can deliver T cell activation signals in an antigen-specific, MHC-restricted manner, both in vitro and in vivo. Such a fusion protein might be useful to study peptide–MHC interactions. While the fusion protein did not promote full T cell activation or T cell immunity, further manipulations may allow for boosting immunity in class I-restricted T-cell-mediated responses, such as to certain viral pathogens and tumors which evade immune recognition by downregulating class I expression.

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ACKNOWLEDGMENTS 28. We thank Drs. S. Thornton, M. Rothenberg, and R. Colbert for helpful discussions and critical review of the manuscript.

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